Chapter 17: Regulation of Gene Expression in Eukaryotes

Base analog

-If you have a base analog (i.e. 5-Azacytosine) instead of cytosine, methylation will not occur -this means that some genes, that are supposed to be turned off, will not be -used to treat sickle cell anemia

CAAT box

-a common proximal promoter -modulates (increases?) levels of transcription -located 70-80 base pairs (bp) upstream from start site -critical to the promoter's ability to initiate transcription -mutations within the CAAT sequence lower the transcription rate

Core Promoter

-a promoter subcategory -determines the accurate initiation of transcription by RNAP II

Proximal Promoter

-a promoter subcategory -modulates the efficiency of basal levels of transcription (in combination with core promoters) -located upstream of the TATA box and BRE Examples of proximal promoters: -CAAT box -GC Box: located at position -110

SWI/SNF Complex

-a type of chromatin remodeling complex -role in nucleosome movement (arrangement) -acts in several ways: 1. can loosen attachment between DNA and histones -causes the nucleosome to slide along the DNA and expose regulatory regions (alteration of DNA protein contacts) 2. can loosen the DNA strand from the nucleosome core (alteration of the DNA path) (DNA is pulled off the nucleosome) 3. Can reorganize the internal nucleosome components (Remodeling of nucleosome core particle-nucleosome dimer forms) -in all cases, the SWI/SNF complex leaves DNA exposed -allows transcription factors and RNAP to associate with the DNA -complex is recruited by activator proteins It is directed to specific DNA (gene) sites in many ways: a. binding to transcription factors b. binding to modify histones c. binding to methylated DNA

Trans-activating/repressing domain

-activates of represses transcription -interacts with other t-factors or RNAP -are distinct from their DNA binding domains

DNA Methylation

-adding a methyl group to the bases and sugars in DNA -inhibits gene expression (silences it) (i.e. the inactivated X chromosome in female cells is highly methylated) -represses transcription by inhibiting the binding of transcription factors to DNA -low methylation = high levels of expression -high methylation = low levels of expression -occurs at the 5' cytosine of GC doublets -these methylated CpG sequences exist in clusters called CpG islands Methylation patterns are tissue specific: -they are heritable for all cells of that tissues -proper patterns of DNA methylation are required for normal mammalian development

Histone Methylation

-adding a methyl to a histone tail will activate gene transcription -makes genes more euchromatic

In vitro experiments of promoters and enhancers

-analyze the effects that specific mutations have on the transcription of clone genes -introduce cloned enhancer elements in various orientations, and then testing their expression Experiments revealed what distinguishes promoters from enhancers: 1. position of an enhancer is not fixed to the gene it regulates -it will function whether it is up/stream, or within a gene 2. inverting the orientation of an enhancer produces no significant effect 3. If an enhancer is moved next to a different gene in the genome, or if an unrelated gene is placed near the enhancer, the transcription of that gene will be enhanced

RNA editing

-another post transcriptional modification -involves base substitutions made after transcription and splicing i.e. the PARA gene in Drosophila has at least 6 alternative splicing sites -leads to 48 different mRNA variants -but also goes through RNA editing -produces more than 1 million different kinds of transcripts -but alternative splicing is more common than RNA editing

DNA-binding domain

-binds to specific DNA sequences present in the cis-acting regulatory site -has various 3D structural motifs: 1. Helix-turn-helix 2. Zinc finger 3. Basic leucine zipper (bZIP)

Regulation of transcription factors

-can be tissue specific (present in only certain cell types) -can respond to environmental clues -can respond temporally (during a specific time in development)

Interchromosomal domain

-channels between chromosomes that contain little or no DNA

Weintraub and Groudine

-concluded that both transcriptionally active and inactive genes are associated with nucleosomes -but nucleosomes associated with transcriptionally active genes are in altered conformation

Restriction Enzymes

-enzymes that cleave DNA at specific regions -each restriction enzymes cuts only at its region, and it that region is not there, it will not cut i.e. HpaII looks for the CCGG site, but only if the cytosines are methylated

Zinc finger motif

-found in many t-factors that regulate gene expression related to growth, development, and differentiation -contains repeating clusters of two cysteines and two histidines -the clusters bind to zinc atoms, fold into loops, and interact with specific DNA

Chromosome territories

-in the interphase nucleus, each chromosome occupies a discrete domain called a territory -it stays separate from other chromosomes -transcriptionally active genes are located on the edge of the chromosome territory, right next to the inter-chromosomal domain

Chromatin remodeling complexes

-large, multi-subunit complexes -use energy from ATP hydrolysis to rearrange nucleosomes on the DNA -rearrangement of nucleosomes allows more accessibility to the chromosome for transcription regulatory proteins and activators (like RNAPII) -One type is the SWI/SNF Complex

Alternative Splicing and Human diseases

-mutations can affect regulation of splicing, leading to several genetic disorders 1. Myotonic dystrophy -caused by spliceopathies: defects in regulation of RNA splicing -severity of symptoms is directly related to the number of copies of the repeat sequence 2. Fragile X-syndrome 3. Huntington's disease

Transcription factory

-nuclear sites that contain most of the active RNA polymerase (RNAP) and transcription regulatory molecules -they form rapidly -by concentrating transcription proteins and actively transcribed genes in specific nucleus locations, the cell may enhance expression of these genes

Changing nucleosome composition

-nucleosomes usually contain the normal histone H2A -presence of variants, like H2A.Z within the nucleosomes affects mobility of nucleosomes and their positioning on DNA -H2A.Z is a variant that keeps promoters repressor free...so they will be activated) -in results, gene promoters of the nucleosomes may be activated or repressed

GAL gene system in yeast

-one of the first model systems used to study eukaryotic gene regulation GAL comprises: -4 structural genes (GAL1, 10, 2, 7) -their protein products transport galactose into the cell -their transcription is inducible -3 regulatory genes (GAL4, 80, 3) -their products positively and negatively control transcription of the structural genes ...how much detail do i need to know

Helix-turn-helix motif

-present in both prokaryotic and eukaryotic transcription factors -characterized by geometric conformation (rather than a distinct amino acid sequence)

Chromatin

-repeating units (nucleosomes) that are wound in 30nm fibers, turning into complex structures -complex structures inhibit some processes -made up of a combination of histones and nonhistone proteins

Basic leucine zipper (bZIP) motif

-the leucine zipper region allows for protein-protein dimerization -when two bZIP cottoning molecules dimerize, the leucine residue zips together -it keeps protein chains together -the dimer contains two basic alpha-helical regions (next to the zipper) that bind to the phosphate residues and specific bases

Metallothionein IIA Gene

-the transcription of the human MTIIA gene is regulated by multiple promoter/enhancer elements and the t-factors that bind to them -product of MTIIA: protein that binds to heavy metals (zinc, cadmium) -products the cell from the toxicity of the metals -normally, MTIIA is expressed at low levels, but levels increase when exposed to metals

DNA sequence elements (promoter elements)

-these (focused) promoter elements bind specific transcription initiation proteins TATA box: -a promoter consensus sequence ------a DNA sequence that indicates where a genetic sequence can be read and decoded. It is a type of promoter sequence, which specifies to other molecules where transcription begins -found in common with other promoters of that same cell -located at -30 relative to the transcription start site (tss) Initiator (Inr): -encompasses the transcription start site, from -2 to +4 from the start site TFIIB Recognition Element (BRE): -found in core promoters either immediately up/downstream of the TATA box Down Promoter Element (DPE): -downstream of the transcription start site @ +28 to +33 Motif Ten Element (MTE): -downstream of the transcription start site @ +18 to +27

Chromatin alteration mechanisms

1. Changing nucleosome composition 2. Histone Modification 3. Chromatin Remodeling

DNA elements that regulate gene expression

1. Cis-acting sequences: a. promoters b. enhancers c. silencers 2. Trans-acting sequences

Features that allow eukaryotes to have many mechanisms for gene regulation

1. Eukaryotic cells have more DNA -DNA is associated with histones (form chromatin) that can be altered 2. mRNAs must be spliced, capped, and polyadenylated prior to transport from the nucleus to the cytoplasm -each of these steps can be regulated 3. Eukaryotic mRNAs have a wide range of half lives (t 1/2) -the types and amounts of mRNA are altered to respond to environmental changes 4. Translation rates can be modified -the way proteins are processed and degraded

Promoter structure

1. Focused promoters -specify transcription initiation at a single specific nucleotide (the transcription start site) -associated with genes whose transcription levels are highly regulated -made up of one or more DNA sequence elements 2. Dispersed promoters -direct initiation from a few weak transcription start sites, located over a 50-100 nucleotide sequence -associated with genes that are transcribed constitutively -low levels of transcription initiation -usually found within CpG islands

mRNA degradation pathways

1. mRNA may be targeted for degradation by enzymes that shorten the poly-A tail -normal length = 200 nucleotides -if shortened to ~30, the mRNA becomes unstable -will become a substrate for exonuclease, which degrades it 2. Decapping enzymes renders mRNA unstable 3. mRNA can be cleaved internally (by endonucleases) -leaves the ends unprotected Examples: -nonsense mediated decay: translation terminates at premature stop codons -removes mutated mRNA -RNA interference

RNAi Pathway (RISC)

1. siRNA or miRNA associate with the enzyme complex called the RNA-induced silencing complex (RISC) 2. Within the RISC, the short double-stranded RNA is denatured, and the sense strand is degraded 3. The RNA/RISC becomes a highly functional and specific agent of RNAi -they seek out the RNA molecules that are complementary to the antisense RNA (that's in the RISC) -If the antisense RNA is perfectly complementary to the mRNA, RISC will cleave it -the cleaved mRNA will be degraded by ribonucleases -if the antisense RNA is not perfectly complementary, the RISC stays bound to the mRNA -this interferes with the ability of ribosomes to translate the mRNA -in this way, RNAi can silence gene expression by affecting either mRNA stability or translation

Trans-acting sequences

A DNA sequence that can influence the expression of a gene on any chromosome

Cis-acting sequences

A DNA sequence that is located on the same chromosome as the gene it regulates

Promoter

A promoter is a region of DNA that recognizes transcription machinery and binds to one or more proteins that regulate transcription initiation -promoters specify the transcription start site (and direction of transcription) -promoters are cis-regulator -located adjacent to the genes it regulates (+1 of tss) -every gene has its own promoter that dictates its transcription by recruiting transcription factors and polymerase to bind -promoters are highly methylated (correlated with low levels of expression b/c in most genes in tissues are turned off) Promoter elements -short nucleotide sequences (within the promoter) that bind specific regulatory factors Promoter subcategories 1. Core promoter 2. Proximal promoter

Post-transcriptional Gene Regulation

After transcription, but prior to translation, eukaryotic mRNA transcripts can be processed by: -removing noncoding introns -splicing together remaining exons -capping 5' end of mRNA -synthesizing a poly-A-tail at the 3' end Each of these steps can be regulated to control quantity, stability, and activity of the protein product Mechanisms: 1. control of alternative Splicing 2. control of mRNA stability 3. control of translation

RNAi technology

Allows researchers to make single-gene defects without inducing inherited gene mutations RNAi mediated silencing is specific and inexpensive -Allows scientists to rapidly analyze gene function Companies manufacture synthetic siRNAs of specific ribonucleotide sequence to be used in research -They can be introduced into cultured cells to knock out specific gene products

Alternative splicing of mRNA

Alternative splicing can generate different forms of mRNA to form identical pre-mRNA molecules -so expression of one gene can give rise to a number of proteins, with similar or different products Changing splicing patterns has effects on the translated protein: -alters enzymatic activity -alters receptor binding capacity -alters protein localization in the cell -alternative splicing increases the number of proteins that can be made from each gene -the proteome is not = to the # of genes in the genome -2/3, or 30-60% of the genes in the human genome can undergo alternative splicing

CpG islands

Clusters of methylated CpG sequences -located at the 5' end of genes (right next to the gene), usually near promoter regions -methylation inhibits expression ~5% of cytosines residues (in the eukaryotic genome) are methylated

Organization of DNA in chromosomes

DNA associates with histones to form nucleosomes -nucleosomes have polypeptide tails sticking out of them -these tails are prone to modifications (having chemical groups added)

Enhancers

Enhancers are DNA sequences that regulate transcription of eukaryotic genes -they control the rate of transcription -have modular structures -located on either side of the gene, or even within a gene (at any distance) -cis-regulator -necessary for achieving the maximum level of transcription -similar to the operator regions of prokaryotes, but more complex -are tissue specific: specific enhancers for different tissues, even of the same gene -control time specific transcription: developmental time: depending on where you are in developmental time, you have different regulatory genes

Gene Regulation in Eukaryotes

Eukaryotes have many mechanisms that control/regulate gene expression -can occur at many levels 1. transcription 2. splicing, processing mRNA 3. transport to cytoplasm 4. mRNA storage/turnover 5. translational regulation 6. chemical modification of protein product/activity

Acetylation

Gene activation: -decreases positive charge on histones, resulting in reduced affinity of the histone for DNA -moves nucleosomes apart, so DNA is available for transcription -catalyzed by HATs (histone acetyltransferase enzymes) complex -HDACs (histone deacetylases) remove acetate groups from histone tails *regulation of these complexes allows you to control transcription of these genes

RNAi as a pharmaceutical

In theory, any disease caused by the overexpression of a specific gene, or even normal expression of an abnormal gene product, could be attacked by therapeutic RNAi Viral infections -RNAi in tissue cultures may reduce severity of viruses like HIV, influenza, and polio In Animals: siRNA molecules have successfully treated viral infections, eye diseases, cancers, etc Human trials: -Phase I (toxicity) has been passed -Phase II and III (efficacy) trials are in progress -Testing the use of siRNAs as a drug to treat age-related macular degeneration -Also in trials to test RNAi in treatment of hepatitis B and respiratory syncytial virus -Other trails for siRNA treatment of influenza and hepC and some tumors

Alternative splicing of the CT/CGRP gene

In thyroid cells: -CT/CGRP gene is spliced so that the mRNA contains only the first four exons -the mRNA is translated into calcitonin peptide hormone that is responsible for regulating Ca In the brain/PNS: -CT/CGRO gene is spliced so the mRNA contains exons 5 and 6, but not 4 -the mRNA is translated into a peptide with hormonal activities in a wide range of tissues -this shows that through alternative splicing one gene can synthesize two peptide hormones, with different structures, locations, and functions

Activators and Repressors

Influence transcription in many ways: 1. bind to chromatin near promoter and recruit chromatin remodeling complexes -can open or repress the complex 2. make direct contacts with general t-factors to enhance or inhibit them -if enhancing, the activators bind to enhancers and interact with co-activators in the enhance some -can inhibit by inhibiting PIC formation by creating repressive domains 3. activators may increase rate of DNA unwinding within the gene -this accelerates release of RNAP from the promoter into the transcribed region of the gene

Enhancesome

Made up activator protein -forms a complex to glue the enhancer (cis-element) (far away) to the gene -maximizes transcription at the locus

Open and closed Chromatin

Open -euchromatin -loosely bound chromatin -open to transcription regulatory factors and enzymes (i.e. RNAP) -transcription can occur Closed Chromatin -tightly bound chromatin (tight association of DNA with nucleosomes and other proteins) -inhibits access of the DNA to proteins, so transcription does not occur -heterochromatin

RNAi Recent studies

RNA induced gene silencing mechanisms operate during normal development -can control expression of batteries of genes involved in tissue specific cellular differentiation Abnormal activities of miRNAs can contribute to: -cancer, diabetes, heart disease

RNAi

RNA interference -repressing translation and triggering degradation of mRNAs Discovery: Fire and Mello Experiment -the presence of a double stranded RNA acts to degrade the mRNA, if the mRNA is complementary in sequence to one strand of the double stranded RNA -only a few molecules of double stranded RNA are needed to bring about the degradation of large amounts of mRNA

RNA-induced gene silencing

RNA molecules that are ~21 nucleotides long can regulate gene expression in the cytoplasm of plans, animals, and fungi; repress translation and triggers mRNA degradation -through RNA interference (RNAi) -or small RNA molecules can also act in the nucleus to alter chromatin structure and repress transcription

RNA Polymerase

RNAPI -in the nucleolus -transcribes rRNA -resistant to a amanitin RNAPII -in the nucleus -transcribes pre-RNA -sensitive to a-amanitin RNAPIII -in the nucleus -transcribes small RNA, such as tRNA -moderately sensitive

Initiating Transcription

RNAPII binds to the promoter to initiate transcription -it recruits clusters of enzymes to work together (creating the holoenzyme) -most of these enzymes are transcription factors (help activate transcription) TFIID is the first to bind -searches and binds to the promoter -a complex within itself, made of TBP (TATA binding protein) and TAF (TBP associated factors), and ~12 other factors -TBP binds to TATA box -TAFs bind to Inr, DPEs, and MTEs -once these factors bind to TFIID, they recruit RNAPII -TFIIF binds to RNAPII **we need open space at the chromosome because there are so many proteins involved!

RNAi repressing transcription indirectly

RNAi's can silence transcription factor mRNAs -when levels of t-factors are reduced in the cell, t-genes, whose expression depends on these factors, is also repressed

Chromatin Remodeling

Repositioning/removal of nucleosomes on DNA -caused by chromatin remodeling complexes -makes genes accessible to transcriptional machinery

Sex determination in Drosophila: a model for regulation of alternative splicing

Sex in Drosophila is determined by the ration of X chromosomes toe sets of autosomes (X:A) - 0.5 ratio = male - 1.0 ratio = female - intermediate ratio = intersex The ratios are interpreted by genes that initiate a cascade of splicing events, resulting in production of female or male somatic cells Regulatory gene = Sxl -only expressed in females Transformer gene = Tra -pre-mRNA of tra is transcribed in both females and males, but only activated if SXL protein is present Double sex gene = Dsx -control point in development of sexual phenotype -produces functional mRNA and protein in both males and females, but it is processed in sex-specific patterns that produce female or male

Silencers

Silencers repress the level of transcription initiation -they affect the rate of transcription initiated from an associated promoter -sometimes they can act in tissue or temporal specific ways to control gene expression -they are cis-regulators

Mechanisms that regulate mRNA stability (half-life)

Specific RNA-sequence elements recruit degrading or stabilizing complexes, like ARE ARE-adenosine-uracil rich element: -ARE-containing mRNAs encode proteins that are involved in cell growth or transcription control, and need to be rapidly modulated in abundance -In cells that are not growing, or require low levels of expression, complexes bind to ARE and bring in shortening of the poly-A tail (leading to degradation) ~ 10 % of mammalian mRNAs contain these instability elements

Functional Domains of Transcription Factors

T-factors are proteins that bind to DNA and activate/repress transcription initiation -this is achieved through the presence of functional domains Functional domains are clusters of amino acids that carry out a specific function 1. DNA -binding domain 2. Trans-activating/trans-repression domain

Histone Modification

The covalent bonding of functional groups on the N-termnal (amino end) tails of histone proteins 1. acetylation -gene activation 2. methylation -gene activation (but methylation of DNA is deactivating) 3. phosphate -these processes are reversible -represent an epigenetic mechanism

Translation and Posttranslational Regulation

The end point of gene expression is the presence of an active gene -the quantity and activity of a gene product can be regulated -Translation of mRNA can be regulated to produce the appropriate quantity -Posttranslational structure modification can affect activity i.e. p53 protein

RNAi in diagnosis and treatment of cancer

The expression profiles of miRNA genes are characteristics of each tumor type Certain types of cancers have defects in miRNA gene expression -Treatment of these tumors with syntetic siRNAs may be able to correct these defects and reverse the cancer phenotype Some cancers are characterized by overexpression or abnormal expression of proto-oncogenes -If RNAi methods can target these specific gene products, that can be used in treatment of cancers that are resistant

Control of mRNA stability

The steady-state level of mRNA -the amount of mRNA in the cell that is available for translation -determined by a combination of -the rate at which the gene is transcribed -the rate at which mRNA is degraded -this turnover is important in regulation of gene expression The lifetime of an mRNA is defined in terms of its half-life or t1/2 -half live can be varied and regulated in response to the needs of the cell mRNA can be degraded along 3 general pathways Example: translational control of tubulin synthesis

Transcription factors

Transcription factors are regulator proteins that bind to cis-acting regulatory sites (promoters, enhancers, and silencers) They effect transcription levels: -activators increase levels of initiation -repressors reduced levels -different t-factors can compete for binding to the same DNA sequence -multiple t-factors that bind to several different enhancer and promoter elements can interact with one another -fine the levels and timing of t-initiation

Molecular mechanisms of RNA-induced Gene silencing

Two types of short RNA molecules that are involved in RNA-induced gene silencing 1. small interfering RNAs (siRNA) 2. microRNAs (miRNAs) -both siena and miRNA are short, double stranded molecules, between 20-25 ribonucleotides long -in the nucleus, short RNA binds to complementary DNA regions, directs methylation and inactivation of genes

Formation of RNAPII Transcription Initiation Complex

general transcription factors assemble in a specific order and form a pre-initiation complex (PIC) -TFIID, TFIIB, TFIIA, TFIIE, TFIIF, TFIIH, and Mediator PIC formation: 1. TFIID binds to a core promoter element (with TFIIA assistance) 2. TFIIB binds to BRE elements on one or both side of the TATA box 3. Once TFIID and TFIIB are bound to the core promoter, the other general t-factors recruit RNAP to the promoter 4. the full formed PIC mediates: -unwinding of promoter DNA at the start site -transition of RNAPII from transcription initiation to elongation -RNAPII clears the promoter as it goes down the DNA template

ribosomal RNA

genes that make rRNA (which will make ribosomes, necessary for all gene translation) -are found on the afrocentric chromosomes -the nucleolus: clump of ribosomal genes

miRNAs

miRNAs = micro RNAs -derived from single stranded RNAs that are transcribed within the nucleus from the cell's own genome and contain a double stranded cell loop -negatively regulate gene expression -nuclease enzymes within the nucleus recognize the stem loop structure and cleaved them from the longer single-stranded structure -the stamp loop fragments are sent from the nucleus into the cytoplasm, where they are processed into short, linear, double stranded miRNAs -more than 1,500 miRNAs have been discovered in the human genome -scientists believe there are more genes that are transcribed into noncoding RNAs, which can regulate the expression of more than half of all protein coding genes

RNAi Pathway (RITS)

miRNAs and siRNAs can also repress transcription of specific genes and larger regions of the genome -through association with the RNA-induced transcription silencing (RITS) 1. the antisense RNA strand within the RITS targets the RITS complex to specific gene promoters or larger regions of chromatin 2. RITS then recruits chromatin modifying enzymes to these regions -the enzymes methylate histones and DNA -results in heterochromatic formation, therefore silencing transcription Because of their effects on chromatin-mediated gene silencing, miRNA molecules are thought to be involved in epigenetics (such as imprinting and X chromosome inactivation)

siRNAs

siRNAs = small interfering RNAs -derived from longer RNA molecules that are linear, double stranded, and located in the cell cytoplasm -arise in cells as a result of virus infection or the expression of a transposon -this will synthesize double stranded RNA molecules as part of their life cycles (RNAi may be a method by which cels recognize double stranded RNAs and inactivate them to protect the organism) -siRNA molecules can be produced in the lab -in the cytoplasm, double stranded RNA (dsRNA) molecules are recognized by an enzyme know as Dicer -Dicer cleaves these RNAs into siRNAs

Alternative splicing ad polypeptides

the Dscam gene in Drosophila encodes protein that guides axon growth -Exons 4, 6, 9, and 17 of Dscam pre-mRNA can be spliced into 95 alternatives, producing 38, 016 possible combinations

p53 Protein

An example of postranslational regulation -p53 is essential to protect normal cells from the effects of DNA damage and other stresses -p53 is a transcription factor protein -it increases the transcription of genes who are involved in: cell- cycle arrest, DNA repair, and programmed cell death -under normal conditions, p53 levels are low/inactive -when cells suffer DNA damage or metabolic stress, levels of p53 increase dramatically -In unstressed cells, p53 is bound to the protein Mdm2 -Mdm2 bings to the transcriptional activation domain of p53, which blocks its ability to induce transcription -Mdm2 also acts as a ubiquitin ligase -Adds ubiquitin residues onto p53 -Ubiquitin tags other proteins for degradation -When cells are stressed, Mdm2 and p53 are modified (by phosphorylation and acetylation), causing Mdm2 to release from p53 -P53 proteins are stabilized and levels increase -P53 can now act as a transcription factor -Negative feedback loop: Induces transcription of Mdm2 gene, which creates more Mdm2, causing p53 to inactivate