MCB 3201 FINAL

Positive selection

type of selective pressure where the ratio of non-synonymous substitutions to synonymous substitutions for a given gene is greater than 1 (dN/dS>1), indicating that certain mutations changing the amino acid composition are selected for. Also known as adaptive evolution.

The zinc-finger domain

the most common DNA binding domain of the human genome •DNA-binding folds organized around one or more stabilizing zinc ions •A single protein will typically contain several zinc fingers, each domain recognizing 2-3bp •Each finger inserts its alpha helix end into the major groove

G protein-coupled receptors (GPCRs)

the most numerous class of receptors found in organisms from yeast to human

H3K9 Methylation

the most studied of histone modifications to date •associated with silent chromatin •HMT for H3K9 was the first to be identified: SUV39H1 (Rea at el., 2000) •H3K9me2 and me3 create binding site for HP1 through its chromodomain •realization it was a methyltransferase through homology to plant methyltransferase

primary (RNA) transcript

the original unmodified RNA product corresponding to a transcription unit

Centromeres are demarcated by

the presence of CENP-A nucleosomes

Strategies for co-opting signaling pathways in development of cancer:

- Overexpression of receptor, Ras GTPase, or MAPK component - Inactivation of negative regulator of MAPK pathway (phosphatase) - Activating mutations in receptor or pathway component

centromere

A constricted region of a chromosome that includes the site of attachment (the kinetochore) to the mitotic or meiotic spindle. - It consists of unique DNA sequences and proteins not found anywhere else in the chromosome.

duplication

A copy of the same sequence generated through errors in replication or recombination

nucleolus

A discrete region of the nucleus where ribosomes are produced.

chromatin is composed of

DNA, PROTEIN and RNA

What factors are involved in promoter-proximal Pausing?

DSIF (DRB Sensitivity Inducing Factor - Spt5,Spt4) NELF (Negative ELongation Factor - 4 subunits) 1) These factors associate with paused Pol II 2) RNAi depletion of NELF or DSIF reduces pausing.

Splicing Is temporally and functionally coupled with transcription

Ordered exit of exons/introns dictates exon pairings

rate-limiting step in transcription

Pausing is a rate-limiting step in transcription after initiation and promoter clearance. • It occurs from ~+20-+50bp downstream of the TSS)

Mechanism of RNA surveillance

TRAMP complex recognizes aberrant RNA - RNA binding and helicase activities • Tramp complex includes poly-A polymerase (PAP) that adds oligo(A) tail - a short poly(A) tail, generally referring to a stretch of less than 15 A's • Substrates for TRAMP-exosome degradation include unspliced or aberrantly spliced pre-mRNAs and improperly terminated RNA Pol II transcripts lacking a poly(A) tail. • Mechanism of recognition by TRAMP not completely clear

Quality Control of mRNA Translation Is Performed by Cytoplasmic Surveillance Systems

TRAMP is part of a nuclear RNA surveillance system. There Nonsense-mediated decay (NMD) tare also cytoplasmic surveillance mechanisms that are coupled to translation.

Regulated recruitment of Trithorax (TRX) / MLL Complex

TRX /MLL can be recuited by: a. A DNA binding domain specific for non-methylated CpGs b. Transcription factors c. ncRNAs d. Binding to its product - H4K4me3

Average human gene structure

The average human gene is 27 kb long and has nine exons, usually comprising two longer exons at each end and seven internal exons

abundance

The average number of mRNA molecules per cell.

DNA microarray technology and whole transcriptome sequencing

allows a snapshot to be taken of the expression of the entire genome in a cell. - for example the differences in expression between a normal cell and a cancer cell can be analyzed using microarrays

When DNA is wrapped around histone octamers to form nucleosomes it becomes: Underwound Over wound

Underwound

unequal crossing over (nonreciprocal recombination)

Unequal crossing over results from an error in pairing and crossing over in which nonequivalent sites are involved in a recombination event. - It produces one recombinant with a deletion of material and one with a duplication.

Cellular hormone receptors have common structure

Variable region, DNA-binding domain, Ligand binding domain

Mechanisms of miRNA mediated gene silencing

Various partners with the RISC complex mediate translation repression or disruption of mRNA closed loop

Pervasive transcription generates a variety of potentially regulatory RNA

Vast tracts of the eukaryotic genome are transcribed - encode estimates ~70% Many RNAs overlap - complete overlap - 3'-overlap - 5'- overlap

Termination factors mimic

amino-acyl tRNAs but are proteins - The eukaryotic termination factor eRF1 has a structure that mimics tRNA

Somatic genes

amount of gene product is influenced by the number of copies of a gene: -haploinsufficiency -excessive gene dosage -> lethality (e.g. trisomies) -aneuploidy and cancer

thalassemias

an inherited blood disorder in which the body makes an abnormal form of hemoglobin. - caused by various deletions that eliminate α- or β-globin genes. - The severity of the disease depends on the individual deletion.

Transcriptional interference (TI) occurs when

an overlapping transcript on the same or opposite strand prevents transcription of another gene.

The role of TRAMP

and the exosome in degrading aberrant nuclear RNAs.

The five snRNPs involved in splicing

are U1, U2, U4, U5, and U6. - named for the snRNA within it. All the snRNPs except U6 contain a conserved sequence that binds the spliceosomal proteins.

Short-interfering RNA (siRNAs)

are complementary to viruses and transposable elements

Poly-A nucleases

are distributive nucleases that chew away at the poly-A tail

Bromodomains

are present within subunits of acetyltransferase and chromatin remodeler complexes are protein motifs that bind acetyl lysines. A bromodomain consists of a cluster of 4 alpha-helices with an acetylbinding pocket at one end. Shown in Gcn5 bound to an H4K16Ac peptide

CTD modifications

are reversible and change during the transcription cycle Main ones are: • Ser-5 is Phosphorylated by Cdk7 (TFIIH) and Cdk8 (Mediator) - early elongation • Ser-7 is phosphorylated by Cdk7 - Occurs during early elongation • Ser-2 is phosphorylated by Cdk9 (PTEFb) - occurs at same time as pause escape

ARE elements (A-rich sequences)

are typically destabilizing.

Recognition of a termination codon

as premature involves unusual 3′ UTR structure or length in many organisms and the presence of downstream exon junction complexes (EJC) in mammals.

Signaling pathways are often misregulated in

cancer

exosome

catalyzes 3′ to 5′ mRNA digestion, is a processive nuclease

Enhancers contain

disease-associated SNPs • Phenotypic traits are not only associated with coding regions • Transcription regulation through enhancers is important for normal and disease phenotype.

Signaling molecules or ligands came in

diverse categories

Alternative splicing increases

diversity of proteins

Intrinsic terminators

do not require additional factors • Intrinsic terminators are common in bacteria and RNAP III genes • They form a stem loop in the RNA transcript • Downstream of stem loop there is a poly(U) stretch in the RNA • DNA/RNA hybrid is destabilized and the polymerase is released

Enhancers produce bidirectional transcription: enhancer RNAs

eRNAs • Short transcripts (< 1kb) • Non-polyadenylated • Rapidly degraded - (difficult to detect Many hypotheses about the roles of enhancer transcription and eRNAs, but function remains largely unknown

Scaffolding proteins can increase

efficiency and specificity of signaling

CENP-A is

essential for centromere activity sufficient for centromere activity

T/F After decapping or deadenylation, an endonuclease efficiently degrades the RNA.

false

T/F Binding of the TRAMP complex to histone 3'-ends allows their degradation to be coordinated with the cell-cycle.

false

T/F Electron Spectroscopy Imaging (ESI) demonstrated the existence of 30nm fibers in intact nuclei

false

T/F H3K4me1, H3K27ac, and Insulator binding are all common characteristic of active enhancers

false

T/F Histone H2A-H2B dimers carry much of the epigenetic information

false

T/F In Drosophila, one X chromosome is inactivated in females

false

T/F The "histone code" has the same predictive power as the genetic code

false

T/F The Spliceosome is held together by RNA interactions and catalysis is performed by the proteins

false

Chiasma

formation can result in the generation of recombinants caused by crossing over between 2 of the chromatids

Processed pseudogenes

forms by reverse transcription and insertion • No regulatory sequences • No introns • Not transcribed

How can the min size of the proteome estimated?

from the number of types of genes

Homologous sequences

genes/sequences that are evolutionarily related.

Oligosaccharides:

glyco-lipids, beta-glucan

Highly repetitive DNA (or satellite DNA)

has a very short repeating sequence and no coding function - occurs in large blocks that can have distinct physical properties.

miRNAs are loaded

into the RISC complex for targeting mRNAs

Nucleosome assembly requires...

histone chaperones •Nucleosome assembly is NOT spontaneous •Factors identified that interact with either H3-H4 tetramers or H2A-H2B dimers and and escort them to sites of nucleosome assembly •they prevent histones from interacting with DNA unproductively

Methylation of H3 and H4 lysines is mediated by

histone methyltransferases (HMTs) - Nearly all share a SET domain - SET= Su(var)3-9, Enhancer of Zeste, Trithorax - These are SET domain HMTs for H3K9, H3K27, H3K4

Cohesin

holds sister chromatids together until anaphase onset

achiasmate non-disjunction

homologs fail to pair and segregate to the same pole

Polymorphic locus

if two or more alleles are present at a frequency >1% in a population

What is the role of N-tails in compaction?

in vitro studies have shown that the presence of N-termini tails compact the 10-nm fiber Positively charged tails contribute to compaction by interacting with linker DNA and histone octamer e.g. histone H4 tail and HFD of H2A

Extension of heterochromatin:

inactivates genes.

Determine nucleosome positions experimentally using....

indirect labeling or using genome-wide approaches.

Histone H1

induces compaction

lariat

is a circular intermediate in RNA splicing created by a 5 -2 bond

XRN1

is a processive 5' to 3' exonuclease that degrades De-capped RNA

Sedimentation coefficient (S)

is dependent on size and density of particles particle velocity/centrifugal force applied

Decay rate

is not always random in the cell: It can be encoded in specific sequences within each mRNA.

Linker DNA

is the region of 8 to 114 bp that is susceptible to early cleavage by nucleases

Specific cis-elements in an mRNA affect

its rate of degradation

Pausing is released by

kinase action of PTEFb (cdk9)

Lack of pausing in c.elegans and yeast is coincident with

lack of discernable NELF homolog

CTD is a

large unstructured domain of Pol II. • Made of many Heptad repeats of the consensus sequence YSPTSPS. • Number of repeats scales with complexity of genomes (i.e. Humans > Drosophila > Yeast) • Degree of consensus varies between organisms

miRNAs are processed from

larger primary micro-RNA transcripts

Where are valuable markers shown in?

linkage studies

Imprinting Control Cegion (ICR)

locus responsible for specifying expression pattern

Differential mRNA stability is an important contributor to

mRNA abundance and therefore the spectrum of proteins made in a cell

An unknown number of mRNAs are targets for degradation or translational repression by

microRNAs (miRNAs).

Termination is coupled to

modifications of Pol II C-Terminal Domain

3'-end formation is coupled to

modifications ofPol II C-Terminal Domain

Do exons change order in mRNA as in DNA?

no

Difference between lipid soluble hormone signaling and typical cell-surface receptor pathway:

no second messenger/effector pathway => no amplification

Core promoter

shortest DNA sequence at which RNA polymerase can initiate transcription. No regulation. Only GTFs present.

reverse genetics, strategy of knocking down gene depends on

organism

Recruitment of HATs and HDACs to

regulate gene activity often part of large complexes that have additional activities

Enhancers (distal elements)

regulate transcription in cis from up to thousands of base pairs upstream or downstream of promoter through recruitment of activators and the action of co-regulators

DNA binding domains are generally

rich in Lysine and Arginine residues (+) and Tyrosines and Serines (H bond donors)

Unequal crossing over can lead to ...

spreading of identical repeats in cis Unequal recombination allows one particular repeating unit to occupy the entire cluster. The numbers indicate the length of the repeating unit at each stage.

Ribonucleases differ in their

substrate preference and mode of attack.

Structure of TBP binding to the minor groove of a TATA promoter element

• TBP binds the minor groove of TATA box or other sequence • Shaped as a saddle it bends the DNA of 80o • Bending allows formation of surface for binding by TFIIB and other TFs

How does compaction occur and why is it necessary?

•Compaction results from bending and kinking of 10nm fibers NOT from organized solenoid formation •Chromatin compaction is necessary to organize DNA so that it can further compact in mitosis to allow segregation

Negative selection

type of selective pressure where the ratio of non-synonymous substitutions to synonymous substitutions for a given gene is less than 1 (dN/dS<1), indicating that selection acts against changes within this protein. Also known as purifying selection.

siRNA pair

with target. May be endogenous or exogenous

Allostery=

when protein activity it's modulated by change in its conformation

If a DNA stretch has equal properties the nucleosomes

will not be positioned

What are regulatory sequences?

within genes for regulation Regulatory sequences are sites in the gene to which regulatory proteins bind

% of genes show signs of imprinting

~1-2

Regulation of translation elongation through codon usage

• Proper positioning of tRNA in A-site allows for peptidyl transfer • Elongation can be regulated: - Codon usage, cell stress - Evidenced by ribosomal footprinting experiments

Determination of all mRNA half lives in a single experiment

• Pulse with 4sU • 4sU analog can be biotinylated • Biotinylated RNAs can be isolated by chromatography with strepavidin beads • Isolated RNA is newly synthesized • Comparing ratios of newly synthesized and pre-existing RNAs can give rates of synthesis and degradation

Types of RNA transcribed by Eukaryotic RNA Polymerases

• RNA polymerase I synthesizes >50% of all rRNA (18S and 28S subunits) in the nucleolus. • RNA polymerase II synthesizes mRNA and most small RNAs. • RNA polymerase III synthesizes 5S rRNA, tRNAs, some small RNAs, snRNAs.

The pre-initiation complex (PIC) components are:

• RNA polymerase II core enzyme (purple) • General transcription factors (blue, green, red) • Promoter+gene The PIC complex is competent to initiate transcription in vitro. In vivo transcription requires additional protein complexes

Cellular response is controlled by expression of receptors

• Radiolabeled progesterone added to cells with or without PR expression • Only cells with PR expression accumulate (and respond to ) progesterone.

Diverse strategies of IRES-mediated initiation

• Recruit complete initiation machinery • Recruit partial initiation machinery • Bypass all canonical initiation machinery

Termination of translation

• Release factors bind termination codons • Release factors hydrolyze the polypeptide-tRNA linkage - peptide release • RRF binds and displaces final tRNA • Translocation by eEF2 dissociates ribosome The RF (release factor) terminates translation by releasing the protein chain.

Two steps Termination of translation

• Release of polypeptide chain • Dissociation of final tRNA, mRNA, and ribosomal subunits

How are genes and other sequences distributed in the genome?

• Repeated sequences (present in more than one copy) account for >50% of the human genome. • The great bulk of repeated sequences consists of copies of nonfunctional transposons. • There are many duplications of large chromosome regions.

Transmittance of histone modifications through generations

• Requires establishment of histone modifications • Maintenance may involve an enzyme that binds it's own substrate allowing propagation and possibly spreading. • Maintenance may involve 'crosstalk' with other pathways. - DNA methyaltion and H3K9me2 are reinforcing

Why is gene regulation important?

• Response to environment - Hormones - Stress - Immune response • Development / differentiation • Cell proliferation - Cell growth - Cell cycle progression • Homeostasis / metabolism

Genes for rRNA Form Tandem Repeats Including an Invariant Transcription Unit

• Ribosomal RNA is encoded by a large number of identical genes that are tandemly repeated to form one or more clusters. • Each rDNA cluster is organized so that transcription units alternate with nontranscribed spacers. • The genes in an rDNA cluster all have an identical sequence A tandem gene cluster has an alternation of transcription unit and nontranscribed spacer and generates a circular restriction map.

Elongation regulation: 5'- ribosome accumulation during translation

• Ribosome footprinting reveals that Ribosomes accumulate at 5'-end. • 5'-accumulation exacerbated during heat stress • Not clear whether these are paused or premature termination intermediates

Direct cytokine regulation of JAK/STAT pathway

• STAT= Signal Transducer and Activtor of Transcription • Cytokine receptors activate Janus Kinases (JAKs) in cytosol. • STAT proteins acre recruited and activated through phosphorylation by JAKs • STAT proteins can travel directly to the nucleus to activate target genes. - no signal amplification

Drivers of birth and advances in genomics

• Shear will / exploration - Impetus for original human genome sequencing project • Benefits for molecular medicine and understanding human evolution - Promise of predicting disease risk and developing therapies. • Advances in sequencing technologies - Second generation or next generation sequencing - Third generation sequencing

Activation of cytokine receptors

• Similar to RTK activation except kinases are in separate complexes that bind receptor. • Ligand-induced dimerization results in activating phosphorylation of cytosolic receptor kinases (step 2) • Activated kinases phosphorylate tyrosines on receptor(step 3)

Alternative splicing connected to transcription rate

• Slow elongation rate favors inclusion of weak or proximal splice sites • Fast elongation rate favors inclusion of strong or distal splice sites

Newly Synthesized RNAs Are Checked for Defects via a

• Some RNAs are problematic before becoming mRNA and must be removed before exiting the nucleus. • Aberrant nuclear RNAs are identified and destroyed by an RNA surveillance system. • The nuclear exosome functions both in the processing of normal substrate RNAs and in the destruction of aberrant RNAs. • The yeast TRAMP complex recruits the exosome to aberrant RNAs and facilitates its 3ʹ to 5ʹ exonuclease activity.

Coupling of transcription and export through nuclear pore

• Some nuclear pore subunits are free in nucleus. • Bind components of transcription machinery • Directed movement or diffusion bring gene locus to the pore (mechnaism unknown). • Facilitates docking and transport through the pore. i.e. rapid nuclear export immediately following transcription

How is chromatin set up for TF binding?

• Some regions have low intrinsic nucleosome binding. - TF binding sites more accessible • Replication disrupts chromatin and allows access • TF cooperativity • Pioneer factors prime the region by opening the chromatin

Non-spliced mRNAs (e.g. most yeast genes

• Somehow sensitive to remaining length of mRNA • Possibly through sensing PABP at Poly- A tail

RNA transport

• Specific factors called receptors/transporters and adapters mediate transport of RNAs through the nuclear pore. • Transport mechanism is distinct for RNA subtypes • And yes, it begins as a transcription coupled process

Meiosis in human female

• Starts during FETAL DEVELOPMENT • oocytes enter a protracted arrest after they initiate homologous recombination (prophase I) • resumption of meiosis occurs years later in mature woman before ovulation (completion of MI, arrest again in MII) • MII completed after FERTILIZATION

Histone mRNAs have unique 3'-ends

• Stem loop binds Stem loop binding protein (SLBP) • SLBP and U7 snRN stimulate cleavage of 3'-end • SLBP coordinates histone mRNA translation and degradation with cell cycle.

Measuring mRNA decay rates

• Stop transcription: - Pol II temperature sensitive mutant - Pol II inhibitor: alpha-amanitin • Measure RNA abundance in a time series - RT-qPCR - Southern blots (hybridization) • Calculate t1/2 Half lives range from 3 to 100 minutes in yeast; Minutes to hours in higher eukaryotes

What is Genomics??

• Study of or determination of the structure, function, and evolution of genomes • Study of genomic causes or responses to development or disease. • Utilizes DNA sequencing, functional genomics assays, and computational biology

What is the baseline used to determine if nonsynoymous changes occur with higher, lower, or similar frequency?

• Synonymous substitutions accumulate ~10× faster than nonsynonymous substitutions.

Signaling Via Cell-Surface Receptors (I)

• Synthesis and release of signaling molecules by signaling cells • Transport of signaling molecules to the target cells • Binding of the signaling molecule with a specific receptor protein on the membrane leading to activation

siRNAs result in

transcript cleavage.

Gases:

NO, ethylene etc

GRO-seq studies reveal Pol II pausing is common in

higher eukaryotes

The capping process takes place during

transcription and may be important for release from pausing of transcription.

What is a molecular clock

A constant rate of sequence divergence is a molecular clock

Outron

'intron' or sequence removed by transcplicing of SL.

Piwi-interacting RNA (piRNA)

(26-31 bases) Regulate gene expression in germ cells and act to silence transposable elements.

Mechanisms of transcription factor repression

(A) Sequestration of activator in cytoplasm (B) Masking of activator Figure 28.F05: A repressor may control transcription various ways (C) 'activation' of sequestered repressor (D) Competition

Mechanisms of transcription factor activation

(A) Synthesis of the activator (B) Activation by post-translational modification (C) Ligand binding (D) Translocation to the nucleus (E) Change in binding partner / Cooperative binding to DNA (F) Cleavage of inactive precursor

Antisense transcription can both repress and activate sense gene transcription

(A) antisense transcription stimulates repression through siRNA-like mechanisms (B) antisense transcription results in chromatin remodeling that de-represses or primes the chromatin for sense transcription

Peptides & protein hormones

(most abundant): e.g., thyrotropin, Gonadotropin releasing hormone (GnRH), growth hormone (GH), prolactn (PRL), insulin etc.

The GU-AG rule

(originally called the GT-AG rule in terms of DNA sequence) describes the requirement for these constant dinucleotides at the first two and last two positions of introns in premRNAs. Splice sites, including the branch site, are more well defined in yeast than multicellular eukaryotes

Composite profile (average signal) of GRO-seq reads relative to all TSSs reveals promoter proximal pausing and more

+50 from TSS Many genes have Paused polymerases -250 from TSS Many genes have an upstream, divergent engaged polymerase (covered soon)

iron-response element (IRE)

- A cis sequence found in certain mRNAs whose stability or translation is regulated by cellular iron concentration.

Autocrine signaling:

- Cell responds to own signals - Examples: Growth factors => stimulate cell growth and proliferaLon • Especially prominent mechanism used by cancerous cells to promote tumor growth

Signaling by plasma membrane-attached proteins:

- Direct cell-to-cell signaling - Ligand and receptor are both part of integral membrane proteins - Examples: • Epidermal Growth Factor (EGF)

Multistep processing leads to final small RNA loading into repressive complex (RISC for miRNA and siRNA, RITS for piRNAs)

- Drosha does preprocessing in the nucleus - Dicer does final processing in cytosol - Small RNAs loading into Argonaute (Ago) proteins within RISC complex

There are a variety of cell signaling mechanisms

- Endocrine - Paracrine - Autocrine - Membrane bound proteins

Definition of CpG island (CGI):

- Greater than 50% GC content - Larger than 200bp - CpG frequency (observed/expected) >0.6

Characterics of Signals: specific and rapid

- Have specifcity: can only be detected by the molecular machinery designed for the detection - easily produced and mobilized => generally small molecules - altered and/or destroyed easily - often involved second messenger systems that act intra- or inter-cellularly

small nuclear RNA (snRNA)

- One of many small RNA species confined to the nucleus; several of them are involved in splicing or other RNA processing reactions.

Signaling Via Cell-Surface Receptors (II)

- Initiating one or more intracellular signal-transduction pathways initiated by the activated receptor - specific change in cellular response (cellular function, metabolic change or gene expression) - removal of the signal to terminate the cellular response-feedback

Many proteins bind RNA in nucleus; Interactions important for:

- Processing - capping, splicing, 3'-end formation - Surveillance - Nuclear pore targeting and transport

Characteristics of ligands-receptors interactions

- Receptor should possess high affinity: higher than 10^-8 M (10 nM) - Receptor should have high specificity to prevent cross binding with un-related ligands - ligand- receptor binding should be reversible - Receptor should have a tissue distribution appropriate to its action (tissue specificity) - receptor binding should be correlated with some biological effects

Paracrine signaling

- Short range signaling => Signaling molecules target only those in the immediate vicinity - Examples: • Neurotransmi^ers: between nerve cells or nerve-muscle cell junctions • Growth factors

Signalling responses can be:

- Short term changes in cytosol - Long term changes in gene expression

pre-mRNA

- The nuclear transcript that is processed by modification and splicing to give an mRNA. May be nascent, may be post-transcriptional

Maintenance of DNA methylation by methyl-C binding protein UHRF1

- UHRF1 binds hemi-methylated DNA after replication - Recruits Dnmt1 DNA methyltransferase - Also interacts with histone modifying enzyme and can help with propagation of chromatin states

Gene "switched on"

- active (open) chromatin - unmethylated cytosines - acetylated histones

Similarities and differences between miRNA and siRNAs

- any dsRNA can act as an siRNA - siRNAs are typically perfectly base paired (unlike miRNA) - Can be made in the nucleus - siRNA target recognition usually results in cleavage

Some pathways are amplification cascades

- cAMP - MAPK

Introns can be detected when genes are compared with their mRNA by:

- gel electroporesis, - restriction mapping, - sequencing, - or EM.

Lysine Acetylation

- removing (+) charge - mediated by histone acetyl transferases - remove histone deacetylation - DNA is more accessible and creates site called bromodomains that bind acetylation - HAT = contains bromodomains ---> add more acetylation in the region important in DNA replication ---> modifications can be passed through the generation level

Endocrine signaling:

- signaling molecule (often a hormone) is synthesized by signaling cells - Signaling molecule travels through circulatory system to target distant cells - Examples: • Hormones: - Insulin secreted by the pancreas - Epinephrine secreted by the adrenal gland

Gene "switched off"

- silent (condensed) chromatin - methylated cytosines (red circles) - Deacetylated histones

Gene expression is influenced by

-Chromatin environment -Position within nucleus -Number of alleles/copies

Genome stability

-DNA repair/apoptosis -accurate segregation -transcriptional repression of transposons and satellites -nuclear organization

How can we assay for silencing?

-It is of great importance to understand which factors and mechanisms contribute to silencing • -Reporter genes (whose transcription can be easily assayed) can be artificially inserted within HC regions. • -In normal conditions, the reporter gene will be OFF • -One can screen for mutations that will activate transcription of the reporter gene -> identify new regulators of HC

Experimental evidence for non-colinearity of eukaryotic genes

1) DNA and RNA extracted 2) Denaturation 3) Hybridization 4) Electron Microscope (EM) observation Conclusion: most genes consist of coding and non-coding sequences

Intron Splicing

1) Goal: stitch together all the exons of a gene and eliminate intron sequences 2) Takes place in the nucleus 3) Carried out by a very large and complex molecular machinery called spliceosome

Effect of acetylation of H3 and H4 lysines

1) increase DNA accessibility 2) recruitment of proteins that bind to acetylated lysines (e.g. bromodomain containing pts)

How do DNA binding proteins gain access to DNA which is wrapped in a nucleosome?

1) spontaneous (no energy required) 2) non-spontaneous (ATP required)

Trans-esterfication reactions in splicing

1. 2'-5' link between 2'-OH at branch site and 5'-P of 5'- splice site 1. 3'-OH of exon1 attacks phosphodiester bond at 3'- splice site 1. Lariat released, exons ligated

General themes in regulation of initiation

1. 5'-cap 2. Hairpin in 5'-UTR can inhibit scanning to find start 3. IRES elements allow cap-independent regulation of initiation. 4. Protein or RNA binding sites in UTRs 5. Upstream ORF prevents initiation from downstream ORF 6. Poly-A tail stimulates translation

Complexes required for activation

1. A large complex, the Mediator (which has more than 20 subunits), is needed to activate many Pol II transcribed genes • The Mediator interacts with PolII CTD on one side and with DNAbound activators on the other 2. Histone modifying enzymes (e.g. HATs and HMTases for H3K4) 3. Nucleosome remodeling enzymes that can slide nucleosomes off the promoter (e.g. SWI/SNF) 4. Incorporation of histone variant H3.3 upstream activating sequences or enhancers

Functions of promoter proximal pausing:

1. Allows maintenance of chromatin structure that is permissive to transcription through competing with nucleosomes. 2. Promotes synchrony in activation. 3. Allows coordinated regulation from multiple transcription factors / signalling pathwa pathways 4. Checkpoint for mRNA quality control - e.g. capping

Possible Mechanisms for Enhancer-Promoter targeting

1. Diffusion: Factors bound to both the enhancer and promoter could interact via free diffusion. 2. Assisted Assembly: (blue circles) bind intervening sequences and organize them to bring enhancer and promoter into proximity 3. Factory: RNA polymerase II, interactions brings them into proximity via association with a common transcription ''factory''.

Functions of eukaryotic Initiation Factors:

1. Form pre-initiation complex with 40S subunit and 5'- end of mRNA 2. Mediate binding of pre-initiation complex with MettRNA complex 3. Enable scanning of mRNA for the first AUG 4. Detecting binding of initiator tRNA to AUG start 5. Mediate joining of 60S subunit

Scaffold protein is critical for

1. Rapid signaling 2. Specificity of pathway

Capping complex adds inverted meGTP to 5'-end

1. Removal of terminal phosphate 2. Addition of guanine 3. Methylation of guanine at position 7

Histone H3.3

1. H3.1 and H3.2 in humans differ by a single amino acid but function identically (canonical H3s) 2. H3.1 and H3.2 are deposited in a DNA replication-coupled (RC) manner 3. H3.3 variant: • differs by only four amino acids • synthesized in S, G1, G2 and G0 and in differentiated cells • It does not require DNA replication for deposition • deposited by histone chaperone HIRA • rapidly incorporated into active genes during transcription

cDNA screen to identify receptors

1. Introduce plasmid with cDNAs into cells. 2. Add radiolabled ligand 3. Detect radiolabled cells with autoradiography 4. Isolate radiolabled cells 5. Identify cDNA with PCR

Coordination of transcription through nucleosomes

1. Paf1 binds CTD Ser-2 2. Bre1 adds Ubiquitin to H2B 3. FACT binding and removal of H2A/B is stimulated 4. SAGA removes Ub, and H2A/B can be recycled

What is the function of promoter-proximal pausing?

1. Paused Pol II is important for establishing permissive chromatin - RNAi depletion of NELF results in a decrease of Pol II pausing (and gene expression), with nucleosomes filling into the pause region - This indicates that paused Pol II competes with nucleosomes at genes to maintain their expression 2. Paused Pol II is important for rapid and/or synchronous activation - Levine group generated transgenic flies that express a yellow reporter gene under the control of an embryonic enhancer upstream of the tup promoter (highly paused) and the pnr promoter (not paused) --->The highly paused Tup promoter showed synchronous activation of yellow reporter in embryos --->The non-paused pnr promoter showed asynchronous activation of yellow reporter in embryos 3. Paused Pol II can allow for integration of multiple regulatory signals - Recruitment of a TF2 (triggers pause release) to Gene 1 (lacks TF1) would not allow for an interaction with TF1 (establishes pause) and thus not activate gene expression - Recruitment of a TF2 (triggers pause release) to Gene 2 would allow for an interaction with TF1 (establishes pause) and trigger gene expression 4. Paused Pol II can act as a checkpoint in early elongation - Several studies has shown functional interactions between capping enzymes (CEC) and pausing factors (DSIF). Thus pausing may provide a time window to ensure proper capping, assemply of RNA processing factors (RPF) and productive elongation

Three major classes of RNA

1. Ribosomal RNAs (rRNA) --MOST ABUNDANT 2. tRNAs 3. mRNAs (2-5% of cytoplasmic RNA

Two physiological functions for IRESs

1. Support low levels of translation initiation for cellular IREScontaining mRNAs with highly structured 5'-UTRs (incompatible with efficient scanning). 2. They support robust translation of cellular mRNAs under a variety of physiological conditions such as mitosis, when capdependent translation is compromised

Regulation of TF binding

1. TF binding is regulated by a variety of mechanisms. (e. g., Synthesis of TF or masking the binding activity. 2. Binding seen in vitro does not always reflect binding in vivo situation: • Chromatin matters. 3. Pioneer Factors can bind chromatin, and the inherent ability of their DBDs to target binding sites displayed on the nucleosome surface is the primary determinant of pioneer factor activity.

formation of the early transcribing complex

1. TFIID recruits TFIIB (single polypeptide) 2. Asymmetrical binding contributes to establishing directionality 3. Recruitment of preinitiation complex (PIC) 4. TFIIB's extended loop (beta finger) extends deep into RNAPII enzyme, with its tip near the active site enhancing the formation of the early transcribing complex TFIIB binds to DNA and contacts RNA polymerase near the RNA exit site and at the active center, and orients it on DNA •The TFIIH complex catalyzes the ATP-powered unwinding of the DNA to expose the template strand

Transposable Elements can cause sequence changes

1. Transposition to a new site could affect gene expression: • Can cause deletions, inversions, or movement of sequences to new locations 2. Cross-over can cause rearrangements

PIWI-interacting RNAs (piRNAs) are specialized to silence transposable elements in the germline: Drosophila

1. Transposons and piRNA clusters are active in the germline - create dsRNA 2. Silencing requires Piwi and Ago3 to process each RNA strand into siRNA 3. Active transposons are then targeted for heterochromatin formation

Name two classes of DNA sequences that you expect to be enriched in a chromatin immunoprecipitation sequencing (ChIP-seq) experiment with antibodies for H3K9methylation.

1. _______satellite DNA_____________ 2. ________transposable elements____________

H3K4Me

1. associated with euchromatin (active genes) 2. H3K4me2/me3 is observed at the 5' end of genes in during activation 3. mediated by the Set1, HMT which is recruited by RNA polymerase II 4. H3K4me2/me3 recruit CHD1 (2 chromodomains), aids in the progression of the transcription fork 5. NURF complex mobilizes nucleosomes in active genes in Drosophila, recognizes K4me through a PHD domain

How do HPTMs influence chromatin function?

1. by affecting chromatin STRUCTURE 2. by disrupting the binding of chromatin-binding proteins 3. by creating binding sites for specific proteins

Two main advantages of Electron Spectroscopic Imaging (ESI)

1.it removes electrons that do not interact with specimen producing a dark-field image (no contrasting heavy atoms needed) 2.element-specific maps can be created (e.g. phosphorus and nitrogen maps)

how many SNPs can be used to identify most of the haplotypes in humans?

100,000

Basics of mature RNA

3′ untranslated region (UTR) - The untranslated sequence downstream from the coding region of an mRNA. 5′ untranslated region (UTR) - The untranslated sequence upstream from the coding region of an mRNA stem-loop - A secondary structure that appears in RNAs consisting of a base paired region (stem) and a terminal loop of single-stranded RNA. - Stem and loop can both vary in size.

Percent of human genes, alternatively spliced

60% Up to 80% of the alternative splices change protein sequence, so the proteome has ~50,000 to 60,000 members.

Chemical structure of mRNA cap

A 5′ cap is formed by adding a G to the terminal base of the transcript via a 5′-5′ link. (inverted 7-methyl GTP cap) The cap blocks the 5' end of mRNA and is methylated at several positions.

nonsynonymous mutation

A change in DNA sequence in a coding region that alters the amino acid that is encoded.

synonymous mutation

A change in DNA sequence in a coding region that does not alter the amino acid that is encoded.

RITS (RNA-induced transcriptional silencing)

A complex that uses short single-stranded siRNA to repress heterochromatin transcription. - analogous role to RISC in miRNA silencing

RNA-dependent RNA polymerase (RDRP)

A component of the RITS complex that copies the heterochromatin ncRNA that is then used to silence heterochromatin transcription.

cytoplasmic cap-binding protein

A component of the eukaryotic initiation factor 4F (eIF4F) that binds the 7-methyl guanosine cap at the 5′ end of eukaryotic mRNA.

internal ribosome entry site (IRES)

A eukaryotic messenger RNA sequence that allows a ribosome to initiate polypeptide translation without migrating from the 5′ end - i.e. independent of a 5'-cap. • Often found in viral RNAs • Form diverse secondary structures with low conservation • Makes them difficult to find / study • Form non-conical interactions with initiation machinery • Infrequent: • Only a few well-demonstrated examples • In silico predictions estimate ~600 or greater in the genome

acentric fragment

A fragment of a chromosome (generated by breakage) that lacks a centromere and is lost at cell division.

overlapping gene

A gene in which part of the sequence is found within part of the sequence of another

Interrupted gene

A gene in which the coding sequence is not continuous due to the presence of introns

housekeeping gene

A gene that is expressed in virtually all cells because it provides basic functions needed for sustenance of all cell types (e.g. DNA replication, cytoskeleton, transcription).

Example of contacts between side chains and bases at the major groove

A hydrogen bond donor on a protein is paired with an acceptor in the DNA, while a hydrophobic side chain might establish van der Waals interaction with the methyl group of thymine

Differential Methylated Region (DMR)

A locus that has differential methylation in alleles

mature transcript

A modified RNA transcript. Modification may include the removal of intron sequences and alterations to the 5′ and 3′ ends

Restriction Fragment Length Polymorphism (RFLP)

A point mutation that affects a restriction site is detected by a difference in restriction fragment lengths.

splicing factor

A protein component of the spliceosome that is not part of one of the snRNPs.

Release factor (RF)

A protein required to terminate polypeptide translation to cause release of the completed polypeptide chain and the ribosome from mRNA.

microtubule organizing center (MTOC)

A region from which microtubules emanate. - In animal cells the centrosome is the major microtubule organizing center.

A regulator RNA

A regulator RNA is a small RNA with a single-stranded region that can pair with a single-stranded region in a target RNA.

synteny

A relationship between chromosomal regions of different species where homologous genes occur in the same order There are extensive syntenic relationships between the mouse and human genomes, and most functional genes are in a syntenic region.

endoribonuclease

A ribonuclease that cleaves an RNA at an internal site(s).

exoribonuclease

A ribonuclease that removes terminal ribonucleotides from RNA. - Exonucleases also have specificity for direction (5'-3' or 3'-5').

Intron

A segment of DNA that is transcribed, but later removed from within the transcript by splicing together the sequences (exons) on either side of it.

gene family

A set of genes within a genome that encode related or identical proteins or RNAs. - The members were derived by duplication of an ancestral gene followed by accumulation of changes in sequence between the copies. - Most often the members are related but not identical.

expressed sequence tag (EST)

A short sequenced fragment of a cDNA sequence that can be used to identify an actively expressed gene.

cDNA

A single-stranded DNA complementary to an RNA, synthesized from it by reverse transcription in vitro. the DNA copy of an mRNA

small nucleolar RNA (snoRNA)

A small nuclear RNA that is localized in the nucleolus. Important for rRNA processing

Which of these statements about human female meiosis is incorrect? A) it starts during puberty B) oocytes arrest in a prophase I for several years C) it is highly prone to errors D) it becomes more error prone with increasing age E) each meiosis produces only one viable oocyte

A) it starts during puberty

Many regulatory factors bind the

A-site

Indicate which phase of transcription or associated process is not influenced by the CTD of Pol II. (2 points) A. Transcription factor binding B. RNA processing C. Escape from pausing D. Histone modifications E. Transcription termination F. None of the above (They are all affected)

A. Transcription factor binding

Interaction between which two proteins is critical for maintaining the closed loop structure of mRNA? A. PABPC1 / EIF4E (cytosolic cap binding protein) B. SR proteins / exon junction complex C. Cap binding protein / 40s ribosome D. CCR4-NOT / DCP1

A. PABPC1 / EIF4E (cytosolic cap binding protein)

Which of these characteristics would make you suspicious that a gene is in fact a processed pseudogene? A. it does not contain introns B. it contains a mutated promoter C. it is expressed at high levels D. it contains nonsense mutation

A. it does not contain introns

You carried out a mutational screen in Drosophila using a line that contains the white reporter gene (which encodes for the red eye pigment) inserted in heterochromatin. You identified a suppressor of variegation, or Su(var), fly mutant. This mutant has _______eye color because it causes_________________of the white gene. An example of such Su(var) gene would be a mutation in_______. HP1= heterochromatin protein 1; HAT= histone acetyltransferase A. red; expression; HP1 B. white; expression; HAT C. red; silencing; HAT D. white; silencing; HP1 E. white; silencing; HAT

A. red; expression; HP1

example of nucleosome preferences for certain sequences

AT di-nucleotides bend easily.

Mechanisms of activation

Activators can activate transcription by: •Direct recruitment of the transcription machinery (not RNA Pol directly, but through specific interactions with either Mediator and/or one of the GTFs, e.g. TFIID) •Recruiting histone modifying enzymes (e.g. HATs and HMTases for H3K4) locally •Recruiting nucleosome remodeling enzymes that can slide nucleosomes off the promoter (e.g. SWI/SNF) •Incorporation of histone variants e.g. H3.3

Advantages and disadvantages of Nuclear run-on

Advantages: • Allows quantification of polymerase location within a gene. Disadvantages: • Low throughput • Challenging experiment • Requires use of radioactivity

Identification of Cell-Surface Receptors

Affinity chromatography: isolation of target protein by affinity to immobilized ligand. 1. Immobilized ligand to beads 2. Flow through crude cellular or membrane extract to bind specific receptor 3. Wash away non-specific proteins 4. Elute purified receptor by competing with excess ligand.

How may Gene duplication contributes to genome evolution?

After a globin gene has been duplicated, differences may accumulate between the copies.

alternative splicing

Alternative Splicing Is a Rule, Rather Than an Exception, in Multicellular Eukaryotes Specific exons or exonic sequences may be excluded or included in the mRNA products by using alternative splicing sites. Alternative splicing contributes to structural and functional diversity of gene products

puromycin

An antibiotic that terminates protein synthesis by mimicking a tRNA and becoming linked to the nascent protein chain • Binds A-site • Takes part in peptidyl transfer, but . . . • Not anchored to P-site (no tRNA) • After transfer nascent peptide floats away = termination of translation

synthetic genetic array analysis (SGA)

An automated technique in budding yeast (haploid) whereby a mutant is crossed to an array of approximately 5000 deletion mutants to determine if the mutations interact to cause a synthetic lethal phenotype - Results: Median is ~25 genetic partners per gene. 10% of genetically interacting genes encode polypeptides that physically interact. - Conclusion: organisms are protected against the damaging effects of mutations by built-in redundancy

Dicer

An endonuclease that processes double-stranded precursor RNA to 21 to 23 nucleotide RNAi molecules

Drosha

An endonuclease that processes double-stranded primary RNAs into short, ~70 base pair precursors for Dicer processing

distributive (nuclease)

An enzyme that catalyzes the removal of only one or a few nucleotides before dissociating from the substrate.

processive (nuclease)

An enzyme that remains associated with the substrate while catalyzing the sequential removal of nucleotides.

Histone chaperone

An escort protein that performs a transfer reaction on a histone, such as 1) deposition onto DNA, 2) eviction from DNA, 3) transfer to another chaperone, 4) histone storage for later use

The exon junction complex (EJC) can initiate RNA export mechanisms

Assembles during transcription and splicing • Remains bound to RNA after splicing • Mediates downstream processes The EJC (exon junction complex) is deposited near the splice junction as a consequence of the splicing reaction.

STARR-seq

Assess function with reporter assays - detects all, functional test - element taken out of context i.e. may have been repressive chromatin and thus active

The major mechanism of RNA degradation in cells is initiated by________? A. Decapping B. Deadenylation C. Endonuclease cleavage D. Hairpin formation

B. Deadenylation

Differential codon usage can affect the rate of which stage of translation? (2 point) A. Initiation B. Elongation C. Termination D. None: it does not affect translation

B. Elongation

Which of these nucleosome activities occurs spontaneously? A. Nucleosome sliding B. Nucleosome DNA unwrapping C. Nucleosome DNA buldging D. Nucleosome transfer E. All of the above

B. Nucleosome DNA unwrapping

Which of the following statements is true regarding promoter-proximal pausing? (2 points) A. Pausing primarily results in gene repression. B. Pausing is important for synchronous activation of genes across cell populations. C. Pausing is critical for transcription termination. D. Pausing is the simplest form of transcription regulation. E. Pausing is enhanced by the activity of P-TEFb kinase.

B. Pausing is important for synchronous activation of genes across cell populations

gene A1 AAT-TAC-CCG-TTA gene A2 AAC-TAT-GCG-TTG What is the dN/dS for these two genes? What type of selection are these two genes under? A. dN/dS >1 B. dN/dS < 1 C. dN/dS = 1

B. dN/dS < 1 dN/dS = 1/3 < 1 C. Negative selecton

Which of the following are not part of the nuclear export pathway? (2 point) A. adapters B. mediator C. transporters D. exon junction complex E. nucleoporins F. none of the above

B. mediator

All of the following examples of epigenetic mechanisms, except? (2 points) A. Allelic imprinting B. DNA methylation C. DNA mutation D. Transmittance of histone modifications

C. DNA mutation

What is the major driver of the advances in the field of genomics? (2 point) A. Interest in genomic medicine. B. Sequencing of the human genome. C. Next generation sequencing technologies. D. Interest understanding human evolution

C. Next generation sequencing technologies.

T/F Enhancers are close (within a few kb) to promoters they regulate.

False

Which statement best explains why long-noncoding RNAs (lncRNAs) display a moderate amount of sequence conservation? (2 points) A. The protein is rapidly evolving. B. lncRNAs can tolerate just as much variation as introns. C. They require some constraint to maintain conserved secondary structures. D. Coding sequences are intolerable to changes.

C. They require some constraint to maintain conserved secondary structures.

Which of these mutations can be cis-acting ? A. homozygous frameshift mutation in transcription factor gene B. homozygous mutation in intron of transcription factor gene C. heterozygous mutation at the promoter of gene A D. silent mutation in exon of gene A E. none of the above

C. heterozygous mutation at the promoter of gene A

Properties of Histone Chaperones: CAF-1 HIRA RCAF NAP-1

CAF-1: four subunits, H3 and H4 histones, interaction with sliding clamp, needs DNA replication HIRA: four subunits, H3 and H4 histones, no interaction with sliding clamp, does not need DNA replication RCAF: one subunit, H3 and H4 histones, no interaction with sliding clamp, does need DNA replication NAP-1: one subunit, H2A and H2B, no interaction with sliding clamp, does need DNA replication

Leucine zippers

Can work with helix-loop-helix to bind DNA and can dimerize •Coiled-coils: pairs of antiparallel alpha helices wound around each other •Leucine zippers: 60 amino acid long helices forming a parallel coiled coil with Leucine residues at the hydrophobic dimer interface •The Leucines "zip" together the two coils and stabilize them •The helical structure forms upon interaction with the correct DNA sequence

Coordination of RNA capping, histone modification, and pause escape (activation)

Cdk7 and Cdk8 kinases phosphorylate Ser-5 and Cdk7 phosphorylates Ser-7 shortly after initiation • Set1 (Histone Methyltransferase) recruited by Ser-5p • Capping enzyme recruited by Ser5-P • Brd4 (Bromo domain) recruits Cdk9 (part of PTEFb), resulting in phosphorylation of Ser-2 and other protein targets

Chromatin is a ___________ structure based on a __________ chromatin fiber type

Chromatin is a UNIFORM structure based on a SINGLE chromatin fiber type

Multiple forms of splicing

Cis: most common Trans (common leader sequence): 100% tryanosomes, 70% worms Trans (between genes) Trans (between alleles of same gene): Rare, function not verified well

Polymorphism

Co-existence of multiple alleles at a locus in a population

Benefits of NGS

Compared to other methods (PCR, microarrays) • Complete genome coverage • No prior knowledge of target necessary • Broader dynamic range • Increased sensitivity

Progeria patient (HGPS) primary fibroblasts rescue

Conclusions: •Expression of normal Lamin A does not rescue the nuclear defects •However, correction of the aberrant splicing event using a modified oligonucleotide targeted to the activated cryptic splice site reversed the phenotype

abundant mRNA

Consists of a small number of individual species, each present in a large number of copies per cell.

Retrotransposons

Copy-and-paste mechanism Requires an RNA intermediate. Can cause reverse transcription of cellular mRNA which can then result in re-insertion of a processed pseudogene (aka retrogene) Only present in eukaryotes

Concept of colinearity

Crick 1958; with collinearity, the number of nucleotides in the gene is proportional to the number of amino acids in the protein

DNA transposons

Cut-and-paste mechanism The transposon's DNA encodes for transposase, required for excision and insertion Also present in bacteria

Genomes Can Be Mapped at Several Levels of Resolution

Cytogenetic/banding maps Genetic mapping (linkage maps) Physical mapping (restriction enzyme maps) - i.e. FISH mapping Direct sequencing

Which of these is NOT a function of chromatin: A) Organize DNA & neutralize its charge B) protect DNA from damage C) efficient transmission of DNA to daughter cells D) shorten DNA to fit into the nucleus E) regulation of gene expression

D) shorten DNA to fit into the nucleus

Which are known or potential functions of adding a methyl-GTP cap to premRNA? A. Triggering releaser of promoter-proximal pausing B. Binding of factors that are important for downstream processes. C. Protection of RNA from degradation. D. All of the above.

D. All of the above.

satellite DNA

DNA that consists of many tandem repeats (identical or related) of a short basic repeating unit

Circle which statement applies to histone lysine methylation: A. it is always associated with active transcription B. it always results in chromatin compaction C. it always results in chromatin decompaction D. it can be associated with active transcription or silencing, depending on the lysine E. it always recruits repressor proteins

D. it can be associated with active transcription or silencing, depending on the lysine

Unwinding DNA by RNA Pol requires ...

DNA to be released from the octamer's surface Even though the promoter has been freed from nucleosomes, nucleosomes are in the path of RNA Pol

Two types of DNA methyltransferases (DNMT)

DNA methyltransferase - An enzyme that adds a methyl group to a specific target sequence in DNA. • De novo methylatranferase: establishes new methylation sites (Dnmt3A, Dnmt3B) • Maintenance / perpetuating methylatransferase: propagates methylation signal stably to next generation (Dnmt1)

Boundary Elements/Insulators

DNA sequences that recruit proteins that create a physical separation between different chromatin domains. They are DNAse I hypersensitive sites.

Destabilizing elements (DEs) vs. stabilizing elements (SEs)

Destabilizing elements (DEs) can accelerate mRNA decay, while stabilizing elements (SEs) can reduce it.

Second pathway of deadenylation-dependent degradation

Destablizing of the 3'-end by CCR4-NOT can also allow Exosome to degrade mRNA from 3' to 5'.

DNA sequencing:

Determining the number and order of nucleotides that make up a given molecule of DNA.

Reversal of DNA methylation

Direct demethylation • 5-meC can be converted to 5-hydroxy- methyl-Cyt. by Tet enzyme • 5hme-C is a substrate for TDG => removes hydroxy-methyl group. Passive demethylation • 5hme-C is not recognized well by maintenance pathway. • Results in lack of maintenance and removal of 5me-C through dilution

Transposable Elements

Discovered in maize by Barbara McClintock, 1940s (Nobel 1983) • They can "jump" around in the genome causing gene disruption or expression changes • About 50% of the human genome is composed of TEs • 85% in Zea mays (corn) • Majority of TEs are non-functional, but functional TEs can mediated the transposition of non-functional ones • Selfish DNA elements. In the big scheme of things: No advantage nor disadvantage to host.

Multiple STAT protein results in specific responses to diverse signaling events

Diversity in regulation: • STATs can be regulated by different pathways. • Have slightly different DNA-binding domains => different specificity • Can form heterodimers • Therefore, can bind many types of DNA binding elements.

T/F Histone H3-H4 tetramers are lost during DNA replication

False

Which of these statements about boundary elements is correct? A) they prevent different types of chromatin from encroaching B) they consist of DNA motifs bound by insulator proteins C) they physically separate domains through looping D) they are DNase1 hypersensitive sites E) all of the above

E) all of the above

Which of these approaches will give you gene mapping information at the base pair level? A. Linkage maps B. Restriction enzyme mapping C. Cytogenetic banding D. FISH mapping E. Direct sequencing

E. Direct sequencing

___________ results in degradation of RNAs when ________ is not removed during translation. A. Non-stop Decay / the Exon Junction Complex B. Nonsense Mediated Decay / a hairpin C. No-go Decay / the TRAMP Complex D. Decapping / the TRAMP Complex E. Nonsense Mediated Decay / the Exon Junction Complex

E. Nonsense Mediated Decay / the Exon Junction Complex

Fast rates of transcription affect splicing patterns by favoring what alternative splicing event? A. exon inclusion B. intron inclusion C. trans-splicing D. intron extension E. exon exclusion

E. exon exclusion

What is the definition of synthetic lethality A. two lethal mutations are viable when combined B. a mutant can no long synthesize a vital product C. two genes have redundant function D. two gene are not always expressed E. two viable mutations are lethal when combined

E. two viable mutations are lethal when combined

chromosome

Each DNA molecule packaged in chromatin

mRNA structure

Each mRNA consists of a untranslated 5' region (5' UTR), a coding region, and an untranslated 3' UTR

Acetylation effects....

Effect on compaction of nucleosomes is reversible

Enhancers bind multiple TFs in various ways

Enhanceosome: highly cooperative binding, all must bind for activity 'all or nothing' Billboard: cooperative binding, additive activity (Various combinations of TFs provide cell specificity) Collective binding: cooperative binding, binding by tethering, additive activity (Various combinations of TFs provide cell specificity)

Enhancer specificity

Enhancers are cell-type specific • Active enhancers vary more between cell types that promoters • Enhancers increase the diversity and specificity of gene regulation

Different sequences in eukaryotic genomes.

Eukaryotic Genomes Contain Both Nonrepetitive and Repetitive DNA Sequences • protein-encoding DNA comes from nonrepetitive portion of genome • A large part of moderately repetitive DNA may be made up of transposons.

Can colinearity be applied to eukaryotes?

Eukaryotic genes contain far more DNA than is required to encode proteins Many large RNA molecules isolated from nuclei are absent from the cytoplasm --> some type of change occurs before RNA export

T/F IRES elements are RNA hairpins that cause arrest or premature termination of translation. Lead to translation initiation independent of cap.

False

T/F In both DNA replication and transcription, only one strand of DNA is being copied

False

T/F Long dsRNAs cannot be used for RNAi in mammals because there is no dicer endonuclease to cut them.

False

T/F Most CpG islands are methylated in the genome, whereas most CpG dinucleotides are not.

False

Splicing Can Be Regulated by

Exonic and Intronic Splicing Enhancers and Silencers

What type of selections are exons usually under vs. intorns?

Exons are usually under negative selection while introns evolve more rapidly

Dscam gene

Extreme case of alternative splicing: Dscam gene in Drosophila

What controls specificity and timing of 3'-end formation?

Factors involved in termination are in the right place at the right time because of interactions with the CTD

Splicing occurs in two stages

First the 5' exon is cleaved off, and then it is joined to the 3' exon.

Epigenetics definition (historical)

First used by Conrad Waddington in the 1940's: • Merging of two terms: - Epigenesis - development of embryo from single cell - Genetics - Study of heritable traits encoded in genetic material Interested in studying the influence of environment on development

Chromosome Territories: FISH

Fluorescence in situ hybridization (FISH) confirmed the Cremer findings. •CTs are present in all higher eukaryotes, less defined in yeast, where chromosomes are less compacted •CTs are irregular in shape and 1-2 micrometers in diameter •CTs are in close proximity and neighboring chromosomes can invade each other's territories and intermingle at their peripheries

Some histone modifications are self-propagating: Repression and activation

For repression: PRC2 binds H3K37me3 => maintains repression by methylating nearby nucleosome For activation: TRX/MLL binds either non- methylated CpG or H3K4me3 => maintains activation by methylating nearby nucleosomes

A prototypical pioneer factor: FoxA

FoxA possesses features of linker histones and conventional transcription factors. • Winged helix DNA binding domain binds minor groove and scans along the DNA • Combined action TransActivation Domain (TAD) and C-terminal domain opens chromatin and allows other TFs to bind major groove.

Pol II CTD interacts with several components of splicing machinery

Functional coupling of transcription and splicing through CTD action as an interaction scaffold

How many different types of genes are present different species?

Gene families result from gene duplications of ancestral unique genes. This create more genes without creating new distinct gene families. Many genes are duplicated, and as a result the number of different gene families is much less than the total number of genes.

Why do genes show a distribution of sizes?

Genes Show a Wide Distribution of Sizes Due Primarily to Intron Size and Number Variation Most genes of S. cerevisiae lack introns while most genes of multicellular eukaryotes contain introns. - Most genes are uninterrupted in yeast, but most genes are interrupted in flies and mammals. Exons are usually short, typically encoding fewer than 100 amino acids (~30- 40 on average). Introns are short in simple eukaryotes but can be many kilobases (kb) in multicellular eukaryotes. The overall length of a gene is determined largely by its introns. - introns range from very short to very long

Satellite DNAs Often Lie in...

Heterochromatin

When are histone modified

Histones are post-translationally modified

Orthologs

Homologous genes found in different species that evolved from the same gene in a common ancestor. Genes A1 and B1 are orthologs. Genes A2 and B2 are orthologs.

Paralogs

Homologous genes in the same organism arising by duplication of a single gene in the evolutionary past. Genes A1 and A2 are paralogs. Genes B1 and B2 are paralogs.

true non-disjunction

Homologs dont separate Sisters dont separate

in situ hybridization

Hybridization performed by denaturing the DNA of cells squashed on a microscope slide so that reaction is possible with an added single-stranded RNA or DNA. - The added preparation is radioactively labeled and its hybridization is followed by autoradiography.

Which of the following is not a determinant of whether a transcription factor will bind a site in vivo. G. Activation state of the transcription factor H. Chromatin environment / architecture I. Presence of insulator proteins J. Binding specificity for the DNA

I. Presence of insulator proteins

What is the pattern of frequency of a mutation?

In small populations, the frequency of a mutation will change randomly and new mutations are likely to be eliminated by chance.

You identified a DNAse I hypersensitive site adjacent to an heterochromatic region. What could the function of this site be? Be specific and brief

It could be a boundary element

Pol II CTD

Major Surface for Interaction With Protein Factors Involved in Transcription Elongation, histone modification, and mRNA Processing provide many potential binding landscapes (10,000 possible states)

How do DNA binding proteins gain access to DNA which is wrapped in a nucleosome?

In vitro studies showed that some DNA binding proteins including RE are able to access specific sequences buried within the nucleosome particle These are passive DNA binding proteins that gain access to DNA without the use of energy (e.g. Restriction enzymes, DNAse) ->Spontaneous nucleosome unwrapping (no energy, no enzymes) -> Facilitated by ATP-dependent remodeling factors that actively unwrap or move the nucleosome (energy + enzymes)

How to contain or target enhancer activity?

Insulators are enhancer blockers

Assisted assembly:

Insulators create domains and bring distal regions together

CstF (cleavage stimulation Factor)

Interactions critical for timing and specificity of 3'-end formation

Heterochromatin Depends on....

Interactions with Histones

Intron or exon definition pairs splice sites

Intron definition (small introns (<300bp) - Nearly all splicing in yeast uses intron definition exon definition (large introns) - SR proteins bind across exon - Mechanism of transfer unknown

Determining nucleosome positioning

Isolate nuclei > induce double-strand breaks in linker DNA with micrococcal nuclease > isolate cleaved DNA and cut with restriction enzyme > Random nucleosome = cleavage at many positions due to random nucleosome positions OR positioned nucleosome = cleavage at specific regions between positioned nucleosomes OR Hybridize to microarray - If a nucleosome is not specifically positioned, micrococcal and restriction digestion result in a smear of bands of DNA of different sizes. - In the absence of nucleosome positioning, a restriction site can lie at any possible location in different copies of the genome.

Activation of Receptor Tyrosine Kinases (RTKs)

Ligand-induced dimerization results in activating auto- phosphorylation (step 2) • Activated RTKs phosphorylate other tyrosine residues on receptor in trans (step 3)

How can recent selective events be indicated?

Low heterozygosity of a gene may indicate recent selective events

Regulation of translation elongation through codon usage

Low-copy tRNAs can create a bottleneck and slow translation at rare codons

When is repression more efficient?

Lysine methylation is reversible but how rapidly is unclear, reversing repression more efficient by replacing H3, only available H3 outside of S phase is H3.3

Nuclear pore is nuclear RNA gatekeeper

Made up of over 30 protein (nucleoporins) NP mass ~ 125mDa Pore diameter = ~125 Channel diameter ~5nm Mediated regulated transport of all RNAs to cytoplasm (mRNA, snRNA, mi RNA, tRNA, rRNA) Small molecules (i.e. NTPs) pass freely

LOTS of mRNA are localized specifically

Mislocalization can sometimes create just as strong of a phenotype as a gene knock out

Gene expression is influenced by

Modifications Activators/Repressors promoter transcription factors

How does morphological complexity evolve?

Morphological complexity evolves by adding new gene functions • In general, comparisons of eukaryotes to prokaryotes, multicellular to unicellular eukaryotes, and vertebrate to invertebrate animals show a positive correlation between gene family number and morphological complexity as additional genes are needed with generally increased complexity. • Most of the genes that are unique to vertebrates function in the immune or nervous systems • Human genes can be classified according to how widely their homologues are distributed in other species.

Distribution of CGIs in mammals

Most CGIs overlap with gene promoters • Intergenic and intragenic CGIs are likely unannotated gene promoters or enhancers

What do functional genetic variation in human species affect?

Most functional genetic variation in the human species affects gene regulation and not variation in proteins

Pioneer factors vs most transcription factors

Most transcription factors; 1. cooperatively allows nucleosome/chromatin binding 2. simultaneous binding with other factors 3. Bind major groove Pioneer transcription factors 1. independent nucleosome/chromatin binding 2. precedes other factors binding 3. some bind major and minor grooves

Pausing is stabilized by

NELF and DSIF

Activation domains

Not as well structurally-defined as the DNA binding motifs Characterized by amino acid context or charge rather than structure (e.g. acidic, proline-rich, etc.)

DNA unwrapping

Nucleosome position is fixed, occurs spontaneously

Which for of nucleosome remodeling does not occur spontaneously and requires energy?

Nucleosome sliding and DNA bulging

What happens to nucleosomes during transcription?

Nucleosomes Are Displaced and Reassembled During Transcription Cartoon of classical experiment showing that the histone octamer is displaced from DNA and rebinds at a new position

Do individual chromosomes occupy defined regions of the nucleus or are they randomly distributed?

Occupy defined regions

Deacylated tRNA

Post transfer tRNA - exits via the E-site

Genomic imprinting

Preferential expression of a maternal or paternal allele - parent-of -origin specific Most often involves methylation of DNA Works in cis

Premature sister separation

Premature sister separation- in MI instead of MII due to defect in cohesion

RNA splicing

Process of excising introns from RNA and connecting the exons into a continuous coding mRNA molecule

RNA interference (RNAi)

Process through which any of the RNAs above inhibit gene regulation. - Endogenous or exogenous RNAs can modulate expression of mRNA by translation inhibition or degradation.

4 stages of transcription elongation

Promoter Escape / clearance - +8nt - Pol II extends the transcript to form a stable RNA-DNA hybrid Promoter Pausing - +20 - +50nt - Pol II elongation is inefficient and pauses in a ratelimiting step Pausing Escape Productive elongation - +50nt - termination - Paused complex is modified to efficiently transcribe across gene

Pol II associates with different factors in different phases

Promoter/TSS = GTFs, Activators, Co-activators Pause region = some GTFs, pause factors, RNA processing Gene body = Elongation factors (speed up pol II and move/remodel nucleosome), splicing factors Gene end = RNA cleavage, 3'-end formation, termination factors

Experiment: Determine transcription factor binding sequences:

Protein Binding-sequencing (PB-seq)

Stem loops

Protein interaction surfaces Secondary structure structure for binding splicing factors and RNA Base pairing can occur between snRNA and pre-mRNA or between snRNAs

Adapter proteins:

Proteins with specialized domains that recognize the cytosolic side of receptor molecules and act as docking sites for for other effector proteins

miRNA action

RNA closed loop structure is a target for miRNA-directed silencing RNA stability AND translational efficiency AND miRNA silencing mediated through interactions at 5'- and 3'-UTR

General mode of function by inhibitory ncRNAs

RNA functions as a regulator by forming a region of secondary structure (either inter- or intramolecular) that changes the properties of a target sequence

Nascent RNA

RNA in the process of being transcribed

RNA polymerase size

RNA polymerase is comparable in size to the nucleosome and might encounter difficulties in following DNA around the histone octamer.

mutations in introns can affect;

RNA processing

RNA closed loop structure important for

RNA stability AND translation efficiency

Antisense RNA

RNA that has a complementary sequence to an RNA that is its target

piRNAs require

RNA-dependent RNA polymerase (RDRP) to produce double- stranded RNA

RISC

RNA-induced silencing complex, a ribonucleoprotein particle composed of a short, single-stranded siRNA and a nuclease that binds and (sometimes) cleaves mRNAs complementary to the siRNA. - It receives siRNA from Dicer and delivers it to the mRNA. - Contains Argonaute proteins (AGO)

Classic example of transcription interference: yeast SER3 gene

Repression: - CHA4 = serine dependent activator. - High serine = active Cha4 - Cha4 activates SRG1 ncRNA transcription - Interferes with SER3 promoter - Serine biosynthesis is OFF • Activation: - Low serine = Cha4 inactive - Activator of SER3 can bind near promoter and activate transcription - Serine biosynthesis is ON

Examples of opposing regulation mediated by CpG dinucleotides

Repression: • MeCP2 - methyl CpG binding protein • Binds methylated CpGs and recruits repressive complexes Activation: - MLL complex binds non-methylated CpGs through CXXC motif - Recruits coactivators (including SET1) - Tri-methylates H3K4 => active region

Epigenetics definition (revised)

Refinement by Robin Holliday and others (80's-90's): • Epimutation - heritable changes in genes that are not due to changes in DNA sequence. • Interested in DNA methylation.

euchromatin

Regions that comprise most of the genome in the interphase nucleus are less tightly compacted than heterochromatin, and contain most of the active or potentially active single-copy genes.

Example of stabilizing element: transferrin mRNA

Regulation of transferring mRNA stability by iron levels. At high Iron levels, there is less need fro transferrin, the the transcripts is degraded

Differences between cis-acting and trans-acting elements

Regulatory proteins are trans-acting. They can act on the expression of both alleles of a gene in the cell -in a gene for a regulatory protein affects both alleles of a gene that it controls Cis-acting mutations only affect the allele harboring the mutation, which can no longer be bound by the regulatory protein - controls expression of adjacent DNA but does not influence the homologous allele

orthologous genes (orthologs)

Related genes in different species.

Two developmental regulators transmit chromatin states through cell lineages

Repressive Polycomb (PcG) Repressive complex: PRC1 and PRC2 complexes PRC2 = HMTase (H3K27me3) Active Trithorax (TRX; Drosophila) MLL (mammals) SET1 = HMTase (H3K4me3) • Highly regulated during development • PcG and MLL complexes are typically mutually exclusive

Detection of elongation intermediates

Ribosome footprinting • Isolate ribosome • Nuclease treat (mRNA within ribosome protected) • 'Footprint' of ribosome reveals precise location. • NGS sequencing provides comprehensive mapping

Site in the genome where individual members of species differ in a single base pair are called

SNPs/single nucleotide polymorphisms

how is the 5' splice site defined

SR proteins complex with U1 to help define the 5'-splice site

You have performed chromatin immunoprecipitation sequencing (ChIP-Seq) with an antibody specific for histone H3K9trime. What type of DNA sequences to you expect to be enriched?

Satellite DNA, transposable elements (because these are found in heterochromatin, where H3K9tri-me is present)

ChIP-seq

Search for histone modifications or transcription factor binding outside of gene promoters - detects all, esp.H3K27ac - need antibodies, not always specific for enhancers

Eukaryotic chromosome

Segregation Device held on the mitotic spindle by the attachment of microtubules to the kinetochore that forms in its centromeric region.

How can you detect selection?

Selection can be detected by measuring sequence variation

In Eukaryotes transcription and translation occur in __________.

Separate compartments - Transcription occurs in nucleus - Translation occurs in cytoplasm In eukaryotes, genes frequently contain introns that are not represented in the polypeptide Introns are removed from the pre-mRNA transcript by splicing to give an mRNA that is colinear with the polypeptide product

mRNA-Specific Half-Lives Are Controlled by

Sequences or Structures Within the mRNA

Sanger sequencing

Sequencing technology for the HGP ddNTPs cause termination during synthesis revealing identity of base

SR proteins:

Serine-Arginine repeats proteins are important splicing mediators

Function of RNA localization in cells

Sets up important gradients important for proper development of body plan. Important for morphology and function of neurons. mRNAs important for development or cell function are brought in close proximity To where the products are needed

JAK / STAT signal cascades in Immunological Disease

Severe Combined Immunodeficiency (SCID) - Affected individuals are compromised in B- and T-cell development. (Bubble boy disease). - Several genetic causes - One form comes from mutation in JAK3 kinase that makes it inactive Autoimmune disease - Numerous types - Gain of function mutations in JAK / STAT pathway can result in hyperactive immune system, inflammation, and cancer.

microRNA (miRNA):

Short (~ 22 bases) RNAs that modulate mRNA expression.

simple sequence DNA

Short repeating units of DNA sequence

Pioneer factor states:

Silent state: chromatin scanning, initial targeting competent state: enable other factors to access (TFs, chromatin remodeler/modifier) Active state: cooperative/stable binding with secondary TFs, acquisition of active histone modifications

Amplification of an External Signal Downstream from a Cell-Surface Receptor

Small amounts of signaling molecules can results in thousands of activated effectors

A second messenger is used in many GPCR pathways:

Small molecules that transmit and often amplify the signal within the cell

snRNPs ('snurps')

Small nuclear ribonucleoproteins (snRNAs associated with proteins).

What can exons correspond to?

Some Exons Correspond to Functional Domains within a Protein Proteins can consist of independent functional modules the boundaries of which, in some cases, can be equated with those of exons

Heterochromatin is found:

Some heterochromatin forms the telomeres that cap the ends of the chromatids, and much is concentrated near the centromere, where sister chromatids are joined.

Splicing Nomenclature check

Splice sites are named for their relationship relative to the intron NOT the exons

SL

Splicing Leader sequence - contains the 7meG cap which is important for stability and translation

What is divergence between any pair of sequences proportional to?

Substitutions may accumulate at a more or less constant rate after genes separate, so that the divergence between any pair of sequences is proportional to the time since they shared common ancestry.

Swi/SNF2

Superfamily is defined by the catalytic subunit • SWI2/SNF2: Catalytic subunit of the SWI/SNF chromatin remodeling complex • SNF: SWitch/Sucrose Non Fermentable • Mutants affecting the expression of the budding yeast SUC2 gene, which encodes the invertase enzyme (sucrose->glucose+fructose) required to ferment SUCROSE as carbon source • Large, 8-15 subunits

Upf proteins

Targeting of NMD substrates requires a conserved set of translation release factors

the Subunit of All Chromatin

The Nucleosome

Non-coding transcription can be important for regulation

The RNA is not always functional - sometimes transcription itself can be a regulatory 'tool'.

genetic hitchhiking

The change in frequency of a genetic variant due to its linkage to a selected variant at another locus.

transcriptome

The complete set of RNAs present in a cell, tissue, or organism. - Its complexity is due mostly to mRNAs, but it also includes noncoding RNAs.

interactome

The complete set of protein complexes/protein-protein interactions present in a cell, tissue, or organism.

proteome

The complete set of proteins that is expressed by the entire genome. - The term is sometimes used to describe the complement of proteins expressed by a cell at any one time

genome

The complete set of sequences in the genetic material of an organism. - It includes the sequence of each chromosome plus any DNA in organelles.

steady state (molecular concentration)

The concentration of population of molecules when the rates of synthesis and degradation are constant

DNA Is Organized in Arrays of Nucleosomes

The core DNA is the length of 146 bp that is found on the core particles produced by prolonged digestion with MNase - Micrococcal nuclease initially cleaves between nucleosomes

divergence

The evolutionary divergence between two DNA sequences is measured by the corrected percent* of positions at which the corresponding nucleotides differ. *corrected for the possibility of convergent mutations (same mutation at the same site in two separate lineages) and true revertants.

constitutive heterochromatin

The inert state of permanently silenced sequences, such as satellite DNA and transposable elements

facultative heterochromatin

The inert state of sequences that also exist in active copies; for example, one mammalian X chromosome in females. Also: HOX gene cluster is repressed and derepressed at different times in development (polycomb heterochromatin)

The intron is released as a

The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together

C-value paradox

The lack of relationship between the DNA content (C-value) of an organism and its coding potential.

min number of genes for a parasitic prokaryote and free-living nonparasitic prokaryote:

The minimum number of genes for a parasitic prokaryote is about 500; for a free-living nonparasitic prokaryote it is about 1500.

number of genes vs. genome size

The number of genes in bacterial and archaeal genomes is proportional to genome size.

The Organization of Introns and Exons of Genes are often Conserved

The positions of introns are usually conserved when homologous genes are compared between different organisms. - The lengths of the corresponding introns may vary greatly

fixation

The process by which a new allele replaces the allele that was previously predominant in a population.

RNA splicing

The process of excising introns from RNA and connecting the exons into a continuous mRNA

Ratio of nonsynonymous to synonymous

The ratio of nonsynonymous to synonymous substitutions in the evolutionary history of a gene is a measure of positive or negative selection.

nucleolar organizer

The region of a chromosome carrying genes encoding rRNA.

Where do sequences of orthologous genes in different species vary?

The sequences of orthologous genes in different species vary at nonsynonymous sites (where mutations have caused amino acid substitutions) and synonymous sites (where mutation has not affected the amino acid sequence)

Haplotype

The specific set of SNPs for a chromosome or part of a chromosome

C-value

The total amount of DNA in the genome (per haploid set of chromosomes) Transposons greatly contribute to C-value of eukaryotic cells (~50% of human genome)

How do some DNA sequences encode more than one polypeptide?

The use of alternative transcription and/or translation initiation or termination codons allows multiple variants of a polypeptide chain. Two proteins can be generated from a single gene by starting (or terminating) expression at different points. Different polypeptides can be produced from the same sequence of DNA when the mRNA is read in different reading frames (as two overlapping genes). - Two genes may overlap by reading the same DNA sequence in different frames. Otherwise identical polypeptides, differing by the presence or absence of certain regions, can be generated by differential (alternative) splicing when certain exons are included or excluded. - This may take the form of including or excluding individual exons, or of choosing between alternative exons.

Which compartment does transcription and translation occur?

Transcription and translation take place in the same compartment in bacteria

PEV in Drosophila was used in genetic screens designed to isolate genes involved in heterochromatin

The white gene responsible of the red pigmentation of Drosophila eyes •When inserted within heterochromatin its expression becomes variegated (ON in some cells-red and OFF in other-white) Genetic screens aimed at the identification of suppressors of variegation (i.e. genes that when mutated cause alleviation of silencing=red eyes)-> Su(var) Examples of Su(var)s: •Su(var)3-9 HMTase for H3K9 •Su(var)2-5 HP1 PEV, wild type Su(var), mutant PEV = position-effect variegation

What type of supercoiling is the wrapping of DNA around a histone?

The wrapping of DNA around a histone octamer introduces a left-handed torsion resulting in negative-supercoiling

Activation of transcription through MAPK cascade

There are many MAPKs and strategies to activate them

mRNA and protein gradients are critical for development

These proteins are often transcription factors or result in activation of transcription factors that activate genes necessary for specific cell types

Histone modification - DNA methylation 'crosstalk'

These repressive modifications are mutually reinforcing Mammalian HP1 is recruited to regions where lysine 9 of histone H3 (H3K9) has been methylated by a histone methyltransferase.

Amino Acid Derivatives:

Thyroid hormone, epinephrine

Nascent RNA sequencing (GRO-seq) or RNA-seq

Track bidirectional eRNA transcription - detection of regulatory elements -detects all - not clear whether all active enhancers are transcribed

How do we measure promoter-proximal pausing?

Transcription assay: Nuclear run-on

T/F Active enhancers vary more between cell types than promoters.

True

T/F DNA methylation can be removed enzymatically or through dilution over generations

True

T/F MicroRNAs primarily result in translational repression rather than RNA degradation.

True

T/F Processes associated with RNA export begin during transcription.

True

T/F Progeria is caused by a spontaneous dominant mutation in the lamin A gene

True

T/F RNA localization can result from directed movement along the actin cytoskeleton

True

T/F The bromodomain is responsible of binding acetylated histone Lysines

True

T/F Translation termination factors are proteins that mimic amino-acyl tRNAs.

True

T/F nucleosomes compact DNA 6-fold

True

Are chromosome territories randomly arranged in the nucleus, or are they organized into patterns?

Two experiments demonstrate that chromosomes are not randomly localized within the nucleus. 1.measuring the distance of a given chromosome from the center of a cell's nucleus, shows that some chromosomes localize toward the periphery, often touching the nuclear membrane, whereas others are located toward the center of the nucleus. 2. when visualizing multiple chromosome territories, there are recurrent clusters of chromosomes. For example, in mouse lymphocytes, chromosome 12 often sits next to chromosome 14, which in turn is adjacent to chromosome 15, thereby forming a triplet cluster

Enhancer bypass:

Two interacting insulators bring enhancer in close proximity to target promoter

synthetic lethal

Two mutations that are viable by themselves but cause lethality when combined

Commitment of pre-mRNA to the splicing pathway - splice site recognition

U1 and SR proteins define the 5' splice site U2AF binds Py tract and 3' splice site; branch point binding protein recognizes the branch site SR proteins bridge the complexes at the 5' and 3' splice sites to form commitment (E) complex In cells of multicellular eukaryotes, SR proteins play an essential role in initiating the formation of the commitment complex.

Base pairing between snRNA and pre-mRNA is essential

U1 pairs with 5'-splice site • Normal pairing = normal splicing • Mutant splice site = defective splicing • Complementary U1 mutations = normal splicing Mutations that abolish function of the 5' splice site can be suppressed by compensating mutations in U1 snRNA that restore base pairing.

initiation of splicing is done by

U1 snRNP initiates splicing by binding to the 5′ splice site by means of an RNA-RNA pairing reaction.

Unequal Crossing Over Rearranges Gene Clusters

When a genome contains a cluster of genes with related sequences, mispairing between nonallelic loci can cause unequal crossing over. - This produces a deletion in one recombinant chromosome and a corresponding duplication in the other. - Gene number can be changed by unequal crossing over.

How do Transcription factors find their targets?

When replication disrupts chromatin structure, chromatin can reform or transcription factors can bind and prevent chromatin formation.

Seven Major Classes of Cell-Surface Receptors

Work through diverse mechanisms - second messengers, effector proteins • Two types of responses: • short term: results in action in cytosol - Calcium release, cytoskeletal movement. • long term: results in gene expression changes - cell proliferation, differentiation

Heterochromatin Propagates from __________

a Nucleation Event

Bacterial Genes are Colinear with their proteins

a bacterial gene consist of continuous length of 3N nucleotides that encodes N amino acids The gene is colinear with both its mRNA and polypeptide products

spliceosome

a complex formed by the snRNPs that are required for splicing together with additional protein factors.

A restriction polymorphism can be used as

a genetic marker to measure recombination distance from a phenotypic marker.

Result of regulation and STAT properties is

a highly specific and diverse targeting of gene activation

branch site

a short sequence just before the end of an intron at which the lariat intermediate is formed in splicing.

SL RNA

a short sequence spliced to the 5′ ends of many precursor mRNAs have a structure resembling the Sm-binding site of U snRNAs. Creation of y-shaped intermediate at branch point - equivalent of lariat provides an exon that is connected to the first exon of an mRNA by trans-splicing.

Heterochromatin accounts for ___________ of the chromatin in both humans and flies.

a third

Bicoid

a translational repressor of caudal, which sets up opposing gradients. Bicoid's special homeodomain allows it to bind both DNA and RNA and regulate transcription and translation

Transport in cytosol is mediated by

adapter proteins that bridge mRNAs with myosin motors

Core promoters are only sufficient for

basal-level of constitutive transcription • Additional upstream regulatory elements (proximal and distal) recruit activators (more regulation) • Regulatory elements can also recruit repressors

CTD Heptad repeat varies

between species

Stabilizing and destabilizing elements

bind factors that influence degradation pathways

How does Lysine methylation (Kme) influence chromatin function?

by creating binding sites for specific proteins

How do DNA sequences evolve?

by mutation and a sorting mechanism

Nucleaotdies

cAMP, cytokinins, 1-methylalanine

Cross-talk between pathways or transcription factors can result in

change in set of activated genes

JAK/STAT pathway is direct form of

cytokine signaling

The helix-turn helix is a

common DNA-binding motif found in all organisms •2 alpha-helices positioned at a fixed angle and connected by a tight bend ("turn") •The second helix is the one that recognizes specific bases in the major groove

decapping enzyme

competes with the translation initiation complex for 5′ cap binding

The commitment complex (or E complex)

contains U1 snRNP bound at the 5′ splice site and the protein U2AF bound to a pyrimidine tract between the branch site and the 3′ splice site

'CTD code'

coordinates RNA processing, histone modifications, and transcription elongation

Deadenylation may be followed either by

decapping and 5′ to 3′ exonuclease digestion, or by 3′ to 5′ exonuclease digestion.

DNase hypersensitivity (DNase-seq)

detects open / accessible regions of chromatin - detects all - not all open regions are enhancers

How to estimate the proportion of expressed genes at a given time in a given cell?

determining the amount of genomic DNA that can hybridize with the mRNA isolated from that cell. In any particular cell, most genes are expressed at a low level. - Yeast: ~4000 (out of 6000 total) expressed genes - Somatic cells of plants and vertebrates: 10,000- 15,000 expressed genes

GPCR pathways

have short term effects in cells by quickly modifying existing proteins or enzymes or ion channels, but also long term effects involving change in gene expression

Small RNAs (siRNAs, piRNAs) can promote

heterochromatin formation

piRNAs result in

heterochromatin formation /silencing of locus (usually transposons ins germline)

Pioneer transcription factors target and open sites with..

high intrinsic nucleosome occupancy

Eukaryotic mRNAs Exist in the Form of

mRNPs from Their Birth to Their Death

Global Run-on sequencing (GRO-seq)

maps polymerase position, density and direction - Br-UTP RNA can be purified efficiently by affinity chromatography

Accurate repair of doublestrand breaks in heterochromatin is challenging, because

most of its DNA consists of short, repeated sequences.

Regulatory proteins

must be able to specifically recognize the correct regulatory sequence Each regulator has a DNA binding domain that recognizes a specific sequence

Variation can be shown through

mutant vs. wild type

Pioneer factors can bind

nucleosomal DNA

Mendelian view of alleles

phenotypes

CTD can be

phosphorylated, acetylated, glycosylated, and prolines can be isomerized

CTD and its modifications serve as a

platform for coordination of various processes such as RNA processing, transcription elongation, nulceosome remodeling and histone modification.

The two major mRNA decay pathways are initiated by deadenylation catalyzed by

poly(A) nucleases

Mutations in exons can affect;

polypeptide sequence

Lipids

prostaglandin & retinoic acid

RNAs are always associated with

proteins

Heterochromatin is created by

proteins that associate with histones.

Traits of Histone N-termini (N-tails)

protrude from the nucleosome at specific positions •Histone N-tails stabilize the DNA wrapping around the octamer (like the grooves of a screw) •the fact that they protrude makes it possible to modify them •modifications will influence the interactions between the octamer and DNA

The Centromere

provides the foundation for kinetochore assembly in mitosis and meiosis

mRNA decay

random or stochastic mRNA degradation - RNA is an unstable molecule

Cell signaling is generally

rapid, specific, and reversible

transesterification

reaction breaks and makes chemical bonds in a coordinated transfer so that no energy is required.

The specific pattern of chemical groups makes

recognition of specific bases possible •C-G and G-C expose different chemical groups on the major groove side but the same on the minor groove side •The same is true for A-T and T-A pairs, they look the same on the minor groove side but different on the major groove side

Chromodomains, Tudor domains, and PHD fingers

recognize methylated lysines domains found in complexes that subsequently mediate activation or repression

miRNAs use a seed sequence (~7nt) near 5'-end to

recognize target. Rest of pairing may involve mismatches / bulges.

•Eukaryotic regulators often control transcription by

recruitment of coactivators or co-repressors These cannot bind DNA alone, but are recruited by specific regulatory proteins

Meiotic segregation errors in humans

relationship between maternal age and the incidence of trisomy Errors occur at several stages during oocyte development • most trisomies are lethal • humans are unusually prone to meiotic missegregation (>35% of all conceptions are aneuploid compared to 2% in mouse!) • 1/3 of all miscarriages are aneuploid (leading known cause of pregnancy loss) • conceptions that survive - trisomy 21 and Sex chromosomes trisomies (47 XXX, or XXY, or XYY) account for 0.3% of live births • If we count still births 4% are aneuploid • If we count spontaneous abortions 35% are trisomic or monosomic • Most common defects in spontaneous abortions: 45,X monosomy (10%), trisomies 16, 21 and 22 together 50% of spontaneous abortions

Enzymatic terminators

require additional factors • Enzymatic terminators require the binding of enzymes to the RNA and are recruited by specific sequences on the transcript • Using ATP hydrolysis they pull the transcript inside the ring, moving it away from the polymerase • E.g. Rho in bacteria

Pioneer factors can alter

response to stimuli Activity of pioneer factor expands the regulation potential of other transcription factors

How may pseudogenes come about?

result from the accumulation of mutations in a duplicated gene Many changes have occurred in a β-globin gene since it became a pseudogene.

What is the name of the class of Transposons that use a reverse transcriptase during transposition?

retrotransposons or retroelements

mRNA instability is due to the action of

ribonucleases

At sites where there is no active regulation of nucleosome positioning, these will position based on

sequence preferences

miRNAs produced by

sequential cleavage steps in the nucleus and cytoplasm

HP1

silent heterochromatin key protein in forming heterochromatin in complex organisms, and acts by binding to di- and trimethylated histone H3. contains a chromodomain and a chromoshadow domain Methylation of histone H3 creates a binding site for HP1 binds to methylated H3, and spreads over neighboring chromatin - a reporter gene inserted within the heterochromatin will be repressed ---> binding of HP1 to methylated histone H3 forms a trigger for silencing

DNA replication slippage model

slippage and reattachment may cause a bubble to form in the new strand Cannot occur in DNA without repeating patterns of bases

At a subset of sites, nucleosomes are positioned .....

specifically via the the interactions of other proteins with the DNA and/or histones

Aminoacyl-tRNA

tRNA charged with amino acid

Peptidyl-tRNA

tRNA that is covalently linked to nascent peptide

Nonsense-mediated decay (NMD)

targets mRNAs with premature stop codons NMD occurs during pioneer round of translation - The first translation event for a newly synthesized and exported mRNA. • Premature stop codons • Retained EJC or long UTR

Steroid hormones:

testosterone, estrogen, cortisone etc.

•Chromosome compaction is achieved by....

the formation of nucleosomes (DNA+histones)

In Vitro Binding of HSF To Genomic DNA (PB-seq) Reveals

the Binding Potential of all HSEs

The cleavage reaction requires

the SLBP to bind to a stem-loop structure and the U7 snRNA to pair with an adjacent single-stranded region. The cleavage reaction is catalyzed by a factor shared with the polyadenylation complex. After S-phase SLBP is degraded resulting in degradation of histone mRNAs

Inheritance of parental H3-H4 tetramers facilitates

the inheritance of chromatin states

DNA binding occurs through

the interaction between specific DNA sequences and proteins •The chemical groups on the base pairs that are exposed in the major and minor grooves of the DNA differ (i.e. are asymmetrical) •Each base has a characteristic set of chemicals groups exposed to the two sides: hydrogen bond donors (red) and acceptors (blue) and a methyl group (for thymine only)

CTD state changes during

the transcription cycle

RNA stability AND translational efficiency mediated

through interactions at 5'- and 3'-UTR

Half life

time in which half of existing mRNA is degraded - Measure of mRNA stability

How are SNPs within a haplotype inherited?

together if they are close

Transcription elongation produces supercoiling that must be relieved by

topoisomerases • The DNA flanking the transcribed region is not free to rotate due to packaging of DNA into chromatin • DNA unwinding is compensated by positive supercoiling ahead of the polymerase and negative supercoiling upstream • Build-up of superhelical tension could lead to stalling and must be relieved by Topoisomerases

The end target of many signaling pathways are

transcription factors (TFs)

Operon:

transcription of multiple genes within the same transcription unit.

CHD1

transcriptional activation

miRNAs result in

translational repression

T/F 5' and 3' UTRs are part of the mRNA

true

T/F Insulators can both block enhancers and create active and repressive domains.

true

T/F The 'torpedo' model for transcription termination requires cleavage of the RNA.

true

T/F during evolution, introns accumulate mutations faster than exons

true

T/F to visualize introns, one can hybridize cytoplasmic RNA and genomic DNA from the same cells and use electron microscopy

true

All GPCR signaling pathways share the following common elements:

• A receptor that contains seven membrane-spanning elements (transmembrane domains) • A coupled trimeric G protein which functions as a switch by cycling between active and inactive forms (activator or inhibitory) • A membrane-bound effector protein • Feedback regulation and desensitization of the signaling pathway

The ribosome has three tRNA-binding sites.

• A site: initial tRNA entry point • P site: binds peptidyl tRNA • Deacylated tRNA exits via the E site.

Termination of RNAP II is coupled to RNA processing

• A) Allosteric Model: Recognition of polyA (or 3'cap) and cleavage of transcripts by RNA processing factors induces change in polymerase causing its release • B) Torpedo Model: RNA downstream of cleavage is digested by 5'-3' ribonuclease until it runs into RNAPII causing its dissociation

Coordination of mRNA export by RNA adapters and transporters

• ALY adapter binds RNA with EJC • Adapter binds transporter • Transporter takes RNA through pore • RNA release from transporter in the cytoplasm

Regulation of RNA stability by Stabilizing and Destabilizing elements

• AU-rich elements (AREs) are most often destabilizing elements in mammals, and are bound by a variety of proteins. • Some DE-binding proteins interact with components of the decay machinery and probably recruit them for degradation. • Stabilizing elements occur on some highly stable mRNAs. • Regulation: mRNA degradation rates can be altered in response to a variety of signals.

lncRNAs can also activate transcription

• Act as scaffolds for activators • Mediate long-range chromatin interactions. - Bind mediator / cohesin • Remove or titrate repressor complexes

•Regulatory proteins are often modular, and can have additional domains that:

• Activate or repress transcription • Interact with other regulators

What determines whether TF will bind a site?

• Activation of TF • Binding specificity for DNA • Chromatin environment / architecture

Coordination of splicing

• All 5'-splice sites can pair with any 3'-splice site.

Subunits of Eukaryotic RNA Polymerase

• All eukaryotic RNA polymerases have ~12 subunits and are complexes of ~500 kD. • Some subunits are common to all three RNA polymerases. • The largest subunit in RNA polymerase II has a CTD (carboxy-terminal domain) consisting of multiple repeats of a heptamer SAD-PAGE separation of isolated RNAP complex. Some subunits are common to all classes of eukaryotic RNA polymerases and some are related to bacterial RNA polymerase.

Elements within a promoter

• All polymerases need the TATA binding protein (TBP, part of TFIID) •The TATA box is present in ~50% of promoters. TBP binds to the TATA box (TATAA consensus sequence) ~25-30bp upstream of the transcription start • Eukaryotic promoters generally have characteristic elements, though these can be varied and hard to predict • Other common elements that may be present include the TFIIB recognition element (BRE), initiator element (Inr) and downstream promoter element (DPE; found downstream of the transcription start) • There are many promoters that do not have any of these elements

Transcriptomics or gene expression analysis

• Analyze when, where and to what degree a gene is 'on'. • E.g. can compare between different tissue types, developmental time points or disease states to identify possible gene function. • Can isolate & evaluate different portions of the RNA pool (e.g. mRNA, miRNA, siRNA, etc.). • For most analyses of RNA expression, the RNA is converted into DNA = cDNA (complementary DNA)

Signal transmittance through G-protein switches

• Binding of GTP creates conformational shift in protein. • Results in active or inactive state (active in this example) • Switch between states is tightly regulated by GAPs and GEFs: • GAPs - accelerate GTP hydrolysis • GEFs - stimulate exchange of GDP for GTP • GTP binding can activate or repress signal transduction • Regulation allows tight control of duration of response

Hormone-Induced Activation and Inhibition of Adenylyl Cyclase

• Binding of ligand to Ga-stim or Ga-inhib protein activates or inhibits synthesis of cAMP by adenylyl cyclase. • Second messenger signaling: cAMP, in turn, activates cAMP-dependent protein kinase that phosphorylate target proteins => transmittal of signal

Pathway of deadenylation-dependent degradation

• CCR4-NOT partially deadenylates mRNA (distributive) • Destabilizes PABP interaction with the poly-A tail • Destabilizes EIF4 interaction with cap • LSM complex binds 3'-end • LSM complex helps to recruit decapping enzyme (DCP) • XRN1 (5'-3' exonulcease) degrades mRNA

3'-end formation is regulated

• CPEB1 can be sequestered in the nucleus or cytoplasm. • Binding to C-elementin RNA favors use of nearby weak poly-A sites (wPAS). • Results in mRNAs with different 3-UTR lengths. • Effects translation and stability of RNA

Regulation of translation initiation

• Cap mediated regulation of translation initiation: - Binding and competition of the 5' and 3' end of the mRNA - Scanning of 5'-UTR until AUG start codon is encountered. • Use of Internal Ribosomal Entry Sites (IRES)

Pre-mRNA capping is co-transcriptional

• Capping can occur after only 20 bases are transcribed • Almost immediately after nascent RNA comes out of RNA Polymerase II • Cap enzymes associate with base of CTD - right outside RNA exit tunnel • Sets the stage for later processing and transport events - Without it, RNA can be degraded - Splicing can be defective - RNA can be sequestered in nucleus

What are enhancers?

• Cis-acting regions of DNA that 'enhance' transcription of a gene • Work independently of position and orientation relative to target gene • Can be a few kilobases to ~1 megabase from target gene

The 3' processing complex consists of several activities:

• Cleavage and polyadenylation specificity facotr (CPSF56) recognizes AAUAAA signal • CPSF73 is endonuclease that cuts RNA • Poly-A polymerase adds ~200 A to 3'-end • PAP is aided by poly-A binding protein (PBP)

Chromosome segregation

• Must be ACCURATE to prevent missegreagtion of chromosomes • Depends on multiple mechanisms that ensure that it is carried out correctly • true for both MITOSIS and MEIOSIS

Where are regulatory sequences located?

• Commonly located upstream they can be proximal or distal to the promoter • Complex regulatory sequences located far from the gene are common in eukaryotes • Multicellular organisms often have dozens of regulatory proteins bound to these sequences

How do you detect sequence variation?

• Comparing the rates of substitution among related species can indicate whether selection on the gene has occurred. A higher number of nonsynonymous substitutions in lysozyme sequences in the cow/deer lineage as compared to the pig lineage.

CpG methylation in the genome

• CpGs dinucleotides are rare: - Present at only 21% of that expected by base content • Result of spontaneous deamination of 5-meC to thymine - 70% are methylated • CpGs within CpG islands are preferentially hypomethylated, but are hypermethylated during regulated silencing.

Key interactions in cytosol:

• Cytosolic PABP replaces nuclear version on Poly-A tail • PABP interacts with translation initiation factors that bind the cap (EIF4E) • Closed circular structure is important for stability and translation

DNA methylation

• DNA methylation: cytosine is methylated at position 5. • Occurs mostly at CpG dinucleotides • Most CpG in the genome are methylated • Generally associated with repression - but is context dependent

Example of imprinting locus in mouse: RasGRF1 protein

• DNA repeats somehow dictate methylation status of DMD (DMR) • DMD and DNA repeats together make up the ICR

Establishment and maintenance of 5-meC

• De novo Dnmt establishes fully methylated sites. • Replication converts a fully methylated site to a hemimethylated site. • Maintenance methyltransferase recognizes hemimethylated sites and restores full methylation (left). • Lack of maintenance results in dilution of methylation (right).

Four other Degradation Pathways Target Specific mRNAs

• Decapping and degradation in absence of de-adenylation • The degradation of the nonpolyadenylated histone mRNAs is initiated by 3′ addition of a poly(U) tail. • Degradation of some mRNAs may be initiated by sequence- specific or structure-specific endonucleolytic cleavage.

Active enhancers produce bidirectional transcription: enhancer RNAs

• Define enhancers as intergenic CBP (histone acetyl transferase) ChIPseq peaks in neuronal cells. • Find stimulus (+KCl) dependent production of transcripts • Transcription appears bidirectional

Various signaling pathways interface with transcription regulation

• Different mechanisms of receptor action • Different signal transduction pathways • Can all lead to activation of TFs and different sets of genes. Transcription factor activation is often final product of signaling pathways

DNase Sensitivity Detects Changes in Chromatin Structure

• Discovered 30 years ago, Hypersensitive sites are found at the promoters of expressed genes, as well as at other important sites such as origins of replication and enhancers. • Hypersensitive sites are generated by the binding of factors that exclude histone octamers. • A chromatin domain containing a transcribed gene is defined by increased sensitivity to degradation by DNase I. Indirect endlabeling identifies the distance of a DNase hypersensitive site from a restriction cleavage site.

MicroRNA discovery: Lin-4 gene

• Discovered by Ambros and Ruvkin labs in 1993. • Lin-4 isolated as a gene important for various development processes in C. elegans. • Lin-4 opposes function of lin-14 • Mapped lin-4 phenotype to 700bp RNA. • Mutation of putative open reading frame (ORF) had no effect on phenotype. • Found 2 'processed' fragments of 61 and 22 bases • Had complementarity to 3'-end of lin-14 UTR • Lin-14 known to be regulated post-transcriptionally via the 3'-UTR Conclusion: lin-4 short RNA regulates lin-14 through complementarity with 3'-UTR

Spliced mRNA

• EJC normally cleared during pioneering round of translation. • If not, UPF2,3 remain at EJC and target mRNA for degradation

Ribosomal RNA Pervades Both Ribosomal Subunits

• Each rRNA has several distinct domains that fold independently. • Virtually all ribosomal proteins are in contact with rRNA Contacts between the ribosomal subunits are mostly made by RNA

Signal amplification through phosphorylation Cascade

• Effector or signaling proteins amplify the signal by activating more downstream proteins • Also offers more points for regulation and crosstalk between signaling pathways.

Elongating polymerase is stably associated with DNA and highly processive

• Elongating polymerase is tightly and stably anchored to DNA • Elongation is highly processive, 20-50nt per second are synthesized • A typical transcription bubble contains around 12-14nt of which 9 are the most recently added ribonucleotides of transcript

Two phases of epigenetic inheritance

• Establishment: mechanisms leading to the initial establishment of chromatin state or epigenetic mark. • Maintenance: Mechanisms that propagate the established chromatin state through generations (cell-divisions)

General model of cap-mediated translation initiation

• Eukaryotic 40S ribosomal subunits bind to the 5′ end of mRNA and scan the mRNA until they reach an initiation site. • A eukaryotic initiation site consists of a 10-nucleotide sequence that includes an AUG codon. • 60S ribosomal subunits join the complex at the initiation site. This process is mediated by Initiation factors (eIFs).

Enzymatic activities involved in 3'-end formation and termination: Torpedo model

• Pre-mRNA is cleaved by endonuclease • Remaining nascent RNA is degradated by an exonuclease called Xrn2 • Poly-A tail is added to 3'- end of pre-mRNA by polyA polymerase

Basal transcriptional apparatus

• Eukaryotic RNA polymerases unable to read DNA • They require the presence of DNA-binding proteins to signal where transcription needs to initiate • Transcription factors (TF) bound to core promoter are called general transcription factors (GTFs) • TF= any protein required for transcriptional initiation that is not part of the RNA pol complex

Receptor tyrosine kinases in Disease

• Example: Anaplastic lymphoma kinase (ALK) is a RTK that is misregulated in a number of cancers. • Physiological role is not well known, but it is not essential in mice. • Activated in cancer by overexpression (gene duplication), translocation (fusion protein), and point mutations

RNA polymerase must separate the two strands of DNA

• For transcription to start, PIC must transition to open complex by denaturing ~14bp of DNA • RNAP II requires TFIIH to unwind. • TFIIH uses ATP hydrolysis to unwind the DNA • For RNAP I and III the open complex forms spontaneously • The unpaired template strand is kept open by regions within the active site of the polymerase • Incoming NTPs enter the active site and are added to the growing RNA chain (blue)

Enhancers can be found in a poised or active state

• H3K27me3 = repressive histone modification • H3K4me1 = mark of previously active enhancers or poised for future activation

lncRNA function: recruit activating and repressive complexes to target loci

• HOX genes are important for body plan and limb development • HOTAIR transcribed from intergenic region of HOXC locus • Binds repressive PRC2 complex and recruits it to target genes • Important for repression of a number of genes, including HOXD locus lncRNAs act as scaffolds to mediate regulatory interactions

How are histones synthesized?

• Histones are synthesized in S phase (Zweidler, 1984) • Histone genes are present in many copies in most genomes to aid in the rapid production of histones during S phase

Lipid soluble hormone signaling

• Hormone permeates cell membrane- binds receptor • Receptor translocates to the nucleus • Receptor binds DNA to affect gene expression • Can be positive or negative regulation • Sometimes receptor is already in the nucleus, just inactive without ligand

Signaling can also directly regulation transcription responses

• Hormone receptors • Cytokine /immune signaling - JAK/STAT pathway

Types of lipid soluble hormone signaling molecules

• Hormones diffuse freely through cell membranes • Bypasses the need for cell-surface receptors

Classic example of imprinting locus in mouse

• Igf2 is a growth factor important for fetal growth • H19 is a ncRNA (miRNA) which moderates / slows growth • Enhancers can target either H19 ncRNA or Igf2 gene • The maternal ICR binds CTCF insulator => Direct enhancers to H19 gene • The paternal ICR is methylated, preventing CTCF binding => enhancers target Igf2 gene

Heterochromatin plays a number of critical functions in genome function and organization

• In a continuum of chromatin states from active to repressed, heterochromatin is found at the extreme-end of repressed • Referred to as "silenced" chromatin • It is primarily associated with DNA rich in satellites and transposable elements contributing to their silencing and stability • It protects repetitive DNA from DNA damage • It suppresses homologous recombination • It maintains genome stability via regulation of centromere and telomere function

TBP's beta finger (or loop) must move out of the way to allow promoter clearance

• Initial RNA synthesis produces only short transcripts 2-9nt long (abortive initiation) • TFIIB is responsible for abortive initiation, blocking transcript elongation via its beta finger • Change in conformation moves the beta finger out of the way, allowing the polymerase to break free (promoter clearance)

Where did (spliceosomal) introns originate from?

• Intron early - Introns were universal and were lost in prokaryotes due to "genome streamlining". • intron late - Introns emerged after the split of eukaryotes and became widespread. • Evidence: Self-splicing group-II introns are widespread. But why are spliceosomal introns present only in eukaryotes? And why are their distribution among different lineages so irregular?

Intronization

• Intronization - Introns originate from exons that contain premature termination codons. By being spliced out these alleles are not selected against and can accumulate mutations that make splicing more efficient. • Evidence: constitutively spliced introns in one species often align to homologous alternatively spliced coding sequences of orthologous genes from another species

Long noncoding RNAs: properties

• Introns of protein coding and lncRNAs are poorly conserved through evolution (red) • Exons of protein coing genes are highly conserved (green) • Exons of lncRNAs show moderat lncRNAs have less overall conservation but have conserved structure - less constraint on structure than coding sequences

Some pathways are direct:

• JAK/STAT • Steroid hormones

Histone Code Hypothesis

• Jenuwein and Allis in 2001 (Science) • It proposed that the information encoded within DNA is in part regulated by the chemical modifications of histones • Histone modifications serve to recruit other proteins by specific recognition of the modified histone by specific binding domains within regulators • Unlike the genetic code, which is universally applicable, the 'histone code' is harder to define due to differences in diverse species, tissues, and the combinatorial effects caused by multiple histone modifications occurring simultaneously.

How do you identify the genes in a genome?

• Knowing the sequence does not mean you automatically know which parts are genes. • Identify ORFs using prediction tools • Can use transcriptome information to identify genes where transcripts originate from • Can use homology with other genes in other organisms

Genomic imprinting is an epigenetic phenomenon

• Known imprinting examples often involve differential CTCF or transcription factor binding at differentially methylated regions (DMRs). • Differential regulation is thought to be the result of parental conflict between the male and female during fetal development. - Imprinting results in male chromosome expressing pro-growth gene (Igf2 or Ras in the examples above). - Female tries to mitigate this by expressing growth-slowing genes (H19) or regressing pro-growth genes (Ras).

specific RNAi in mammals requires small RNAs

• Large dsRNAs shut down translation and cause degradation of all RNAs - related to recognition of viral RNA. • Small RNAs evade this mechanism and can target specific mRNAs • This is not a concern in flies or nematodes dsRNA inhibits protein synthesis and triggers degradation of all mRNA in mammalian cells, as well as having sequence-specific effects.

Meiosis in human male

• MI: segregation of the homologs from each other • MII: segregation of the sister chromatids from each other (like mitosis) • Starts in PUBERTY • cells progress from MI to MII without delays • New sperm continuously produced

Translation initiation can be regulated through competitive interactions

• Many repressing interactions are mediated through the 3'-UTR • Mechanisms include: - Sequestering capbinding complex - Competitive binding with eIF4G or eIF4E

Gene activation through GPCR-MAPK pathway

• Mating factor (pheromone) stimulates mating pathway through binding GPCR. • Exchange of GTP for GDP in Ga releases of b and g subunits. • Scaffold protein, Ste5, recruits components of MAPK pathway. • Fus3 (MAPK) activates Ste12 in nucleus => activation mating genes

How do How do micro-RNAs target mRNAs? micro-RNAs target mRNAs?

• MicroRNAs regulate gene expression by base pairing with complementary sequences in target mRNAs • Base pairing is rarely perfect and multiple bulges or open ends are allowed • Recognition starts with pairing of a 'seed' sequence in 5'-end of miRNA • RNA interference triggers degradation or translation inhibition of mRNAs complementary to miRNA or siRNA. - It can also lead to mRNA activation

TBP is a universal factor

• Name comes from TATA box to which TBP binds ( ~50% of RNA Pol II promoters have it) • TBP binds to other factors and acts as a RNA Pol positioning factor • All 3 polymerases require TBP • The factor for RNA polymerase II is TFIID, which consists of TBP and ~14 TAFs, with a total mass ~800 kDa RNA polymerases are positioned at all promoters by a factor that contains TBP.

Signal transduction through protein phosphorylation

• Nearly all signaling pathways utilize phosphorylation to regulated protein activity • Phosphorylation often activates proteins, but can also be inhibitory • Phosphorylation is counteracted by phosphatases

Functions of 3'-end formation and polyadenylation

• Necessary for termination • Cleavage and polyadenylation can be regulated through interactions with the Pol II CTD or by binding of sequence-specific factors (CPEB1). • Influences: - Stability - Transport - Translation

Three forms of Cytoplasmic surveillance

• Nonsense-mediated decay (NMD) targets mRNAs with premature stop codons. • Nonstop decay (NSD) targets mRNAs lacking an inframe termination codon and requires a conserved set of SKI proteins. • No-go decay (NGD) targets mRNAs with stalled ribosomes in their coding regions

How many genes are essential?

• Not all genes are essential. In yeast, deletions of <50% of the genes have detectable effects. • When two or more genes are redundant, a mutation in any one of them may not have detectable effects. Essential yeast genes are found in all classes. 50% of genes involved in protein synthesis are essential. ~19% of yeast genes are essential. Note yeast is haploid We do not fully understand the persistence of genes that are apparently dispensable in the genome. Redundancy of function may provide selective advantage - A systematic analysis of loss of function (RNAi) for 86% of worm genes shows that only 10% have detectable effects on the phenotype.

Some histone modifications are self-propagating

• Nucleosomes are randomly segregated to daughter chromosome strands. • Epigentic modifiers (i.e. histone modifying enzymes) recognize their own substrate. • EMs then spread histone modification to neighboring nucleosomes.

Exons in the genome

• Only 1% of the human genome consists of exons. • The exons comprise ~5% of each gene, so genes (exons plus introns) comprise ~25% of the genome.

Receptor tyrosine kinases ALK in Disease

• Overexpression of wildtype receptor makes cells especially sensitive to growth factors • Activating mutations result in constitutive signal in absence of ligand • Fusion proteins that result in dimerization in the cytosol are constitutively active

Allosteric and torpedo models for termination

• PCF11 binds CTD of Pol II (ser-2P) and causes conformational change in Pol II that results in release from the DNA • Xrn2 is recruited as a complex to Pol II (ser-2P) and degrades the RNA from the 5'-end These models are not mutually exclusive (they can happen at the same time)

Recruitment of Polycomb Repressive Complex

• PRC can be recruited by Polycomb Repressive Element. • PRC can be recruited by ncRNAs (e.g. HOTAIR). • PRC2 can also bind it's product (H3K27me3) Recruitment is the first step in establishing silencing

DNA Methylation Is Responsible for Imprinting

• Paternal and maternal alleles may have different patterns of methylation at fertilization. • Methylation is usually associated with inactivation of the locus - but can cause activation of nearby genes (see below). • When genes are differentially imprinted, the functional allele is provided by the parent with the unmethylated allele. • Imprinting is reset in the germline

Heterochromatin formation requires small RNAs: S. pombe

• Pol II transcribes centromeric DNA. • RNA -dependent RNA polymerase (RDRC) makes dsRNA. • Dicer processes dsRNA into siRNA. • siRNA loaded into RITS and targeted to centromere • RITS complex induces heterochromatin formation and spreading through H3K9me and HP1 binding Analogous to miRNA silencing, except genomic locus is target instead of mRNA.

No-Go decay:

• Targets stalled ribosomes • Endonuclease recruited • Followed by 5' to 3' and 3' to 5' decay

The Y Chromosome Has Several Male-Specific Genes

• The Y chromosome contains ~60 genes that are expressed specifically in the testis. • The male-specific genes are present in multiple copies in repeated chromosomal segments. • Gene conversion between multiple copies allows the active genes to be maintained during evolution. The Y chromosome consists of X-transposed regions, X-degenerate regions, and amplicons

TFIID nucleates assembly of the RNA polymerase II pre-initiation complex

• The basal transcriptional machinery is competent for transcription but the rate of transcription is low • Additional activators and co-activators must be recruited to stimulate transcription • The mediator is not part of the PIC and is recruited by regulatory proteins • Additional activators include transcription factors, HATs, and chromatin remodeling complexes

The spliceosome assembly pathway: key steps

• The commitment complex progresses to pre-spliceosome (the A complex) in the presence of ATP - U1 and U2 SNRPs bound. • Recruitment of U5 and U4/U6 snRNPs converts the A complex to the mature spliceosome (the B1 complex). • The B1 complex is next converted to the B2 complex in which U1 snRNP is released to allow U6 snRNA to interact with the 5′ splice site. • When U4 dissociates from U6, U6 snRNA can pair with U2 snRNA to form the catalytic active site (C-complex). • Catalysis and release ensues

Unique processing of histone 3'-ends

• The expression of histone mRNAs is replication dependent and is regulated during the cell cycle. • Histone mRNAs are not polyadenylated; their 3′ ends are generated by a cleavage reaction that depends on the structure of the mRNA. • Several factors involved (SLBP, U7 snRNP) are unique to this process

Transcription is terminated by a variety of mechanisms

• The polymerase must stop transcribing at the proper position • Must release transcript and DNA • Must prevent read-through • Makes the polymerase available for additional rounds of transcription • Transcription stops at defined DNA sequences called terminators • Termination is a regulated step

what is the probability of a mutation influenced by?

• The probability of a mutation is influenced by the likelihood that the particular error will occur and the likelihood that it will be repaired

RNA stability

• There are multiple pathways to degradation of mRNAs • mRNA stability is dictated by RBPs that bind specific sequences and RNA secondary structures • Stability mechanisms are tied directly to translation

General Structure of G-Protein-coupled Receptor

• There are several sub-families of G protein-coupled receptors • G-protein coupled receptors (GPCR) consists of hydrophobic amino acids that allow proteins to be stably anchored in the hydrophobic core of the membrane (seven membrane spanning domains) • Loops C3 and C4 are involved in binding to G protein.

The origin and types of histones variants

• They originate by random mutation and fixation through acquisition of new functions from the duplicated original canonical histone • the changes can be minimal (a few amino acid difference) from substantial • Add another level of complexity in interpreting the outcome of a particular chromatin state

Gene Activators

•Activators have distinct and separable DNA binding and activating functions. •Well-studied activator is the Gal4 protein, which activates the genes responsible to utilize galactose as a carbon source in S. cerevisiae (e.g. the gene GAL1)

General properties of histones

•At least 20% of residues are either Lysine or Arginine i.e. positively charged •small in size, highly conserved through eukaryotes

Interactions within nulceosome assembly..

•CAF-1 interacts with PCNA near the fork •deposits new and old H3-H4 tetramers onto the DNA, the H3-H4 tetramers stay with the DNA •NAP1 deposits new and old H2A-H2B dimers from the soluble pool

Pervasive transcription generates a variety RNA

• Transcription can displace activators • Transcription can displace / prevent new transcription initiation • Polymerases can pause over regulatory sites, occluding them In these cases, the RNA itself is not believed to be important

TREX complex is critical for preparing RNA for export

• Transcription coupled Exporter (TREX). • Includes multiple proteins (THO) in addition to adapter (ALY) • Assembles on RNA during transcription. • Important prior to adapter binding. • Important for packaging of mRNA RNP for transport. • Without it, mRNA accumulates in nucleus.

What is the molecular consequence of capping?

• Transcriptional regulation: - Release from pausing - Cap binding factors may help trigger release. • Blocks 5'-end from exonucleases - increases stability of RNA • Unique binding surface for Cap Binding Complex (CBC) and other proteins important for: - efficient splicing - RNA export - translation.

Receptor tyrosine kinases (RTKs):

• Transmembrane receptors • Generally monomeric • Dimerize upon ligand binding • Dimerization activates intrinsic kinase activity on cytosolic side • Tyrosine phosphorylation on receptor transmits activation signal • Example processes: cell division, metabolism

Cytokine receptors:

• Transmembrane receptors • Generally monomeric -dimerize upon ligand binding • Kinase activity resides in separate complex (e.g. JAK kinases) • JAK kinases activated by receptor dimerization • Phosphorylate receptor tyrosines to transmit signal • Examples processes: cell differentiation, proliferation, immune response

Mechanism of G-protein coupled receptor activation

• Trimeric G-proteins are covalently linked to lipids in the cell surface. • In the trimeric form, Ga is bound with GDP and is inactive. • Binding to of ligand to receptor stimulates conformational change • Receptor then binds Ga subunit. • Binding stimulates release of GDP. • GTP binding by Ga releases Ga from Gbg. (step 3) • Ga is now activated and can bind to effector proteins: transmit signal on cytosolic side of membrane. (step 4) • Activation ceases after GTP hydrolysis and release of effector protein (step 5) • Activation cycle can begin again

Detection of DNA methylation

• Unmethylated C converted to U by bisulfite treatment. C=>T transition • Methylated C protected

Long noncoding RNAs: Discovery

• Using histone mark of active transcription, identified regions outside of genes as potential novel genes - H3K4me3 = active promoter - H3K36me3 = active gene region • Identified >1000 new ncRNAs in a single cell type. • Integration of RNA-seq data and multiple cell types has increased the number to ~15,000 lncRNAs - outside of or overlapping protein coding

Example of Natural antisense transcription regulation of splicing

• Zeb gene has an IRES element in an intron. • Normally spliced out leading to inefficient translation • can pair with the splice site, inhibiting splicing • IRES included in mRNA => efficient translation

The origin of Histones

• all organisms wrap their DNA in some kind of architectural protein • eukaryotic nucleosomes likely evolved from archaeal nucleosomes • Archaeal nucleosomes 1. are tetrameric 2. have histone folds (no tails) similar to (H3-H4)2 tetramers 3. wrap one turn of DNA

Transcriptional regulators

• bind to specific DNA targets modular proteins that can recruit additional factors that regulate transcription

Transcription in eukaryotes: overview

•Catalyzed by RNA polymerases •Unlike DNA polymerase, it does not require a primer •Multiple RNA pol can transcribe a gene at the same time (synthesize large numbers of transcripts from a single gene) •The process is less accurate than that of DNA replication •the genomic profile of 2 cells from same organism is identical but the TRANSCRIPTION profile is often not

Cap mediated scanning of 5'-UTR

• eIF4E andeIF4G are critical for forming the 48S complex or preinitiation complex. • 48s complex scans the 5'-UTR until AUG start codon is reached. • Large, 60S, subunit joins to make the 80S ribosome. After GTP hydrolysis, the complex is competent for translation. Mediated by eIF5.

Long noncoding RNAs

• lncRNA = non-coding RNA >200bp in length • No coding potential • Generally have moderate levels of conservation - implies functionality • Like protein-coding genes, many have tissue specific expression - implies functionality • Not all RNAs are necessarily functional

Non-stop decay

• mRNAs without stop codon • Exosome recruited by SKI

lncRNA

• non-coding RNA >200bp in length • No coding potential • Generally have moderate levels of conservation - implies functionality • Can act as scaffolds to recruit activator or repressors into complexes or specific loci in the genome. • Antisense transcription can lead to RNA pairing and alternative splciing (Zeb2 gene example) • Not all RNAs are necessarily functional - Sometimes transcription itself is function through transcription interference (yeast SER3 gene example).

Small interfering RNAs (siRNAs) are also captured by RNAi machinery

• siRNAs can be generated from a number of precursors • Recognized by RNAi machinery in cytosol • Stimulate mRNA cleavage

Features associated with CpG islands

• ~29,000 CGIs in the human genome • Typically overlap with gene promoters • Promoters of nearly all constitutively express genes (those expressed in all cell types) overlap a CGI • 40% of tissue specific genes have promoter overlap with CGI • CGIs maintain a permissive transcription environment • CGIs are generally refractory to methylation

Structure of RNA Polymerase II

•"crab claw" •RBP1 - active center cleft •RBP2 - active center cleft

Single-nucleotide polymorphisms (SNPs)

•(snips)=sites in the genome where individual members of a species differ in a single base pair •2 random humans are 99.9% identical at the DNA level •Human Genome is ~3 billion bp so 2 individual will differ in about 3 million bp •SNPs arise through mutations and are inherited as any allele

How do rates differ and compare in the rate of evolution for different proteins?

•3 different rates for these 3 proteins •Rate is constant for individual proteins

Significance of chromatin compaction?

•DNA's negatively charged phosphate backbone produces electrostatic repulsion limiting DNA folding upon itself •histone octamer neutralizes 60% of this negative charge allowing folding •nucleosomes introduce 6-fold compaction •formation of mitotic nucleosomes require 10,000 fold shortening

Inheritance of chromatin states

•During DNA replication there will be a DNA bound fraction and a soluble fraction of histones •histone mods remain at specific chromosomal regions •specific histone binding proteins will bind old mods and target modifying enzymes to add mods to adjacent new histones

Cryo-EM

•Form of transmission EM •widely used in structural biology •the sample is studied at cryogenic temperatures (generally liquid nitrogen temperatures) •advantage: it allows the observation of specimens that have not been stained or fixed in any way, showing them in their native state and fully hydrated •disadvantage: high noise, images must be extensively modified. Information from other methods (e.g. X ray crystallography) must be used to reconcile models

Gene-rich and gene-poor CTs

•Gene-poor chromosomes localize preferentially near the nuclear periphery whereas gene-rich chromosomes are found more towards the center of the nucleus

Why do we need nucleosome remodeling?

•Genome is packaged in nucleosomes (genome organization and way to regulate DNA-access) •Many DNA-binding activities cannot access nucleosomal DNA (e.g. transcription factors) •Catalytic enzymes using ATP-hydrolysis can remodel nucleosomes to expose DNA •These enzymes have been implicated in virtually all DNA-based processes including replication, DNA damage repair and transcription

Chromosome Territories Hypothesis

•Hypothesis 1: Chromosomes are randomly intermingled ("spaghetti" model) •Hypothesis 2: Chromosomes occupy defined territories •Test: Marking of a sub-region of the nucleus would affect many chromosomes (top) or only a subset of chromosomes (bottom)

Nucleosome positioning -Are there intrinsic preferred positions where nucleosome are placed within a genome?

•Isolate nucleosome core particles from chicken red blood cells •Extract DNA •Clone DNA fragments, sequence •align all sequences in the middle, •apply statistical methods to determine the probability that a particular dNTP is in a given position •align all sequences in the middle, •apply statistical methods to determine the probability that a particular dNTP is in a given position

Determining the size of nucleosomal DNA

•Microccoccal nuclease (MNase) •unbiased nuclease, cleaves linker DNA •by changing incubation time or MNase concentration can get partial digestion •extensive digestion reveals the length of the minimum DNA length possible •147bp is the length of DNA that wraps around the histone octamer •Mononucleosome, di, tri, tetra... •147bp is an invariant feature of nucleosomes, linker length is variable (20-60bp)

What is the biological significance of supercoiling?

•Negative supercoil=unwind •Way to store free energy to aid processes that require DNA denaturation •DNA is negatively supercoiled within cells •NUCLEOSOMES (DNA wrapped around histone octamers) introduce negative supercoling

Example of gene regulated at the PIC recruitment step, yeast PHO5

•Pho5 is an acid phosphatase induced by Pi starvation •Pho2 (activator) and NuA4 (HAT) already bound •Starvation leads to accumulation of unphosphorylated activator Pho4 in the nucleus •Pho4 binds the UAS and interacts with Pho2. •SAGA (HAT) is recruited, histones are acetylated then remodeled by Swi/Snf •PIC recruited, gene is activated

Electron Spectroscopic Imaging (ESI) Properties

•Phosphorus: highly abundant in DNA (and RNA), low in protein •Nitrogen: high in both •phosphorus and nitrogen maps can be combined to reveal the composition of the interchromatin space. •pseudo-colored image: blue indicates protein, yellow, chromatin (protein+nucleic acids)

Histone variants

•Provide an additional level of specification of specific chromatin regions •serve to demarcate specific chromosomal regions and to confer specific functions to the nucleosomes that contains them

how are the old histones redistributed?

•Repackaging of the DNA is tightly linked with DNA replication. •1/2 of the histones will be "old" and 1/2 will be new •old H3-H4 tetramers remain bound to either daughter strand of DNA and are randomly segregated to either sister chromosome •H2A-H2B dimers leave the DNA and become part of a soluble pool that contains both old and new H2AH2B dimers •H3-H4 tetramers and H2A-H2B dimers are composed of all new or all old histones (no mixing) •randomly segregating old and new H3-H4 tetramers ensures that epigenetic marks are inherited by both sister chromosomes H2A-H2B dimers and H3-H4 tetramers are "all new" or "all old" no mixing

Nucleosome Remodeling

•Requires ATP •mediated by large complexes •All can do the sliding •A subset can transfer

The FACT complex

•The FACT (Facilitates Chromatin Transcription) complex destabilizes the nucleosome •H2A-H2B dimers are released •This is sufficient for RNA Pol to transcribe through •H2A-H2B dimers are re-assembled (FACT?) •Loss of histones is not necessary for transcription (crosslinking experiment)

Example of a gene that is activated at the pause-escape step, Drosophila Hsp70

•The Hsp70 promoter is free of nucleosomes from the start •Activator GAGA factor maintains this nucleosome-free state and keeps RNAPII in a paused state •Heat shock causes activator HSG to trimerize and bind upstream of gene •HSF recruits co-activators, more nucleosomes are lost, RNAPII enters productive elongation •Phosphorylation and release of pausing factors (purple and pink) allows recruitment of more RNAPII that will not be paused

Aneuploidy and cancer

•The large majority of cancers cells contain an incorrect number of chromosomes i.e. they are ANEUPLOID

The nucleosome

•The smallest unit of chromatin •Composed of DNA and a histone octamer •Chromatin formation results in a sixfold compaction • Full compaction is of 1,000-10,000 fold •(H32-H42) 1 tetramer •(H2A-H2B)2 2xdimers

How do boundary elements provide the separation?

•Through recruitment of specific proteins they could work by: •organizing chromatin in loops whereby anchoring chromatin at the base of the loops would separate domains •they could attach DNA to the nuclear envelope again to create separate domains

Chromosome Territories: the Cremer experiment

•Use a narrow UV laser to induce localized DNA damage in interphase •Cells with were pulse-labeled with 3H-thymidine •Analyze the marked regions in mitosis when chromosomes are distinguishable and condensed, using autoradiography •Clear outcome: only a few chromosomes affected in support of the existence of chromosome territories (CTs)

Role of chromatin?

•balance charge of DNA •folding of DNA •NOT compaction to fit DNA in the nucleus

•Packaging DNA into chromosomes provides several advantages:

•compact and organized form •protect from DNA damage •efficient transmission to daughter cells •regulation of gene expression •recombination during meiosis

Methylation of H3 lysines

•depending on which lysine is methylated, it can have positive or negative effects on transcription •multiple methylated states: -mono, -di, -tri •many known sites, combinatorial effects

Nuclear diseases laminopathies e.g. Progeria

•extremely rare genetic condition, only ~45 cases in the world •Caused by a spontaneous mutation in the LMNA gene during the earliest cell divisions of the zygote •Causes the activation of a cryptic splice site that results in a truncated form of the Lamin A protein

Limitation of transmission EM

•the images created are low contrast •heavy-atom contrasting agents are needed to provide sufficient contrast (e.g. Uranium or Lead salts) •they function by coating biological material with large precipitates of stain -> limits spatial resolution •heavy atoms salts do not interact uniformly with with different biochemical components -> artifacts can be created that give false impressions of electron dense regions and mass-depleted

How are nucleosome assembled?

•to duplicate a chromosome, at least ½ of the nucleosomes on each sister chromosome must be newly synthesized