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 V. Chromatin remodeling 2005-10-29 Chaoqun Wu, Fudan University 3 Nucleosome Essential Cell Biology by Alberts et al. 1997 2005-10-29 Chaoqun Wu, Fudan University 4 What is Chromatin remodeling Chromatin remodeling(染色质重构) is an important epigenetic mechanism. Chemical modification of the protruding histone tails — by the addition of acetyl, methyl or phosphate groups — can alter chromatin structure, which in turn influences the activity of adjacent genes. 2005-10-29 Chaoqun Wu, Fudan University 5 The remodeling of chromatin allows transcription factors to bind their cognate DNA sites. Associated proteins with structural, modifying and/or transcriptional activities propagate and/or stabilize the actively transcribing chromatin structure, leading to expression of the gene. The temporal order of interactions between cis and trans elements can vary. 2005-10-29 Chaoqun Wu, Fudan University 6 General model for gene regulation RNA polymerase II holoenzyme Chromatin Modifying Factors Chromatin Remodeling Factors DNA is packaged in higher order chromatin structure Activators bind to specific DNA sequences 2005-10-29 Chaoqun Wu, Fudan University 7 1. Nucleosome Positioning ? Nucleosome positioning is the phenomenon where nucleosomes assume specific positions on a DNA molecule ? This position has two attributes: – a rotational setting – a translational setting ? Nucleosomes have defined translational and rotational positions 2005-10-29 Chaoqun Wu, Fudan University 8 A nucleosome can assume one of several different positions along a DNA molecule This is referred to as translational positioning Nucleosome DNA Position 1 Position 2 Position 3 2005-10-29 Chaoqun Wu, Fudan University 9 ?This causes certain cis-elements to be located in nucleosomes at positions where the trans-acting factors cannot recognize and bind to them ? There is therefore a requirement to make nucleosomally wrapped DNA of some gene promoters more accessible ? This is a function performed by the chromatin remodelers 2005-10-29 Chaoqun Wu, Fudan University 10 2. Supposed remodeling steps ? Dissociation of H1 ? Acetylation and partial decondensation of the chromatin ?recruitment of select transcriptional activators, which, in turn, recruit ? Chromatin remodelers(染色质重构复合物) ? Additional histone modification enzymes ? The order of recruitment is not known 2005-10-29 Chaoqun Wu, Fudan University 11 The nucleosome can be restructured by two mechanisms 1. the modification of core histones by histone acetyltransferases, deactylases, methyltrans- ferases, and kinases. 2. the movement of nucleosomes along DNA which is carried out by ATP-dependent chromatin remodeling complexes. The purpose of the chromatin remodeling proteins is to alter the nucleosome architecture such that genes are exposed to or hidden from the transcriptional machinery. 2005-10-29 Chaoqun Wu, Fudan University 12 Chromatin Modification and Remodeling ?ATP-independent, covalent modifications Ac Ub Pi Ac CH3 CH3 ?HAT’s, HDAC’s ?Methylases ?Kinases ?Ubiquitin Ligases ATP ADP + Pi ?ATP-dependent remodeling Chromatin remodelers 2005-10-29 Chaoqun Wu, Fudan University 13 2005-10-29 Chaoqun Wu, Fudan University 14 Ac Ac Ac Histone 1 Cis elements TATA box Early events in preparation for the expression of a gene in a mitotic chromosome likely include: ? Dissociation of H1 ? Acetylation and partial decondensation of the chromatin 2005-10-29 Chaoqun Wu, Fudan University 15 Ac Ac Ac Ac Ac Chromatin remodeler Histone acetylase Ac Ac Ac Activator The partially decondensed chromatin probably allows recruitment of select transcriptional activators, which, in turn, recruit ? Chromatin remodelers ? Additional histone modification enzymes ? The order of recruitment is not known 2005-10-29 Chaoqun Wu, Fudan University 16 ? The chromatin remodeler changes the local structure of chromatin, allowing binding of factors to their sites previously occluded by nucleosomes ? The remodeling may cause nucleosome loss, or transient exposure of previously obscured DNA sequences Pol II Ac Ac Ac Ac TFIIB TFIID Ac Ac Ac Ac 2005-10-29 Chaoqun Wu, Fudan University 17 Next to remodeling ?The chromatin remodeler changes the local structure of chromatin, allowing binding of factors to their sites previously occluded by nucleosomes ?The remodeling may cause nucleosome loss, or transient exposure of previously obscured DNA sequences 2005-10-29 Chaoqun Wu, Fudan University 18 A simplified version of the events that lead from a silent to an actively transcribing from chromatin. In this version, a remodelling complex, guided by a pioneer factor, associates with the silent chromatin. The remodeling complex changes the chromatin structure — for example, through associated ATPases, leading to nucleosome displacement, or through associated histone acetyltransferases (HATs) that modify the histones locally. 2005-10-29 Chaoqun Wu, Fudan University 19 How does a chromatin remodeler function? 2005-10-29 Chaoqun Wu, Fudan University 20 Mechanism of ATP-dependent Chromatin Remodeling Complex Two-step model of SWI/SNF and RSC action in chromatin remodeling. The binding of the remodeling complex to chromatin is ATP independent (A) Binding of remodeling complexes to DNA and nucleosomes. (B) ATP-dependent nucleosome disruption. (C) Chromatin remodeling. 2005-10-29 Chaoqun Wu, Fudan University 21 Linker histones are ubiquitous components of cellular chromatin that constrain the entry/exit DNA of the nucleosome and incorporate another 20 bp of DNA into a particle called a chromatosome. Linker histone or members of the polycomb group (PcG) of proteins are conserved from flies to mammals, and they are required for virtually elimination of remodeling activities of yeast SWI–SNF, human SWI–SNF, ACF and Mi-2 complexes . Linker histones might brake the chromatin remodeling machine (EMBO Reports 3, 4, 319–322 (2002) ) 2005-10-29 Chaoqun Wu, Fudan University 22 3. Chromatin remodelers (染色质重构复合物) ? Chromatin remodelers acquire energy through ATP hydrolysis to remodel chromatin ? Numerous chromatin remodeling complexes exits ? They differ in the core DNA-dependent ATPase subunit ? There is some sharing of factors between complexes and homologs between species ?There are 3 main classes (more could follow) 2005-10-29 Chaoqun Wu, Fudan University 23 Chromatin-remodeling complexes carry out key enzymatic activities, changing chromatin structure by altering DNA–histone contacts within a nucleosome in an ATP-dependent manner. These complexes can be divided into three classes on the basis of the similarities of their ATPase subunits to the Swi2/Snf2, ISWI, and Mi-2 proteins. 1. The SWI2/SNF2 group. 2. The ISWI group 3. The Mi-2 group: chromatin-remodeling and deacetylase complexes 2005-10-29 Chaoqun Wu, Fudan University 24 First identified in yeast – Yeast complex known as SWI/SNF – Human complexes are BRM and BRG1 – 2~12 subunits protein complex ? Function suppressed by histone and other chromatin components ? Relieves chromatin-mediated suppression of a set of inducible genes ? Important for genes expressed in late anaphase – When chromatin condensation is not yet fully reversed after mitosis ATP-dependent Chromatin Remodeling Complex 2005-10-29 Chaoqun Wu, Fudan University 25 Chromatin Remodeling Complexes SWI/SNF: – Yeast: SWI/SNF, RSC – Drosophila: dSWI/SNF – Human: Brm, Brg ISWI: – Yeast: ISW1, ISW2 – Drosophila: NURF, CHRAC, ACF – Human: WCRF/ACF, RSF, NORC Mi-2/CHD-1 – Drosophila: dNuRD – Human: NuRD ATP ADP + Pi Swi/SNF, ISWI, Mi-2 2005-10-29 Chaoqun Wu, Fudan University 26 The three major identified nucleosome remodeling ATPases 2005-10-29 Chaoqun Wu, Fudan University 27 Mammalian ATPase-dependent chromatin-remodeling complexes (Current Opinion in Genetics & Development 2004, 14:308–315) 2005-10-29 Chaoqun Wu, Fudan University 28 Representative members of the SWI–SNF, ISWI and Mi-2/CHD subclasses of chromatin remodeling enzymes that are found in human cells. 2005-10-29 Chaoqun Wu, Fudan University 29 The three major identified nucleosome remodeling ATPases, their domains, homologues, and complexes in which they are found. Each ATPase core domain has seven, highly conserved subdomains. This ATPase core-domains are braced by domains that differ considerably between the three types of nucleosome remodeling ATPases 2005-10-29 Chaoqun Wu, Fudan University 30 Interactions of nucleosome and some proteins in nucleosome-remodeling complexes 2005-10-29 Chaoqun Wu, Fudan University 31 2005-10-29 Chaoqun Wu, Fudan University 32 Transcription activation Transcription repression Chromatin remodeling Ac -acetylated histones; mC-methylated Cytosine HDAC -histone deacetylases: Pol II- RNA polymerase II GTF- general transcription factors HAT -histone acetyltransferases; MBD -methylated DNA binding domain 2005-10-29 Chaoqun Wu, Fudan University 33 ATP-dependent Chromatin Remodeling Contributes to Many Cellular Functions ? Proliferation ? Differentiation ? DNA replication ? Stress responses 2005-10-29 Chaoqun Wu, Fudan University 34 Chromatin- Remodeling Complexes and The Cell Cycle Cell cycle regulation of the SWI/SNF complex. The activity of hSWI/SNF is regulated, at least in part, by phosphorylation of some of its subunits. The complex is activated after G1 by a cyclin E/cdk2- dependent phosphorylation event of BAF155 and BRG1. Phosphorylation toward the end of G2, which might also occur at the level of BRG1, inactivates it, while a dephosphorylation even occurring late in G2 also seems to have an activating role in SWI/SNF function. The question marks denote the lack of information concerning how these two apparently contradictory sets of data are related. 2005-10-29 Chaoqun Wu, Fudan University 35 Chromatin Remodeling and HAT Complexes The remodeling complexes are directed to sites in chromatin via their interactions with transcriptional regulatory factors. The available data do not distinguish between the two models presented. It is possible that the factor binds the DNA first and then acts as a docking pad for the remodeling complex. Alternatively, it is possible that the interaction between the factor and the remodeling complex takes place in solution and the DNA-binding domain of the transcription factor directs the remodeling complex to chromatin in a later step. Targeting of the SWI/SNF complex. 2005-10-29 Chaoqun Wu, Fudan University 36 Chromatin- Remodeling Complexes Regulating DNA Replication The function and dysfunction of some human disease-related chromatin remodeling factors regulating DNA replication. 2005-10-29 Chaoqun Wu, Fudan University 37 The SWI/SNF complex SNF2 SWI1 SNF5 SWI3 SWp82 SNF12/SWP73 ARP7 ARP9 SNF6 ANC1/ TGF3 SNF11 ? 11 subunits ?~1 MDa ? Snf2 is an ATPase SWI (switch) SNF (sucrose non fermenting) 2005-10-29 Chaoqun Wu, Fudan University 38 Subunit Size (kDa) Function SWI2 / SNF2 194 DNA dependent ATPase SWI1 148 AT-rich interaction domain (ARID) for non- specific binding, Zn-finger protein SNF5 103 Assembly and catalytic functions of the complex SWI3 93 SWp82p ~82 SNF12 / SWP73 64 ARP7 54 Actin-related protein ARP9 53 Actin-related protein SNF6 38 ANC1 / TFG3 27 SNF11 19 Interacts with the N-terminal D1 region of SWI2 2005-10-29 Chaoqun Wu, Fudan University 39 The Mi-2/RSC (remodels the structure of chromatin) complex ? 12+ subunits ?~1 MDa ? Sth1 (Snf two homolog) is an ATPase Sth1 Rsc1 Rsc30 Rsc3 Rsc2 Rsc4 Rsc9 Rsc8 Rsc6 Sfh1 ARP7 ARP9 Subunits with no known homolog in Swi/Snf 2005-10-29 Chaoqun Wu, Fudan University 40 Subunit SWI/SNF homolog Size (kDa) Function Sth1 Rsc1 107 Rsc2 102 Rsc3 102 Rsc30 101 Rsc4 72 Rsc9 65 Rsc8 Sfh1 Rsc6 Arp7 Arp9 SWI2 / SNF2 157 DNA dependent ATPase SWI3 63 Subunit assembly, binds through C- terminal coiled coil domain SNF5 49 Assembly and catalytic functions of the complex SWP73 54 ARP7 54 Actin-related protein ARP9 53 Actin-related protein 2005-10-29 Chaoqun Wu, Fudan University 41 Multiple types of ISWI complexes exits 2005-10-29 Chaoqun Wu, Fudan University 42 In summary, the importance of chromatin remodeling in human biology, Mendelian disease, and somatic tumors has become increasingly apparent during the past few years. The current studies of model organisms excitingly suggest that heritable epigenetic variation will account for a portion of the phenotype in complex diseases and raise questions concerning the contribution of epigenetic variation to quantitative traits in general. In particular, do the increasing incidences of diseases such As asthma, syndrome X, and some neoplasias arise in part from environmental influences and the selection for epigenetic traits? 2005-10-29 Chaoqun Wu, Fudan University 43 CHD-1: chromodomain-helicase-dna-binding protein 1 HP-1: heterochrmatin binding protein 1 NuRD: nucleosome remodelling ISWI : Imitation SWItch ATRX: -thalassemia X-linked mental retardation BRG1: brahma-related gene1 BRM: brahma CBP: CREB-binding protein ERCC6: excision repair cross-complementing rodent repair deficiency, complementation group 6 ETV6: ETS variant gene 6 MECP2: methyl-CpG-binding protein 2 RAR: retinoic acid receptor SMARCAL1: SWI/SNF-related matrix-associated, actin-dependent regulator of chromatin, subfamily A-like protein 1 SMARCB1: SWI/SNF- related, matrix-associated, actin-dependent regulator of chromatin, subfamily B, member 1. 2005-10-29 Chaoqun Wu, Fudan University 44 Exciting recent developments Swi/Snf machinery can remove nucleosomes Swi/Snf machinery deposits histone variants for specialized functions Histone methylation and DNA methylation interdependent RNAi silencing machinery and histone methylation interdependent 2005-10-29 Chaoqun Wu, Fudan University 45 4.Boundary elements(边界子 边界子 ), or insulator elements( (隔离子) 边界子和 边界子和隔离子 的隔离功能 的隔离功能 ： ： 1、封阻末梢增强子对启动子的作用。 、封阻末梢增强子对启动子的作用。 2、防止染色质位置效应（ 、防止染色质位置效应（ CPE)。 。 2005-10-29 Chaoqun Wu, Fudan University 46 Boundary elements in chromatin Selective recognition of methylated Lysine 9 on histone H3 by the HP1 Chromo domain Andrew J. Bannister et al. Nature 2002 2005-10-29 Chaoqun Wu, Fudan University 47 ‘ON’ ‘OFF’ Active euchromatin Silenced and HP1-coated heterochromatin modified from Bannister et al. Nature 2001 Ac Ac Ac Ac BrD BrD 2005-10-29 Chaoqun Wu, Fudan University 48 2005-10-29 Chaoqun Wu, Fudan University 49 Insulators are chromosomal elements that can shelter genes from the effects of silencers and enhancers. Genes are usually subject to regulation by long-distance-acting elements, which either enhance (enhancers) or repress expression (silencers). a: Such elements can be shared, for example by two reporter genes. b: However, interposition of an insulator will specifically block communication between the enhancer or silencer and the downstream gene, without affecting the capacity of the enhancer or silencer to regulate the second reporter gene. BioEssays 26:523– 532, 2004. 2005-10-29 Chaoqun Wu, Fudan University 50 Confining Gene Expression Some of the organisational properties of the eukaryotic genome reside in the ability of chromatin to establish autonomous units that specify levels and patterns of gene expression. i.e. enhancers act on a promoter in a specific domain, but are unable to act on a promoter in a separate domain. 2005-10-29 Chaoqun Wu, Fudan University 51 The candidates charged with the function of establishing and delimiting domains of expression are boundary or insulator elements. These set up independent territories of gene activity. A few of these sequences have been characterised in Drosophila and vertebrates and the gypsy retrotransposon is one that has been well characterised. 2005-10-29 Chaoqun Wu, Fudan University 52 How does a gene with its own programmed pattern of expression defend itself against its neighbors? With boundary/ insulator elements What is a boundary/ insulator elements Two properties: ? insulators have the ability to act as a "positional enhancer blocker“ ? insulators have the ability to protect against position effects enhancer Gene Insulator Active expression enhance Gene Insulator enhancer Gene Insulator No expression Active expression X Effect is position specific 2005-10-29 Chaoqun Wu, Fudan University 53 Can boundaries be modulated? ?DNA methylation has been shown to prevent the binding of the CTCF, a protein shown to be responsible for enhancer blocking activity in insulators Human/mouse Imprinted Igf2/H19 locus ICR Igf2 (OFF) H19 (ON) ?This results in the loss of enhancer blocking activity x CTCF Igf2 (ON) H19 (OFF) x Me Me Me Me 2005-10-29 Chaoqun Wu, Fudan University 54 2005-10-29 Chaoqun Wu, Fudan University 55 Properties of chromatin insulators Insulators buffer transgenes from chromosomal position-effects. Insulators interfere with enhancer-promoter interactions in a directional manner as positional enhancer blocker . 2005-10-29 Chaoqun Wu, Fudan University 56 Insulators buffer transgenes from chromosomal position-effects. 2005-10-29 Chaoqun Wu, Fudan University 57 Protection against position effect ?Gene randomly inserted into chromatin are subject to the surrounding element ?There expression can either be downregulated or unregulated ?Frequently genes are silenced Silent chromatin Active gene Insulator protect against silencing of chromatin Silent chromatin Active gene Silent chromatin spreads into gene an silences it Insulator absent Silent gene 2005-10-29 Chaoqun Wu, Fudan University 58 Insulators interfere with enhancer- promoter interactions in a directional manner. 2005-10-29 Chaoqun Wu, Fudan University 59 2005-10-29 Chaoqun Wu, Fudan University 60 2005-10-29 Chaoqun Wu, Fudan University 61 2005-10-29 Chaoqun Wu, Fudan University 62 2005-10-29 Chaoqun Wu, Fudan University 63 Multiple effective models of insulator 2005-10-29 Chaoqun Wu, Fudan University 64 果蝇中最典型的两个隔离子 果蝇中最典型的两个隔离子 隔离子 隔离子 位置 位置 蛋白组分 蛋白组分 scs/scs’ hsp70末端 末端 BEAF-32A BEAF-32B gypsy gypsy逆转 逆转 录转座子中 录转座子中 su[Hw] mod[mdg4 2005-10-29 Chaoqun Wu, Fudan University 65 脊椎动物中首先发现的隔离子 脊椎动物中首先发现的隔离子 隔离子 隔离子 位置 位置 蛋白组分 蛋白组分 cHS4 β β － － 珠蛋白基 珠蛋白基 因和叶酸受体 因和叶酸受体 基因之间 基因之间 CTCF 果蝇 果蝇 eve启动子 启动子 (GAGA) 果蝇 果蝇 eve启动 启动 子 子 Trl蛋白 蛋白 2005-10-29 Chaoqun Wu, Fudan University 66 隔离子的作用机制 隔离子的作用机制 a.增强子环的干涉诱导模型 增强子环的干涉诱导模型 2005-10-29 Chaoqun Wu, Fudan University 67 b.增强子出轨模型 增强子出轨模型 2005-10-29 Chaoqun Wu, Fudan University 68 c.核区室化模型 核区室化模型 2005-10-29 Chaoqun Wu, Fudan University 69 d.简化的环状区域模型 简化的环状区域模型 2005-10-29 Chaoqun Wu, Fudan University 70 6. Nucleus Compartment (核区室化 ) What is a chromatin domain? Eukaryotic genes are located in separate domains on each chromosome which contain all the appropriate regulatory elements for their correct expression 异染色质与常染色质的主要区别是能否被DAPⅠ染 色。 2005-10-29 Chaoqun Wu, Fudan University 71 2005-10-29 Chaoqun Wu, Fudan University 72 Nuclear genome organization As the nucleus reassembles after mitosis, distinct chromosomal bands segregate into different regions, giving rise to polar chromosome territories. Alignment of polar chromosome territories results in the establishment of distinct higher-order genome compartments, with functionally distinct chromatin fractions. 2005-10-29 Chaoqun Wu, Fudan University 73 Chromatin changes in senescent cells. 2005-10-29 Chaoqun Wu, Fudan University 74 What are Chromosomal territories and how do they influence gene regulation Eukaryotic chromosome are specifically compartmentalized to form a distinct nuclear architecture 2005-10-29 Chaoqun Wu, Fudan University 75 What do lampbrush chromosomes suggests? Provides cytological evidence chromosome is subdivided into a series of discrete and topologically independent domains Note loops Lampbrush chromosome from an amphibian oocyte 2005-10-29 Chaoqun Wu, Fudan University 76 What does heat shock puff in polytene chromosome suggest? Although individual fibers not readily visualized the distinctive banding pattern suggest discrete domains. 2005-10-29 Chaoqun Wu, Fudan University 77 What does this data suggest Chromatin is organized in discrete domains Each topological domain is likely to be specified by the underlying DNA sequence Each domain corresponds to a functionally autonomous genetic unit Domain can be changed 2005-10-29 Chaoqun Wu, Fudan University 78 An example of a domain 4 3 2 1 ρ εβ A β H 5’ HS sites 3’HS β/ε HSA Folate receptor gene COR 3’β1 COR 3’β2 5 kb All the element necessary for proper regulation and expression of genes are present Two boundaries separate the domain form the rest of the genome Regulatory element control development expression 2005-10-29 Chaoqun Wu, Fudan University 79 How are chromosome organized in a nucleus Chromosomes occupy discrete territories in the cell nucleus Chicken nuclei Staining pattern results in different color specific for each chromosome 2005-10-29 Chaoqun Wu, Fudan University 80 Features of human chromosome territories Note specific location of p and q arms and specific territories Active ANT2 on surface inactive ANT2 buried Transparent view Three dimensional view of chromsome territories Same specific territories for the active and inactive X chromosome 2005-10-29 Chaoqun Wu, Fudan University 81 Where are gene-rich and gene-poor regions of chromosome located Gene-poor chromosome located at nuclear periphery Gene-rich chromosome located in nuclear interior So generally silent regions of chromosomes are located at the nuclear periphery 2005-10-29 Chaoqun Wu, Fudan University 82 Where are actively expressing genes located The interchromatin compartment (IC) contains various types of non-chromatin domains with factors for transcription, splicing, DNA replication and repair. This suggest active gene expression in these compartments Nonsplicing factors Overlay 2005-10-29 Chaoqun Wu, Fudan University 83 Subnuclear domains 2005-10-29 Chaoqun Wu, Fudan University 84 Subnuclear domains The Cajal bodies (CBs) are involved in transport and maturation of snRNPs and in the assembly of the transcription machinery. Oct1/PTF/transcription (OPT) domains constitute a compartment where a specific group of genes is brought together, making transcriptional regulation more efficient. Subset of promyelocytic leukemia (PML) bodies and the perinucleolar compartment (PNC) are putative sites of transcriptional activity. 2005-10-29 Chaoqun Wu, Fudan University 85 Human polycomb group complex (PcG), specifically localized at heterochromatin regions, is considered to play a role in the constitutive repression of transcription. The synthesis and processing of hnRNAs occur at the splicing-factor compartments (SFCs), that include the interchromatin granule clusters and the perichromatin granules . The spatial organization of nuclear processes suggests the existence of a dynamically regulated architecture in the nucleus. 2005-10-29 Chaoqun Wu, Fudan University 86 7. Nuclear structure in cancer cells Nuclear architecture — the spatial arrangement of chromosomes and other nuclear components — provides a framework for organizing and regulating the diverse functional processes within the nucleus. There are characteristic differences in the nuclear architectures of cancer cells, compared with normal cells, and some anticancer treatments restore normal nuclear structure and function. Nature Review Cancer, 4:677, 2004 2005-10-29 Chaoqun Wu, Fudan University 87 Nuclear shape changes associated with cancer 2005-10-29 Chaoqun Wu, Fudan University 88 Nuclear structure in normal and cancer cells. Nuclei can become irregular and begin to fold, Coarse heterochromatin aggregates Perinucleolar compartment (pink) in tumour cells 2005-10-29 Chaoqun Wu, Fudan University 89 2005-10-29 Chaoqun Wu, Fudan University 90 Rnomics — “The Modern RNA World” Part VI. 2005-10-29 Chaoqun Wu, Fudan University 91 The past four years researches have seen an explosion in the number of detected RNA transcripts with no apparent protein-coding potential. This has led to speculation that non- protein-coding RNAs (ncRNAs) might be as important as proteins in the regulation of vital cellular functions. However, there has been significantly less progress in actually demonstrating the functions of these transcripts. Alexander H, Peter S and Norbert P: Trends in Genetics, 21(5):289-297, May 2005 2005-10-29 Chaoqun Wu, Fudan University 92 Some numbers Increasing – 45% of the genome derived from retrotransposition – 41-60% of multiexon genes have alternative spliceforms Decreasing – Estimated number of protein coding genes: 24,500 2005-10-29 Chaoqun Wu, Fudan University 93 Haploid genome (Mbases) Cell types Genes Neurons Prokaryotes E.coli ~ 4.6 ~ 15 ~ 100 ~ 100 ~ 120 ~ 3300 1-2 470-4.000 unicellular eukaryot Yeast 1-3 ~ 6.000 Plant Arabidopsis ~ 30 ~ 24.000 Worm C. Elegans ~ 50 ~ 18.500 ~ 300 neurons Fruit fly Drosophila ~ 50 ~ 13.500 Human ~ 120 < 30.000 10x10 9 neurons Some numbers 2005-10-29 Chaoqun Wu, Fudan University 94 Complexity and non-protein coding DNA Ryan, Mattick et al. 2003 Non-coding DNA/total genomic DNA enucleate nucleate unicell nucleate multicell Enucleate Nucleate unicellular Plant Invertebrate vertebrate 2005-10-29 Chaoqun Wu, Fudan University 95 The rapidly increasing number of mammalian ncRNAs and ncRNA candidates from 1999 to 2004. 2005-10-29 Chaoqun Wu, Fudan University 96 Genomic space for the discovery of novel ncRNAs in higher eukaryotes. 2005-10-29 Chaoqun Wu, Fudan University 97 The number of known ncRNAs and putative ncRNAs of unknown function has increased dramatically in the past few years. Moreover, particularly in higher eukaryotes, only a fraction of the genome (i.e. ~ 1.4% in humans) is translated into proteins,whereas ~ 27% is transcribed as introns and UTRs but not translated. In addition, ~ 25% of mammalian genomes are predicted to be transcribed but not translated, further increasing the space for potential novel ncRNA genes. 2005-10-29 Chaoqun Wu, Fudan University 98 RNomics (RNA Genomics ) – What is a non-coding RNA (ncRNA)? – Classes of known ncRNA types. – Finding novel ncRNA sequences. –RNAi. 2005-10-29 Chaoqun Wu, Fudan University 99 ? Study of RNA expression and function. ? Non-coding RNA (ncRNA) sequences: – RNA sequences that function without being translated into a protein. – Wide variety of ncRNA are known and there are probably many that have yet to be discovered. 2005-10-29 Chaoqun Wu, Fudan University 100 NONCODE http://noncode.bioinfo.org.cn/index.htm 2005-10-29 Chaoqun Wu, Fudan University 101 Central Dogma of Biology DNA RNA Protein 2005-10-29 Chaoqun Wu, Fudan University 102 The construction of an unbiased, general purpose cDNA library (upper right) of ncRNAs or a more specialized library encoding RNAs form a specific ncRNA subclass, for example, snoRNAs (upper left). 2005-10-29 Chaoqun Wu, Fudan University 103 Experimental RNomics Novel snmRNAs in A. thaliana: A schematic overview and classification of 140 candidates for small non-messenger RNAs in A. thaliana. Current Biology, 12:2002–2013, 2002, 2005-10-29 Chaoqun Wu, Fudan University 104 Hypothesis RNA creates a regulatory network required for the increase in complexity RNA drives evolution – Extensive RNA processing and regulation gives rise to phenotypic diversity – RNA-directed rewriting of DNA enables dissemination of ‘selfish’ RNA associated with successful outcomes 2005-10-29 Chaoqun Wu, Fudan University 105 RNA is an active player RNA subunit of telomerase DNA pre mRNA Self splicing intron mRNA Translation tRNA+ rRNA in Ribosoms protein Transcription Splicing sncRNA Protein after translational modification tRNA 2005-10-29 Chaoqun Wu, Fudan University 106 Sites of transcription of polyadenylated and nonpolyadenylated RNAs for 10 human chromosomes were mapped at 5-base pair resolution in eight cell lines. Nonpolyadenylated transcripts comprise the major proportion of the transcriptional output of the human genome. Of all transcribed sequences: Polyadenylated sequences — 19.4%, Nonpolyadenylated sequences — 43.7%, Bimorphic sequences — 36.9% Half of all transcribed sequences are found only in the nucleus, most part are unannotated. Cheng J et al. Science. :1149-54. 2005, 2005-10-29 Chaoqun Wu, Fudan University 107 Transcription of non-protein-coding RNA is far more widespread than was previously anticipated. Although some ncRNAs act as molecular switches that regulate gene expression, the function of many ncRNAs is unknown. New experimental and computational approaches are emerging that will help determine whether these newly identified transcription products are evidence of important new biochemical pathways or are merely ‘junk’ RNA generated by the cell as a by-product of its functional activities. 2005-10-29 Chaoqun Wu, Fudan University 108 Two different kinds of RNAs in the cells from all known organisms: ? mRNAs, which are translated into proteins; ? nmRNAs (non-messenger RNAs), also referred to as non-coding RNAs (ncRNAs), which are not translated into proteins. 2005-10-29 Chaoqun Wu, Fudan University 109 gene chromosome transcription Primary transcript RNA plicing intronsAssembled exons mRNA tranlation Protein (Structure, catalytic Signaling, regulatory) ncRNA (Various functions) processing + snoRNA (RNA editing) microRNA (regulatory functions) processing Other RNA? 2005-10-29 Chaoqun Wu, Fudan University 110 nmRNAs nmRNAs range from very large, for example,~17 kb as Xist RNA, to extremely small (21–23 nt) as microRNAs (miRNAs). In general, the sizes of the majority of known nmRNAs vary from about 20 nt to 500 nt, well below the size of the majority of mRNAs and are therefore termed snmRNAs (small,non-messenger RNAs ). 2005-10-29 Chaoqun Wu, Fudan University 111 Most snmRNAs can be grouped into specific RNA classes on the basis of size, structure or sequence motifs, protein partners, or subcellular location. § tRNAs that serve as essential components of the protein synthesizing machinery, § snRNAs (Small nuclear RNAs ) required for splicing of premRNAs, § snoRNAs (small nucleolar RNA ), which are involved in modification of other RNAs. § miRNAs (microRNA), of 21–23 nt, inhibitors § siRNAs (small interfering RNAs ), of 21–23 nt , inhibitors. 2005-10-29 Chaoqun Wu, Fudan University 112 Classes of nmRNA ? Group I Introns – A class of RNA introns that catalyze their own splicing. ? miRNA – (micro RNA) Post-transcriptional gene regulation by RNAi pathway. ? Riboswitches – Cis-acting regulatory sequences that respond to the environment. ? RNase P RNA – Component of RNase P, which edits tRNA. ? rRNA – (ribosomal RNA) Ribosomal RNA is responsible for peptide bond formation in the ribosome. 2005-10-29 Chaoqun Wu, Fudan University 113 ? snoRNA – (small nucleolar RNA) Direct rRNA modification. ? SRP RNA – (signal recognition molecule) Involved in the transport of secreted proteins to the endoplasmic reticulum. ? tRNA – (transfer RNA) that serve as essential components of the protein synthesizing machinery ? Telomerase RNA – Structured RNA that provides sequence template for telomere sequences. ? tmRNA – (tRNA- mRNA-like) Rescues stalled ribosomes and tags the protein product for degradation. 2005-10-29 Chaoqun Wu, Fudan University 114 Abbreviations for different classes of non-coding RNA ? fRNA: Functional RNA — essentially synonymous with non-coding RNA ? miRNA: MicroRNA — putative translational regulatory gene family ? ncRNA: Non-coding RNA — all RNAs other than mRNA ? rRNA: Ribosomal RNA ? siRNA: Small interfering RNA — active molecules in RNA interference ? snRNA: Small nuclear RNA including spliceosomal RNAs ? snmRNA: Small non-mRNA — essentially synonymous with small ncRNAs ? snoRNA: Small nucleolar RNA — most known snoRNAs are involved in rRNA modification ? stRNA: Small temporal RNA — for example, lin-4 and let-7 in Caenorhabditis elegans ? tRNA: Transfer RNA 2005-10-29 Chaoqun Wu, Fudan University 115 Nuclear introns Splicing of nuclear introns requires neither a free guanine nucleoside nor an extensive conserved secondary structure in the target RNA. Instead, splicing of nuclear introns requires a spliceosome which consists of 44 or more proteins and a series of small nuclear RNAs (snRNAs). The snRNAs appear to play the role of the conserved secondary structures used in Group I and II introns. There are sequence requirements for splicing of nuclear introns. 2005-10-29 Chaoqun Wu, Fudan University 116 Group I introns Group I introns are the only class of introns whose splicing requires a free guanine nucleoside. They also have a conserved secondary structure which is different from that of group II introns. Group I introns have been demonstrated to self-splice. Group I introns are found in RNA transcripts of protozoa, fungal mitochondria, bacteriophage T4 and bacteria. 2005-10-29 Chaoqun Wu, Fudan University 117 Group II introns The lariat pathway is used in splicing of group II introns. Some group II introns are spliced in vitro without the addition of any proteins. The conserved secondary structure probably participates in catalysis. The 2'OH is sterically poised for the attack by being attached to a single bulged base in an RNA double helix. The base, adenine, intercalates in the helix, thus holding the sugar in the correct orientation for catalysis. Group II introns are found in fungal mitochondria, higher plant mitochondria and plastids. 2005-10-29 Chaoqun Wu, Fudan University 118 Transplicing, splicing between two separate RNAs, is analogous to group II intron splicing Location Mitochondrial introns, in genes encoding components of the electron transport system (mt mRNA genes of Cox1 and Cob genes) and large rRNA genes (mt rRNA). Chloroplastic introns, mainly in large rRNA and tRNA genes. Nuclear introns, in both large and small rRNA genes. Phage introns, in genes encoding proteins involved in DNA metabolism (mRNAs). Bacterial introns, in tRNA genes. Size: Variable From 68 over 3000 nt. Most are over 400 nt. 2005-10-29 Chaoqun Wu, Fudan University 119 Splicing mechanism: Self- splicing introns ?Most are able to splice themselves in the absence of proteins, i.e. the RNA itself is catalytic ("ribozymes"). ?Initiate splicing with an external G nucleotide (cofactor). ? Uses a phosphoester transfer mechanism, i.e. two successive transesterification steps catalysed by RNA in vitro. ? Not all group I introns are truly catalytic. Splicing of some group I introns in vivo is modulated by a number of proteins encoded either by various genes 2005-10-29 Chaoqun Wu, Fudan University 120 Finding Novel nmRNA ? It has been estimated that as much as 97-98% of the transcriptional output of the human genome is nmRNA sequences (Mattick. BioEssays. 2003. 25:930.) ? Most nmRNA sequences have been found as part of the study of a cellular process using standard genetic methods. ? There is interest in finding novel nmRNA sequences computationally in genomic sequences. 2005-10-29 Chaoqun Wu, Fudan University 121 Structure is Conserved, Although Sequence is Not RD0260 GCGACCGGGGCUGGCUUGGUAAUGGUACUCCCCUGUCACGGGAGAGAAUGUGGGUUCAAAUCCCAUCGGUCGCGCCA RE6781 UCCGUCGUAGUCUAGGUGGUUAGGAUACUCGGCUCUCACCCGAGAGAC-CCGGGUUCGAGUCCCGGCGACGGAACCA ^^^^^^^ ^^^^ ^^^^ ^^^^^ ^^^^^ ^^^^^ ^^^^^^^^^^^^ 2005-10-29 Chaoqun Wu, Fudan University 122 How do nmRNA Sequences Differ from Other Genomic Sequences? ? Rivas & Eddy. BMC Bioinformatics. 2001. 2:8. ? Comparative analysis of alignment of two genomes: 2005-10-29 Chaoqun Wu, Fudan University 123 Reading ? Washietl, Hofacker, Stadler. “Fast and reliable prediction of noncoding RNAs.” Proceedings of the National Academy of Sciences, USA, 2005, 102: 2454-2459. ? Hypothesis: Non-coding RNA alignment fragments from genome sequence alignments have significantly lower predicted secondary structure formation free energy change than other sequences. 2005-10-29 Chaoqun Wu, Fudan University 124 RNA directed readout 2005-10-29 Chaoqun Wu, Fudan University 125 RNA-directed RNA readout RNA-directed DNA readout RNA-directed rewriting of RNA RNA-directed rewriting of DNA 2005-10-29 Chaoqun Wu, Fudan University 126 RNA directed RNA readout: dsRNA plants metazoan 2005-10-29 Chaoqun Wu, Fudan University 127 RNA directed RNA readout small silencing RNA 2005-10-29 Chaoqun Wu, Fudan University 128 RNA directed RNA readout dsRNA (many sources) ? small silencing RNAs – Processed by RNAse III dicer into siRNA – siRNAs incorporated into RISC – RISC degrades RNA complementary to siRNA – Spread of RNAi by RdRP Evolution: related genes will escape cosuppression by sequence divergence 2005-10-29 Chaoqun Wu, Fudan University 129 RNA directed RNA readout 2 Translational inhibition – First discovered in C.elegans – lin-4, let-7 microRNA – Some microRNA target RNAs to the RNAi pathway (plants + some animals) – Other microRNAs are though to bind to elements important for translational control 2005-10-29 Chaoqun Wu, Fudan University 130 RNA directed RNA readout Transcriptional gene silencing - switch to promotor outside silenced region (drosophila bithorax) Bidirectional promotor (cardiac myosin HC6+7) - transcribe one - switch another off 2005-10-29 Chaoqun Wu, Fudan University 131 RNA directed DNA readout Transcriptional gene silencing(TGS) – Mechanisms not well understood – Trans: examples from plants: dsRNA homologous to promotor ? TGS – Associated with methylation. – RISC might be involved since deletions lead to diminished methylation – Cis: Good evidence from S.pombe 2005-10-29 Chaoqun Wu, Fudan University 132 Silent chromatin & RNAi CREDIT: KATHARINE SUTLIFF/SCIENCE ? Centromers contain repeats and are often heterochromatic (silenced) ?Finding: Deletion of RNAi machinery causes desilencing centromeric regions 2005-10-29 Chaoqun Wu, Fudan University 133 RNA directed DNA readout (sense antisense transcription units, SATs) Far more frequent than earlier anticipated – ~ 1,600 human SATs – 2481 SATs in the mouse FANTOM2 set Effective gene-regulation – Both active ? dsRNA ? histone modification ? TGS (Transcriptional gene silencing) 2005-10-29 Chaoqun Wu, Fudan University 134 RNA-directed rewriting of RNA Regulate alternative splicing Ex.: thyroid receptor alpha SATs (sense antisense transcription units) RNA-editing: Frequency unknown Common in trypanosomes mitochondria insert and delete uridines restore reading frame editing can change splice sites splicing can prevent editing 2005-10-29 Chaoqun Wu, Fudan University 135 RNA-directed rewriting of DNA 45% of the human genome is derived from retrotransposition!! 2005-10-29 Chaoqun Wu, Fudan University 136 Control Architecture Genome Transcriptome Proteome Imprinting – methylation Splicing Regulation by proteins Regulation by RNA RNAi *New phenotypes: Newly generated RNA extract different subsets of information from the genome. *Not dependent on mutation of protein coding genes. Exon- shuffling not dependent on maintaining reading frame. * ? Faster evolution Ribozymes 2005-10-29 Chaoqun Wu, Fudan University 137 Part VII. Epigenetics and Diseases 2005-10-29 Chaoqun Wu, Fudan University 138 Marie and Jo — not their real names — are twins, genetically identical and raised in a happy home. similar in almost every respect. But as adults, their lives and personalities diverged: Marie was diagnosed with schizophrenia. Such examples have long baffled geneticists. Despite sharing the same DNA and often the same environment, 'identical' twins can sometimes show striking differences. Now some researchers are beginning to investigate whether subtle modifications to the genome that don't alter its DNA sequence, known as epigenetic changes, may provide the answer. 2005-10-29 Chaoqun Wu, Fudan University 139 Dr Alan Wolffe (1999) “Epigenetics is heritable changes in gene expression that occur without a change in DNA sequence” Epigenetics is ingenious system to selectively utilize genome information, through activating or inactivating functional genes. Identified epigenetic processes involved in human disease: 1. DNA methylation 2. imprinting 3. histone modifications Each of these processes influences chromatin structure and Thus regulates gene expression and DNA methylation, replication, recombination and repair. 2005-10-29 Chaoqun Wu, Fudan University 140 Changes in both chromatin structure and DNA methylation result in complex diseases: MECP2 chromatin lose ability to remodelling speak and walk DNA methyl- DNA methylation ICF syndrome (for transferase 3B immunodeficiency, centromere instability and facial anomalies ATRX Epigenetic mental retardation, urogenital abnormalities and a form of anaemia Indicating: Some features of complex diseases are easier to explain in terms of epigenetic changes than through conventional genetics. 2005-10-29 Chaoqun Wu, Fudan University 141 The idea that epigenetics underpins many of the world's health scourges is still highly speculative. "Most geneticists believe that the essence of all human disease is related to DNA-sequence variation," says Arturas Petronis, a psychiatrist at the University of Toronto in Canada. But with the genomics revolution having yet to yield the hoped-for avalanche of genes that confer susceptibility to common diseases, Petronis is not alone in believing that it's time to revisit the problem under the spotlight of epigenetics. Nature 421, 686 - 688 (13 February 2003) 2005-10-29 Chaoqun Wu, Fudan University 142 Nakao M, 2001 2005-10-29 Chaoqun Wu, Fudan University 143 Genetic diseases associated with chromatin remodeling 2005-10-29 Chaoqun Wu, Fudan University 144 Disorders associated with aberrant chromatin remodeling activity. Mutations in several nucleosome remodeling ATPases (SWI/SNF) and HATs have been associated with inherited diseases; to date, mutations in HDACs have not been identified, although deregulated function of histone deacetylases secondary to mutation of other genes has been associated with inherited disease. The top row lists genes that when mutated cause aberrant chromatin remodeling and consequently human disease, the middle row lists the mechanism by which the mutant gene products cause aberrant chromatin remodeling, and the bottom row lists the consequent diseases. 2005-10-29 Chaoqun Wu, Fudan University 145 The function and dysfunction of some human disease-related chromatin-remodeling factors regulating transcriptional initiation 2005-10-29 Chaoqun Wu, Fudan University 146 The function and dysfunction of some human disease-related chromatin- remodeling factors regulating transcriptional initiation 1. BRG1 and BRM 2. ATRX 3. MECP2 2005-10-29 Chaoqun Wu, Fudan University 147 Williams syndrome是一种先天性疾病，患儿有典型的脸 部外观，身体瘦小，有轻、中度的智能发展迟缓，牙 齿通常长得很慢且小而稀疏；友善而爱说话的个性也 是他们的另一个特征。此症病儿常合并先天性心脏病 ，尤其是主动脉狭窄、肺动脉狭窄或肺动脉瓣狭窄。 2005-10-29 Chaoqun Wu, Fudan University 148 (i) BRG1 and BRM: These two SWI/SNF-related proteins and their interacting proteins form the WINAC complex, which is recruited by the transcription factor BAZ1B for vitamin D-dependent regulation of transcription. With decreased dosage of BAZ1B secondary to hemizygous deletion of the BAZ1B gene in Williams syndrome, the targeting of the WINAC complex to promoters is inadequate to maintain transcription. 2005-10-29 Chaoqun Wu, Fudan University 149 The features in ATR-X syndrome There are 3 principal features in the ATR-X syndrome: ?learning difficulties ?a characteristic facial appearance ?an unusual form of anaemia known as a thalassaemia. Other features such as abnormal genital development may also be present The anaemia can be detected by a specific blood test and this forms the basis of a laboratory test for this condition. 2005-10-29 Chaoqun Wu, Fudan University 150 (ii) ATRX: The interaction of this SWI/SNF-related protein with Daxx and repetitive DNA suggests that ATRX represses transcription (TF, transcription factor). With the loss of functional ATRX, therefore, the transcription complex either would not form or form but be unable to repress transcription. 2005-10-29 Chaoqun Wu, Fudan University 151 (iii) MECP2: Binding of methylated-DNA by MECP2 recruits HDAC and mSin3A to suppress transcription of genes such as BDNF. When MECP2 is phosphorylated, it is released from DNA, and the transcription of BDNF is initiated. Deficiency of functional MECP2 causes insufficient recruitment of the HDAC-mSin3A complex to adequately repress transcription. A, acetyl group; P, phosphate. 雷得症候群（ Rett Syndrome） :属于退化性障碍，症状包括失语、出现重复挥手 和摇摆身体的动作；社交退缩、严重智能不足。 2005-10-29 Chaoqun Wu, Fudan University 152 Ac -acetylated histones H3 Lys9 CpG-Me - methylated Cytosine HDAC -histone deacetylases DNMT -DNA methyltransferase HMT-histone methyltransferase MBD -methylated DNA binding domain HDAC deacetylates lysine residues as the prerequisite for methylation HP1 protein recognizes MeK9, binds also HMT and heterchromatin can spread 2005-10-29 Chaoqun Wu, Fudan University 153 Genes Mechanims where involved SIOD - Schimle immuno-osseous dysplasia COFS - cerebro-oculo-facio-skeletal syndrome CBS - Cockayne syndrome type B RTS - Rubinstein Taybi syndrome Diseases Huang et al., 2003 2005-10-29 Chaoqun Wu, Fudan University 154 Chromatin remodelling disorders 1.ATRX, SNF2-family helicase (a-thalassemia X-linked mental retardation) mutations: Causes several mental retardation disorders, facial, skeletal, an urigenital abnormalities, a - thalassemia and microcephaly ATRX protein resides predominantly in repetitive DNA, ribosomal gene clusters, pericentromeric heterochromatin. In ATRX cells, the ribosomal DNA repeats are hypomethylated. 2005-10-29 Chaoqun Wu, Fudan University 155 2. ERCC6 gene (excision repair cross- complementing rodent repair deficiency, compelentation group 6): (a)COFS (cerebro-oculo-facio-skeletal) syndrome: failure of multiple systems and premature death (b) (b) Cockayne syndrome: UV-sensitivity, dwarfism, skeletal abnormalities, mental retardation etc. Both cellular phenotypes include increased sensitivity to oxydative and UV- induced DNA-damage and failure to recover RNA synthesis after UV irradiation. ERCC6 plays key role in transcription coupled DNA repair, presumably opens the chromatin allowing access of the DNA repair apparatus to the DNA 2005-10-29 Chaoqun Wu, Fudan University 156 3. SMARCAL1 (SWI/SNF-related matrix- associated, actin-dependent regulator of chromatin, subfamily A-like protein 1): Schimke immuno-osseous dysplasia characterized by T-cell immunodeficiency, renal failure, hypothyroidism, bone-marrow failure etc. SMARCAL1 probably regulates a subset of genes necessary for cellular proliferation. 2005-10-29 Chaoqun Wu, Fudan University 157 Epigenetics and human diseases (be continued) 2005-10-29 Chaoqun Wu, Fudan University 158 2005-10-29 Chaoqun Wu, Fudan University 159 Cancer epigenetics Feinberg and Vogelstein (1983): loss of DNA methylation in cancer cells compared to normal tissues Feinberg and Tycko, 2004 2005-10-29 Chaoqun Wu, Fudan University 160 2005-10-29 Chaoqun Wu, Fudan University 161 2005-10-29 Chaoqun Wu, Fudan University 162 Genes altered through methylation in cancer 2005-10-29 Chaoqun Wu, Fudan University 163 Nature reviews CANCER 4:1, 2004 2005-10-29 Chaoqun Wu, Fudan University 164 Histological and epigenetic progression in the development of squamous-cell carcinoma of the lung. The morphological changes defined for the development of squamous cell carcinoma are accompanied by a progressive increase in the prevalence for inactivation of the CDKN2A gene by promoter hypermethylation. Nature reviews CANCER 4:1, 2004 2005-10-29 Chaoqun Wu, Fudan University 165 Hypomethylation and cancer 1. Hypomethylation can lead to gene activation (e.g. HRAS, which is normally expressed only in testis) — Overexpression of: cyclin D2 in gastric carcinoma MN/CA9 in renal-cell carcinoma S100A4 metastasis associated gene in colon cancer HPV16 in cervical cancer 2. A cellular ‘methylator phenotype’ has been linked to mismatch repair (Lengauer et al) — Hypermethylation of the mismatch-repair gene MLH1 is commonly found in mismatch- repair-defective tumors 2005-10-29 Chaoqun Wu, Fudan University 166 3. Hypomethylation in cancer is related to chromosomal instability — Frequent unbalanced chromosomal translocations with breakpoints in pericentromeric satellite sequences (otherwise highly methylated) 4. Hypomethylation is a mechanism of drug, toxin and viral effects in cancer — MDR1, multidrug resistance gene correlates with increased expression and drug resistance in acute myelogenous leukemia — Cadmium inhibits DNA methyltransferase activity and leads to acute hypomethylation, which is followed by hypermethylation of dna after chronic exposure to this “epigenic’ carcinogen — Arsenic induces Ras hypomethylation in mice — cervical cancer latency is caused by hyper- methylation of HPV16 genome 2005-10-29 Chaoqun Wu, Fudan University 167 Hypermethylation and cancer Promotor CpG hypermethylation of tumor supressor genes: Retinoblastoma gene RB Cyclin-dependent kinase inhibitor (INK4A,p16, CDKN2A) Mismatch repair gene MLH1 Von Hippel-Lindau (VHL) tumour supressor E-cadherin Is the initial silencing hypermethylation, or is hyperpemthylation a consequence? — Probably it is part of “programmed” silencing, but is not per se responsible for inactivation of a gene 2005-10-29 Chaoqun Wu, Fudan University 168 Alternative models for CpG methylation in cancer 2005-10-29 Chaoqun Wu, Fudan University 169 sporadic germline Loss of imprinting in cancer BWS is fetal overgrowth disorder due to deregulation of imprinted genes at 11p15: paternally expressed IGF2, KCnQ1OT1 & maternally expressed H19, CDKN1C, KNCQ1 Wilms tumour: hypermethylation of H19 due to LOI of IGF2 leading to biallelic expression and twofold increase in doses 2005-10-29 Chaoqun Wu, Fudan University 170 Summary 2005-10-29 Chaoqun Wu, Fudan University 171 1. Chromosomes occupy discrete territories in the cell nucleus and contain distinct chromosome-arm and chromosome-band domains. 2. Chromosome territories (CTs) with different gene densities occupy distinct nuclear positions. 3. Gene-poor, mid-to-late-replicating chromatin is enriched in nuclear compart- ments that are located at the nuclear periphery and at the perinucleolar region. 2005-10-29 Chaoqun Wu, Fudan University 172 4. A compartment for gene-dense, early-replicating chromatin is separated from the compartments for mid-to-late-replicating chromatin. 5. Chromatin domains with a DNA content of 1 Mb can be detected in nuclei during interphase and in non-cycling cells. 6. The interchromatin compartment (IC) contains various types of non-chromatin domains with factors for transcription, splicing, DNA replication and repair. 2005-10-29 Chaoqun Wu, Fudan University 173 7. The CT–IC model predicts that a specific topological relationship between the IC and chromatin domains is essential for gene regulation. 8. The transcriptional status of genes correlates with gene positioning in CTs. 9. A dynamic repositioning of genes with respect to centromeric heterochromatin has a role in gene silencing and activation. 2005-10-29 Chaoqun Wu, Fudan University 174 How far will epigenetics go past transcriptional effects ? Emerging evidence indicates that epigenetic alterations influence ? programmed DNA rearrangements ? imprinting phenomena ? germ line silencing ? developmentally cued stem cell divisions ? overall chromosome stability and identity 2005-10-29 Chaoqun Wu, Fudan University 175 Epigenetics imparts a fundamental regulatory system beyond the sequence information of our genetic code "Mendel's gene is more than just a DNA moiety ” 2005-10-29 Chaoqun Wu, Fudan University 176 Genetics Epigenetics Immortal Chromatin! 2005-10-29 Chaoqun Wu, Fudan University 177 References: 1.Korber, P., and Horz, W. (2004). Cell 117, 5–7. 2.John Tamkun and David Stillman. (2003). Current Opinion in Genetics & Development, 13:136–142 3.Cheng Huang, Emily A Sloan et al (2003). Current Opinion in Genetics & Development, 13:246–252 4.Eberharter A, Becker PB.(2002), EMBO Rep. 3(3):224-9. 5.Craig L. Peterson EMBO reports (2002)vol.3 no.4 319-322 6.Peter B Becker Wolfram, Horz. (2002) Annual Review of Biochemistry 71: 247 7.En Li (2002) . Nature Reviews Genetics 3, 662 –673 8.Thomas Jenuwein, C. David Allis(2001), Science, Volume 29 3, Number 5532, 9.Alan P Woffe. Oncogene (2001) 20, 2988 – 2990 10.Patrick Varga-Weisz. Oncogene (2001) 20, 3076 – 3085 11.Caroline Demeret, Yegor Vassetzky et al. Oncogene (2001) 20, 3086 - 3093 12. N. M. Maraldi, G. Lattanzi b , P. Sabatelli b , A. Ognibene and S. Squarzoni (2002), Neuromuscular Disorders 12( 9): 815-823 2005-10-29 Chaoqun Wu, Fudan University 178