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We nowknow that many of these proteins bind to nucleosomes, and their abundancemight suggestthat histones are more than just packaging proteins.A second reason to challenge the view that histones were inconsequential togene regulation was based on the amazingly slow rate of evolutionary change inthe sequences of the four core histones. The previously mentioned fact thatthere are only two amino acid differences in the sequence of mammalian andpea histone H4 implies that a change in almost any one of the 102 amino acidsin H4 must be deleterious to these organisms.\iVhattype of process could makethe life of an organism so sensitive to the exact structure of the nucleosome corethat only two amino acids had changed in more than 500 million years of random variation followed by natural selection?Last but not least, a combination of genetics and cytology had revealed thata particular form of chromatin silencesthe genesthat it packageswithout regardto nucleotide sequence-and does so in a manner that is directly inherited byboth daughter cells when a cell divides.
It is to this subiect that we turn next.Heterochromatinls HighlyOrganizedand UnusuallyResistanttoGeneExpressionLight-microscope studies in the 1930sdistinguished two types of chromatin inthe interphase nuclei of many higher eucaryotic cells: a highly condensed form,called heterochromatin, and all the rest, which is less condensed, calledeuchromatin.
Heterochromatin representsan especially compact form of chromatin (see Figure 4-9), and we are finally beginning to understand importantaspects of its molecular properties. Although present in many locations alongchromosomes, it is also highly concentrated in specific regions, most notably atthe centromeres and telomeres introduced previously (seeFigure 4-21). In a typical mammalian cell, more than ten percent of the genome is packaged in thisway.The DNA in heterochromatin contains very few genes, and those euchromatic genes that become packaged into heterochromatin are turned off by thistype of packaging. However, we know now that the term heterochromatinencompassesseveraldistinct types of chromatin structures whose common feature is an especially high degree of compaction.
Thus, heterochromatin shouldnot be thought of as encapsulating "dead" DNA, but rather as creating differenttlpes of compact chromatin with distinct features that make it highly resistantto gene expression for the vast majority of genes.lvhen a gene that is normally expressedin euchromatin is experimentallyrelocated into a region of heterochromatin, it ceasesto be expressed,and thegene is said to be silenced.Thesedifferences in gene expression are examples ofposition effects, in which the activity of a gene depends on its position relativeto a nearby region of heterochromatin on a chromosome.
First recognized inDrosophila, position effects have now been observed in many eucarvotes,including yeasts,plants, and humans.221THEREGULATIONOFCHROMATINSTRUCTURE12345b ar r i e rIIgenes_a---12345heterochromatin euchromatin12345trI1T."I{tr;iffiTlTi_T: jl12345Ie a r l yi n t h e d e v e l o p i n ge m b r y o ,h e t e r o c h r o m a t i fno r m s a n d s p r e a d si n t o n e i g h b o r i n geuchromatinto different extents in different cellsIr--r-ICHROMOSOMETRANSLOCATION12345T:'t-TrI12345heterochromatin euchromatinr--r[rT-r]c l o n eo f c e l l sw i t hgene 1 inactive(A)c l o n eo f c e l l sw i t hg e n e s1 , 2 , a n d 3 i n a c t i v ec l o n eo f c e l l sw i t hn o g e n e si n a c t i v a t e d(B)Figure4-36 The causeof position effect variegationin Drosophild.(A)Heterochromatin(green)is normallypreventedwhichwe shalldiscussshortly.sequences,from spreadinginto adjacentregionsof euchromatin(red)by specialbarrierDNAthis barrieris no longerpresent.(B)Duringthe earlyIn fliesthat inheritcertainchromosomalhowever,rearrangements,for differentDNA,proceedingdevelopmentof suchflies,heterochromatincan spreadinto neighboringchromosomalpatternof heterochromatinis inherited,so thatdistancesin differentcells.Thisspreadingsoonstops,but the establishedandlargeclonesof progenycellsareproducedthat havethe sameneighboringgenescondensedinto heterochromatinis(hencethe "variegated"therebyinactivatedappearanceof someof theseflies;seeFigure4-37).Although"spreading"the term may not beexistingheterochromatin,usedto describethe formationof new heterochromatincloseto previouslycan"skipover"someregionsof chromatin,whollyaccurate.heterochromatinThereis evidencethat duringexpansion,sparingthe genesthat lie withinthem from repressiveeffectsThe position effects associated with heterochromatin exhibit a featurecalled position effectuariegation,which in retrospect provided critical clues concerning chromatin function.
ln Drosophila, chromosome breakage events thatdirectly connect a region of heterochromatin to a region of euchromatin tend toinactivate the nearby euchromatic genes.The zone of inactivation spreadsa different distance in different early cells in the fly embryo, but once the heterchromatic condition is established on a gene, it tends to be stably inherited by all ofthe cell's progeny (Figure 4-36). This remarkable phenomenon was first recognized through a detailed genetic analysis of the mottled loss of red pigment inthe fly eye (Figure 4-37), but it shares many features with the extensive spreadof heterochromatin that inactivates of one of the two X chromosomes in femalemammals (seep. 473).Extensive genetic screenshave been carried out in Drosophila, as well as infungi, in a search for gene products that either enhance or suppress the spreadof heterochromatin and its stable inheritance-that is, for genes that whenmutated serve as either enhancers or suppressorsof position effect variegation.In this way, more than 50 genes have been identified that play a critical role inthese processes.In recent years, the detailed characterization of the proteinsproduced by these genes has revealed that many are nonhistone chromosomalproteins that underlie a remarkable mechanism for eucaryotic gene control, oneWhite geneat normallocationbarnerheterochromatinrare cnromosomeIn v e r s t o nDarFigure4-37 The discoveryof positioneffectson gene expression.TheWhitegene in the fruit fly Drosophilacontrolseyepigmentproductionand is namedafterthe mutationthat firstidentifiedit.Wild-typeflieswith a normal Whitegene(White+)havenormalpigmentproduction,which givesthem red eyes,but if the Whitethegeneis mutatedand inactivated,mutantflies(White-)makeno pigmentand havewhiteeyes.Infliesin whichanormalWhite+gene has been moved nearthe eyesarea regionof heterochromatin,mottled,with both red and whltepatches.fhe white patchesrepresentcell lineagesin which the White+gene hasbeensilencedby the effectsof theIn contrast,the redheterochromatin.patchesrepresentcelllineagesin whichthe White+gene is expressed.Earlyinwhen the heterochromatindevelooment,isfirstformed,it spreadsinto neighboringto differentextentsineuchromatindifferentembryoniccells(seeFigure4-36).The presenceof largepatchesof redand whitecellsrevealsthat the stateofactivity,asdeterminedbytranscriptionalthe packagingof this geneinto chromatinin thoseancestorcells,is inheritedby alldauqhtercells.222Chapter4: DNA,Chromosomes,and Genomes( A ) L Y S I NAEC E T Y L A T I OANN D M E T H Y L A T I OANR EC O M P E T I NRGE A C T I O N SHO-N-IHHOrtlC-C--N-ICH,Htaut.-lysrnel\Ht-fn'CHtCH,IN-,.+ICH,+t-ICHtH:Ct-CH,I'H^C|t t- Cl -n fn,ICHrt|CHtC:O-N-CCHt+HHOtllC-CI II.\cn./ \cn.|H,c-HH-monomethyllysinedimethyl lysineCH:CH:acetyllysineFigure4-38 Someprominenttypesof covalentamino acid side-chainmodificationsfound on nucleosomalhistones.(A)Threedifferentlevelsoflysinemethylationareshown;eachcan be recognizedby a differentbindingproteinand thuseachcan havea differentsignificancefor the cell.Notethat acetylationremovesthe pluschargeon lysine,and that,mostimportantly,an acetylatedlysinecannot be methylated,and viceversa.(B)Serinephosphorylationaddsa negativechargeto a histone.Modificationsnot shownherearethe mono-or di-methylationof anarginine,the phosphorylationof a threonine,the additionof ADP-ribosetoa glutamicacid,and the additionof a ubiquityl,sumoyl,or biotingrouptoa lysine.trimethyl lysine( B ) S E R I NPEH O S P H O R Y L A T I O NHOHOttlttl-N-C-CH-N-C-C-CH?lOHSenneH+CH,t-oO-IP:OIthat requires the precise amino acid sequencesof the core histones.
This mechanism of gene control therefore helps to explain the remarkably slow change inthe histones over time.TheCoreHistonesAreCovalentlyModifiedat ManyDifferentSitesThe amino acid side chains of the four histones in the nucleosome core are subjected to a remarkable variety of covalent modifications, including the acetylation of lysines, the mono-, di-, and tri-methylation of lysines, and the phosphorylation of serines (Figure 4-38). A large number of these side-chain modifications occur on the eight relatively unstructured N-terminal "histone tails" thatprotrude from the nucleosome (Figure 4-39). However, there are also specificside-chain modifications on the nucleosome'sglobular core (Figure 4-40).Ail of the above types of modifications are reversible.The modification of aparticular amino acid side chain in a nucleosome is created by a specificenzyme, with most of these enzymes acting only on one or a few sites.A different enzyme is responsible for removing each side chain modification.
Thus, forexample, acetyl groups are added to specific lysines by a set of different histoneacetyl transferases (FIATs)and removed by a set of histone deacetylase complexes (HDACs).Likewise,methyl groups are added to lysine side chains by a setof different histone methyl transferases and removed by a set of histonedemethylases. Each enzwe is recruited to specific sites on the chromatin atdefined times in each cell'slife history. For the most part, the initial recruitmentof these enz).rnesdepends on gene regulatory proteins thatbind to specific DNAsequencesalong chromosomes, and these are produced at different times in thelife of an organism, as described in chapter 7.
But in at least some cases,thecovalent modifications on nucleosomes can persist long after the gene regulatory proteins that first induced them have disappeared,thereby carrying a memory in the cell of its developmental history. very different patterns of covalentmodifications are therefore found on different groups of nucleosomes, according to their exact position on a chromosome and the status of the cell.ophosphoserine223THEREGULATIONOFCHROMATINSTRUCTURE# 'ift- #W|H2As c n e x o c e r . q n a x a l s t R s s R A GL Q F P v G R V - i r1315915119PMA.A lelIriA'fI'1.."4l-rH2B.
"ffi'\ Yprrat<sapeprrcs;rtxevrfteQ(i<ocxxn20 232412 141s5120A.&M$A':{.Ml?YYY MlM MIPYr x n o * l $ A p A r c GV K - KA * r K e r A R K S r G G K An * l o369 r014 1718 " o 23 2627282 4PMAAAAMAAttltttllS,:Rl'XCCXI:Y79MML GKaIG1rKRHRKVLIT.DNT !G iL'-K135812162079N-terminaltailsM methylationP phosphorylationIbottom viewSl"brbr.domainsffiacetvlation UubiquitylationI(B)(A)highlightingthe locationFigure4-39 The covalentmodificationof core histonetails.(A)Thestructureof the nucleosome(B\Well-documentedof themodificationsof the first30 aminoacidsin eachof its eight N-terminalhistonetails(green).Althoughonly a singlesymbolis usedfor methylationhere(M),eachlysine(K)orfour histonecoreproteinsareindicated.arginine(R)can be methylatedin severaldifferentways.Notealsothat somepositions(e.9.,lysine9 of H3)can be modifiedsmallmoleculeshownadd a relativelyeitherby methylationor by acetylation,but not both.Mostof the modifications(seeFigureonto the histonetails;the exceptionis ubiquitin,a 76 aminoacidproteinalsousedfor othercellprocessesWith permissionfrom Elsevier')6-92).
(Adaptedfrom H. Santos-Rosaand C.Caldas,Eur.J. Cancer41:2381-2402,2005.The modifications ofimportant consequences.tends to loosen chromatinlysine removes its positivethe histones are carefully controlled, and they haveThe acetylation of lysines on the N-terminal tailsstructure, in part because adding an acetyl group tocharge, thereby reducing the affinity of the tails forH 3t a i l s-tItop vlewa acetylationO methylationS phosphorylationu b i qu i t y l a t i o nI acetylationor methylations i d ev i e wFigure 4-4OA map of histone modificationson the surfaceof the nucleosomecoreparticle.As noted,the histonetails havebeenomittedhere(comparewith Figure4-39).The functionsof most of thesecoremodificationsare not yet known.(Adaptedfrom M.S.Cosgrove,J.D.BoekeandC.Wolberger,Nat.Sttuct.Mol.
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