2024 Gene Expression Notes PDF

Burman University, BIOL 374, Gene Expression, p.1 Learning Objectives: After completing this module, you will be able to 1. Compare and contrast gene expression in prokaryotic and eukaryotic organisms. 2. Compare the eukaryotic RNA polymerases and the genes they transcribe. 3. Describe RNA po...

Burman University, BIOL 374, Gene Expression, p.1 Learning Objectives: After completing this module, you will be able to 1. Compare and contrast gene expression in prokaryotic and eukaryotic organisms. 2. Compare the eukaryotic RNA polymerases and the genes they transcribe. 3. Describe RNA pol II promoters and the general transcription factors associated with them. 4. Define the role of cis-acting elements, promoter-proximal elements and enhancers, in tissue-specific transcription. 5. Describe the different categories of transcription factors that act at the cis-acting elements. 6. Describe the epigenetic regulation of genes by DNA methylation and lncRNA inactivation of the X chromosome. 7. Define the terms activation, repression, cis-element, trans factors, enhancesome, hyper- and hypo-acetylation, condensation and decondensation of chromosomes, mediator complex, gene silencing, epigenetics, lncRNA, DNase 1 footprinting, EMSA, CpG islands, CTD domain of RNA pol II and more. 8. Compare transcription initiation in RNA Pol I and III with Pol II. Text: Lodish et al. (2021), Ch. 8 Verse: But God gives it a body as he has determined, and to each kind of seed he gives its own body. Not all flesh is the same: People have one kind of flesh, animals have another, birds another and fish another. 1 Cor 15:38-39 (NIV) The accepted molecular biological basis of speciation is genome changes that facilitate the natural selection of species members that are more adapted to the prevailing environment. While this understanding is reasonable, there are some problems, specifically, 1) it would take a long time to accumulate significant mutations to allow the genetic program, or blue print, to be different enough to account for those adaptations, and 2) the inability to explain high similarities and identities in the genomes of divergent species. So rapid adaptations (in terms of a few decades) is not possible with those subtle base pair changes. However, a natural disaster could trigger the Darwinian concept of adaptive radiation (where organisms diversify rapidly from an ancestral species into a multitude of new forms due to a sudden change/environmental challenge). The “Darwin's finches” are often used as an example of adaptive radiation. These are a group of about 18 species of passerine birds that are well known for their remarkable diversity in beak form and function. The finches’ beaks and bodies changed allowing them to eat certain types of foods such as nuts, fruits, and insects. Recent work showed that changes in epigenetics is a better explanation to how Darwin’s finches respond to rapid environmental changes. In one study, very little genetic variations were detected in rural and urban populations of two species of Darwin’s finches but substantial epigenetic differences were seen that could be related to environmental differences resulting from urbanization (McNew et al., 2017). Likewise, in another study, epigenetic alterations in LW2024 Burman University, BIOL 374, Gene Expression, p.2 DNA methylation patterns (also referred to as epimutations in this study) were more common in five different species than polymorphic genome variations (Skinner, et al., 2014). Furthermore, the number of epimutations increased with phylogenetic distance compared to those from genetic mutations (the number of genetic mutations did not consistently increase with phylogenetic distance). As environmental factors are known to result in heritable changes in the epigenome, it is possible that epigenetic changes (DNA methylation, histone modifications, chromatin remodeling, and lncRNA regulation and all those great concepts we are learning) contribute to the molecular basis of the evolution of Darwin’s finches. While the Bible text from last week describes God’s gift of this “seed” or genomic design for all creatures. Considerable physical variations can be obtained by epigenetic control of this genome. It also could explain the high degree of genomic sequence similarities between species because it may not be the DNA sequence which defines a species but its epigenome (epigenome is usually defined as the complete description of the chemical modifications to DNA and histone proteins that regulate the expression of genes within the genome). References: McNew, S.M., Beck, D., Sadler-Riggleman, I. et al. Epigenetic variation between urban and rural populations of Darwin’s finches. BMC Evol Biol 17, 183 (2017). https://doi.org/10.1186/s12862-017- 1025-9 Michael K. Skinner, Carlos Gurerrero-Bosagna, M. Muksitul Haque, Eric E. Nilsson, Jennifer A.H. Koop, Sarah A. Knutie, Dale H. Clayton, Epigenetics and the Evolution of Darwin’s Finches, Genome Biology and Evolution, Volume 6, Issue 8, August 2014, Pages 1972–1989, https://doi.org/10.1093/gbe/evu158 Introduction (Topic 7) § Gene Expression – Entire process whereby the information encoded in a particular gene is decoded into a particular protein. – Includes: 1. Transcription initiation [F8-3] 2. RNA Polymerization/Elongation 3. Transcription termination § Constitutive vs. Inducible 1. Constitutive or housekeeping genes are always active in growing cells, during all stages of the cell cycle, e.g., ribosomes, cytochrome B, b-actin and others. 2. The synthesis of inducible or regulated genes is controlled in response to the needs of a cell or organism. Results in differential gene expression, e.g., in developmental proteins. LW2024 Burman University, BIOL 374, Gene Expression, p.3 7.1 Overview of Eukaryotic Gene Control and RNA Polymerases § In bacterial cells and most single cell eukaryotes, gene control serves mainly to allow these cells to adjust to changes in its environment so that growth and division can be optimized. § However, gene control in multicellular organisms is important for development and differentiation, and is generally not reversible. With exception, relatively few eukaryotic genes are reversibly regulated by environmental conditions. § The primary purpose of gene control in multicellular organisms is the precise execution of developmental decisions, i.e., proper genes are expressed in the proper cells/time during development and cellular differentiation. § Transcriptional control is the primary means of regulating eukaryotic gene expression. § Protein-binding regulatory DNA sequences, or cis-control elements, are associated with genes. § Some of these cis-elements may be kilobases away from the transcription start site (e.g., Pax6 genes; F8-4). § Specific proteins, or trans factors, that bind to a gene’s regulatory sequences (i.e., cis elements) determine where transcription will start, and either activate or repress its transcription. § Some trans factors that position Pol II at transcription initiation start sites and help separate the DNA strands, thus required by all Pol II-promoters, are called general transcription factors. § Inactive genes are assembled into condensed chromatin, which inhibits the binding of RNA polymerases and general transcription factors required for transcription initiation. § Activator proteins bind to control elements close to transcription start sites, as well as kilobases away, to promote chromatin decondensation and RNA polymerase binding. § Repressor proteins bind control elements, causing condensation of chromatin and inhibition of polymerase binding [F8-3]. § Three eukaryotic polymerases catalyze formation of different RNAs [T8-1, T9-2 (old text)]. – Purified RNA Polymerases are eluted at different salt concentrations during ion- exchange chromatography [F8-6]. – Also differ in their sensitivity to a-amanitin (poisonous cyclic octapeptide produced by some mushrooms). Pol I is insensitive, Pol II is very sensitive, and Pol LW2024 Burman University, BIOL 374, Gene Expression, p.4 III has intermediate sensitivity. 1. RNA Pol I: pre-rRNA (ribosome components, protein synthesis) 2. RNA Pol II: mRNA, snRNA (small nuclear RNAs required for splicing) siRNA (chromatin-mediated repression, translation control) and miRNA (translation control) 3. RNA Pol III: tRNAs, 5S rRNA, small stable RNAs (protein synthesis, ribosome components, etc) § Subunit structure of yeast and E. coli RNA polymerases [F8-7]. – All three yeast polymerases have 5 core subunits homologous to the b’, b, two a and w subunits of E. coli RNA polymerase. – The largest subunit of RNA Pol II also contains an essential C-terminal domain (CTD) [F8-8]. The CTD has a repeated heptapeptide (Tyr-Ser- Pro-Thr-Ser-Pro-Ser; in vertebrates there are 52 repeats) in the C-terminal domain (CTD). The Ser (and some Tyr) residues of the CTD become phosphorylated right after transcription initiation and remains phosphorylated during active transcription. – Both RNA Pol I & III have the same two nonidentical a-like subunits, whereas RNA Pol II have more distantly related nonidentical a-subunits. – All three share the same w-like subunit and 4 other common subunits. – In addition, each yeast RNA polymerase contains 3 to 7 unique subunits. – In the transcribing complex, a common clamp domain LW2024 Burman University, BIOL 374, Gene Expression, p.5 closes on the template and the newly formed DNA-RNA hybrid, enabling RNA Pol II to transcribe long stretches of DNA [F8-9]. This domain is part of the b’, or RPB1, subunit. Together the factor DSIF (F8-3), a transcription elongation factor, the clamp converts the polymerase to be a processive enzyme, polymerizing ribonucleotides rapidly until transcription is terminated. 7.2 RNA Pol II Promoters and General Transcription Factors § Protein-coding genes are transcribed by RNA pol II. § Expression of these genes is regulated by multiple protein- binding DNA sequences, generically referred to as transcriptional control regions. § These control regions are cis-acting elements which only affecting expression of genes on the same DNA molecule. § Transcription is initiated at a specific base pair, +1 (that corresponds to the 5’-cap in mRNA) and is controlled by the binding of trans-acting proteins (transcription factors) to cis-acting regulatory DNA sequences. § Eukaryotic cis-acting elements are often much further from the promoter they regulate (many kilobases from start sites), and transcription from a single promoter may be regulated by binding of multiple transcription factors to alternative control elements. § Transcription control sequences can be identified by analysis of a 5¢-deletion series [F7-13; old text]. § Most eukaryotic genes are regulated by multiple cis-acting control regions: 1. Promoters determine the site of transcription initiation and direct binding of RNA pol II. 2. Promoter-proximal elements, close to the start site, help regulate eukaryotic genes (discussed later in 7.3). 3. Enhancers, distant from the transcriptional start site, can stimulate transcription by RNA pol II (discussed later in 7.3). LW2024 Burman University, BIOL 374, Gene Expression, p.6 A. Promoters 1. TATA box – Most common in rapidly transcribed genes. – Similar to E.coli Pribnow box. – The TATA box is a highly conserved promoter in eukaryotic DNA [F9-16]. ~ 26 to 31 bases upstream from the +1 start site. Deletion of the spacing between the TATA box and +1 site result in transcription initiation at a new site ~ 25 bp downstream from the TATA box. 2. Initiators – Less common than TATA box. – Most have a cytosine (C) at –1 and an adenine (A) at +1 transcription start site. – Degenerate initiator consensus sequence: 5’-YYA+1N(T/A)YYY-3’ where Y = C or T, N = any base, A at +1, and A or T at +3. 3. CpG islands – Generally, for genes that are transcribed at low rates, e.g., “house-keeping” genes. – Tend to have ill-defined initiation site, anywhere over a 100-1000 bp region. As a result, mRNAs tend to have several alternative 5’-ends. – Contain a CG-rich stretch of 20-50 bases within ~ 100 bp upstream of the initiation site. – CG-rich regions/clusters, or CpG islands, are rare in mammalian genomes. – Cs in CpG islands are unmethylated (most Cs followed by Gs in the genome are methylated; 5-methyl C). – CpG islands coincide with nucleosome-free regions of DNA. – CpG island promoters are divergently transcribed, however, RNA molecules transcribed in the wrong direction, i.e., resulting in (-) standed RNA, are terminated at ~1-3 kb from the +1 site. B. General Transcription Factors at the Promoters – Initiation by Pol II requires general transcription factors (TFIIs, e.g., TFIIB), which position Pol II at initiation sites and are required for transcription of most genes transcribed by this polymerase. – Most general transcription factors are multimeric and highly conserved. – TFIIs together with Pol II, called the preinitiation complex (or PIC), assemble in a specific order in vitro but most of the proteins may combine to form a holoenzyme complex in vivo. – Stepwise assembly of Pol II transcription-initiation complex in vitro [F9-19] 1. Locating the start site – Involves the binding of TFIID, which binds to the TATA-box through its TATA-binding protein subunit, TBP. LW2024 Burman University, BIOL 374, Gene Expression, p.7 – TFIID is ~ 750 kDa; single TBP (38 kDa) and 13 TAFs (TBP-associated factors). – TBP The conserved C-terminal domain (180 aa) of TBP binds to TATA-box DNA. TBP is saddle-shaped monomer with dyad symmetry. TBP interacts with the minor groove in DNA, bending the helix (F5-5). Only TBP (but not TAFs of TFIID) is required for in vitro RNA Pol II initiation. 2. TFIIA and TFIIB join TBP – TFIIA associates with TBP and DNA on the upstream side of the TBP-TATA box complex. – C-terminal of TFIIB contacts both DNA (on either side of TATA-box) and TBP. – Additionally, TFIIB binds to the TFIIB recognition element BRE upstream of the TATA-box in “strong” promoters [F9-16]. – N-terminal of TFIIB assists Pol II in melting the DNA at the transcription start site. – TAF subunits also contact the DPE element (downstream promoter element; ~30 bp downstream from the transcription start site) present in TATA-less genes like the initiator element. – These additional interaction increases TFIID binding. 3. Formation of core preinitiation complex (PIC) – Preformed complex of TFIIF and Pol II binds, positioning the Pol II over the start site. 4. Closed PIC – TFIIE tetramer joins preformed complex; contains the docking site for TFIIH. – TFIIH joins to form closed PIC. LW2024 Burman University, BIOL 374, Gene Expression, p.8 5. Open PIC – TFIIH has 10 subunits. – Helicase activity of one of the TFIIH subunits unwind DNA (using ATP) at start site, allowing Pol II to form the open PIC complex. 6. Pol II starts transcribing mRNA. – The kinase subunits of TFIIH phosphorylate the Pol II CTD at multiple sites (phosphorylated CTD is a docking site for other entities needed later). – All of initiation TFs but TBP dissociate and are replaced by elongation factors. 7.3 Regulatory Sequences in Protein-Coding Genes and Activators/Repressors that Modulate Them. § Transcription factors, often cell-type specific, (i.e., functioning only in differentiated cell types; as opposed to the general TFs that bind to promoter sequences in S9.3B) can stimulate or repress transcription. § They do so by binding to promoter-proximal elements and enhancers in eukaryotic DNA. § The human genome codes for ~ 2000 different transcription factors, however, their concentrations very low in the cell (often 20 bp) may decrease transcription. – Cell-type specific. LW2024 Burman University, BIOL 374, Gene Expression, p.9 – Transcription-control elements can be identified with linker scanning mutants [F9-22]. Linkers, used to introduce mutations, are inserted in the control regions in an overlapping fashion, hence linker scanning mutation. B. Enhancers – Multiple ~6- to 10-bp control elements, each a protein-binding site and contributing to the overall activity of the enhancer, within a region of ~200 bp. – Located 200 to 50,000 bp upstream or downstream from a promoter, within an intron, or downstream from the final exon of a gene, and in either orientations (i.e., on + or – strand of DNA) [F9-23a]. – Again cell-type specific. – SV40 enhancer element (~100 bp) stimulates transcription of all mammalian promoters that have been tested. C. Yeast transcription control regions § In yeast, upstream activating sequences (UASs) function similarly to promoter- proximal and enhancer elements in higher eukaryotes [F9-23c]. D. Trans-acting elements – Repressors and activators that bind to these cis- elements (i.e., transcriptional control regions) are trans-acting. – Trans-acting elements affect expression of regulated genes no matter on which DNA molecule in the cell these are located, i.e., on the same or different DNA molecule. – Trans-acting elements, or transcription factors, are diffusible proteins. – Protein-DNA interactions can be detected by footprinting and gel-shift assays. 1. In DNase I footprinting, a region of DNA is protected from digestion by the nuclease DNase I when it is bound by a protein [F9-24a; middle fig]. Fig 9-24b (right fig) illustrate a DNase I experiment of a promoter with general transcription factors. LW2024 Burman University, BIOL 374, Gene Expression, p.10 2. In the gel-shift assay, or electrophoretic mobility shift (EMSA, or band-shift), the electrophoretic mobility of a DNA fragment is retarded when it is complexed to protein, causing a shift in the location of the fragment band [F9-25; top left]. – Once a transcription factor is isolated and purified, its partial amino acid sequence can be determined and used to clone a gene or cDNA encoding it. The isolated gene can then be used to test the ability to activate or repress transcription in an in vivo transfection assay [F9-26]. E. Activators are modular § Transcriptional activators are modular proteins composed of at least 2 distinct functional domains [F9-28]: 1. DNA-binding domain bind to specific DNA sequences. 2. Activation domain interact with other proteins to stimulate transcription from a nearby promoter. – Modular domains are connected by flexible regions in the protein chain that allow activation domains in different activators to interact even when their DNA-binding domains are bound to sites separated by tens/hundreds of bp. – Roles of modular domains, i.e., DNA- binding or activation, can be demonstrated by using deletion constructs, e.g., Gal4 deletion mutants demonstrating functional domains [F9-27]. F. Repressors – Repressors are functional converse of activators, i.e., they inhibit gene transcription. – Repressors are also modular, with DNA- binding domains and repression domains. – Many eukaryotic genes are subject to simultaneous activation and repression. LW2024 Burman University, BIOL 374, Gene Expression, p.11 The outcome is determined by which one wins – the activator or repressor. – Repression domains can also function by interacting with other protein domains located far away. – Some repressors interfere with the binding of an activator to its cognate site. – Others allow activators to bind to their cognate cis-elements, however, by binding next to the activators inhibit the activator via its repression domain so that the gene is repressed. G. DNA-binding domains § DNA-binding domains contain a variety of structural motifs that bind specific cis- sequences. § The binding usually occurs by noncovalent interactions between amino acid side chains one or more a-helices in the DNA-binding domain and the bases within a major grove in its cognate site. § e.g., Bacteriophage 434 repressor and DNA [F9-29] Binds as a dimer, where each monomer of the repressor binds to a half-site (half-sites are located at adjacent major grooves). The a-helix is called the recognition helix or sequence- reading helix. The DNA-binding domain of the bacteriophage 434 repressor has a helix-turn-helix motif. – DNA-binding domains in eukaryotic transcription factors can be classified into numerous structural types/motifs: 1. Homeodomain proteins 2. Zinc-finger proteins 3. Leucine-zipper proteins 4. Basic helix-loop-helix (bHLH) proteins – Homeodomain proteins 60-residue DNA-binding motif, called the homeodomain (or “homeobox” motif) 1st found in the homeotic genes (e.g., antennapedia and ultrabithorax) in Drosophila. Conserved regions were diagrammed in a box when sequences were compared. DNA-binding domain has a helix-turn-helix motif similar to bacteriophage repressor (F9-29). – Zinc-finger proteins The zinc-finger motif has a polypeptide chain folded around a central Zn2+ ion, to resemble a finger. Several types including the C2H2 type, which has Zn2+ ion bound by 2 Cys and 2 His residues (most common DNA- binding motif in mammals; F9-30a). C2H2 zinc-finger proteins LW2024 Burman University, BIOL 374, Gene Expression, p.12 § Most transcription factors contain multiple C2H2 zinc fingers that bind as a monomer wrapping around the DNA double helix (F9-30a; bottom left). § Each C2H2 finger has an a-helix that is inserted in the major groove of DNA. C4 zinc- finger proteins § The Zn2+ ion is bound by 4 Cys. § This includes the steroid receptor superfamily, which has 2-finger units that bind as hetero- or homo-dimers [F9-30b; top right]. – Leucine-Zipper Protein Fig F9-30c shows the interaction of a homodimeric leucine-zipper protein and DNA. Hydrophobic Leu stripe, called the Leucine-Zipper, along one side of the a helix is required for dimerization. The coiled-coil dimerization is stabilized by hydrophobic interactions. Two extended a helices grip the DNA like a pair of scissors at 2 adjacent major grooves. Belong to the general basic zipper (bZip) proteins because any hydrophobic aa can be used in the zipper region. Most bZips are heterodimeric. – Basic helix-loop-helix (bHLH) proteins [F9-30d] The DNA-binding domain of basic helix-loop-helix (bHLH) proteins is very similar to the Leucine Zipper proteins. Basic amino acids interact with the DNA, hence basic Helix-Loop-Helix (bHLH). A nonhelical region (loop) separates two a helices. One pair of the a helices form “bZip” coiled-coils. H. Activation/repression domains – Activation and repression domains in transcription factors exhibit considerable structural diversity, i.e., a variety of amino acid sequences and 3-dimensional structures. – Some have acidic activation domains, rich in acidic amino acids. LW2024 Burman University, BIOL 374, Gene Expression, p.13 – Ligand binding induces conformational change that allow activation domain to interact with other proteins [F9-31]. Some exist as unstructured, random-coiled regions until they interact with a co- activator/ligand, e.g., CREB with cAMP (F9-31a). Binding of ligand (estrogen) creates a domain that can bind a co-activator (F9-31b). The antagonistic ligand (tamoxifen) block the interaction with the coactivator by stabilizing an alternate, non-activating structure (F9-31c). I. Multiple transcription factor binding sites. § The transcription-control regions of most genes contain binding sites for multiple transcription factors. § Transcription of such genes varies depending on the repertoire of transcription factors that are expressed and activated in a particular cell at a particular time. J. Heterodimeric transcription factors – Transcription factors bZip and bHLH can form heterodimers, i.e., of two different monomers to form a functioning heterodimer. – Heterodimeric transcriptional factors increase gene- control options (F9-32). – Combinatorial activation/repression domains can be formed with homo- [F9-32a] & hetero-dimers. – In heterodimeric transcriptional factors, each composite binding site is divided into two half-sites, each one bound by a monomer in the heterodimeric transcription factor [F9-32bc]. – The alternative combinations of these monomers bring together many different combinations of activation domains and DNA sites recognized. – Combinatorial complexity can also result from cooperative binding of two unrelated transcription factors on neighboring sites [F9-33]. LW2024 Burman University, BIOL 374, Gene Expression, p.14 K. In vivo Polymerase II Initiation – Enhancesome Enhancers generally contain multiple clustered binding sites for transcription factors [F9-34]. Cooperative binding of multiple activators to nearby sites in an enhancer forms a large nucleoprotein/multiprotein complex called an enhancesome. Assembly of enhancesome requires small proteins (called architectural proteins, e.g. HMG1) that bind to the DNA minor groove and bend the DNA sharply, allowing proteins to bind on either side of the bend to interact more readily [F7-30; old text]. – In vivo assembly of Pol II initiation complex requires the Mediator complex. The multiprotein Mediator complex is large, with ~ 30 proteins and as large as a ribosome [F9-39]. ~ 20 mediator subunits, comprising of the “head and middle” portions, may be involved in the binding to Pol II. The rest bind to activation domains in various activator proteins. The mediator complex forms a bridge between activators bound to its cognate site on the DNA and Pol II on the promoter. Multiple weak interactions are enhanced because the transcription factors are bound to neighboring DNA sites indirectly. The mediator is a co-activator; forms a molecular bridge between activation domains of transcription factors and RNA Pol II. By binding several activators simultaneously, the mediator help integrate the effects of multiple activators on a single promoter [F9-40]. Activators bound to a distant enhancer can interact with transcription factors bound to a promoter because DNA is flexible and the intervening DNA can form a large loop. – Cell-type Specificity in Transcription Initiation. The highly cooperative assembly of preinitiation complexes in vivo generally requires several activators [Fig; old text]. LW2024  Burman University, BIOL 374, Gene Expression, p.15 A cell must produce the specific set of activators required for transcription of a particular gene in order to express that gene. Therefore, tissue-specific proteins are expressed because of the presence of tissue- specific transcription factors, or a combination of a specific set of activators. L. Elongation and Termination of Transcription § Different mechanisms of transcription termination are employed by each of the eukaryotic nuclear RNA polymerases. § Once RNA pol II transcribes protein-coding genes beyond ~200 bases, further elongation is highly processive and does not terminate until the cleavage and polyadenylation site is transcribed. § Termination then occurs 0.5 -2 kb beyond the poly A site. 7.4 Molecular Mechanisms of Transcription Activation & Repression § Occurs at 3 levels: 1. Changes in chromatin structure directed by activators and repressors 2. Modulation of the levels and/or activities of activators and repressors (see 9.6) 3. Direct influence of activators and repressors on assembly of transcription-initiation complexes (see S7.3K) § Heterochromatin § Heterochromatin are condensed regions of chromatin. § In these regions, DNA is relatively inaccessible to transcription factors and other proteins. § Formation of heterochromatin “silences” (or represses) gene expression at telomeres, near centromeres and other regions. § In the life cycle of S. cerevisiae, mating-type control in yeast is controlled by gene silencing [F9-35]. – When the a or a sequences are present in the MAT (mating-type) locus, they can be transcribed into mRNAs whose encoded proteins specify the mating- type phenotype of the cell. LW2024 Burman University, BIOL 374, Gene Expression, p.16 – The amino acid residues (~20-60) at the N-terminus of each histone extend from the surface of the nucleosome [F8-26a]. – These amino acids are rich in Lysine residues, which can be reversibly modified by acetylation. – Heterochromatin is hypoacetylated. – Schematic model of silencing at yeast telomeres [F9-36d] – The interactions of several proteins with each other and the hypoacetylated N-termini of histones H3 and H4 are responsible for the repressing chromatin structure in these regions. – RAP1 – binds directly to telomeres (HML & HMR), and to each other. – SIR 2, 3 & 4 (silent information regulators) – bind to hypoacetylated histones H3 & H4, and to each other. – Assembly of multiprotein repressing complex result in the formation of heterochromatin. § Repressors/Activators & Acetylation – Some repressors interact with histone deacetylase complexes, resulting in the deacetylation of histones in nucleosomes near the repressor-binding site [F9-37a]. – Deacetylated histones lead to condensed chromatin, inhibiting the interaction of general transcription factors and promoter DNA, thereby repressing transcription initiation. – Conversely, activators result in hyperacetylation by stimulating histone acetylase complexes, thereby stimulating transcription initiation [F9-37b]. § Chromatin-remodeling complexes – In addition to histone acetylase complexes, chromatin- remodeling complexes are also required for activation at many promoters. – Homologous to helicases, these multisubunit complexes can transiently dissociate DNA from histone cores in an ATP-dependent reaction, or decondense chromatin. – Thus, promotes the binding of DNA-proteins needed for initiation to occur. LW2024 Burman University, BIOL 374, Gene Expression, p.17 7.5 Regulation of Transcription-Factors Activity § Transcription factors can be in an active or inactive conformation. § Conformational change that modify their interactions with other proteins can be achieved by: 1. Noncovalent binding of a ligand, e.g., lipid-soluble hormone binding, or 2. Covalent modification, e.g., via phosphorylation. § Lipid-soluble hormones control the activities of nuclear receptors [F9-42]. § Secreted from one cell type and travel through extracellular fluids to affect the function of cells at a different location. § Can diffuse through plasma and nuclear membranes. § Bind to and regulate members of the nuclear- receptor superfamily. All nuclear receptors share a common domain structure [F9-43] Unique N-terminal domain that can function as the activation domains. Conserved DNA-binding domain that bind to nuclear- receptor response elements on DNA control regions. The hormone-binding (or ligand- binding) domain, at the C- terminal, contains a hormone- dependent activation domain. § Response elements are DNA sites that major nuclear receptors bind to [F9-44]. – Estrogen receptor response element (ERE) – 6-bp inverted repeats – Receptors bind as homodimers. – Thyroid hormone receptor response element (TRE) – 6-bp direct repeats of ERE separated by 4 bp – Receptors bind as heterodimers with a common nuclear-receptor monomer called RXR, e.g., RXR-TR. § Mechanism of hormonal control of nuclear-receptor activity – For heterodimeric nuclear receptors, e.g., RXR-TR, when bound to their response elements, act as repressors or activators of transcription depending LW2024 Burman University, BIOL 374, Gene Expression, p.18 on whether hormone occupies the ligand- binding site. - hormone à represses à histone deacetylation + hormone à activates à histone hyperacetylation – For homodimeric nuclear receptors, e.g., ER, their activity is regulated by controlling their transport from the cytoplasm to the nucleus in a hormone-dependent nuclear translocation [F9-45a, d]. § Polypeptide hormone binding results in phosphorylation of some transcription factors. – Peptide or protein hormones cannot diffuse through the lipid bilayers. – Instead they bind to cell surface receptors, which pass the signal to proteins in the cytoplasm (process called signal transduction; Chapters 15 & 16). – Usually leads to transcription-factor phosphorylation, which could either activate or deactivate the transcription factor, e.g., JAK-STAT Pathway [F16-12a]. LW2024 Burman University, BIOL 374, Gene Expression, p.19 7.6 Epigenetic Regulation of Transcription § Epigenetic control of transcription refers to repression or activation that is maintained after cells replicate as the result of DNA methylation or post-translational modification of histones, especially histone methylation. § Methylation of CpG sequences in CpG island promoters in mammals generates binding sites for a family of methyl-binding proteins (MBDs) that associate with histone deacetylases, inducing hypoacetylation of the promoter regions and transcriptional repression. § Lysine 9 on histone H3 when di- and trimethylation creates binding sites for the heterochromatin-associated protein HP1, which results in the condensation of chromatin and transcriptional repression. – These post-translational modifications are perpetuated following chromosome replication because the methylated histones are randomly associated with the daughter DNA molecules and associate with H3K9 HMT that methylate H3 lys 9 on newly synthesized histone octamers assembled on the daughter DNA [F9-47]. – Post-translational modification (methylations, phosphorylations, and unbiquinations) of histone tails are associated with active and repressed genes [T9-3]. LW2024 Burman University, BIOL 374, Gene Expression, p.20 § lncRNA and X-chromosome – Long noncoding RNA (lncRNA) direct epigenetic repression in metazoans. – X-chromosome inactivation in female mammals requires XIST lncRNA from the Xist gene that is transcribed from the X-inactivation center of one X chromosome and then spreads by a poorly understood mechanism along the length of the same chromosome [F9-50c]. – The active X chromosome produces another lncRNA called TSIX that represses the inhibitory effect of XIST [F9-50a]. – In differentiated female cells, the inactive X chromosome (Xi) is associated with XIST lncRNA. – The XIST lncRNA only acts in cis, i.e., remaining with the inactive X and does not interact with the active X, or other chromosomes. – During very early embryogenesis, TSIX lncRNA is expressed in both the X chromosomes, thus preventing the expression of XIST lncRNA (present on the complementary strand of the DNA). As cells differentiate, the Tsix gene (gene for TSIX lncRNA) is repressed in one of the X chromosomes, allowing the transcription of the XIST lncRNA. – Accumulation of XIST lncRNA leads to X inactivation of that X chromosome (the process of choosing which X chromosome is inactivated is random, i.e., Xp from sperm or Xm from egg). – XIST lncRNA interacts with a co-repressor that binds a histone deacetylase and repression complexes (e.g., PRC2; F9-50d] at an early stage of embryogenesis, initiating X inactivation. – X inactivation is maintained throughout the remainder of embryogenesis and adult life by continued association with repression complexes and DNA methylation of CpG island promoters on the inactive X. § Some lncRNAs have been discovered that lead to repression of other genes in trans (i.e., on another chromosome), as opposed to the cis inactivation imposed by XIST. – e.g., HOTAIR, a lncRNA in the HOXC locus of the Hox genes, can repress the HOXD locus on another chromosome. – Repression is initiated by their interaction with repression complexes. LW2024 Burman University, BIOL 374, Gene Expression, p.21 7.7 Other Transcription Systems § Transcription initiation by Pol I and Pol III is analogous to that by Pol II. § All RNA polymerases use some promoter sequences and TBP. § Differences: 1. Different general transcription factors. 2. RNA Pol I & III do not require ATP hydrolysis during transcription initiation. 3. No defined enhancers or promoter-proximal elements. A. RNA Pol I [F9-51] – General multimeric transcription factors, UBF and Pol I/TBP/TAFs, bind to upstream control element (UCE; -155 to -60) and core element (-40 to +5), respectively. B. RNA Pol III [F9-52] – Internal promoter elements (A-, B- and C- boxes) located downstream from the transcription start site. – Also, in snRNA promoters with TATA and upstream promoter element (PSE). C. Mitochondrial/Chloroplast DNAs – Mitochondrial DNA is transcribed by a nuclear- encoded RNA pol composed of 2 subunits - one homologous to the bacteriophage T7 RNA pol and the other resembles bacterial s factors. – Chloroplast DNA is transcribed by a chloroplast- encoded RNA pol homologous to bacterial RNA pol, except that it lacks a s factor. LW2024