2005-10-29 Chaoqun Wu, Fudan University 1 Epigenetics — Chromatin based gene control Chaoqun Wu School of Life Sciences, Fudan University 2005-10-29 Chaoqun Wu, Fudan University 2 Part II. Chromatin has important role in gene regulation 2005-10-29 Chaoqun Wu, Fudan University 3 Multiple levels of gene regulation 2005-10-29 Chaoqun Wu, Fudan University 4 RNA polymerase transcribes genes - How is this process regulated? How is specificity achieved? Key players – transcriptional activator proteins -bind to specific sites on DNA and turn on the expression of nearby genes. Two adhesive surfaces – one binds DNA, the other interacts with, and recruits RNA polymerase. 2005-10-29 Chaoqun Wu, Fudan University 5 Enzyme specificity – Many enzymes are capable of acting on a common motif found in many different proteins - Example - kinases General solution – recruitment through specific protein- protein interactions. Regulated localization - locator proteins 2005-10-29 Chaoqun Wu, Fudan University 6 How is transcription initiated in eukaryotes TFIID binds to TATA box (TFIID composed of TBP{TATA-binding protein} and more than eight other subunit{TAFs}) Inhibitors can bind blocking binding of other general transcription factors TFIIA prevents inhibitor binding TFIIB binds to D-A complex Preformed complex RNA polymerase II and TFIIF bind TFIIE, TFIIH, and TFIIJ add to complex in that order now transcription can begin 2005-10-29 Chaoqun Wu, Fudan University 7 Where does eukaryote transciption occur? Modifications act as code for the recognition of other factors/ regulator factors 2005-10-29 Chaoqun Wu, Fudan University 8 Eukaryotic transcription occurs in the context of chromatin Modification of chromatin is a common theme in modulation of eukaryotic gene regulation Histone modifications ◆Acetylation of histone tail generally associated with active gene expression ◆Unacetylated histone tails generally associated with gene silence ◆Methylation of histone tail may be associated with active or inactive genes ◆Phosphorylation generally associated with active gene expression though currently unclear 2005-10-29 Chaoqun Wu, Fudan University 9 Transcriptional regulation in the context of chromatin The basics of eukaryotic gene regulation 2005-10-29 Chaoqun Wu, Fudan University 10 Enhancesome and a repressosome Enhanceosome Involves the cooperative assembly of a multiprotein complex containing several factors/activators These complexes recruit CBP which activates transcription β-interferon enhanceosome Repressosome Involves a multiprotein complex containing several factors/repessors These factors recruit Groucho which blocks formation or function of the basal transcription complex by interacting with TFIIE Groucho repressosome CBP is an acetylase Groucho may associate with deacetylase 2005-10-29 Chaoqun Wu, Fudan University 11 Histon modification 2005-10-29 Chaoqun Wu, Fudan University 12 H3 H4 H2A H2B H3 ‘tail’ The tails are required for transcription Luger et al., Nature 1997 2005-10-29 Chaoqun Wu, Fudan University 13 Gene Activation in Chromatin T A T A Act HAT Ac Histone Acetyltransferase Activator recruitment of histone modifiers Adaptor TATA TBP RNA Pol II Transcription Nucleosomes altered and transcription Act 2005-10-29 Chaoqun Wu, Fudan University 14 Histone Deacetylation correlates with gene repression HML α HMR aMAT Inactive InactiveActive Sir1 Sir3 Sir2 Trancriptional silencing in yeast is associated with reduced nucleosome acetylation Mirian Braunstein, Alan B. Rose, Scott G. Holmes, C. David Allis and James R. Broach. GENES & DEVELOPMENT 7:592-604 ? 1993 2005-10-29 Chaoqun Wu, Fudan University 15 An example of transcriptional activation in the context of chromatin The promoter is assembled into a nucleosomal structure that is transcriptionally inactive. Interaction of a sequence-specific DNA binding protein (A) recruits a chromatin-remodeling complex (SWI/SNF), which results in stabilized binding of protein A through an ATP-dependent perturbation of nucleosomal structure. In some promoters, a partial initiation complex (TFIIB, TBP) may also be bound at this stage. After remodeling, a histone acetyltransferase complex (HAT) is targeted to the promoter, where it acetylates nucleosomes (in green) and facilitates the binding of a second transcriptional activator (B). A complete initiation complex (TFIID, RNAP II) may be formed at this stage on some promoters. Protein B engages a mediator/coactivator complex (Mediator) and induces a particular structural conformation, which imparts specificity to its interaction with components of the initiation complex. This results in RNAP II release and activated transcription. 2005-10-29 Chaoqun Wu, Fudan University 16 Part III. DNA methylation 2005-10-29 Chaoqun Wu, Fudan University 17 2005-10-29 Chaoqun Wu, Fudan University 18 2005-10-29 Chaoqun Wu, Fudan University 19 DNA甲基化 真核生物的 真核生物的 DNA甲基化主要是胞嘧啶的 甲基化主要是胞嘧啶的 第 第 5位碳原子上加上甲基，催化这一反 位碳原子上加上甲基，催化这一反 应的为 应的为 DNA甲基化转移酶（ 甲基化转移酶（ Dnmt)。 。 真核生物的 真核生物的 DNA维持较低水平的甲基 维持较低水平的甲基 化，且大部分甲基化集中在 化，且大部分甲基化集中在 CpG序列 序列 上。 上。 2005-10-29 Chaoqun Wu, Fudan University 20 DNA Methylation ?Cytosines in palindromic CG dinucleotides are subject to methylation at the carbon 5 position in plants and vertebrates ?Methylation does not alter base pairing ?CpG methylation does not occur in yeast or Drosophila ?~70% of cytosines in CpGs are methylated in vertebrates ?CpG methylation is regulated tightly during development and is associated with gene silencing, X-inactivation, and allele-specific imprinting 2005-10-29 Chaoqun Wu, Fudan University 21 CpG Islands: ??? CpG is under-represented throughout most of genome but is found at expected frequencies in short ~1 KB stretches. ??? Enrichment of CpG in these regions led to designation as CpG islands. ??? ~45,000 CpG islands in genome, and these are mostly located at promoters within first exons of genes. ??? CpG islands are unmethylated in normal cells Methylation of CpGs near tumor suppressor genes (p53 or p16) often related to silencing of these genes in tumors. 2005-10-29 Chaoqun Wu, Fudan University 22 1. DNA methylation Cytosine is methylated by a several enzymes Maintenance of methylation allows the modification to become heritable Note during replication the parent strands will be methylated In mammals CpG is the site for methylation Methylated promoters are associated with silent genes 2005-10-29 Chaoqun Wu, Fudan University 23 DNA methylation refers to the transfer of a methyl (CH3 group) to one of the bases that constitute DNA. The reaction is catalyzed by a DNA methyltransferase (Mtase), and uses S- Adenosyl Methionine (SAM) as a methyl donor. In humans, normal DNA methylation is limited to the Cytosine base 2005-10-29 Chaoqun Wu, Fudan University 24 * CpG islands: >200 bp stretches of DNA that have a significantly higher concentration of 5’-CpG;3’ dinucleotides than the bulk of the genome * Cytosine resudue in complementary 3’-GpC-5’ that makes a basepair, is also methylated symmetrically, and these two methyl groups show a three-dimentional structure prominent in the major groove of the dsDNA * 50-60% of human genes have CpG islands in front of and covering core promotor and transcription start site * 70-80% of CpGs in the genome is methylated 2005-10-29 Chaoqun Wu, Fudan University 25 * CpG islands in front of genes are mostly unmethylated exceptions: imprinted genes and X-linked genes * CpG island are divided into several classes: (1) methylated on both alleles in all tissues located in high CG isochores (2) differentially methylated and located in low CG (<0.5) isochores *genomic methylation pattern is stable and heritable *genome-wide methylation patterns are reprogrammed in mammalian germ cells and in pre-implantation embryos 2005-10-29 Chaoqun Wu, Fudan University 26 Three types of DNA Methylation enzymes De novo DNA methyltransferases Perpetuation DNA methyltransferases DNA demethylase 2005-10-29 Chaoqun Wu, Fudan University 27 Three types of reaction could demethylate DNA 2005-10-29 Chaoqun Wu, Fudan University 28 Methylation is Reset each generation 2005-10-29 Chaoqun Wu, Fudan University 29 DNA甲基化转移酶 甲基化转移酶 DNA甲基化转移酶 甲基化转移酶 三类甲基化转移酶 三类甲基化转移酶 Dnmt1, Dnmt2,Dnmt3a/3b ● Dnmt1在 在 DNA复制时维持 复制时维持 DNA甲基化。 甲基化。 ●● Dnmt2可能与可能与 DNA上特异位点结合，但具体作上特异位点结合，但具体作 用还不是很清楚 用还不是很清楚 ; ● Dnmt3的功能可能主要是对着丝粒卫星 的功能可能主要是对着丝粒卫星 DNA的 的 重复序列起甲基化作用，但 重复序列起甲基化作用，但 Dnmt3a, Dnmt3b 作用并不重叠 作用并不重叠 。 。 2005-10-29 Chaoqun Wu, Fudan University 30 Mammalian methyltransferases: DNMT1 - maintenance DNA methyltransferase — methylates hemi-methylated DNA providing methylation pattern to the newly replicated daugther strand, based on parent strand — represses transcription in complex with histone deacetylases 2005-10-29 Chaoqun Wu, Fudan University 31 DNMT3a, DNMT3b - de novo methylases — add a methyl group to unmethylated CpG base pairs, resulting in creation of a new hemi- methylated and then fully methylated CpG — de novo methylation is implicated in cell growth and differentation, and in altered methylation in tumorigenesis. — DNMT3b - mutated (common splice variant) in patients with ICF syndrome (immunodeficiency in association with centromere instability of chromosome 1, 9, 16, and facial anomalies): hypomethylation of pericentromeric satellite sequences Methyl-CpG binding proteins: MeCP2, MBD1-4 2005-10-29 Chaoqun Wu, Fudan University 32 DNA去甲基化转移酶 去甲基化转移酶 一种观点： 一种观点： DNA复制时，如果缺乏 复制时，如果缺乏 DNA甲基化 甲基化 转移酶，就导致去甲基化，同时， 转移酶，就导致去甲基化，同时， C-C共价键 共价键 是高能键，一旦胞嘧啶甲基化后，就不能被 是高能键，一旦胞嘧啶甲基化后，就不能被 打断 打断 。 。 另一种观点： 另一种观点： Moshe Szyf认为生物化学中没 认为生物化学中没 有不可逆的过程，酶能解决能量问题 有不可逆的过程，酶能解决能量问题 。他从 。他从 肺癌细胞系分离到具有甲基基团转移活性的 肺癌细胞系分离到具有甲基基团转移活性的 酶，称之为去甲基化转移酶 酶，称之为去甲基化转移酶 （ （ dMTase） ） ， ， 但 但 进一步分析证明他所分离的 进一步分析证明他所分离的 dMTase等同于 等同于 MBD2b－ － 甲基化 甲基化 CpG结合蛋白。 结合蛋白。 2005-10-29 Chaoqun Wu, Fudan University 33 Model methylation reaction: Cytosine nucleotide (red) is flipped out of the DNA double helix by a methyltransferase (white), so it can be methylated. The end product after the methyl group has been transferred to the DNA is pictured in green. DNA cytosine methylation is a chemical modification of the DNA in which a single carbon, in the form of a methyl group (M), is added to certain cytosines (C). This altered cytosine, known as methyl-C, acts like a fifth base, and is a critical factor in gene regulation. The enzyme responsible for DNA methylation in humans is known as DNA cytosine methyltransferase. 2005-10-29 Chaoqun Wu, Fudan University 34 *Methylated DNA is replicated later than actively transcribed DNA *Monoallelically expressed genes (imprinted) have coordinated replication timing along human chromosomes Non-replicated (silenced) genes Replicated (active) genes Ensminger and Chess, 2004 FISH analysis with imprinted gene pairs selected from one chromosome 2005-10-29 Chaoqun Wu, Fudan University 35 2. DNA silencing by DNA methylation Plants and filamentous fungi share with mammals enzymes responsible for DNA methylation. In these organisms, DNA methylation is associated with gene silencing and transposon control. However, plants and fungi differ from mammals in the genomic distribution, sequence specificity, and heritability of methylation. Transposons play a role in establishing methylation patterns and the epigenetic consequences of their perturbation Martienssen, Science 2001 2005-10-29 Chaoqun Wu, Fudan University 36 Methylation usually silences gene expression. Normally, about 70% of all CpG dinucleotides in the mammalian genome are methylated. The remainder, clusters near the 5' end of genes known as CpG islands, are protected from it. Too little methylation across the genome or too much methylation in the CpG islands can cause problems, the former by activating nearby oncogenes, and the latter by silencing tumor suppressor genes. 2005-10-29 Chaoqun Wu, Fudan University 37 DNA methylation tags cytosine, one of the four chemical bases that make up the genetic code, with a methyl group. Although not a hard-and-fast rule, DNA methylation is generally associated with silencing of gene expression, whereas active genes are usually unmethylated. . A methylating enzyme (white) binds to its target site (red) on DNA; the methyl donor is shown in green. 2005-10-29 Chaoqun Wu, Fudan University 38 Methylation of DNA in certain control regions in our genome can cause genes to be inappropriately silenced. Up to 65% of all cancers appear to involve such methylation abnormalities. 2005-10-29 Chaoqun Wu, Fudan University 39 The pattern of DNA methylation in a cell dramatically affects the function of the DNA by switching genes on or off. Abnormal methylation events occur during aging and in the development of many cancers. 2005-10-29 Chaoqun Wu, Fudan University 40 The anti-cancer drug development is based upon the inhibition of methylation as a therapeutic strategy to treat such cancers. 2005-10-29 Chaoqun Wu, Fudan University 41 3. Inheritance of methylation states Replication of methylated DNA (a) Results in hemimethylated progeny DNA (b) Maintenance methylization enzymes, such as DNA methyltransferases, methylate cytocine (c) Based on the hemimethylated atates of symmetric CG or CXG motifs * C and G represent cytosine and guanine respectively, whereas X is any nucleotide. Other mechanisms of methylation inheritance are also known. 2005-10-29 Chaoqun Wu, Fudan University 42 What are epigenetic modifications Methylation can be maintained through replication making it heritable. Some proteins specifically interact with methylated DNA 2005-10-29 Chaoqun Wu, Fudan University 43 Genetic Imprinting Remember that DNA methylation can be maintained through replication. This allows the packing of chromatin to be passed on - just like a gene sequence. – However, differences in chromatin packing are not as stable as gene sequences. Heritable but potentially reversible changes in gene expression are called EPIGENETIC phenomena – Vertebrates use these differences in chromatin packing to IMPRINT certain patterns of gene regulation. – Some genes show MATERNAL IMPRINTING while other show PATERNAL IMPRINTING. The alleles of some genes that are inherited from the relevant parent are methylated, and therefore are not expressed. 2005-10-29 Chaoqun Wu, Fudan University 44 Imprinted Genes in the Mouse Data from http://www.mgu.har.mrc.ac.uk/research/imprinted/imprin.html Red: Maternal allele. Blue: Paternal allele. 2005-10-29 Chaoqun Wu, Fudan University 45 4. Properties DNA methylation Repeats may serve as trigger to induce DNA methylation and so silence gene expression, but exceptions exist DNA methylation especially in plants and animals DNA methylation and histone acetylation are antagonistic mechanisms in chromatin modulation 2005-10-29 Chaoqun Wu, Fudan University 46 Chromocenters in Arabidopsis 2005-10-29 Chaoqun Wu, Fudan University 47 Example of CpG Islands in the Retinoblastoma gene region. The dotted line represents the statistically expected frequency of CpG sites (1/16), while the solid line represents the measured frequency of CpG sites in the 180 kb of DNA sequence that encompass the Rb gene exons and introns. The location of two CpG islands is indicated by arrows. Only the most 5' CpG island corresponds to the promoter of the gene. 2005-10-29 Chaoqun Wu, Fudan University 48 5. DNA甲基化过程是一个累进的过程 人类乳房上皮细胞瘤的 人类乳房上皮细胞瘤的 p16CpG岛为例 岛为例 2005-10-29 Chaoqun Wu, Fudan University 49 p16CpG岛的甲基化 岛的甲基化 2005-10-29 Chaoqun Wu, Fudan University 50 2005-10-29 Chaoqun Wu, Fudan University 51 2005-10-29 Chaoqun Wu, Fudan University 52 2005-10-29 Chaoqun Wu, Fudan University 53 2005-10-29 Chaoqun Wu, Fudan University 54 2005-10-29 Chaoqun Wu, Fudan University 55 p16CpG岛的 岛的 47个 个 CpG位点的甲基化并不具有 位点的甲基化并不具有 位点特异性，但也不是完全的随机性，最初 位点特异性，但也不是完全的随机性，最初 出现在三个离散的区域，逐代累积，并向附 出现在三个离散的区域，逐代累积，并向附 近扩散。形成三个甲基化离散区的原因还不 近扩散。形成三个甲基化离散区的原因还不 清楚，可能是因为这三个区域的 清楚，可能是因为这三个区域的 DNA一级结 一级结 构，二级结构，染色质的结构及 构，二级结构，染色质的结构及 DNA结合蛋 结合蛋 白等使得 白等使得 DNA甲基化转移酶靠近或阻碍 甲基化转移酶靠近或阻碍 DNA去 去 甲基化转移酶靠近。 甲基化转移酶靠近。 2005-10-29 Chaoqun Wu, Fudan University 56 6. Methyl-CpG-binding proteins Methyl-CpG-binding domain (MBD), consisting of about 70 residues, possesses a unique /- sandwich structure with characteristic loops, and is able to bind single methylated CpG pairs as a monomer. MeCP2, MBD1, MBD2, MBD3 and MBD4 constitute a family of vertebrate proteins that share the methyl-CpG-binding domain (MBD). 2005-10-29 Chaoqun Wu, Fudan University 57 Sequence alignment of the MBD of human MeCP2, MBD1, MBD2, MBD3, Xenopus MBD3 and human MBD4. Positions of -strands (arrows), loops (thick lines), and the -helix (rectangle) defined by the solution structures of MeCP2 and MBD1 are indicated above the alignment. MeCP2 numbering is located above human MeCP2 sequence. General numbering for MBDs is located below all the sequences. Conserved residues are shaded and those essential for binding to methylated DNA are indicated by an asterisk. Four Rett syndrome mutants occuring in the MBD are indicated by grey circles. 2005-10-29 Chaoqun Wu, Fudan University 58 MBD proteins are involved in recruiting histone deacetylases to methyl CpG-enriched regions in the genome to repress transcription. MBD1 represses transcription through the co- operation of the MBD, CxxC motifs and TRD . MBD2b represses transcription in a deacetylase- dependent manner. MBD3 is a component of a deacetylase complex with a nucleosome remodelling activity, known as the Mi-2/NuRD complex . MBD4 can efficiently remove thymine or uracil from a mismatch CpG site in vitro, suggesting that this enzyme may function to minimize mutation at methyl-CpG. 2005-10-29 Chaoqun Wu, Fudan University 59 MeCP2 as the archetypical methyl-CpG- binding protein A short region of MeCP2 containing about 70 residues located within its N-terminal third retained the ability to bind selectively methylated DNA. Distribution of MeCP2 along the chromosomes parallels that of methyl-CpG. 2005-10-29 Chaoqun Wu, Fudan University 60 Model for the interaction between MBD and methylated DNA. Co-ordinates for the MeCP2 MD and the CpG helix were used to construct this model (accession numbers 1qk9 and 329G, respectively). Four Rett MBD mutations are shown. Esteban Ballestar 1 and Alan P. Wolffe (2001) Eur. J. Biochem. 268, 1-6 2005-10-29 Chaoqun Wu, Fudan University 61 MBDs target corepresor complexes to methylated DNA. Different situations that the MBDs may recognize. Empty circles represent unmethylated CpGs, whereas full circles correspond to their methylated status. (A) corresponds to hypomethylated DNA with occasional hemi-methylated CpGs. (B) corresponds to fully methylated sequence with a low density of CpGs. (C) and (D) are two sequences with a high number of CpGs but with different organizations that may be recognized by different MBDs. (E) includes the structure of the chromatin. Eur. J. Biochem. 268, 1-6 (2001) 2005-10-29 Chaoqun Wu, Fudan University 62 7. DNA Demethylation Most biological modifications such as protein phosphorylation are reversible, and enzymes exist that can catalyze either the modification or its removal. This reversibility is essential for their functioning as biological signals that can respond to changing physiological cues. DNA methylation has been considered to be an exception because removal of a methyl group from DNA must involve a cleavage of a carbon–carbon bond, which has been considered an unlikely reaction. Direct demethylation of DNA has in the past been considered highly unlikely, but some dMTase-like Proteins were reported. 2005-10-29 Chaoqun Wu, Fudan University 63 Active DNA demethylation. Two pathways are illustrated. (A) The 5-methylcytosine demethylase hydrolyzes 5-methylcytosine to cytosine and water . (B) The 5-methylcytosine DNA glycosylase abstracts 5-methylcytosine from the phosphodiester backbone, which then is repaired by using endonuclease 2005-10-29 Chaoqun Wu, Fudan University 64 Passive DNA demethylationr eplication- coupled DNA demethylation (A)Normally, each strand of a symmetrically methylated CpG dinucleotide (M) would be segregated after replication daughter chromatids, leaving one strand of DNA methylated (M) and the other not . The maintenance methyltransferase activity of Dnmt1 then would restore symmetrical methylation. (B)Regulatory nucleoprotein complexes might occlude Dnmt1 leading to loss of methylation . (C)Histone acetylation might repel or inhibit Dnmt1, leading to loss of methylation. PNAS Vol. 96, Issue 11, 5894-5896, May 25, 1999 2005-10-29 Chaoqun Wu, Fudan University 65 2005-10-29 Chaoqun Wu, Fudan University 66 Demethylation by demethylase 2005-10-29 Chaoqun Wu, Fudan University 67 Demethylation by DNA replacement 2005-10-29 Chaoqun Wu, Fudan University 68 Part IV. Histone modifications 2005-10-29 Chaoqun Wu, Fudan University 69 Low salt Physiological ionic strength 10 nm fiber 30 nm solenoid nucleosomes: ‘beads on a string’ in different stages of condensation... 2005-10-29 Chaoqun Wu, Fudan University 70 Histones are highly conserved proteins that are intimately associated with the DNA in chromatin – small in size – carry a large number of basic residues – complexed into a particle termed a nucleosome 2004-10-26 44Chaoqun Wu, Fudan Universityun 2005-10-29 Chaoqun Wu, Fudan University 71 1. Histones The basic unit of chromatin is the nucleosome core particle, which contains 147 bp of DNA wrapped nearly twice around an octamer of the core histones. The histone octamer is composed of a central heterotetramer of histones H3 and H4, flanked by two heterodimers of histones H2A and H2B 2005-10-29 Chaoqun Wu, Fudan University 72 Each of the core histones has a related globular domain that mediates histone–histone interactions within the octamer, and that organizes the two wraps of nucleosomal DNA. Each histone also harbors an aminoterminal 20–35 residue segment that is rich in basic amino acids and extends from the surface of the nucleosome. Histones are subject to an enormous number of post- translational modifications, including acetylation and methylation of lysines (K) and arginines (R), phosphorylation of serines (S) and threonines (T), ubiquitylation and sumoylation of lysines, as well as ribosylation 2005-10-29 Chaoqun Wu, Fudan University 73 Characteristics of Histones from Calf Thymus DNA Histone Type Lysine Arginine amino acids Molecular WT H1 29% 1% 215 23,000 H2A 11% 9% 129 13,960 H2B 16% 6% 125 13,775 H3 10% 13% 135 15,340 H4 11% 14% 102 11,280 2005-10-29 Chaoqun Wu, Fudan University 74 Nucleosome: – Proposed by Roger Kornberg in 1974 – Structural unit for packaging DNA – Consists of histone core (octamer) plus ~ 200 of DNA – Compaction: ~ 7-fold Nucleosome core particle – histone core + 146 bp of DNA associated with the octamer Chromatosome (染色质小体 ) – Nucleosome + H1 Histone core H1 H2A H2A H2B H2B H3 H3 H4 H4 11 nm 6 nm Histone octamer 2005-10-29 Chaoqun Wu, Fudan University 75 nucleosome core particle: Octamer of: ?H2A ?H2B ?H3 ?H4 ? ~ 200 bp DNA wrapped around twice (7 fold compaction) 2005-10-29 Chaoqun Wu, Fudan University 76 Further compaction can be achieved by formation of a solenoid of nucleosomes... H1 required for formation of 30 nm fiber... 2005-10-29 Chaoqun Wu, Fudan University 77 Histones are subject to a complex and dynamic set of covalent modifications that are thought to be involved in the modulation of transcription during development, in X chromosome inactivation in female mammals, and in genome stability and meiotic chromosome dynamics. Histone modifications reported to date include acetylation, phosphorylation, methylation, ADP ribosylation, and ubiquitination 2. Histone modifications 2005-10-29 Chaoqun Wu, Fudan University 78 Histone Modification Residue Acetylation Lysine Methylation Lysine Arginine SerinePhosphorylation LysineUbiquitylation 2005-10-29 Chaoqun Wu, Fudan University 79 -CCH 3 O -CCH 3 O -CCH 3 O -CCH 3 O -CH 3 -CH 3 -CH 3 CH 3 - -PO 4 PO 4 - -PO 4 -Ub -Ub Acetylation Methylation Phosphorylation Ubiquitination H3 H4 H2A H2B Histone tails are the sites of covalent modifications 2005-10-29 Chaoqun Wu, Fudan University 80 The amino termini of histones contain a diversity of posttranslational modifications. The most promonent of them are acetylation and methylation of Lysine (K) residues in the highly concerved H3 and H4 Histone tails Histone fold domain Grewal and Moazed, 2003 ACETYLATION TRANSCRIPTION 174 bp of Acetyl modifications Methyl modifications 2005-10-29 Chaoqun Wu, Fudan University 81 Modifications: Ac = Acetylation Me = Methylation U = Ubiquitination P = Phosphorylation A schematic of the core histone octamer (centre) with the DNA superhelix (blue) and the core histone tails extended to their full length. Lysine residues that can be modified by acetylation are indicated with an asterisk. (From Wolffe AP and Hayes J , Nucleic Acids Research 27: 711, 1999.) 2005-10-29 Chaoqun Wu, Fudan University 82 Modification sites in histones 2005-10-29 Chaoqun Wu, Fudan University 83 Post-translational modifications of the core histones Current Biology Vol 14 No 14, R548, 2004 2005-10-29 Chaoqun Wu, Fudan University 84 2005-10-29 Chaoqun Wu, Fudan University 85 2005-10-29 Chaoqun Wu, Fudan University 86 CENPA: centromere protein A 2005-10-29 Chaoqun Wu, Fudan University 87 Histone Covalent Modifications Histone H3 K4 K14 K18 K23 K27K9 K79 S28 S10 K36 R17 Histone H2B K123 phosphorylation ubiquitylation acetylation methylation 2005-10-29 Chaoqun Wu, Fudan University 88 2005-10-29 Chaoqun Wu, Fudan University 89 2005-10-29 Chaoqun Wu, Fudan University 90 3. Histone-modifying enzymes. This group of enzymes adds or removes post- translational modifications to amino-terminal histone tails. Modifications to the tails function to decondense chromatin and/or recruit other enzymes or proteins to the nucleosomes. 2005-10-29 Chaoqun Wu, Fudan University 91 The enzymes group into two broad classes: — First, the ATP-dependent nucleosome remodeling enzymes (Sudarsanam and Winston, 2000) — Second, the enzymes that covalently modify the amino-terminal tails of core histones (Bradbury, 1992). This latter class includes histone acetyltransferases and deacetylases, histone kinases and phosphatases and histone methyltransferases. 2005-10-29 Chaoqun Wu, Fudan University 92 (1) Set domain containing proteins SET domain is a conserved sequence containing 115 amino acids, named from three fruit fly genes, PEV inhibitor Su (var) 3-9, Enhancer of zeste and Trithorax, which contain the domain. The evolutionarily conserved SET domain is found in a large and rapidly increasing number of proteins. At present, >350 proteins are known to contain SET domains, most of which are methyltransferases. 2005-10-29 Chaoqun Wu, Fudan University 93 Many SET domain methyltransferases have been shown to possess HMT activity towards specific lysine residues on histone tails, leading to positive or negative regulation of gene expression. Currently, all but one of the known histone lysine residues that are methylated are modified by SET domain proteins. J.R. Min, Q. Feng, Z.Z. Li, Y. Zhang and R.M. Xu, (2003), Cell 112, pp. 711–723. 2005-10-29 Chaoqun Wu, Fudan University 94 (2) Bromodomain containing proteins The bromodomain comprises a roughly 110 amino acid region that is most often present once per protein, but sometimes occurs twice and very rarely more than twice. Ronen Marmorsteina, Shelley L. Berger (2001) , Gene 272: 1-9 2005-10-29 Chaoqun Wu, Fudan University 95 Function of the bromodomains during transcription: 2005-10-29 Chaoqun Wu, Fudan University 96 Function of the bromodomains during transcription: (a)Hypothetical role of acetylation in the interaction of bromodomains during transcriptional activation. A FAT (factor acetyltransferase) acetylates a transcriptional activator, which binds to DNA. The activator recruits first the Swi/Snf remodeling complex and second the HAT domain complex through sequential acetyl-lysine interactions with bromodomains in the complexes. Finally, the acetylated histones within the promoter region, through bromodomain interactions, recruit the TFIID complex containing TBP to the TATA box resulting in increased transcription by RNA polymerase II. 2005-10-29 Chaoqun Wu, Fudan University 97 (b) Hypothetical role of HAT complexes in multiple acetylation of nucleosomes. A HAT (HAT-1) is recruited primarily by interactions with the DNA- bound activator resulting in acetylated histone H4. A second HAT (HAT-2) is recruited primarily by interaction of its bromodomain with acetyl-H4, resulting in acetylated histone H3. 2005-10-29 Chaoqun Wu, Fudan University 98 (3) Chromodomain containing proteins The chromodomain (CD) is a domain of 40– 50 amino acids long contained in various proteins involved in chromatin remodeling and the regulation of gene expression in eukaryotes during development (Cavalli and Paro 1998) Chromodomain-containing proteins can be classified into families based on their broader characteristics. 2005-10-29 Chaoqun Wu, Fudan University 99 The chromodomain is a highly conserved sequence motif that has been identified in a variety of animal and plant species. In mammals, chromodomain proteins appear to be either structural components of large macromolecular chromatin complexes or proteins involved in remodelling chromatin structure. Recent work has suggested that apart from a role in regulating gene activity, chromodomain proteins may also play roles in genome organisation. (BioEssays 22:124-137, 2000.) 2005-10-29 Chaoqun Wu, Fudan University 100 Chromodomain containing proteins family Khairina Tajul-Arifin,1 Rohan Teasdale, et al (2003) : Genome Research 13:1416–1429 . 2005-10-29 Chaoqun Wu, Fudan University 101 4. Histone acetylation enzymes HISTONES in transcriptionally active genes are often ACETYLATED. Acetylation is the modification of lysine residues in histones. – Reduces positive charge, weakens the interaction with DNA. – Makes DNA more accessible to RNA polymerase II Enzymes that ACETYLATE HISTONES are recruited to actively transcribed genes. Enzymes that remove acetyl groups from histones are recruited to methylated DNA. – There are additional types of histone modification as well, such as methylation of the histones. 2005-10-29 Chaoqun Wu, Fudan University 102 Histone deacetylases（ HDAC） 2005-10-29 Chaoqun Wu, Fudan University 103 Mammalian HATs and HDACs. (Current Opinion in Genetics & Development 2004, 14:308–315) 2005-10-29 Chaoqun Wu, Fudan University 104 The most well-understood enzymes in this class are the histone acetyltransferases (HATs) and the histone deacetylases (HDACs),which alter the acetylation state of specific lysines in the amino terminal tails of histones H3 and H4. 2005-10-29 Chaoqun Wu, Fudan University 105 DNA 甲基化和组蛋白去乙酰化 甲基化和组蛋白去乙酰化 甲基化 甲基化 DNA微注射 微注射 转录抑制 转录抑制 HDAC抑制剂 抑制剂 转录激活 转录激活 2005-10-29 Chaoqun Wu, Fudan University 106 The histone acetylation switch. Targeted HAT and HDAC activities negotiate the acetylation of chromatin. Acetylation establishes a structure that permits ATP- dependent chromatin remodeling factors to open promoters. Deacetylation, frequently followed by histone methylation, may form a solid base for hughly repressive structures, such as heterochromatin. 2005-10-29 Chaoqun Wu, Fudan University 107 5. Histone methylation enzymes PRMT1 CARM1 SET domain proteins or Dot1 HMTase 2005-10-29 Chaoqun Wu, Fudan University 108 Histone Methyltransferases HMT Histone Sites Organisms Set1 H3 K4 + S. cerevisiae Set2 H3 K36 + S. cerevisiae Clr4 H3 K9 + S. pombe G9a H3 K9, 27 +Human Eu Suv39h1,h2 H3 K9 +Murine He Set9 H3 K4 + Human Eu Dot1 H3 K79 - S. cerevisiae Eu PR-Set7 H4 K20 + Human He Ezh2 H3 K27 + Drosophila He Chromatin SET domain Science. 2003 Apr 4;300(5616):131-5 2005-10-29 Chaoqun Wu, Fudan University 109 Lysine Methyltransferases usually contain SET domain Proteins bearing the widely distributed SET domain have been shown to methylate lysine residues in histones and other proteins. The SET domain contains the catalytic center of lysine methyltransferases that target the N- terminal tails of histones and regulate chromatin function. Nature Structure Biology 21 October 2002 2005-10-29 Chaoqun Wu, Fudan University 110 Cross-Regulation of Modifications in H3 and H4 tails: 2005-10-29 Chaoqun Wu, Fudan University 111 Is there histone demethylases? Enzymes that covalently modify histones generally come in pairs that have opposing effects on gene expression, such as acetylases and deacetylases or kinases and phosphatases. Notable exceptions are the enzymes that methylate histones on lysine or arginine residues. No demethylases (HDMases) had been identified that remove these potent and very stable epigenetic marks, until now. 2005-10-29 Chaoqun Wu, Fudan University 112 On the basis of thermodynamic principles alone, methyl groups, in particular methyl-lysine, have a considerably lower turnover than do acetyl or phosphoryl groups. The latter two modifications can be removed from histone tails by the activity of HDACs or phosphatases, whereas histone demethylases (HDMases) have yet to be characterized. If HDMases do not exist, histone lysine methylation would be a nearly perfect long-term epigenetic mark for maintaining chromatin states. 2005-10-29 Chaoqun Wu, Fudan University 113 Some proteins were reported to be involved in histone demethylation Wang et al. show that the enzyme peptidylarginine deiminase 4 (PAD4), which converts unmodified arginine residues to citrulline in histones, can also convert methylated arginine residues in histones to citrulline, thereby removing the methyl mark ("demethylimination"). PAD4 can modulate the expression of genes known to be regulated by arginine histone methylases, which suggests that at least one of the elusive histone demethylases may have been identified. Science 306, 279-283 (2004). 2005-10-29 Chaoqun Wu, Fudan University 114 (A)Described protein modules of histone-modifying enzymes that have been shown to interact with site-specific methylation (chromodomain) or acetylation (bromodomain) marks in histone NH 2 - termini. A protein module that would selectively recognize phosphorylated positions is currently not known. HMT, histone methyltransferase; HAT, histone acetyltransferase; HDM, histone demethylase; PPTase, protein phosphatase; HDAC, histone deacetylase. Science, 293( 5532): p. 1074 2001, 2005-10-29 Chaoqun Wu, Fudan University 115 (B) Proposed histone tail interactions for a "reversed" histone code, showing a chromodomain- containing HAT (e.g., Esa1) and part of a nucleosome-remodeling complex that may comprise a bromodomain-containing, inactive HMTase (dashed lettering), such as the trx-G protein HRX. 2005-10-29 Chaoqun Wu, Fudan University 116 (C) Possible functional interactions between Su(var) and Pc-G proteins or between histone- and DNA- methylating enzymes that could be induced or stabilized by site-selective combinations of methylation marks. 2005-10-29 Chaoqun Wu, Fudan University 117 6. Physiologic effects of histone modifications Acetylation Transcriptional activation Histone deposition DNA repair Transcription elongation DNA replication Euchromatin 2005-10-29 Chaoqun Wu, Fudan University 118 Methylation Transcriptional activation (tri-Me) Transcription elongation (tri-Me) Active euchromatin (tri-Me) Permissive euchromatin (di-Me) Transcriptional silencing (tri-Me) DNA methylation (tri-Me) Transcriptional repression Imprinting Transcriptional silencing (mono-Me) Heterochromatin (tri-Me) 2005-10-29 Chaoqun Wu, Fudan University 119 Phosphorylation Mitosis Chromatin assembly? Transcriptional repression Apoptosis Transcriptional activation Immediate-early activation Ubiquitylation Meiosis Transcriptional activation Euchromatin Spermatogenesis 2005-10-29 Chaoqun Wu, Fudan University 120 Histone stablization SUMO(small ubiquitin-related modifier)是泛素 (ubiquitin)类蛋白 家族的重要成员之一。 尽管 SUMO的生化反应途径与泛素相似 ，但不像泛素那样诱导底物蛋白 降解。SUMO化能够使蛋白质更加 稳定，进而调节许多关键的细胞 活动。 Sumoylation 2005-10-29 Chaoqun Wu, Fudan University 121 Chromatin modifications at Lysine sites 2005-10-29 Chaoqun Wu, Fudan University 122 Histone modifications that alter the charge of a residue, such as lysine acetylation or serine phosphorylation, will disrupt histone–DNA interactions leading to ‘open’ or ‘active’ chromatin structures, that is chromatin remodeling. Specific histone modifications control the binding of nonhistone proteins to the chromatin fiber. These nonhistone proteins then elicit the function that is associated with a particular histone mark. A hallmark of many proteins that bind to histone tails is the presence of small histone binding modules. 2005-10-29 Chaoqun Wu, Fudan University 123 Long and Short range repression Long range repression Long-range repressor such as Groucho or Sir3/Sir4 may recruit histone deacetylases to nearby histone tails resulting in an altered chromatin structure. The corepressors may then spread along chromatin by virture of their ability to bind the modified histones Short range repression Short range corepressors may also recruit histone deacetylase. This may result in the local deacetylation of nucleosomes, forming an altered chromatin structure that may displace neighboring activators 2005-10-29 Chaoqun Wu, Fudan University 124 ╳ A hypermethylated promoter is silenced via the targeting of histone deacetylase. (Robertson KR and Wolffe AP: Nature Reviews Genetics 1: 11, 2000. ) 2005-10-29 Chaoqun Wu, Fudan University 125 7. RNA interference (RNAi) pathway 1.Required for HT formation and H3 Lys9 methylation in S. pombe : Argonaute (ago1), member of PAZ/Piwi family Dicer (dcr1), RNaseIII-like protein RNA-dependent RNA polymerase (rdp1) 2. Centromeric repeat sequences that are transcribed at low levels and produce ds RNA are sufficient to recruit HT at an ectopic site 3. Small HT RNAs provide specificity for targeting histone modifying activities and epigenetic modification of the genome through homology recognition 4. The role of RNAi in epigenetic gene silencing appears to be concerved among diverse species 2005-10-29 Chaoqun Wu, Fudan University 126 Small HT RNAs S. cerevisiae S. pombe How are heterochromatin complexes targeted to a specific chromosomal domain? Evidence suggests a role for repetitive DNA elements and non-coding RNAs in regional targeting of HT complexes. Grewal and Moazed, 2003 2005-10-29 Chaoqun Wu, Fudan University 127 1. 2. 3. RISC- RNA induced silencing complex Grewal and Moazed, 2003 2005-10-29 Chaoqun Wu, Fudan University 128 8. Maintance of histone modification –There are mechanisms to maintain histone modification such that they become heritable. –Modified histones are partitioned with parent strands. –Stable modifications such as histone methylation may be maintained 2005-10-29 Chaoqun Wu, Fudan University 129 No histone demethylases has been reported On the basis of thermodynamic principles alone, methyl groups, in particular methyl-lysine, have a considerably lower turnover than do acetyl or phosphoryl groups. The latter two modifications can be removed from histone tails by the activity of HDACs or phosphatases, whereas histone demethylases (HDMases) have yet to be characterized. If HDMases do not exist, histone lysine methylation would be a nearly perfect long-term epigenetic mark for maintaining chromatin states. 2005-10-29 Chaoqun Wu, Fudan University 130 How to remove histone lysine methylation In contrast to DNA methylation--where the methylated imprint can be removed by nucleotide excision followed by repair--DNA replication and semiconservative nucleosome distribution appears as the sole means to "dilute" histone lysine methylation below a critical threshold level A potential mechanism for removing methylation marks from histone tails is proteolytic processing. Histone NH 2 -termini are exposed and labile to proteolysis, and portions of certain histone tails are known to be clipped at precise stages in the cell cycle or at specific stages of development. R. Lin, R. G. Cook, C. D. Allis (1991), Genes Dev. 5, 1601 2005-10-29 Chaoqun Wu, Fudan University 131 A proteolytic model to remove "stable" methylation marks from histone H3. 2005-10-29 Chaoqun Wu, Fudan University 132 H3 Lys 9 acetylation+ H3 Lys4 methylation= STOP heterochromatin Model for formation of silenced chromatin domains E-histone-modifying Enzyme SF- silencing factor BE- boundary element Deacetylation and methylation of H3 Lys9 are followed by deacetylation of H3 Lys 14 and create a binding site for Swi6 silencing factor