Regulation of Histone Gene Expression in Budding Yeast

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We review emerging evidence that histone proteins and their associated covalent modi-fications contribute to a mechanism that can alter chromatin structure,thereby leading to inherited differences in transcriptional“on-off”states or to the stable propagation of chromosomes by defining a specialized high-er order structure at centromeres.Under the assumption that a histone code exists,at least in some form,we discuss potential mecha-nisms for how such a code is“read”andtranslated into biological functions.Throughout this review,we have chosenepigenetic phenomena and underlying mecha-nisms in two general categories:chromatin-based events leading to either gene activation orgene silencing.In particular,we center our dis-cussion on examples where differences in“on-off”transcriptional states are reflected by dif-ferences in histone modifications that are either“euchromatic”(on)or“heterochromatic”(off)(Fig.1A).We also point out that,despite manyelegant genetic and biochemical insights intochromatin function and gene regulation in thebudding yeast Saccharomyces cerevisiae,someof the heterochromatic mechanisms(e.g.,HP1-based gene silencing)discussed here do notexist in an obvious form in this organism.Thus,we will need to pursue other model systems,such as Schizosaccharomyces pombe,Caeno-rhabditis elegans,Drosophila,and mice,to“crack”the histone code.Chromatin Template and HistoneCodeIn the nuclei of all eukaryotic cells,genomicDNA is highly folded,constrained,and com-pacted by histone and nonhistone proteins ina dynamic polymer called chromatin.Forexample,chromosomal regions that remaintranscriptionally inert are highly condensedin the interphase nucleus and remain cytolog-ically visible as heterochromatic foci or as the“Barr body,”which is the inactive X chromo-some in female mammalian cells(2).Thedistinct levels of chromatin organization aredependent on the dynamic higher order struc-turing of nucleosomes,which represent thebasic repeating unit of chromatin.In eachnucleosome,roughly two superhelical turnsof DNA wrap around an octamer of corehistone proteins formed by four histone part-ners:an H3-H4tetramer and two H2A-H2Bdimers(3).Histones are small basic proteinsconsisting of a globular domain and a moreflexible and charged NH2-terminus(histone“tail”)that protrudes from the nucleosome.Itremains unclear how nucleosomal arrays con-taining linker histone(H1)then twist and foldthis chromatin fiber into increasingly morecompacted filaments leading to defined high-er order structures.Central to our current thinking is thatchromatin structure plays an important regu-latory role and that multiple signaling path-ways converge on histones(4).Althoughhistone proteins themselves come in genericor specialized forms(5),exquisite variation isprovided by covalent modifications(acetyla-tion,phosphorylation,methylation)of the hi-stone tail domains,which allow regulatablecontacts with the underlying DNA.The en-zymes transducing these histone tail modifi-cations are highly specific for particular ami-no acid positions(6,7),thereby extendingthe information content of the genome pastthe genetic(DNA)code.This hypothesis pre-dicts that(i)distinct modifications of the1Research Institute of Molecular Pathology(IMP)atthe Vienna Biocenter,Dr.Bohrgasse7,A-1030Vi-enna,Austria.E-mail:jenuwein@nt.imp.univie.ac.at2Department of Biochemistry and Molecular Genetics,University of Virginia Health Science Center,Char-lottesville,VA22908,USA.E-mail:allis@Among the modifier genes identified in the above model systems,one subclass suppresses variegation[the Su(var)group]and comprises gene products such as histone deacetylases (HDACs),protein phosphatases(PPTases),and S-adenosylmethionine(SAM)synthetase(17), as well as chromatin-associated components that are best characterized by the heterochroma-tin protein HP1[Su(var)2-5](18).In addition to the Su(var)group of genes,an antagonizing class of PEV modifiers enhances variegation [E(var)group](12)and counteracts the Su(var)-induced silent chromatin state.Several E(var) gene products are components of adenosine triphosphate(ATP)–dependent nucleosome-re-modeling machines,such as the SWI/SNF and brahma complexes(19,20),which increase overall nucleosome mobility.Extending these parallels even further, Su(var)and E(var)gene products contain several conserved protein domains—the bro-mo-,chromo-,and SET domains—that are also shared with two other classes of antag-onizing chromatin regulators:the Polycomb (Pc-G)and trithorax(trx-G)groups.The Pc-G and trx-G genes are important for main-taining the expression boundaries of the ho-meotic selector genes and several other key developmental genes(21,22),presumably by modulating the chromatin structure of their target loci.The bromodomain(23)is found inSNF2,TAFII 250,and mammalian trithorax(HRX/Mll);the chromodomain(24,25)is shared between Polycomb and HP1;and the SET domain(26)is found in Su(var)3-9,in the Pc-G member E(z),and in trithorax. These modules have been widely used during evolution to generate a considerable function-al diversity among proteins specialized in modulating chromatin structure.Histone acetylation(27,28)and histone phosphorylation(29)modification systems have been characterized in detail.A further class of enzymatic activities that regulate the site-specific addition of methyl groups to hi-stones has recently been described.Original-ly identified as the PEV modifier Su(var)3-9 in Drosophila,homologs from fission yeast (Clr4)to human(SUV39H1)have been shown to encode histone methyltransferases (HMTases)that selectively methylate histone H3at Lys9(30).The HMTase function in the Su(var)3-9family maps to the highly con-served SET domain but also requires adjacent Cys-rich regions.Notably,generation of the H3-Lys9methyl epitope induces a hetero-chromatic affinity for HP1proteins that rec-ognize this epigenetic signal through their chromodomains(31,32).These results pro-vide a strong link among Su(var)function, gene-silencing activity,and the assembly of heterochromatin(31–35).By contrast,an enzymatic HMTase func-tion has not yet been demonstrated for Pc-G and trx-G proteins.Instead,E(z)has been associated with a Pc-G complex containingHDAC activity(36),and trx or HRX havebeen shown to interact with components ofchromatin-remodeling machines(37).In gen-eral terms,Su(var)and Pc-G gene functionwould be characterized by transducing theaddition of heterochromatic marks and theremoval of euchromatic marks on the chro-matin template.Conversely,the antagonizingactivity of E(var)and trx-G gene functionwould involve the establishment of euchro-matic signals(e.g.,increased nucleosomemobility)and destabilize or degrade(see be-low)heterochromatic“imprints”(Fig.1B).Translating the Histone CodeThe histone code hypothesis predicts that themodification marks on the histone tailsshould provide binding sites for effector pro-teins.In agreement with this notion,the bro-modomain has been the first protein moduleto be shown to selectively interact with acovalent mark(acetylated lysine)in the his-tone NH2-terminal tail(23,38,39).In addi-tion to the proteins discussed above,the bro-modomain is also present in many transcrip-tional regulators having intrinsic histoneacetyltransferase(HAT)activity(e.g.,GCN5,PCAF,TAFII250).Consistent with the sec-ond prediction of the histone code(that therebe combinatorial readout),TAFII250,whichitself harbors several histone-modifying ac-tivities,contains two tandem copies of thebromodomain.In this configuration it pref-erentially binds diacetylated histone pep-tides presenting acetyl-lysine moieties thatare appropriately spaced(40).Use of theSimple Modular Architectural ResearchTool(SMART;http://smart.embl-heidel-berg.de)indicates that there areϳ75bro-modomain-containing proteins in humans.Several of these proteins,such as humanpoly-bromodomain protein1,exhibit manycopies(six)of regularly spaced bromodo-mains,which could conceivably bind to aspecific combination of acetyl groups pre-sented on one or several histone tails.Chromodomains,on the other hand,ap-pear to be targeting modules for methylationmarks.The chromodomain of HP1is highlyselective for methylated H3at Lys9,and littleif any binding is observed with H3peptidescontaining a methylated Lys4position(32).Thus,although chromodomains are highlyconserved,it seems likely that not all chro-modomains—nor their methyl targets—be-have similarly.In support,chromodomainswapping experiments have not uniformlyindicated functional conservation in silencingassays(41,42).Interestingly,Su(var)3-9HMTase family members also contain a chro-modomain,whose integrity is critical for si-lencing in vivo(33,43).Several repressivechromatin-remodeling complexes comprisecomponents such as the Mi-2/CHD ATPasesubunit of the NuRD complex(44),whichharbors two chromodomains and might con-ceivably recognize dimethylated histone tailsin a manner analogous to double bromodo-mains.In this regard,we note that Lys9andLys27in the H3tail are embedded in similarsequence motifs,and both positions are“hotspots”for methylation by the SET domain–containing HMTase G9a(45).Finally,a hallmark property of all HP1proteins is the combination of a chromodo-main with a chromoshadow domain that areseparated by a short but variable hinge re-gion.Because the chromoshadow domain ofHP1appears to self-dimerize in solution(46,47),it is tempting to infer that full-lengthHP1may assemble intermolecular chromo-domains,thereby generating a bifunctionalcross-linker that is likely to stabilize the morerigid higher order structure of heterochroma-tin(35,48).Combinations and SwitchesThe above examples provide support for mod-ification-induced recruitment of chromatin-as-sociated proteins to acetylated and methylatedhistone NH2-termini(Fig.2A),and it is likelythat other modules exist that specifically recog-nize phosphorylation marks.Consistent withthe second prediction of the histone code hy-pothesis,all four NH2-termini of the core his-tones contain short“basic patches”that oftencomprise acetylation,phosphorylation,andmethylation marks in close proximity on oneindividual tail(4).All three of these modifica-tions can be found both in active or silencedchromatin regions,which raises the question ofhow combinatorial specificity is used in defin-ing an imprint for euchromatin or heterochro-matin(Fig.1,A and C).Some evidence is emerging about a pos-sible combinatorial code.For example,thehistone H3NH2-terminus appears to exist intwo distinct modification states that are likelyto be regulated by a“switch”between Lys9methylation and Ser10phosphorylation(Fig.1D).Ser10phosphorylation inhibits Lys9methylation(30)but is synergistically cou-pled with Lys9and/or Lys14acetylation dur-ing mitogenic and hormonal stimulation inmammalian cells(49–51).In this phos-phorylated-acetylated state,the modified H3tail marks transcriptional activation(Fig.1C).H3phosphorylation is also important for mi-totic chromosome condensation(52),whereit may be linked to other secondary signal(s)such as the nucleosomal incorporation of thepericentric H3analog Cenp-A(53).Con-versely,aberrant Lys9methylation antagoniz-es Ser10phosphorylation,leading to mitoticchromosome dysfunction(30,54).Further,deacetylation of Lys14in H3(33)is requiredto facilitate subsequent Lys9methylation bythe Clr4HMTase,again highlighting an or-dered interplay to establish distinct histoneImmortal ChromatinThe importance of chromatin in the informa-tion storage and decoding processes of the eukaryotic genome is reinforced by the growth in our knowledge about covalent modifications of histone proteins,and about the enzyme systems that transduce or remove these imprints.Moreover,histone modifica-tions may also be a“sensor”of the metabolic state of the cell.For example,the Sir2en-zyme uses an essential metabolic cofactor (nicotinamide adenine dinucleotide)to regu-late the activity of a family of silencing-associated HDACs(84).Will HDMases be uncovered only when the correct cofactor, itself possibly a direct product from interme-diary carbon metabolism,is added to the test reactions?The lessons learned from the Sir2 paradigm lead to an attractive new concept: Because chromatin is the physiological tem-plate of eukaryotic cells,are genomes pro-grammed to“open”and“close”on demand by enzyme complexes that evolved to re-spond directly to metabolic cues?If correct, we anticipate that further insights will be gained as we systematically investigate chro-matin changes during different physiological or pathological states.To what extent does a histone code link directly to our genetic code,or are these codes separate indexing mechanisms?Will we find evidence of interdependence between histone methylation and DNA methylation, similar to the interplay between histone deacetylation and DNA methylation(44)? Intriguingly,a“chromo-methylase”has re-cently been described in Arabidopsis that combines a chromodomain with a DNA methylating activity(85),and one member of the SET domain family contains a methyl CpG binding motif(35)(Fig.2C).Histone methylation may also help to explain poorly understood chromatin effects where deacety-lase inhibitors and/or5-aza-cytosine fail to cause reversal of previously silent genomic regions(86).Indeed,transcription of many genes is regulated by histone acetylation in organisms(e.g.,in yeast and flies)that exhib-it little DNA modification.Further,X chro-mosome inactivation in mammals correlates with hypoacetylation of histones,except for a few X-linked loci that escape this silencing mechanism(87).In addition,in some branch-es of mammalian evolution(e.g.,marsupials), no allele-specific DNA methylation has been observed.Could histone methylation be one of the conserved mechanisms substituting for the apparent absence of DNA methylation in these organisms,and to what extent is the inactive X chromosome hypoacetylated(88) because it may be hypermethylated at distincthistone NH2-termini?How far will epigenetics go past transcrip-tional effects?Emerging evidence indicates that programmed DNA rearrangements(89),imprinting phenomena(90),germ line si-lencing(57),developmentally cued stemcell divisions(91),and overall chromo-some stability and identity(52,92)are allinfluenced by epigenetic alterations of theunderlying chromatin structure.In keepingwith the distinct qualities of accessible andinaccessible nucleosomal states,could it bethat“open”(euchromatic)chromatin repre-sents the underlying principle that is syn-onymous for the character of progenitor,immortal,and young cells?Conversely,is“closed”(heterochromatic)chromatin thereflection of a developmental“memory”that stabilizes lineage commitment andgradually restricts the self-renewal poten-tial of our somatic cells?As pointed out byothers(93),epigenetics imparts a funda-mental regulatory system 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F.M.Peters, D.O’Carroll,T.Jenuwein,unpublished data.93.A.S.J.Klar,Trends Genet.14,299(1998).94.We thank G.Reuter and members of our laborato-ries for allowing us to cite unpublished observa-tions.We are particularly grateful to S.Rea for hisassistance in preparing thefigures.Supported bythe IMP through Boehringer Ingelheim and bygrants from the Austrian Research Promotion Fundand the Vienna Economy Promotion Fund(T.J.),and by NIH grant GM53512and an NIH MERITaward(C.D.A.).This article is dedicated to the memory of AlanWolffe,an inspirational leader to all of us whohave pondered the mysteries of chromatin andgene regulation.V I E W P O I N TRNA:Guiding Gene SilencingMarjori Matzke,1*Antonius J.M.Matzke,1Jan M.Kooter2In diverse organisms,small RNAs derived from cleavage of double-strand-ed RNA can trigger epigenetic gene silencing in the cytoplasm and at thegenome level.Small RNAs can guide posttranscriptional degradation ofcomplementary messenger RNAs and,in plants,transcriptional gene si-lencing by methylation of homologous DNA sequences.RNA silencing is apotent means to counteract foreign sequences and could play an impor-tant role in plant and animal development.RNA silencing is a new field of research that has coalesced during the last decade from inde-pendent studies on various organisms.Scien-tists who study plants and fungi have known since the late1980s that interactions between homologous DNA and/or RNA sequences can silence genes and induce DNA methylation(1). The discovery of RNA interference(RNAi)in Caenorhabditis elegans in1998(2)focused attention on double-stranded RNA(dsRNA)as an elicitor of gene silencing,and indeed,many gene-silencing effects in plants are now known to be mediated by dsRNA(3).RNAi is usually described as a posttranscriptional gene-silenc-ing phenomenon in which dsRNA triggers deg-radation of homologous mRNA in the cyto-plasm(4).However,the potential for nuclear dsRNA to enter a pathway leading to epigenetic modifications of homologous DNA sequences and silencing at the transcriptional level should not be discounted.Although the nuclear aspects of RNA silencing have been studied primarily in plants,there are hints that similar RNA-directed DNA or chromatin modifications might occur in other organisms as well.Here we adopt a broad definition of RNA silencing that encompasses effects in the cytoplasm and the nucleus,and consider their possible devel-opmental roles and evolutionary origins. RNA Guiding Homologous RNA DegradationAlthough they may differ in detail,RNAi in animals and the related phenomena of post-transcriptional gene silencing(PTGS)in plants and quelling in Neurospora crassa re-sult from the same highly conserved mecha-nism,indicating an ancient origin(5–10).Thebasic process involves a dsRNA that is pro-cessed into shorter units that guide recogni-tion and targeted cleavage of homologousmRNA.dsRNAs that trigger PTGS/RNAican be made in the nucleus or cytoplasm in anumber of ways,including transcriptionthrough inverted DNA repeats,simultaneoussynthesis of sense and antisense RNAs,viralreplication,and the activity of cellular or viralRNA–dependent RNA polymerases(RdRP)on single-stranded RNA templates(Fig.1).InC.elegans,dsRNAs can be injected or intro-duced simply by soaking the worms in asolution containing dsRNA or feeding thembacteria expressing sense and antisense RNA(10).Genetic and biochemical approaches are be-ing used to dissect the mechanism of PTGS/RNAi.Putative RdRPs,putative helicases,andmembers of the PAZ/Piwi family are some ofthe common proteins identified in geneticscreens in N.crassa,C.elegans,and Arabidop-sis(3,5,8,10).Although these proteins provideclues about dsRNA synthesis and processing,the most detailed insight into the two-step RNAdegradation process has come from biochemi-cal experiments with cytoplasmic extracts fromDrosophila(11–15)(Fig.1).The first step in-volves a dsRNA endonuclease[ribonuclease III(RNase III)–like]activity that processes dsRNAinto sense and antisense RNAs21to25nucle-otides(nt)long.These small interfering RNAs(siRNAs),which were first described in a plantsystem(16),are generated in Drosophila by anRNase III–type protein termed Dicer.Orthologsof Dicer,which contains a helicase,dsRNAbinding domains,and a PAZ domain,havebeen identified in Arabidopsis, C.elegans,mammals,and Schizosaccharomyces pombe(15).In the second step,the antisense siRNAsproduced by Dicer serve as guides for a differ-ent ribonuclease complex,RISC(RNA-inducedsilencing complex),which cleaves the homolo-gous single-stranded mRNAs.RISC from Dro-sophila extracts cofractionates with siRNAsthat guide sequence-specific mRNA cleavage(12).RISC cuts the mRNA approximately inthe middle of the region paired with antisensesiRNA(14)(Fig.1),after which the mRNA isfurther degraded.Although most protein com-ponents of RISC have not yet been identified,they might include an endonuclease,an exonu-clease,a helicase,and a homology-searchingactivity(6,10).A candidate for a3Ј,5Ј-exonu-clease is C.elegans MUT7,an RNase D–likeprotein recovered in a screen for RNAi mutants(10).Another component of RISC is a proteinof the PAZ/Piwi family(17),which could in-teract with Dicer through their common PAZdomains(18)to incorporate the siRNA intoRISC(17).Genes encoding members of thePAZ/Piwi family(Arabidopsis:AGO1;N.crassa:QDE2;C.elegans:RDE1),which arehomologous to the translation factor eIF2C,have been shown to be required for PTGS/RNAi in several mutant screens(3,5,8,10).A putative RdRP was the first cellular pro-tein shown to be required for PTGS/RNAi ingenetic screens(N.crassa:QDE1;C.elegans:Ego1;Arabidopsis:SGS2/SDE1)(3,5,8,10),but its exact role is unclear and the predictedenzyme activity remains to be established.Thisprotein might be dispensible when largeamounts of dsRNA are produced from trans-genes or when viral RdRPs are present(5).RdRP might be needed only when dsRNA issynthesized to initiate silencing—for example,from“aberrant”sense RNAs that are prema-turely terminated or processed improperly(19).RISC-cleaved mRNAs may also be used astemplates and converted into dsRNA,increas-ing the level of siRNAs and enhancing PTGS/RNAi(Fig.1).Putative helicases are another class of en-zyme found repeatedly in mutant screens(N.crassa:QDE3;C.elegans:SMG-2;Chlamy-domas:MUT6;Arabidopsis:SDE3)(3,5,8,10).Those recovered so far are not highlyrelated and have not yet been characterizedbiochemically.A DNA helicase(QDE3)andmembers of two RNA helicase superfamilies(MUT6and SMG2/SDE3,respectively)have1Institute of Molecular Biology,Austrian Academy of Sciences,A-5020Salzburg,Austria.2Department of Developmental Genetics,Vrije Universiteit,Amster-dam,Netherlands.*To whom correspondence should be addressed.E-mail:mmatzke@imb.oeaw.ac.at。

Chromatin/Epigenetics Resources
Overview of Chromatin / Epigenetics
Chromatin regulation refers to the events affecting chromatin structure and therefore, transcriptional control of gene expression patterns. Epigenetics, specifically, refers to the heritable modifications which result in altered gene expression and are not known to be encoded in DNA. The nucleosome, made up of four histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have more recently been shown to be dynamic proteins, undergoing multiple types of post-translational modifications (PTMs) and interacting with regulatory proteins to control gene expression. Protein acetylation plays a crucial role in regulating chromatin structure and transcriptional activity. Histone hyperacetylation by histone acetyltransferases (HATs) is associated with transcriptional activation, whereas histone deacetylation by histone deacetylases (HDACs) is associated with transcriptional repression. Hyperacetylation can directly affect chromatin structure by neutralizing the positive charge on histone tails and disrupting nucleosome-nucleosome and nucleosomeDNA interactions. In addition, acetylation creates binding sites for bromodomain-containing chromatin regulatory proteins (histone modification readers). Unlike acetylation, methylation does not alter the charge of arginine and lysine residues and is unlikely to directly modulate nucleosomal interactions required for chromatin folding. Methylated arginine and lysine residues are major determinants for formation of active and inactive regions of the genome. Methylation facilitates binding of chromatin regulatory proteins/histone modification readers that contain various methyl-lysine or methyl-arginine binding domains (PHD, chromo, WD40, Tudor, MBT, Ankyrin repeats, PWWP domains). Recruitment of co-activator and co-repressor proteins is dependent on the specific lysine residue that is modified. The modulation of chromatin structure is an essential component in the regulation of transcriptional activation and repression. One strategy by which chromatin structure can be modulated is through disruption of histone-DNA contacts by ATP-dependent chromatin remodelers, such as the NuRD, Polycomb, and SWI/SNF complexes, which have been shown to regulate gene activation/repression, cell growth, the cell cycle, and differentiation. Chromatin structure is also modulated through other PTMs such as phosphorylation of histone proteins, which affects association with DNA-interacting proteins and has been recently identified to play a role in coordinating other histone modifications. Furthermore, methylation of DNA at cytosine residues in mammalian cells affects chromatin folding and is a heritable, epigenetic modification that is critical for proper regulation of gene silencing, genomic imprinting, and development. Three families of mammalian DNA methyl-transferases have been identified, DNMT1/2/3, that play distinct roles in embryonic stem cells and adult somatic cells. In addition to the core histone proteins, a number of histone variants exist that confer different structural properties to nucleosomes and play a number of specific functions such as DNA repair, proper kinetochore assembly and chromosome segregation during mitosis, and regulation of transcription. Chromatin and epigenetic regulation is crucial for proper programming of the genome during development and under stress conditions, as the misregulation of gene expression can lead to diseased states such as cancer.

JOURNAL OFGENETICS AND GENOMICS J. Genet. Genomics 35 (2008) 403−412Tackling the epigenome in the pluripotent stem cellsXiaodong Zhao, Yijun Ruan, Chia-Lin Wei *Genome Technology and Biology Group, Genome Institute of Singapore , 138672, Singapore Received for publication 14 April 2008; revised 23 May 2008; accepted 24 May 2008AbstractEmbryonic stem cells are unique in their abilities of self-renewal and to differentiate into many, if not all, cellular lineages. Transcrip-tional regulation, epigenetic modifications and chromatin structures are the key modulators in controlling such pluripotency nature of embryonic stem cell genomes, particularly in the developmental decisions and the maintenance of cell fates. Among them, epigenetic regulation of gene expression is mediated partly by covalent modifications of core histone proteins including methylation, phosphoryla-tion and acetylation. Moreover, the chromatins in stem cell genome appear as a highly organized structure containing distinct functional domains. Recent rapid progress of new technologies enables us to take a global, unbiased and comprehensive view of the epigenetic modifications and chromatin structures that contribute to gene expression regulation and cell identity during diverse developmental stages. Here, we summarized the latest advances made by high throughput approaches in profiling epigenetic modifications and chromatin con-formations, with an emphasis on genome-wide analysis of histone modifications and their implications in pluripotency nature of embry-onic stem cells.Keywords : epigenetics; histone modification; ChIP-chip; ChIP-PET; embryonic stem cellIntroductionEmbryonic stem (ES) cells are pluripotent cells derived from inner cell mass of blastocyst in the pre-implantation embryo. These cells are featured by their unique properties of self-renewal and pluripotency, which allow them to ex-pand almost indefinitely in vitro while possess the poten-tial to differentiate into a large variety of cell types that make up organisms (Smith, 2001). These unique properties make ES cells an ideal model for developmental biology and hold a great promise for cell-based therapy in regen-erative medicine (Donovan and Gearhart, 2001).Extrinsic signaling, transcriptional regulation network and epigenetic modifications are the critical components in modulating stem cell pluripotency. Remarkable progress has been made to understand how the extrinsic signaling molecules influence transcription in ES cells. Among them, leukemia inhibitory factor (LIF), bone morphogentic pro-teins (BMPs) and WNT signaling pathways play central* Corresponding author. Tel: +65-6478 8074; Fax: +65-6478 9059. E-mail address : weicl@.sgroles in the maintenance of pluripotential stem cell pheno-type (Smith et al., 1988; Ying et al., 2003; Sato et al., 2004). For example, the binding of LIF to its receptor ac-tivates STAT3 through phosphorylation and the phos-phorylated STAT3 functions as a transcription factor to activate downstream target genes. On the other hand, WNT binds Frizzled receptor on cell membrane, which prevents β-catenin from degradation. Accumulated β-catenin trans-locates into the nucleus and binds TCF/LEF to regulate expression of downstream target genes, including ES-spe- cific transcription factors Oct4, Nanog and Rex1 (Sato et al., 2004).Besides these signaling pathways, intrinsic transcription factors (TFs) are also essential for specifying the undiffer-entiated state of ES cells. To identify the TF genes impor-tant in defining ‘stemness’, DNA microarray was adopted to compare the expression profiles of key stem cells and their differentiated counterparts (Ivanova et al., 2002; Ra-malho-Santos et al., 2002; Fortunel et al., 2003) This re-sulted in more than 300 ‘ESC-enriched genes’ as. ES mo-lecular signature. Among them, transcription factors Oct4, Nanog, Sox2 and Sall4, were further characterized as404 Xiaodong Zhao et al. / Journal of Genetics and Genomics 35 (2008) 403− 412master regulators in undifferentiated ES cells (Scholer et al., 1989; Nichols et al., 1998; Avilion et al., 2003; Chambers et al., 2003; Zhang et al., 2006). Oct4, encoded by Pou5f1, is a POU domain-containing transcription fac- tor known to be essential to ES cells and early embryonic development. Oct4 interacts with Sox2 to co-target multi- ple genes (Boyer et al., 2005). Recent works indicated that Oct4 and Sox2, along with c-Myc and Klf4, were suffi- cient to reprogram fibroblasts to induced pluripotent stem cells (iPS) which were functionally similar to ES cells (Takahashi and Yamanaka, 2006; Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007). Hence, these tran- scription factors can exert a dominant role in reconstruct- ing the transcriptional regulatory network of ES cells. Moreover, even in the presence of very similar local envi- ronmental cues, ES cells could be induced into multiple differentiated cell types (Keller, 1995; Smith, 2001). This phenomenon underscores the importance of epigenetic influence in cell fate specification. It is known that the combination of various epigenetic modifications provides signatures for the underlying properties of chromatin asso- ciated with different segments of the genome (Surani et al., 2007; Zhao et al., 2007). Furthermore, long range DNA looping resulted from epigenetic influence has been shown in several well characterized imprinting loci in ES cells (Zhao et al., 2006). Therefore, chromatin structure and the combinatorial epigenetic modification status play critical roles in defining transcriptional competency during differ- ent development stages.In recent years, many efforts have been focused to elu-cidate how epigenetic modifications mold overall chroma-tin structure in ES-cell genome and how they contribute to self-renewal, maintenance of pluripotency and lineage commitment of ES cells. Here, we reviewed the various genome wide, high-throughput technologies used to profile epigenetic modifications in the mammalian genome land-scape. And we summarized the applications of various genome wide approaches in profiling the epigenetic land-scape and their dynamics in the differentiations, the inter-rogation of long range interactions and higher order chro-matin structures in ES cells and insights in epigenetics directed transcription regulation and its impacts on stem cell development.Epigenetics and histone modificationEpigenetics refers to the non-sequence based structural changes of chromosomal regions that can alter the gene expression activities in response to the external signals. Major types of epigenetic modifications include DNA me- thylation, histone modifications, chromatin remodeling and noncoding RNAs (Bernstein and Allis, 2005). Equiva- lent to ‘genetic code’ underlining in genomic DNA se- quence, distinct histone modifications which carry regula- tory information were proposed as ‘histone code’ (Strahl and Allis, 2000).In eukaryotes, the genomic DNA is packaged by an oc-tamer of four core histones into nucleosome, the basic building block of chromatins. The histone proteins (H2A, H2B, H3 and H4) are each composed of a global domain and a tail domain, which are subjected to a spectrum of posttranslational modification on their unstructured N-terminal tails (Fig. 1). Histones are the subjects for over 8 major distinct classes of modifications with dynamic natures and consequences (Kouzarides, 2007). Such cova-lent modifications play fundamental roles in chromatin condensation, replication, DNA repair, and transcriptional regulation (Martin and Zhang, 2005; Kouzarides, 2007). Among them, methylation and acetylation are the two best- studied histone modifications. Histone acetylation is gen- erally associated with actively transcribed genomic do- mains and the degree of modification correlates with the level of transcription (Schubeler et al., 2004), whereas histone methylation can have different effects on gene ex- pression depending on which residue is modified (Martin and Zhang, 2005). Among the diverse histone modifica- tions, methylations in histone 3 lysine 4 and lysine 27 are of particular importance. These modifications are cata- lyzed by Trithorax (TrxG) and Polycomb (PcG) group com- plexes respectively, which establish developmental deci- sions and mediate the expression programs of lineage- specific genes (Ringrose et al., 2004). Lysine 4 methyla- tion positively regulates gene expression by serving as a binding platform to recruit nucleosome remodeling en- zymes (Santos-Rosa et al., 2003), whereas lysine 27 me- thylation negatively regulates gene expression by generat- ing a compact chromatin domain (Cao et al., 2002; Rin grose et al., 2004). Similarly, H3K36me3 is associated with actively transcribed regions whereas H3K9me3 is involved in inactive genomic domains. Particularly, H3K4me3 is regarded as a hallmark for the promoters of actively transcribed genes and H3K36me3 is an indicator of transcription elongation (Bannister and Kouzarides, 2005; Pokholok et al., 2005). The different preferences of these histone modification occupancies on genome-wide landscape could be used to define transcription units (Mikkelsen et al., 2007; Zhao et al., 2007).The mechanism adopted by histone modifications to regulate transcription and chromatin organization is not clearly defined. An attractive hypothesis is that epigenetic factors, including modifying enzymes or remodeling fac-tors, could potentially tether chromatins to mediate cis and trans interactions (Yang and Kuroda, 2007). Such interac-tions presumably cause the structural changes of underly-ing DNA and chromatins. Conformation changes could even be mediated by specific protein complexes recruited by these modifications. These interactions can alter the physical property of the chromatin and affect its high order structures. For example, H4K16 acetylation was shown toXiaodong Zhao et al . / Journal of Genetics and Genomics 35 (2008) 403-412 405Pluripotent cellgenomeFig. 1. Epigenetic regulations in pluripotent cell genome. Epigenetic components include DNA methylation, histone post-translational modifications, chromatin remodeling and non-coding RNA targeting. These components and their interplays contribute to the regulations of crucial nuclear activities like DNA replication, repair and transcription. The nucleosome subunit below describes the type of histone modifications characterized. The colored shapes represent known post-translational modifications at the core histone tails. They could be methylated at lysines and arginines (pink circle), acetylated (green hexagon) and ubiquitylated (yellow pentagon) at lysines, phosphorylated at serines and threonines (blue triangle).inhibit the compaction of the chromatin fibers as well as cross-fiber interactions (Shogren-Knaak et al., 2006). An-other example is polycomb mediated DNA looping in gene silencing. The DNA loops between polycomb repressive elements (PRE) with Fab-7 and Ubx regions were used to establish contacts with promoter bound transcription ma-chinery to mediate silencing in Drosophila (Cleard et al., 2006; Comet et al., 2006). These and many other recent data suggested that structural changes mediated by histone modifications can have consequences to many aspects of genome functions (Mahy et al., 2002; Xu et al., 2006). Therefore, nuclear organization and spatial interactions implicated through epigenetic processes are emerging as a gene regulatory mechanism (Yang and Kuroda, 2007).ChIP based technologies for profiling epigenetic modi-ficationsMany approaches were developed to determine and compare the epigenetic landscapes of pertinent histone modifications, their associated genes, genomic features, functional outcomes and resulted higher order structures. These methods rely heavily on a powerful procedure called chromatin immunoprecipitation (ChIP). In ChIP, chroma-tin is immunoprecipitated with specific antibody against modified histones or chromatin associated proteins to en-rich genomic sites with the modifications of interest (Fig.2A). In conventional ChIP assay, the enriched regions can either be detected by Southern blot hybridization with probes against the specific regions of interest or real-time q-PCR (ChIP-qPCR) against the specific regions of inter-est (Guccione et al., 2006). Using ChIP with the antibody recognizing modified histones, the distribution of histone modifications can be determined across the entire gene locus (Hebbes et al., 1994) or associated with sequence specific transcription factor binding sites at selective re-gions (Guccione et al., 2006). Although specific, the con-ventional ChIP assay is limited by its scale and can not be used for genome wide characterization of all the epigenetic modifications at an unbiased fashion. For global analysis, tiling array (Bernstein et al., 2005; Boyer et al., 2006) and high-throughput sequencing (Barski et al., 2007; Mikkel-sen et al., 2007) were developed as alternative readouts to facilitate a more unbiased and genome wide detection.ChIP-on-chip for profiling histone modifications The combination of ChIP with array based analysis (ChIP-on-chip) enables researchers to detect epigenetic modifications in large scale and global manner. In ChIP-on-chip, the ChIP-enriched DNA is amplified, la-beled and hybridized onto DNA microarray (chip) con- taining DNA sequences of interest (Fig. 2B). The hybridi-zation signal for each probe is then normalized by signal406Xiaodong Zhao et al . / Journal of Genetics and Genomics 35 (2008) 403−412Fig. 2. ChIP-based analysis of histone modification. A: chromatin immunoprecipitation procedures: chromatin DNA is sonicated to smaller fragments. Antibody to specific histone modification was used to enrich the DNA fragments carrying the modification of interest. B: ChIP-chip procedure: ChIP DNA and input DNA are amplified and labeled by fluorescent dyes, respectively. The labeled probes are hybridized to the array and the signal enrichment fold between the ChIP DNA and input DNA control for each position is plotted to define the modification site. C: ChIP-SAGE is carried out by dividing the steptavidin bead (SA) bound ChIP fragments and ligating them separately with 2 different linkers. Upon type IIs restriction enzyme digestion, the linker containing fragments are ligated and PCR amplified followed by concatenation and sequencing analysis. D: ChIP-PET: pair end ditag from each individual ChIP fragment is generated for sequence analysis. The regions mapped by overlapped PETs will be defined as modification sites. E: ChIP-seq: ChIP DNA is ligated with specific adaptors followed by solid phase PCR and direct short tag based end sequencing.generated from input control DNA and regions containing signal intensities significantly higher than the background are defined as the modified sites. This strategy has been successfully applied for whole genome localization analy-sis of DNA-protein interactions (Ren et al., 2000) and was subsequently applied in global analysis of histone H3 ace-tylation and methylation patterns in yeast (Bernstein et al., 2002). With the same strategy, Kurdistani et al. (2004) and Schubeler et al. (2004) mapped the genome-scale locations of histone acetylation and methylation in both yeast and Drosophila, respectively. Although successful in simple genomes like yeast and fly, the complexity and size of mammalian genomes make it very challenging to apply the ChIP-on-chip analysis on mammalian epigenomes to en-able robust survey. Nevertheless, the ChIP-on-chip tech-nology for mammalian systems has been developed on a limited scale, for example, promoter regions (Li et al., 2003), coding sequences or intergenic regions with high conservations (Bernstein et al., 2006). Since then, ChIP- on-chip was frequently applied in profiling epigenetic modifications (Kirmizis et al., 2004; Bernstein et al., 2005;Pokholok et al., 2005; Huebert et al., 2006; Shivaswamy and Iyer, 2007) and has significantly advanced our under-standing of histone modification landscape in eukaryotic genomes. Despite considerable success, array based read-out of ChIP signal does suffer from several limitations. Firstly, this hybridization-based platform is unable to de-tect histone modification in repeat regions and distinguish the different modifications within the allelic variants be-cause of the cross-hybridization; secondly, it requires a large amount of ChIP DNA (>1 microgram). Therefore, PCR has to be adopted for amplification which potentially introduces bias. In addition, the ChIP-on-chip approach is dependent on the construction of whole genome arrays. For certain complex genomes, they are not commercially available or economically practical for many laboratories.Sequencing-based technologies for profiling epigenetic modificationsAlternatively, immunoprecipitated DNA fragments canXiaodong Zhao et al. /Journal of Genetics and Genomics 35 (2008) 403-412 407be sequenced to determine their identities. However, a significant amount of non-enriched background DNA will still be present in the ChIP DNA material. With a limited survey of the ChIP DNA pool, it is difficult to distinguish between genuine modification sequences and noise. How-ever, if the sampling of the DNA pool can be increased, the genuine ChIP enriched sites can be defined by multiple overlapping ChIP fragments, whereas the nonspecific re-gions will be only covered by random ChIP singletons. The bona fide sites can then be inferred by multiple mapped sequenced fragments. Compared with ChIP-on- chip, the sequencing-based approach is an unbiased and open system for identifying all DNA segments enriched by ChIP. The depth of coverage can be enhanced by short tag based sequencing strategies like serial analysis of gene expression (SAGE), originally developed for counting transcripts levels and later was applied to genome scanning for transcription factor binding site and histone modifica-tions (Fig. 2C) (Roh et al., 2004). However, this monotag approach suffers from mapping ambiguity and is unable to differentiate the amplification bias. Therefore, such ap-proach has limited utility to profile epigenetic modifica-tions in a genome with high accuracy. To exploit the effi-ciency of short tags sequencing, to enhance the accuracy in mapping to the genome and to increase the information content, a paired-end-ditag (PET) method has been devel-oped. Like SAGE, PET approach was initially used for transcriptome analysis and genome annotation (Ng et al., 2005; ENCODE Project Consortium, 2007; Ruan et al., 2007). For comprehensive characterization of the tran-scripts diversity in given cells or tissues, PET is generated from 18 bp of 5′ end and 18 bp of 3′ end of a full length cDNA molecule, respectively and subjected to high throughput sequencing. These 36 bp PETs can accurately demarcate the transcript boundaries when mapped to ge-nomic DNA. This paired-end di-tagging strategy can be applied to characterize the ChIP enriched DNA fragments to analyze DNA-protein interaction and profile histone modifications in highly complex genomes (Loh et al., 2006; Wei et al., 2006; Zhao et al., 2007) . In ChIP-PET, the ge-nomic DNA associated with histone modifications is en-riched by ChIP and subsequently converted into PETs for ultra-high-throughput sequencing. Each PET sequence was mapped onto genome and the locations of modification regions can be determined by overlapping-PET defined clusters (Fig. 2D). With this method, H3K4me3 was found to be a prevalent mark in human ES cell genome and lo- cated in close proximity to the promoters. H3K27me3 is largely co-localized with H3K4me3. The different combi-nations of these two histone modifications are associated with genes from distinct functional categories, which con-tribute to ES cell fundamental properties.With the advance in ultra high throughput and multiplex short tag based sequencing method, direct sequencing the ChIP fragments (ChIP-seq) has proved to be a simple and robust method for globally profiling the transcription fac-tor binding sites and epigenetic modifications (Barski et al., 2007). In ChIP-seq, the ChIP DNA is ligated to adaptors and subject to cluster amplification by bridging PCR and sequencing by synthesis (SBS) analysis. From each run of sequencing experiment, 25 to 36 nucleotides from either end of ChIP DNA fragments will be determined with high accuracy and millions of high quality reads can be gener-ated within days. Based on their mapping locations, re-gions with high number of clusters of ChIP tag sequences will be defined as ChIP enrichment sites (Fig. 2E). Through ChIP-seq, the comprehensive analyses of histone modifications were performed both in pluripotent cells and differentiated cells (Barski et al., 2007; Mikkelsen et al., 2007). With the rapid progress in sequencing technology, the scientific community now has the robust tool to profile all relevant epigenetic marks on a genome-wide scale in a wide variety of normal cells and developmental states. As is reflected by the recent launching of the Epigenomics Roadmap (http://nihroadmap.nih. gov/epigenomics/), such knowledge will be translated to understand important epi-genetic mechanisms, their roles in the regulation of gene expression and their impacts on clinically important condi-tions.Characterization of histone modifications in ES cells With the availability of whole genome and unbiased approaches to define epigenetic modifications, our knowl-edge of their genomic features, landscape, associated genes and expression activity have been drastically ad-vanced in the last years. Here, we summarized what we have learnt collectively on the dynamics of epigenetic marks in ES cells and their impacts on gene expression and lineage differentiation.Histone modifications and genomic DNA methylation are involved in chromatin structure and transcriptional activity. In ES cells, the overall nuclear architecture is globally decondensed and condensation reoccurs during differentiation (Meshorer and Misteli, 2006). Specific changes in histone modifications have been shown to ac-company ES cell differentiation and mammalian develop-ment (Lee et al., 2004; Margueron et al., 2005; Squazzo et al., 2006), in which acetylation level of histone H3 and H4 decreases and histone H3 lysine 9 methylation increases (Meshorer and Misteli, 2006). In nuclear fusion of somatic T cells with murine ES cells, which confers the T cells pluripotency, there are global hypermethylation and hy-peracetylation of histone H3 and H4 in such repro-grammed somatic genome (Kimura et al., 2004). During the differentiation of ES cells, the active chromatin marks such as acetylation of histones H3 and H4 decrease, whereas the repressive marks (i.e., methylation on histone H3 at lysine 9) increase in a differentiation-dependant408 Xiaodong Zhao et al. / Journal of Genetics and Genomics 35 (2008) 403− 412manner (Keohane et al., 1996; Lee et al., 2004). For exam-ple, the acetylation in promoter region of ES-specific gene Rex1 (Zfp-42) reduces during ES cell differentiation. In a study to investigate histone modifications as signatures for the underlying properties of chromatin provided by Lander and colleagues (Bernstein et al., 2006), 56 highly con-served noncoding elements (HCNEs) with 60.3 Mb in size were surveyed for the key active H3K4me3 and repressive H3K27me3 marks by ChIP-on-chip approach in mouse ES cell genome. They uncovered 109 domains that showed overlapping opposing histone modification marks, termed ‘bivalent domains’ where large regions of H3K27me3 harbor smaller regions of H3K4me3. The bivalent domains located in proximal regions of the regulatory elements of genes encoding developmental transcriptional factors ex-pressed in low level. These bivalent modifications are proposed to keep the key developmental genes in poised states for later activation as they are resolved during ES cell differentiation. Based on these observations, they proposed a histone modification-dependent mechanism for maintenance of pluripotency. The role of histone modifica-tions in both murine and human ES cell genomes (Boyer et al., 2006; Lee et al., 2006) was interrogated using genome- wide location analysis with DNA array containing the promoter regions of more than 15,000 annotated genes. The target genes occupied by SUZ12, core component of PcG complexes responsible for methylation of H3K27 were identified. SUZ12 is found to distribute across over hundreds of genes encoding key developmental regulators and, as expected, these genes are also simultaneously modified with H3K27me3. These PRC2 target genes are transcriptionally repressive in ES cells and preferentially activated during differentiation. These studies cast the first light on how epigenetic landscape is involved in the speci-fication of ES cells. However, it remains to be determined that what other histone modification patterns in addition to H3K4 & K27me3 are functionally involved in ES cell ge-nome. Bernstein and colleagues extended the global and high-resolution chromatin maps of histone H3 trimethyla-tion at K4, K9, K27, K36, histone H4 trimethylation at lysine 20 (H4K20me3) and RNA polymerase II binding in mouse ES cells, neural progenitor cells (NPCs) and em-bryonic fibroblasts (MEFs) (Mikkelsen et al., 2007). They found H3K4me3 and H3K27me3 effectively distinguish genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage po- tential. Particularly, they found that ‘bivalent domains’ do not widely occur in mouse ES cells. However, whether there are different mechanisms that function between mouse and human ES cells is not clear (Ginis et al., 2004; Wei et al., 2005). Specifically, whether the ‘bivalent do- main’ exists in human ES cells and plays a role in an epi-genetic mechanism to determine human ES cell identity is not known. Using ChIP-PET to profile genome-wide of H3K4me3 and H3K27me3 and the transcription analyses of the genes affected in human ES cells, H3K4me3 was found to be a prevalent mark which occurs in nearly 70% annotated genes’ promoters (Zhao et al., 2007). H3K27- me3 appears less occupied in promoter regions and forms ‘bivalent domain’ by co-marking 10% of genes with H3K4me3 (Fig. 3). A large portion of genes important for mesoderm development, neuroectoderm and other devel-opmental processes are within the genes co-modified by H3K4me3 and H3K27m3. The genes with diverse histone modification patterns and the associated transcriptional status (actively transcribed for H3K4me3 only marked genes, permissively transcribed for genes with ‘bivalent domain’ and stably repressed for genes with no H3K4- or K27-me3) would be functionally classified into different compartments. This report further highlights how the in-terplay of H3K4me3 and H3K27me3 contributes to the cellular proliferation, maintenance of pluripotency and terminal lineage commitment in human ES cells. Compar-ing the global distribution of H3K4me3 and H3K27me3 during diverse developmental stages, it seems that ‘biva-lent domain’ is not unique in ES cell genome and can be also found in other lineage specified cells like NPCs, MEFs and T cells. These two antagonizing groups of marks play critical roles in lineage specific developmental programs (Ringrose et al., 2004). Although H3K4me3 is extensively distributed both in undifferentiated cells and differentiated cells, H3K27me3 appears to play critical role in maintaining the undifferentiated status of ES cells (Boyer et al, 2006; Lee, 2006). This PRC2-mediated re- pressive mark occupies the genomic regions in a cell-type- specify manner (Squazzo et al., 2006). Interestingly, from a ChIP-on-chip based study, a similar modification pattern of H3K4me3 and H3K27me3 was observed in humanFig. 3. Epigenetic regulation of gene expression during ES cell differ-entiation. In ES cell genome, different categories of genes represented by carrying either only H3K4me3 (pink circle), both H3K4me3 and H3K27me3 (yellow circle) and no mark resolve into either H3K4me3 or H3K27me3 associated genes in different stages of ES cell differentiation.。

酵母转录激活实验原理酵母转录激活实验原理是一项重要的生物学实验，在研究基因表达调控机制以及相关疾病的发生发展方面具有重要的意义。

本文将从人类的视角出发，详细介绍酵母转录激活实验的原理和应用。

酵母是一种单细胞真核生物，因其具有简单的细胞结构和易于操作的特点，成为研究基因调控机制的理想模式生物。

转录激活是指转录因子结合到基因启动子上，促进RNA聚合酶的结合和启动转录的过程。

而酵母转录激活实验即通过人工构建的实验体系，模拟和研究这一过程。

在酵母转录激活实验中，首先需要选取感兴趣的基因和调控因子。

基因是生物体内蛋白质合成的基本遗传单位，而转录因子则是一类能够与DNA特定序列结合的蛋白质，能够调控基因的转录过程。

通过在酵母中引入感兴趣的基因和转录因子，可以模拟真实生物体内的基因调控过程。

接下来需要构建实验体系，使转录因子能够与基因启动子结合并促进转录。

常用的方法是将基因启动子与转录因子编码序列共同构建到一个表达载体中，然后将该载体导入酵母细胞中。

在酵母细胞内，转录因子能够与基因启动子结合，并通过激活转录复合物的形成，促进RNA聚合酶的结合和启动转录。

为了验证转录激活的效果，需要使用一些检测手段。

常用的方法包括荧光素酶报告基因系统、荧光素酶酶活性测定、RT-qPCR等。

这些方法可以定量检测基因表达水平的变化，并评估转录激活实验的结果。

酵母转录激活实验的应用非常广泛。

例如，通过研究转录因子与基因启动子的相互作用，可以揭示基因调控网络的结构和功能。

同时，该实验也可以用于筛选和鉴定新的药物分子，以及研究与疾病发生发展相关的基因和转录因子。

酵母转录激活实验是一种重要的生物学实验，通过模拟和研究基因转录激活的过程，揭示了基因调控机制的一部分。

该实验在基础科学研究和应用研究中具有广泛的应用前景，对于深入理解生物体内基因表达调控的机制以及相关疾病的发生发展具有重要意义。