Advanced Bio Midterm 2
PRC1 and PRC2
- 1 is H3K27me reader - 2 is H3K27me writer -like HP1
DNMT
- DNA methyl transferase - DNA methylation is established by a de novo DNA methyl transferase (DNMT), which puts a methyl group at the 5 position of cytosines found in CpG sequences.
co-transcriptional silencing
-"kills the baby in the cradle" -Ago recognizes gene we're transcribing with siRNA and kills the mRNA -IN NUCLEUS
HP1/Swi6
-"locks" chromatin down by protein-protein interactions -HP1/Swi6 binds to H3K9, chromoshadow domain is the "link" between the histones -can move on and OFF rapidly
S. cerevisiae "point centromere"
-1 CenpA nucleosome is sufficient to attach to 1 microtubule -forces must remained balances to prevent breaking of the chromatin while maintaining tension along the chromatin -microtubules exert outward force (towards spindles) while chromatin maintains an inward force that is flexible enough to accommodate microtubule based tension
small-double stranded RNAs
-15-30 bp in length -regulatory modules
Components of CRISPR in the lab
-2 components: CAS9 and sgRNA for targeting mechanism: -sgRNA targets enzyme to site of cleavage -simple cleavage will lead to non-homologous end joining. -BUT, if we provide an additional donor sequence, homology-driven repair can make insertions, mutations, or any other custom-engineered outcomes -basically causes a DS break
long noncoding RNA (lncRNA)
-A class of relatively long RNA molecules found in eukaryotes that do not code for proteins but provide a variety of other functions, including regulation of gene expression. -just another RNA-homing system
lamina-associated domain (LAD)
-A region of heterochromatin associated with the nuclear lamina -creates a type of facultative heterochromatin
kinetochore
-A structure of proteins attached to the centromere that links each sister chromatid to the mitotic spindle -CenpA connects with the kinetochore -CenpA nucleosomes coordinate the protein network of the CCAN which recruits outer kinetochore (KMN network) that attaches to microtubules -protein adapter between microtubules and CenpA chromatin -outer and inner kinetochores
How much of human genome is C≡G?
-About 42% -Expected frequency of CpG is 4% but actual is ~ 1%
CATD
-CenpA Targeting Domain -creates a surface that defines CenpA
How is CenpA kept where it belongs?
-Centromeric CenpA nucleosomes are stable, centromere-associated proteins block degradation. -Mislocalized CenpA nucleosomes are UNSTABLE.
CpG islands
-DNA regions rich in C residues adjacent to G residues higher than anywhere else in the genome. -Especially abundant around promoters or housekeeping genes and are NOT methylated, these regions are where methylation of cytosine usually occurs
Dnmt3 vs DNMT1
-DNMT3: methylates unmethylated DNA (de novo) -establishment: by either direct recruitment to euchromatin, recruitment by transcription factors, or recruitment by gene silencing (RNAi) -DNMT2: maintenance methyltransferase; adds "matching methyl to strand during DNA replication -is targeted by having a "hemi" methylated state during replication -if methylation doesn't occur by next S phase, mark can be lost
Polycomb proteins
-Family of proteins that bind to condensed nucleosomes, keeping the genes in an inactive state -polycomb group (PcG) proteins which are highly conserved regulatory factors -PcG works through histone methylation (H3K27) (like HP1, but that one works with H3K9) -best known for their role in maintaining silent expression states of Hox genes during development, very strongly shuts OFF expression! -linked to MacroH2A recruitment (X chromosome inactivation) -helps establish differentiation and cell lineages (liver cells do liver things)
CenpA
-H3 variant only found in centromeres, maintains kinetochore attachments -essential for viability -necessary but not sufficient for building kinetochore -assembly is independent of DNA replication -NO methylation! prevents HC from forming -N-terminal tail not required for CenH3 identitiy -NOT assembled during S phase because because then half of CenpA would be lost (half goes to leading strand other half goes to lagging) -CenpA is assembled after mitosis, CenpA is at full capacity (100%) during late G1 phase.. in other phases its like 50%
HP1 (heterochromatin protein 1)
-HP1 bindings to tri- methylated H3 -The inactive region is then extended by the ability of further HP1 molecules to interact with each other -platform for many activities: transcriptional silencing, activation, histone modifiers... etc -involved in a cycle of modifications on pericentromeric heterochromatin -associates with CAF to promote heterochromatin maintenance through DNA replication
Phosphor-methyl switch
-HP1 binds to H3K9me -Phosphorylation of H3S10 disrupts HP1 binding, sending HP1 off the chromatin specifically in mitosis -this allows a window of transcription in the centromere -Aurora kinase phosphorylates S10H3, removing Swi6/HP1
constitutive heterochromatin
-HP1 dependent -chromosomal regions that remain condensed in heterochromatin at most times in all cells -HP1 binding to H3K9me, methylation is established by RNAi, can spread and be inherited -RNAi is required to recruit Clr4 (HMTase) in peri-centromere heterochromatin -HP1/Swi6 is a heterochromatin reader: adapter protein
What are the 3 types of heterochromatin?
-HP1 dependent (pombe pericentromere): H3K9me -Polycomb dependent (differentiation): H3K27me -X-chromosome inactivation: Xist RNA dependent or Constitutive- genes are stably repressed Facultative- genes can convert between states, but once its heterochromatin, it stays OFF (liver cells not doing brain things) X-inactivation
Msp1 & HpaII (DNA methylation) what do they do?
-Msp1: cleaves CCGG and CmeCGG; cuts whether DNA is methylated or not -HpaII: cleaves CCGG only; won't cut where it's methylated -detection of methylation to protect CpG islands
Explain Brain cancer with PRC2
-PRC2 adds H3K27me3 which activates PRC2 (and turns off gene expression) -If H3K27 isn't methylated and methionine placed instead (H3K27M, a mutation), PRC2 doesn't recognize (or is inhibited by) the DNA. -this is associated with a brain cancer
XIST gene
-Produced by inactive X chromosome only -allows for methylation and deacylation of inactive X chromsome -a long ncRNA required for X-inactivation -contains several repetitive elements -contribute to localization on the X chromosome -recruits PRC2 for silencing -once silencing is established, Xist is no longer required and no longer active
RISC complex
-RNA induced silencing complex enzyme -attaches to the small interfering (siRNA) and micro RNAs (miRNA) after they've been processed by dicer -mRNA is then targeted to be degraded or not transcribed -cytoplasm?
Argonaut
-RNA nuclease -binds small RNAs and uses them to home them on an mRNA -primary component of the RISC complex -after RNA is cleaved, this protein helps (along with dicer) to choose the correct "guide" strand of the RNA produced by DICER -This protein then uses that guide strand to find its target -many of these proteins
PTGS (post transcriptional gene silencing)
-RNAi work to PREVENT gene expression post-transcriptionally (after the mRNA is produced) -thus, miRNA genes are repressors (negative regulator) -But, presence of miRNA and siRNA in RISC can formally target DNA/RNA in the nucleus
spreading by coupling
-Reader/writer coupling -Reader/eraser coupling -Work together on neighboring histones
Facultative Heterochromatin
-Regions that can interconvert between euchromatin and heterochromatin -Polycomb (PcG) proteins***
small RNAs (sRNAs)
-Short RNA molecules that help to regulate gene expression. -tiny pieces of nucleic material responsible for critical roles in regulating cell activities beyond a simple, primary protein sequence. -Can recognize 8 base pairs easily -KNOWN TO BE AN IMPORTANT REGULATORY MECHANISM -they use complimentary of small RNAs to identify large mRNAs and target them for destruction or inactivation -make up large part of the transcribed genome in higher eukaryotes
CpG dinucleotides
-Side by side cytosine and guanine nucleotides n the same DNA strand -in most eukaryotes, methylation on DNA is limited to these nucleotides
sgRNA
-Single guide RNA -An RNA molecule engineered to be complementary to the target to be edited -fused RNA that has a targeting sequence adjacent to a hairpin-forming structure
Microtubules
-Spiral strands of protein molecules that form a tubelike structure
Heterochromatin
-The genes in heterochromatin are generally inaccessible to enzymes and are turned off -defines long term cellular memory
protospacer
-The sequence in the target DNA (viral or other) that matches the crRNA/spacer -20 nucleotide long period of a guide rna that is complimentary to the target sequence -the sequence that will be used to program the system
What's the difference between DNA methylation (deamination) making a Uracil and Thymine?
-Uracil can be easily recognized and repaired -Thymine can only be repaired my mismatch repair, not as easy as Uracil
C. Elegans experiment to view RNAi in action
-Worm was fed RNAi against GFP (green fluorescent protein) -The one that had the RNAi had the GFP silenced so there was no green light on his stomach -The other worm that had an RNAi mutation still expressed GFP in the stomach, as a bright green light was seen.
Antisense RNA
-a single-stranded RNA molecule complementary to, and thus targeted against, an mRNA of interest to block its translation (gene expression is silenced) -funny thing: double stranded antisense RNA worked better at knocking down gene expression than single stranded.. WHY?
phase separation
-associated with proteins containing disordered regions and low content of hydrophobic residues -these interact with each other to form a loose assemblage that phase-separates from the whole (oil & vinegar) -Lots of "granules" in the nucleus define nonmembrane bound structures with diff. functions
Having 2 centromeres:
-bad, could be lethal -chromosome breakage when they're pulled apart
Centromere structure:
-central core flanked by repetitive heterochromatin
Metacentric vs acrocentric
-centromere in middle- meta -centromere towards one end of the chromosome - acrocentric
Compartmentalization (condensate)
-clustering of proteins with a common function
What is the next level after TAD?
-compartments -different topological domains with similar epigenetic signatures are characterized by stronger inter-domain interactions and are organized into compartments -if TADs acquire different marks they can switch compartments -compartments can be changed simply by changing gene expression or condensation
Establishment of methyl mark
-de novo placement of a mark where it has NEVER been marked. like... establishing constitutive heterochromatin @ centromere during each cell cycle (after division) lineage-specific repression by polycomb (liver do liver thangs), and random X-chromosome inactivation
Imprinting
-depends on different methylation of paternal and maternal alleles -affects at least 90 genes in humans -also found in neighboring plants -suggests paternal and maternal alleles play different roles during development -only one copy of gene is active at a time: NO BACK UPS!
Which strand is chosen in the RISC complex?
-depends on the stability. -the 5' end with LESS stability (more A-T pairs over G-C)
How is disruption of phase separation associated with cancer?
-elevated gene expression is seen and impaired cell fate determination. -basically, genes are transcribed too much or too fast?
Concentration (condensate)
-enrichment of proteins to reduce effect of space and volume
How to stop HP1 spreading?
-essential or else the whole genome would be heterochromatin 1. temporal controls: via adjacent modification (boundaries) 2. spatial controls (boundaries): fixed boundary elements or fluid boundary chromatin
Dicer
-found in cytoplasm -two substrates: long dsRNA (siRNA) & short hairpin RNA (miRNA) to generate short dsRNA fragments -assembles the correct strand of SIRNA** into the RISC complex -1-2 dicer proteins in different species
Drosha
-found in nucleus -Processes long primary miRNA's to pre miRNA hairpin structures -first cut: cuts miRNA in the nucleus into a hairpin -dsRNA nuclease specific to miRNA
H2AZ
-gene expression, silencing, and chromosomal segregation -containes unique C-terminal tail -barrier to heterochromatin in some cases -required for centromere heterochromatin -disassemble more easily -assembled by SWR1 histone remodeler -H2AZ nucleosomes interspaced with CenpA -pericentric chromatin & centric chromatin: Centric is euchromatin, contained H2AZ & H3K4me <-- associated with gene expression!
What is DNA methylation in eukaryotes usually associated with?
-gene repression! -also linked to histone modifications
EZHIP Protein
-involved with H3K27M... inhibits PRC2 by mimicking H3K27M (NOT H3K27me!!) -found only in placental mammals -associated with brain cancer -upregulated PFA brain cancer
Protospacer Adjacent Motif (PAM)
-is a 3-5 bp DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system -Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence -PAM next to the protospacer targets nuclease (CAS9) to degrade invading DNA (there's a hair pin) -NGG
CTCF
-key protein for TAD formation: "master weaver" of the genome -An architectural protein involved in regulating the organization of chromatin -zinc finger (DNA binding protein) conserved in higher eukaryotes -functions as an insulator preventing transmission of signals -fixed boundary ? -using cohesin to make a TAD: CTCF has directionality and cohesin makes loops in DNA. A finished loop will have two CTCF proteins -deletion of this boundary can affect TADs -CTCF is also a transcription factor, insulator, and RNA binding protein)
Selective partitioning (condensate)
-localization of proteins that are required and exclusion of those that are not
Levels of interaction in "conformation capture":
-loops: ie. enhancer promoter, gene loop, polycomb mediated loop (cohesin holds these loops)
miRNA
-micro RNAs -TRANSCRIBED IN ONE DIRECTION (from single stranded transcripts to make small dsRNA) -a class of functional RNA that regulates the amount of protein produced by a eukaryotic gene -more than 1000 miRNA genes are in humans -single stranded & form loops -no open reading frames (no start codons?) -Imperfect match to it's target!** -down regulate target genes -separate genetic loci from the genes they regulate -encoded by their own locus -double stranded RNA
RITS
-modified ARGONAUT/RISC complex found in nucleus & is associated w/ heterochromatin -this RNA mediated silencing complex induces formation of heterochormatin -argonaut complex -remains in nucleus to act directly on chromatin -can also contribute to transcriptional repression by heterochromatin formation
RdRP (RNA-dependent RNA polymerase)
-necessary for production of virus genome and proteins in negative sense viruses -uses single strand RNA to generate a complementary RNA strand (not found in flies or vertebras)
HJURP
-novel chaperone that recruits CenpA
Chromatin Droplets
-nuclear HP1alpha conc. is increased locally as a result of binding to H3K9me. -chromatin bound HP1 lead to formation of HP1 droplets (DNA inside?). Preformed droplets may directly associate with H3K9me domains -liquid-like fusion of HP1a droplets with heterochromatic domains results in formation of larger domains (and droplets)
Human centromeres
-outer kinetochore, inner kinetochore, centromere (CenpA chromatin) and pericentric heterochromatin -many loops of heterochromatin & cohesin -CenpA/CenH3 nucleosomes are oriented towards the kinetochore
nascent transcript model
-paradox: you have to turn ON transcription to turn it back OFF -transcription generates dsRNA, which is then processed by RNAi to direct argonaut back to the site of transcription -this then localizes a complex w/ histone methyltransferase Clr4 (which is associated with a chromodomain protein to enrich is locally).
X chromosome inactivation
-process that occurs in female mammals in which one of the X chromosomes is randomly turned off in each cell -occurs randomly in early development -requires long non-coding RNA encoded by Xist gene -maintenance requires polycomb and variant histone macroH2A -reversible in germ cell lineage
Pericentric heterochromatin
-provides structural rigidity & promotes kinetochore orientation -HP1 & cohesion stabilize the loop, which helps orient it. -helps reduce the change of chromosome missegregation & damage! -important, but not essential for CEN function. CenpA chromatin IS ESSENTIAL THO -provids a safety area for centromere "drift"
Rabl configuration
-said centromeres and telomeres fall in the periphery of the cell: nuclear organization
topologically associating domains (TADs)
-self-interacting genomic region, meaning that DNA sequences within a TAD physically interact with each other more frequently than with sequences outside the TAD (basically cluster of loops) -domains on EACH chromosome typically 100s of kb to a few Mb -preferential contacts between loci -insulated by adjacent TADs -active loci tend to associate with active loci -inactive loci tend to associate with each other at the nuclear periphery
Types of boundaries:
-sequences that oppose nucleosome assembly making gaps that prevent heterochromatin from spreading -factors that promote nucleosome turnover blocking heterochromatin domain expansion. -conflicting modifications (ie. histone is already acetylated) via transcription
S. pombe "regional centromere"
-several CenpA nucleosomes -flanked by heterochromatin repeats -3-4 microtubules per centromere
Variegation
-silencing by chance -not a specific boundary (fluid border) -tend to be stable (heritable), identical sibling cells in identical environment may have different epigenetic states
siRNA
-small interfering RNAs -TRANSCRIBED IN BOTH DIRECTIONS (from double stranded transcripts, PERFECT MATCH) opposing promoters -derived from same locus? -found in diverse eukaryotes and viruses. -down regulate target genes by promoting mRNA cleavage (destruction) -perfect match to it's target** -class of double-stranded RNAs about 23 nucleotides in length that silence gene expression; act by either promoting the degradation of mRNAs with precisely complementary sequences or by inhibiting the transcription of genes containing precisely complementary sequences -CAN BE AMPLIFIED (RDRP): Requires RISC and RNA poly. (RNA that makes RNA), this is found in plants and yeast but not animals
Models for folding of BLANK:
-solenoid (spiral) -layer (boustrophedon) -loop
Maintenance of methyl mark
-spreading or stability of a mark through cell division like.. spreading of constitutive heterochromatin inheritance of constitutive heterochromatin in non-centromere domains inheritance of inactive facultative heterochromatin through cell division
CRISPR/Cas9
-targeting system from bacteria's basic immune system -a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence -a bacterial system that can be used either to produce a mutation in a specific gene or to correct a mutation that is already present -came from adaptive immunity found in 40% of bacterial genomes -memory consists of sequences from pathogens interspersed with CRISPR repeats -relies on integration of targeting sequences between inverted repeats in the genome, then expression of this region allows programming of enzyme with targeting RNA, that directs towards invading DNA (key is PAM) -allows precise gene targeting
Centromere
-the region of the chromosome that holds the two sister chromatids together during mitosis -bound to microtubules -sister chromatids released and pulled apart by tension when cohesion is destroyed
Robertsonian translocation (ROB)
-translocation in which the long arms of two acrocentric chromosomes become joined to a common centromere, resulting in a chromosome with two long arms and usually another chromosome with two short arms. -fusion of centromere -human Chr2 -problem is in meiosis
What happens when we disrupt TADs?
-very bad, can lead to potentially deleterious changes in gene expression -linked to limb malformation -deletion: brachydactyly short digits -inversion: f-syndrome syndactyly -duplication: polydactyly (extra toes)
Principles of organization:
1. Chromatin dynamics: chromatin undergoes local dynamic motion 2. Polymer-polymer interactions: mediated by chromatin binding proteins, promote formation of chromatin loops and shape overall conformation of fiber 3. Heterogeneity & stochasticity: behavior of individual chromatin regions and genes, give rise to heterogeneity in genome organization 4. Architectural elements: Non-random localization: physical interaction of chromatin with elements such as the nuclear envelope limit degrees of freedom of a genome region 5. Phase separation: ie. euchromatin and heterochromain CPHAP
What are the mechanisms for polycomb recruitment? How do they stop expression?
1. DNA binding protein (transcription factor) 2. small noncoding RNA by chromatin compaction, holding RNA pol II, or not letting swi/snf in
Constitutive Heterochromatin mechanism:
1. Phosphorylation of H3S10 leads to removal of Swi6/HP1 2. allows for expression of non-coding RNA's 3. RNAs are processed by Dicer and argonaut, then sent back to the site of expression via RITS (which remains in nucleus) 4. nascent RNAs are made into dsRNA by RDRP 5. RITS then recruits Clr4 HMTase which methylates H3K9 (its also binds H3K9me) 6. H3K9 recruits Swi6/HP1, transcription is shut-off and H3S10 is dephosphorylated until next M phase
Testing Swi6/HP1 binding by ChIP assay:
1. antibody on Swi6/HP1 2. PCR fragments where Swi6/HP1 was attached (fragments that make uracil from previous example) 3. looking at the ChIP results, we see that the band with heterochromatic (uracil enzyme OFF) contained more Swi6/HP1 than the DNA with the gene encoding for enzyme on. So... decreased expression of marker gene reflects increased Swi6 association
S. Pombe Heterochromatin paradigm:
1. pericombe centromere has many repeats & no protein encoding genes 2. gene that encodes uracil enzyme is inserted... 3. wild-type eventually shows silencing of uracil growth 4. cells with deleted swi6/HP1 keep on expressing Uracil.
Experiment to distinguish between establishment & maintenance***
1. temporarily delete the writer, loss of modification on histone tails leads to loss of heterochromatin 2. replace writer and ask if heterochromatin is reassembled 3. if factor "x" is required to establish heterochromatin, then in X-minus cells you can't reassemble heterochromatin when writer is replaced.
Genomic DNA is methylated on what residues on many species?
A & C! G≡C A=T
Identifying associations "conformation capture":
Big Q: how can I know two regions in genome interact? 1. crosslink DNA (if they're close together) 2. cut with restriction enzyme (two DNA strands still cross linked together) 3. fill ends and mark with biotin 4. ligate the ends (making a circular DNA loop still attached to crosslinker 5. purify and shear DNA 6. sequence using paired endj? -can only be done on same chromosome (ie. chromosome 14) -we get these red maps with the dark diagonal in the middle -darker signal= most likely they associate -signal off the diagonal means association of different regions
Histones involved with centromeres:
CENPA: not methylated= not heterochromatin H2AZ: easy to disassemble nucleosome , associated with expressed genes, centromere stability
random monoallelic expression
Ex: X inactivation by the lncRNA Xist -it's not just for X-chromosomes! -creates cellular heterogeneity
guide strand vs passenger strand
GUIDE STRAND: -An RNA single strand that is produced by RISC that is biologically active -this strand is used to TARGET mRNA -it MUST be complimentary to the target mRNA PASSENGER STRAND: -the "other" strand that needs to be eliminated because it is not complimentary to the mRNA
Pericentromer - Centromere - Pericentromere: which histones are where?
H3K9me3 - H3K4me2, CenpA, .. , .. - H3K9me3
Are there a lot of inter-chromosome interactions within nucleus?
NO, there are very few -chromosomes occupy distinct territories even in interphase -territories correlate with gene expression (ie. gene rich chromosomes are in middle 19, gene poor are in periphery) 18
Steps of degradation for proteins vs RNA?
Proteins: mRNA exportation for translation, and the the mRNA and protein need to be degraded to turn off expression. -when proteins have a mutation (although many are silent) it's bad. The polypeptide could be shortened. RNA: quickly degraded after it's made -RNA is malleable, and more tolerable to mutations.
RNAi
RNA interference -injecting double stranded RNA into a cell turns off expression of a gene with the same sequence as the RNA -works in a variety of eukaryotes if they have an endogenous RNAi system (budding yeast don't) -depends on well designed dsRNA & tested for off-target effects (success depends on the stability of the target protein; controls like scrambled targets? irrelevant dsRNA??)
What's an efficient way to target nucleic acid sequence?
RNA! -way better than proteins which require 105-117 base pairs of genomic sequence (about 35-39 AA's) to recognize a single RNA base with specificity.
Neocentromere
Spontaneously form without alpha satellites. satellites not necessary for centromeres.
T or F: Is centromere silencing disrupted and re-established in each cell cycle?
True, important to make sure mark is reestablished, residual Swi6/HP1 also promotes replication in the centromere domain
What does presence of Swi6/HP1 indicate?
gene is SILENCED (heterochromatinized) -decreased expression of marker gene reflects swi6/HP1.
