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Chromatin structure Genome organisation at the DNA level Genome is plastic Genes may be available for expression in some cells but not in the others (or at some time in the development but not others) Genes may be amplified or made more available than usual under some conditions Change in physical arrangement of DNA (levels of DNA packing) affect gene expression – genes in heterochromatin and mitotic chromosomes are not expressed. 1 Genome organisation at the DNA level DNA in eukaryotic genomes is organised differently from that in prokaryotes In prokaryotes, most DNA codes for protein (mRNA), tRNA or rRNA, and coding sequences are not interrupted. In eukaryotes, most DNA does not encode protein or RNA, and coding sequences may be interrupted by noncoding DNA (introns). Chromatin structure Chromatin Structure is based on successive level of DNA packing. Prokaryotic DNA is: Usually circular Much smaller – small nucleoid region Associated with only a few protein molecules Less elaborately structured and folded: DNA-protein loops anchored to the plasma membrane 2 Chromatin structure Chromatin Structure is based on successive level of DNA packing. Eukaryotic DNA is: Complexed with a large amount of protein to form chromatin Highly extended and tangled during interphase Condensed into short, thick, discrete chromosomes during mitosis Enormous amount of DNA requires an elaborate system of DNA packing to fit all of the cell’s DNA into the nucleus Genome packaging: Chromosomes - DNA and associated protein, which together are called chromatin. chromatin Two types of proteins in chromatin: histones and nonhistone proteins. Nonhistone proteins: diverse structural, enzymatic, and regulatory proteins. Histones: Packaging of eukaryotic DNA depends on histones. Approx. 10% of the chromatin remains in a condensed, compacted form throughout interphase. This compacted chromatin is seen at the periphery of the nucleus. 3 Chromatin Structure and Transcription Transcriptionally active chromatin has distinctive properties compared to inactive chromatin. Active chromatin is more accessible to enzyme degradation (DNAse I), at hypersensitive sites. These sites are found typically in the regulatory regions of actively transcribed, but they are absent from the same regions of genes that are transcriptionally silent. Chromatin Structure Composition: DNA + associated proteins 1. Histones (small, basic; more details below) 2. Nonhistone regulatory proteins States of chromatin 1. In all states have proteins attached 2. Usually differences are due to different states of folding after histones added, not removal of histones 4 Two Basic States of Chromatin Heterochromatin 1. Darkly stained, relatively condensed, genetically inactive 2. Two kinds of (interphase) heterochromatin •Constitutive heterochromatin -- always heterochromatic (ex: chromatin at centromeres, telomeres). Usually repeating in sequence, non coding. •Facultative heterochromatin- sometimes heterochromatic (depends on tissue, time etc.). Example: inactive X. 3. All DNA (chromatin) is heterochromatic during mitosis. •Constitutive heterochromatin remains in the compacted state in all cells at all times (DNA that is permanently silenced). The bulk of the constitutive heterochomatin is found in and around the centromere of each chromosome in mammals. The DNA of constitutive heterochromatin consists primarily of highly repeated sequences and contains relatively few genes. When genes that are normally active are transposed into a position adjacent to heterochromatin, they tend to become inactivated. •Facultative heterochromatin is chromatin that has been specifically inactivated during certain phases of an organism’s life. Although cells of females contain two X chromosomes, only one of them is transcriptionally active. The other X chromosome remains condensed as a heterochromatic clump called a Barr body. 5 Euchromatin 1. Stains more lightly, less condensed. 2. Capable of genetic activity (transcription). Normal state of most DNA during interphase. 3. Euchromatin is often divided into several distinguishable states of folding (although tightness of folding is probably really continuous from relatively loose to relatively tight). Correlation between folding and function is complex. Histones •5 types: 9H2A, H2B (slightly lys rich), 9H3, 9H4 (arg rich) 9H1 (lys rich). All relatively small proteins. •Per 200 bp of DNA: 2 molecules each of H2A, H2B, H3, H4 and one molecule of H1. 6 Model for Chromatin - Beads on string level 1. Octamer of 2 each of H2A, H2B, H3, H4 (+ some DNA) one bead. 2. DNA wound 2X around (on outside of) each octamer. 3. Linker DNA between cores - 50-60 bp; most exposed and most sensitive to Dnase enzyme 4. H1 is on outside of DNA/octamer 5. Nucleosome - repeating unit - 200 BP DNA + octamer (H1 opt). Nucleosomes & Higher Levels of Structure (requires H1). 1. Nucleosomes. DNA + histones - Chain of nucleosomes (1/7) 2. Solenoid. Chain of nucleosomes - 30 nm fiber (supercoil or solenoid 3 beads across; 6 beads/turn) - (1/42). Need tails (of histones) and H1 to form 30nm fiber. 3. Loops. 30 nm fiber - loops about 300nm in diameter (1/750 orig. length). Different sections may be tighter or looser. NB: Individual loops are stretched out (probably to beads-on-astring stage) when actually transcribed. 4. Higher Orders of Folding. Looped structure folds further heterochromatin (not transcribable) • Form structures/fibers about 700 nm across (per chromatid) • At metaphase = tightest = 1/15,000-1/20,000 orig. length Chromosome is 4-5 microns long but contains 75 mm of double helical DNA. 7 Levels of chromatin packing (a) DNA and histone molecules form "beads on a string," consisting of nucleosomes in an extended configuration. Each nucleosome has two molecules each of four types of histone, around which the DNA wraps. The fifth histone, called H1, can bind to DNA adjacent to a "bead." Levels of chromatin packing (b) With the help of histone H1, the string of nucleosomes coils to form a chromatin fiber that is 30 nm in diameter. (c) At the next level are looped domains of the 30-nm fiber. The loops are attached to a scaffold of nonhistone proteins. 8 Levels of chromatin packing (d) The chromatin folds further, resulting in the maximally compacted chromosome seen at metaphase. Nucleosomes Proteins called histones are responsible for the first level of DNA packing in eukaryotic chromatin. Histones have high proportion of positively charged amino acids (lysine and arginine), and they bind tightly to the negatively charged DNA. The basic unit of DNA packing is a nucleosome. 9 Nucleosomes Nucleosomes in electron micrographs look like beads on a string. The nuclosome bead consists of DNA wound around a protein core composed of two molecules each of four different types of histone: H2A, H2B, H3 and H4. The fifth histone, called H1, attaches to the DNA near the bead when the chromatin undergoes the next level of packing. Ribbon diagram of the nucleosome H2A – yellow; H2B – red; H3 – blue, H4 - green 10 Space filling model of the nucleosome H2A – yellow; H2B – red; H3 – blue, H4 - green Solenoid model of the 30nm condensed chromatin fiber in a side view. 11 Histone Acetylation Histone Acetylation is a way the nucleosome may be restructured by addition of acetyl groups to the lysine residues of the core histones. Acetylated lysine side chains no longer bear a positive charge and lose their affinity to DNA Acetylation of the lys at the N terminus of histone proteins removes positive charges, thereby reducing the affinity between histones and DNA. This makes RNA polymerase and transcription factors easier to access the promoter region. Therefore, in most cases, histone acetylation enhances transcription while histone deacetylation represses transcription. Histone acetylation is catalyzed by histone acetyltransferases (HATs) and histone deacetylation is catalyzed by histone deacetylases (denoted by HDs or HDACs) 12 DNA methylation and gene repression One out of 100 nucleotides bears and added methyl group, which is always attached to carbon 5 of cytosine in the 5’-CG-3’ rich island that are often located in or near transcriptional regulatory regions. DNA methylation serves more to maintain a gene in an inactive state than as a mechanism for initial inactivation. The extent of DNA methylation varies significantly among eukaryotes, being strong in mammals and higher plants but rare in yeast and Drosophila. X-chromosome inactivation in female mammals. The non-gamete cells of females each contain two copies of the X chromosome, one inherited from each parent. Early in development, one X chromosome in each existing cell is randomly inactivated by condensation into a tight mass of heterochromatin. The inactivated X chromosome is strongly methylated and does not participate in transcription initiation. After X chromosome inactivation in embryonic cell - all cellular descendants inactivate the same X chromosome. 13 Methylation plays a role in most complex human diseases. Information derived from methylation but not from any of the known genomic methods are: • The methylation status of a gene informs us about its current and its past activation status • The methylation of the promoter of a gene can provide information as to how easily a promoter can be activated Methylation patterns are not only different between the tissues of one individual, but - as known from animal studies - between different populations • • The overall methylation level of a cell`s DNA strongly influences genome stability, allowing the prediction of malignancy of tumor cells • As viral sequences are silenced by methylation, the activity of viruses may be predicted and gene-therapeutic vectors be optimized From Epigenomics Inc. Selected interactions in gene silencing that involve DNA methylation. Inverted repeats (marked by an inverted set of arrows) could trigger methylation either by production of transcripts (represented by wavy lanes) that can form dsRNA or by DNA–DNA interactions. Methylation (marked by lollipops) can accumulate in the promoter or coding regions of a gene. Hypermethylation of a promoter correlates with TGS. Increased levels of methylation in a coding region (the open reading frame [ORF]) may not have immediate consequences. From Paszkowski and Whitman, 2001, Current Op Plant Biol 14 Selection of mutants. Primary selection of mutant candidates: hygromycinresistant seedling after 8 days selection on 15 mg/l hygromycin (a), Northern (b), and Southern (c) blot analysis of the parental line A with the silent hpt gene and the mutants som1-som8. Both membranes were probed with the hpt coding region. Genomic DNA was digested with BamHI. From Mittelsten-Scheid et al., PNAS, 1998 Methylation analysis. The same Southern blot of DNA cut with HpaII was probed with the hpt coding region (a), or with a fragment containing the centromeric 180-bp repeat. Lanes: 1, pGl2 plasmid used to generate the parental line A; 2, wt; 3, parental line A; 411, som 1-8, 12, mutant ddm1/ddm1 with reduced methylation (20); 13, ddm1/A; 14, METIas T3 #10.5 transgenic for a methyltransferase antisense construct (21); 15, METIas T3 #10.5/A 15 Effects of histones on transcription •Core histones: H2A, H2B, H3 and H4 had caused a mild repression of genetic activity. •TFs had no effect of this repression •Core +H1 – strong repression of genetic activity •This repression could be blocked by activators In vitro transcription of reconstituted chromatin Set-up: •Plasmid DNA with Drosophila Krueppel gene •Core histones at various ratios •Polyglutamate used as a vehicle to to help histones deposit on DNA •Primer extension was performed to measure transcription 16 Outcome: •core histone inhibit the transcription is a dose-dependent manner. •Transcription of reconstituted chromatin with av. Of one nucleosome per 200 bp exhibits 75% repression relative to naked DNA. •Remaining 25% is due to promoter sites not covered by nucleosome cores. •Activators, like Sp1, can not counteract the repression caused by nucleosome core formation Why 75% repression? Nucleosomes slow the polymerase? Or 25% of promoters might be left free ? Experiment to check this: Hypothesis: if a promoter is not blocked by nucleosomes and is available for polymerase, it should also be available for the restriction enzyme. Gene has XbaI site downstream of the start site, this enzyme should cut at the genes not protected by nucleosomes. This would render gene inactive. 17 Histone H1 Role-nucleosome cross-linker Addition of it adds to inhibition of transcription caused by core histones This repression can be counteracted by transcription factors Some TFs –Sp1, GAL4 act as antirepressors –prevent repression by H1 and as transcription activators. Some – like GAGA factor – are just antirepressors. They may compete with H1 for binding sit5es of DNA template. 18 Nucleosome positioning Nucleosome –free zones – there is evidence of the nucleosomefree zones in control regions of active genes. Active genes tend to have DNAse-hypersensitive control regions – this is at least due to the absence of nucleosomes 19 •Some active genes have an array of precisely positioned nucleosomes in their control regions •The same control regions, in tissues where these genes are not active, do not have positioned nucleosomes •The positioned nucleosomes can overlap the binding sites for activators •The activators and nucleosomes can coexist in the same place. •Activators have a role in positioning nucleosomes Histone acetylation •Occurs in cytoplasm and in nucleus •HAT B enzyme in cytoplasm prepares histones for incorporation into nucleosomes •Acetyl groups are removed in the nucleus •Nuclear acetylation – HAT A •Correlates with transcription activation •A variety of activator have HAT A activity, allowing them to loosen the association of nucleosomes with the gene’s control region, thereby enhancing transcription 20 • Histone deacetylation Deacetylation – repression event • Known transcription repressors, such as nuclear receptors w/o ligands, interact with corepressors, which interact with histone deacetylases. • DATs remove acetyl groups from histone tails, tightening the grip of nucleosomes and DNA A heterodimer of retinoic acid receptor and retinoic acid receptor X binds to an enhancer. In the absence of ligand - RA – the receptor binds to co-repressor, which binds histone deacetylase (HDAC1) HDAC1 removes acetyl groups –tight association with DNA. 21 Activation and repression by the same nuclear receptor: Normal: No receptor dimer is bound to hormone response element. Histones moderately acetylated. Repressed: Receptor bound to TRE w/o hormone. It interacts with corepressors, they interact with deacetylase. Active: receptor, hormone, coactivators All activators have the HAT A activity Chromatin remodelling: Swi/Snf family – disrupt the core histones of nucleosomes Iswi family – move nucleosomes 22 Reading: p. 386-421. 23