Chromatin Structure and its Effects on Transcription

Transcription

1 Chromatin Structure and its Effects on Transcription Epigenetics 2014 by Nigel Atkinson The University of Texas at Austin From Weaver 4th edition and Armstrong 1st edition

2 What is the point? DNA is not naked. Histones and other proteins organized it into a fantastically compact unit. The presence of nucleosomes can interfere with the binding of TFs to enhancers and with the preinitiation complex to the promoter. = Repression When other proteins (simple TFs) are bound to the DNA they can prevent the histones from binding. This is competitive inhibition of histone binding. Called de-repression or antirepression in the book by Weaver.

3 Eukaryotes five different Histone Classes Histone size amino acids Molecular weight H2A ,000 H2B ,770 H ,400 H ,340 H1 (H5) ,500 Eukaryotes contain many copies of each histone gene in mice, 100X in Drosophila human chromosome about 10 cm long. nucleus 10 umeter 2m DNA in a human cell.

4 Details A)General information about the histones 1) Basic Histones rich in K and R which gives them a basic charge at neutral ph. Positive charge at neutral ph means that they are resistance to acid extraction (this is unusual) and migrate in the opposite direction during electrophoresis than most proteins. Eukaryotes contain many copies of each histone gene in mice, 100X in Drosophila Positive charge helps them interact with the negatively charged DNA. 2) Histones are evolutionarily ancient and strongly conserved molecules Histones evolved very early during the history of life on our planet. In fact, histone H3 and H4 are the most conserved proteins known. In most organisms, their amino acid sequence is identical or only slightly changed. For example, histone H4 from cows and from Mendel's pea plant are identical in all but 2 amino acids. Furthermore, these two changes are conservative amino acid changes. This indicates that the sequence of histone H4 has not significantly changed in the 10 9 years since plants and animals diverged (Freifelder and Malacinski, Essentials of Molecular Biology 2nd ed). 3) Compaction A chromosome is much more compact that the DNA molecule it contains. For example, if stretched out the DNA in just a single human chromosome would be about 10 cm long. A human cell contains about 2m of DNA. But a eukaryotic cell is only about 3-10 um in diameter and the nucleus is smaller than this. 4) Interaction with DNA DNA wraps around histone core 1.65 times The amount of DNA that interacts with the core is 147 bp Histones H-bind to the DNA Histones organize DNA into a nucleosome structure Linker joins the nucleosomes to each other - core + linker = ~200 bp (length of the linker is species dependent)

5 Details REVIEWS 5) Nucelosome (fig. from Tsankova et al 2007) 6) Compaction Naked -> Beads on a string -> 30 nm fiber -> etc 7) Histone H1 8) 30 nm fiber and the Tetranucleosome 9) Loops! as regulatory units A It is only recently that we have started to think about chromosome shapes and M organization as a regulatory M M M A M M M mechanism. Manipulation of chromosome conformation may A be the M M most important regulatory mechanism. M Subnuclear localization is also an important manipulation (for instance - heterochromatin is often associated with M the nuclear envelope). M M HAT M H3 A A M HDM M M K4 H3 A M Acetylation (activating) Deacetylation K9 A Histone tail H2B M Demethylation Methylation (activating) H2A S10 HDAC Histone A H4 Transcription factor + A HMT K14 A DNA K18 K23 A PK HDM A M M K27 A M Phosphorylation (activating) A Demethylation Methylation (repressing) Dephosphorylation S28 Acetylation Methylation Phosphorylation PP K36 A A HMT M K79 Overview of epigenetic mechanisms Chromatin is the complex of DNA, histones and nonhistone proteins in the cell nucleus. Remodelling of chromatin is a dynamic process that modulates gene expression. The fundamental unit of chromatin is the nucleosome, which consists of ~147 base pairs of DNA wrapped around a core histone octamer (~1.65 turns). Each octamer contains two copies each of the histones H2A, H2B, H3 and H4 (FIG. 1a). The nucleosomal structure of chromatin allows DNA to be tightly packaged into the nucleus by organized folding 5. Intricate chromatin remodelling mechanisms ensure that DNA remains accessible to the transcriptional machinery. These epigenetic mechanisms alter gene activity by modulating DNA protein interactions without changing the genetic code. In simplified terms, chromatin exists in an inactivated, condensed state, heterochromatin, which does not allow transcription of genes, and in an activated, open state, euchromatin, which allows individual genes to be transcribed (FIG. 1b). The opening of chromatin is associated with acetylation of nearby histones, although it remains unclear whether acetylation mediates or reflects chromatin decondensation. In reality, chromatin can exist in many states in between Figure 1 General scheme of chromatin remodelling. a Picture of a nucleosome showing a DNA strand wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. The amino (N) termini of the histones face outward from the nucleosome complex. b Chromatin can be conceptualized as existing in two primary structural states: as active, or open, euchromatin (top left) in which histone acetylation (A) is associated with opening the nucleosome to allow binding of the basal transcriptional complex and other activators of transcription; or as inactive, or condensed, heterochromatin where all gene activity is permanently silenced (bottom left). In reality, chromatin exists in a continuum of several functional states (active; permissive (top right); repressed (bottom right); and inactive). Enrichment of histone modifications such as acetylation and methylation (M) at histone N-terminal tails and related binding of transcription factors and co-activators (Co-Act) or repressors (Rep) to chromatin modulates the transcriptional state of the nucleosome. Recent evidence suggests that inactivated chromatin may in some cases be subject to reactivation in adult nerve cells, although this remains uncertain. c Summary of common covalent modifications of H3, which include acetylation, methylation and phosphorylation (P) at several amino acid residues. H3 phosphoacetylation commonly involves phosphorylation of S10 and acetylation of K14. Acetylation is catalysed by histone acetyltransferases (HATs) and reversed by histone deacetylases (HDACs); lysine methylation (which can be either activating or repressing) is catalysed by histone methyltransferases (HMTs) and reversed by histone demethylases (HDMs); and phosphorylation is catalysed by protein kinases (PK) and reversed by protein phosphatases (PP), which have not yet been identified with certainty. K, lysine residue; S, serine residue. Panels a,c modified, with permission, from Nature Rev. Neurosci. REF. 62 (2005) Macmillan Publishers Ltd. 356 MAY 2007 VOLUME 8

6 Nucleosome 1.75 Images from Watson et al Molecalr Biology of the Gene 6th edition

7 Compaction Double helix Beads On A string zig-zag Snake The Solenoid PR nm fiber Probably involve SARS Figure from

8 Two competing models Images from Watson et al Molecular Biology of the Gene 6th edition tetrasome model

9 Histone H1 Images from Watson et al Molecalr Biology of the Gene 6th edition

10 Tetranucleosome Fig 13.7

11 Tetranucleosome

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14 35-80 kb loops Each loop is very independent (e.g. independently supercoiled).

15 Histones repress transcription Activators are responsible for much of the variation in levels of transcription of different genes. Why? See above!

16 Histones represses transcription Natural break on runaway transcription Naked DNA can be transcribed. Histones repress transcription. A difference between prokaryotes and eukaryotes? prok require active repression Histones act as basal repressors. Euk require activation to remove basal repression Figure Weaver 4th edition

17 Moving them can be an act of activation!!! - Remodeling means to move them. Modifying them can make them moveable. The cell does this Example given: swi/snf proteins

18 Histone acetylation amino groups of lysine side chains unacetylated histones tend to repress transcription acetylated histones tend to activate transcription Histone acetyl transferase (HAT) Histone deacetylase (HDAC)

19 HAT type A Nuclear, involved in regulating gene expression Have bromodomain (HAT B s do not) Binds aceylated lysines. So HAT A s can recognize partially acetylated histone tails. Think of it like this: the work of one HAT can attract other HATs. Examples p55, Gcn5p CBP/p300, TAFII250 What mechanism does this suggest? In the context of TAFII250 and core promoter recognition?

20 HAT type B cytoplasmic, acetylate newly synthesized histones H3 and H4 so that they can be assembled into a nucleosome. These acetylations will later be removed in the nucleus. no bromodomain NOT thought to be used in the regulation of gene expression.

21 Acetylation continued Acetylation of histone tails neutralizes some of the positive charge, causing them to relax their grip on the DNA. Reduces nucleosome cross-linking. That is; the interaction between histones in neighboring nucleosome. eg. basic N-terminal tail of H4 in one nucleosome and an acidic pocket in H2A- H2B dimer in the next nucleosome

22 Acetylation continued Also some TFs recognize acetylated histones. eg. TAFII250 has a double bromodomain and recognizes low level acetylated histones. Once bound it is a HAT and increases acetylation. low level acetylation of histones occurs in inactive chromatin.

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24 Deacetylation Most eukaryotic repressors recruit corepressors Most corepressors complexes include histone deacetylases Example, NCoR/SMRT in mammals

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28 Controlling DNA accessibility Histones are repressors - by regulating how tightly histones bind the DNA we regulated access to transcriptional control regions Acetylation helps to make DNA more accessible but by itself it is not enough. Also need additional chromatin remodeling. Acetylation is a form of chromatin modification that can make remodeling easier.

29 Chromatin remodeling Clearest definition ATP-dependent mobilization movement of nucleosomes so that DNA is accessible. Weaver pg 382 Armstrong pg 30

30 Chromatin remodeling List of protein complex families SWI/SNF - SwitchSniff ISWI - iimitation switch CHD - Chromodomain and helicase-like domain ATPases (NuRD) INO80 Produce nucleosome free regions around enhancers & promoters pg 382 in Weaver 4th edition Armstrong Chapter 4

31 Constituitive heterochromatin, Facultative heterochromatin, Euchromatin

32 Additional levels of organization that are regulatory in nature

33 45-80 kb loops

34 Insulators Provides a barrier through which activation or repression cannot pass. Some also block the encroachment of condensed chromatin. Some do one, some the other. What we know is based on very few examples. Probably many different types exist. There are probably many different mechanisms.

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36 Haini Cai & Ping Shen One way that they are thought to work

37 45-80 kb loops