yuowdojaneg ul uolssesdxy Com Latta]Jo sjoo4. o1Seg ULL sonouag requawdojaned J aime 26 ce ysucoeang upceede euep muna el34 Chapter 2 constitutes about half the weight of chromatin and is com posed largely of histones. The nucleosome is the basic unit So how does the same genome give rise to aif of chromatin structure (FIGURE 2.2,B),Ieis composed of an types? To understand this, one needs to underst octamer af histone proteins {two molecules each of histones anatomy of the genes. One of the fundamental differen H2A, 128, Hi, and F4) wrapped with two loops containing, Differential Gene Transcription lstinguishing most eukaryotic gones from prokaryotic genes approximately 147 base paits of DNA (Kornberg and Thomas is that eukaryotic genes are contained within a complex of 4), Histone HI is bound to the 60-80 or so base pairs of DNA and protein called chromatin. The protein component “Linker” DNA between the nucleosomes (Weintrau 198, FIGURE 2.2 Nuceosome and sreomatin stucture. (A) (A) Mode! of nucleosome stucura a8 seen oy X09 cnstalogranhy a e resolution of 1.9.8. Histones 3) Nucleosome,, Ih and M280 low arr rere Histone pape aa ery ws octamer trund fhe pot oo Tee ere a at tend ro taco a th sos aaa and rethaten whi nay pt or subza,onee a thay tho torrston of nuclocsome assembiages. (B Histone Hi can draw nacieosomes tog Compact forms, About 147 ease pars of DNA enc de each histone octamer and abut 60-80 base als of GNA ink th eucleosores together (©) Mode for the arengement of pucleo sores inthe highy compacted sae noida chromatin eit. Hs tals protrucing rom the nucleosome Ssounits allow forthe attachment of hericat groups. (D) Math greups ‘condense rucleasomes mors ight, reverting access to promoter ste And thus prevertng gene ‘ran tin. Avetation wocane nck jacking, exposing the ONA to Fi polymerase an transonat {ors that wil activate the ganes. after Davey ea OO Liser DNA © Histonecore Histone octamer DNA oC nucleosome s oy Condensed nucleosomes: Histone tails largely methylated Uncondensed mucleasames: Histone tal and acetylatedcage | nonspone!| anscipion Transcriptional longgtion Histone _ respon | mer camer ge 41985). There are 14 points of contact between the DNA and the histones (Luger etal, 1997; Bartke et al. 2010}, ‘Whereas classical geneticists have likened genes to “beads ‘ona string,” molecular geneticists liken genes to “string on the beads," an image ia which the beads are nucleosomes. Much of the time, the nucleosomes appear to be wound into tight structures ealled solenoids that are stabilized by iis~ tone Hl (FIGURE 2,20). This FiI-dependent conformation ‘of nucleosomes inhibits the transcription of genes in somatic cells by packing adjacent nucleosomes together into tight amtays that prevent transcription factors and RNA polymer ages from gaining access to the genes (Thoma et al. 1979; Schlissel and Brown 1984), Anatomy of the gene: Active and repressed chromatin HISTONES AS AN ON/OFF SWITCH Histones are critical because they appear to be responsible for ether facilitating 0: forbidding gene expression (FIGURE 2.2D), Repression and activation are controlled toa large extent by modifying the “ails” of histones H3 and Hd with two small organic groups: methyl {CH,) and acetyl (COCH,) residues. In gen: eral, histone acetylation—the addition of negatively charged acetyl groups to histones—neuiralizes the basic charge of Iysine and loosens the histones. This activates transcription Enzymes known as histone acetyltransferases place ace- ‘yl groups on histones (especially on lysines in H3 and H8), destabilizing the nucleosomes so that they come apart eas- lly. Asmight be expected, then, enzymes that remove acety] _tCups histone deacetylases—stabiize the nucleosomes and prevent transcription Histone methylation, the eddition of methyl groups #0 histones by histone methyltransferases, can either activate or further repress transcription, depending on the amino «cid being methylated and the presence of other methyl or acetyl groups in the vicinity (see Strabl and Allis 2000; Cos- stove ot al, 2004), For instance, acetylation ofthe tals of HB and Halong with the addition of three methyl groups on the lysine a position 4 of H3 (ie. H3k4me3; remember that r Differential Gene Expression in Development 35 Sileat beterochromatio FIGURE 23 Hstone matifations on histor 3, The tal of histone HS is amino-most sequence, a the begining ofthe aoten} I Ske out rom the ruciaosome ands caceble ae of being methylated or acetyated. Here, vanes sateon (Gah can be mettyised and recognized by parteusr Proteins, Metryated lysine residues at postions 44,38, and 79 are aasooeted with gone cise HS sl tin, wneraas methyated shes at pastons 9 and 27 ae associated wth repression. The pro teins bndeg tee stas rot shown to scale) ate regrasaried above the math troup. (ir Kevizards ard Berger 2007) Kis the abbreviation for Iysine) is usually associated with actively transcribed chromatin. In contrast, a combined lack of acetylation ofthe H3 and Fi tails and methylation ofthe lysine in the ninth position of H3 (H3KS) is usually assoc ated with highly repressed chromatin (Norma etal, 2001) Jdeed, lysine methylations at H3K9, HSK27, and HAK20 are cofven associated with highly repressed chromatin, FIGURE 2.8 depicts a nucleosome with residues on its H3 teil. Modi- fiations of such residues regulate transesption if methyl groups at specifi places on histones repress transcription, thon getting rid of these methy! moieties should be expected to permit transcription, This has been shown to be the case in the activation ofthe Hox genes, a family of genes that are eitical in giving cells their ident- ties along the anterior-posteior axis In early development, Hox genes are repressed by H3K27 trimethylation (the lysine at position 27 on histone 3 has three met groups: HaKZ7me3), However, in differentiated cells, a demeshyl- ase specific for FI3K27me3 is recruited to those promoters, eliminating the methyl goups and enabling the gene to Be tcanscribed (Agger etal. 2007; Lan et al. 2007). The effects of methylation in controlling gene transcrio- tion are extensive. Sofas, we have documented transcrip- tional regulation by histone metiation. Later in this chapter ‘we will discuss the exciting research on the contol of tran- scription by DNA methylation Anatomy of the gene: Exons and introns Another fundamental difference between prokaryotic and ‘eukaryotic genes (along with the fact that eukaryotic genes are contained within chromatin) is that eukaryotic genes are rot co-linear with their peptide products, Rather, the single rmacleic acd strand of eukaryotic mRNA comes from noncon- tiguows regions on the chromosome, Between exons—the regions of DNA that code for 2 protein*—are intervening sequences called introns that have nothing whatsoever to “The teem exon refers to a nucleotide sequence whase RNA “exits” the nucleus. has taken on the functional definition of 2 protein-encoding nucleatide sequence. Leader sequences and 3° [UTR sequences are algo derived from exons, even though they axenot translated into protein36 Chapter 2 ) ‘Transition ‘Trandation Poly Transeription ‘Transcription inition codon terminator codon adition terination initation (aminoacid) .ing sed aumben Site sequence Promoteyregion AP SELENCe) po eee tee ro | ow | Gi a EW) Soe, RNAPI ‘Been intron! Fon ntron2 Bron { Upateam Bite eee promoter at Leader Sequence aime region UTR) 3 Upszeam promoter eseecemeeae NABI Dining ste cexeetrheasaastaeRacapegc ane ENENLEC EEE roy tcagggcagageetctattgctt {CRGACACAACTUTSTTCACTASCAACCTEAMACAGACACCIRESrasaon initiation codon VallisleuThrProGluGlulysSerAlaVatThrAlaLTrpGlytysValAsnValAspGuValGlyGlyGla LGTGCACCTACTOCTeAGAGAAGTCTGOGTIACTGCECTTGAGCCAMRTTGAACCSGCATEAACTTEGHCETCAG AlaLevGyay i SELES cocadrecancAcarractacesinchiaciosto tit Iron 1 co, LeubeuValvaltye Aacteteainccicaoronencmt octet TRON EHEoCTECTCATOGTCTAC ProTrpThuGinArgPhePheGhiPheGlyAspLeuSerThtProAspAlaValMetGlyAsnProlysVallys py COcTTEGAOCCAGAGetTCTTTCAGTUCTT TGC ATCTETOCACTECTEATGCTETTATGECAACECTANGCTGAAG EXOM 2 AlatlisGlyLysLysValLeuGiyAlaPheSerAspGiyleuAlaHisLenAspAsoLeulysGlyThePhealaThr (CCTEATEGCAAGAAAGTGCTUGETGCCTTTAGIGATGUCCTGUCTCACCTOCACAMCCKCANGICCACCTTTECCACA euSerGluLenHisCysAsplysteubisValaspProGluAsnPhear CTGAGTGAGCTCCACTETEACAACCTELACCTOGNOCTEACHACTICAGHoncCtaredeacocenGaToreT ‘ff feecotrerinedafcariacricmioarasashasecacaucrcisccipenorrtacaatioacine scaccaaTandriscetcastorccanciicaccattirttaxeittemrrinteraciotcithasatntérme ‘enrterenuncttocrfePirremriyerictecccamerreucistemot natoecuctcarachy _ASCanaACAAINATETC A AGIMCTTASIAAadAAbiTiNCACAIOTECOTAG CE RAC ‘Tactanitetotistoertattieckterrextaamenccomcert tatty tort eEt Treat ICAIRCAIAAROX Intron 2 ‘rancanarttitecora A chon marreatinerioncacatarncaccamroansenantianckrt ‘obiaet-rhriwslalecetrenctictamibicrstHinetristetarmctixnctenccomsticert Gh Tonecch moxie castsonicatacrscr ica ccaseaadGaaiaacacncomal eens -scdeauisaoniziattcacstataasrarrrchacaratsagtethacratotiabacortTearertate *rcisbincnaccuocsccaticsacrere rm aoatTaatiscceaTcar ariCasiate Re j LeuleuGiyasnvalLewalCysVlLeuala escrito ineTEASASREAGCTCCTOGCAACOTUCTECTCTGTGTOCTAGL HisHisPheGlyysGluPheThePrnProValGlnAlaAlaTVysGlnLysValvalAlaGlyValAlaAsoAleLew ‘AICACTITGGCAMAGRATICACCEKACKAGTECAGLCTOXCTATCAGAAMGTGGTOGCTEGTUTCGCTAATECCCTS pation AlaHisLys Tyo termination codon GocaclutacciloctoscrrTcrnecTeTocAATTCTATANaGTTCCTTTGTCCCTAAGTOCAACTAC "TAAACTGOGGGATATTATGAAGGGOCTTGAGCKTCTGGATTCTGCCTIRMEAAACATTIATTTFCATIGC Polya addition site Exon sum FIGURE 2.4 Nucleotide sequece ofthe human fralabin gens. (W Schomate repeeontaion of the locations of te promoter region, transcrgton ination sit (cap sequence, 5" untanciated region foader sequence}, axons, rians, and 3" urranelated region. ‘Exons are shown in coir, te numbers tanking ther inca tha mao ac postions each exon encodes in gk. (3) Te nucle bie sequence shown rom the 5 en to the 3 end ofthe RNA, ‘The colors correspond to ther clagrammatic representation in ( The promoter sequences are boxed. as are the translation ition ‘and termination codes ATG and TAA, The age capital laters boxed In color are the bases ofthe exons, withthe amino ackis for wien thay cade abbreveted above ther. Smaler canal lear cate the intron bases. The codons after the trelaton terminaton ste ‘exis n B-gobin mRNA but are not translated into proteins, Wn this croup isthe sequence thought tobe neadled for solace ‘ton. By convention, only the RNA-iko stand ofthe DNA douse hes shown. afer Lawn etal. 1980)lo with the amino acid sequence of the protein. The structure of atypical eukaryotic gene can be illustrated by the human Brglobin gene (FIGURE 2.4). This gene, which encodes part of the hemoglobin pratein ofthe red blood cell, consists of the following elements: + A promoter region, wish is responsible for the bind ing of RNA polymerase I! and for the subsequent intia~ tion of transcription. The promoter region of the human B-globin gene has three distinct units and extends from 95 to 26 base pairs bafore ("upstream from") the tran scription initiation site (.e, from 95 to -26). The TBP site binds the basal transcription factor (TBP) that helps anchor RNA polymerase II to the promoter. ‘+ ‘The transcription initiation site, which éor human B-globin is ACATTTG. This site is often called the cap Sequence because it represents the 5" end of the RNA, which will receive a "cap" of modified nucieotides soon after itis transcribed. The specific cap sequence vaties among genes. + Thes’ untranslated region (S' UTR), often called the leader sequence, This s the sequence of 50 base paits intezvening between the initiation points of transcription and translation, The 8” UTR can determine the rate at which translation is initiated, + The translation initiation site, ATG. This codon (which becomes AUG in mRNA) is located 50 base pairs alter ‘the transcription initiation site in the human Bi-giobin _gene (although this distance differs greatly among cif- ferent genes). This begins the fisst exon. + The protein-eneading portion ofthe first exon, which contains 90 base pairs coding for amino acids 1-30 of suman pr-globin protein, ‘+ Anintron containing 130 base pairs with no coding ‘sequences for B-globin. However, the structure ofthis Intron is important in enabling the RNA to be processed into mRNA and ext from the nucleus. ‘+ An exon containing 222 base pairs coding for amino acids 31-104 + A large intron 880 base paits—having nothing to do with B- globin protein structure, ‘+ An exon containing 126 base pairs coding for amino acids 105146 ofthe protein, A translation termination codon, TAA. This becomes UAAin the mRNA. When a ribosome encounters this codon, the tihosome dissociates and the protein is released A¥ untranslated region (3’ UTR} that, although transcribed, is not translated into protein. This region includes the sequence AATAAA, which is needed for polyadenylation, she insertion of a “tal” of some *By convention, upstream, downstream, and 3’ directions are specified in relation to the RNA, Thus, the promoter is upstream ofthe gene, near 1 and “before” ts 5 end. Differential Gene Expression in Development 37 200-300 adenylate residues on the RNA transcrip, about 20 boses downstream of the AAUAAA sequence, ‘This polyA tall (2) confers stability on the mRNA, @} allows the mRNA to exit the nucleus, and (@) permits the mRNA to be translated into protein. + Ateanscription termination sequence. Teanscrip- tion continues beyond the AATAAA site for about 1000, nucleotides before being terminated. ‘The original transeription product is called nuclear RNA (aRNA), sometimes called heterogeneous nuctear RNA (hnRNA) or pre-messenger RNA (pre-mRNA). Nuciear RNA contains the cap sequence, the 5° UTR, exons, introns, and the 5° UTR. Both ends of these transcripts are madified before these RNAs leave the nucleus. A cap consisting of ‘methylated guanosine is placed on the 5’ end af the RNA in opposite polarity to the RNA itself. This means there is no free 5’ phosphate group on the nRNA., The 5 cap is neces~ sary for the binding of mRNA to the ribosome and for subse- (quent translation (Shatkin 1976}. The ¥ terminus is usually modified in the fucleus by the addition of @ polyA tail. The adenylate rescues in this tall are put together enzymatically ‘and are added to the transcrip they are not part of the gene ‘sequence, Both the 5° and 3 modifications may protect the mRNA from exonuclease that would otherwise digest it (Gheiness and Darnell 1973; Gedamu and Dixon 1978). The modifications thus stabilize che message ang! ts precursor ‘As the nRNA leaves the nucleus, its introns are removed and the remaining exons spliced together. In this way the coding regions of the mRNA~that is, the exons—are brought together to form a single transcrip, and this tran= scripts translated into a protein, The protein can be further ‘modified to make it functional (FIGURE 2.8), ‘Anatomy of the gene: Promoters and enhancers Sn addition to the-protein-eneoding region ofthe gene, there are regulatory sequences that can be located on either end of ‘he gene (or even within it. These sequences—the promot- cers and enhancers—are necessary for controlling where and when a particular gene is transcribed. Promoters ate sites where RNA polymerase I binds to the DNA segitence to initiate transcription. Promoters of genes that synthesize messenger RNAs (i.e, those genes that encode proteins#) are typically located immediately ‘upstream from the site where RNA polymerase If initiates transcription. Most of these promoters contain a stretch of ‘Hn the case of protein-encoding genes, RNA polymerase lis tsed for transerption, There are several types of RNA that do nol encode protein, These incluce the ribosomal RNAs and transfer RNAs (which are used in protein synthesis) and the small miclear RNAS (which are used in RNA processing), In faction, thece ore regulatory RNAc (euch asthe microRNAs and Jong noncoding RNAs tost we will discuss later inthis chapter, ‘which are invoived in regulating gene expression and are tot translated into peptides. These often are transcribed by other RNA polymerases.88 Chapter 2 ‘rapslation ‘Transition Polya ination codon (4G) terminator codon addition Transcription Promote region Transcription initiation cod: (RNA polymerase II | (TAA) terminates binding) See (NA) GUE as ron Oa Beton TR NUCLEAR RNA, RNA) | Procening [Tien Ban 2p zon 3 [Aus J UAA 28? Gyppac se —— je} a (mRNA) Cap Te “ta | Transation S-GLOBINPROTEIN H.W eeeeveoseszonseessesenee COOL Posttranslational smodifiiton Belobin. HEMoctomny Faces oGteen Ne poduton ot tin andtemoatcn, atone, Tansionon oocomes uses the MANA to erode Tertotoion othe Bdtin genecreatesaruciarRacowaney ater Tre B-dotin rota singe uni ts me et tons anc ito, as wal ase can, ah ana! are une complexed wih a gobi and hea ta become active honceicon ‘edo Prossiang enue ANAIniomesoege Faraone, ge host 2000 base pics that isrich inthe sequence CpG (@C enzymes euchae Fistone acetyttensterases) that break up the a G connected through the notmal phosphate bond). suclenena the area or Q stabilizing the transesption int pas agpBians ate caled CpG Islands (Down and Hub tation comrlen ene above. Ths, transcription factors aN 2002; Deaton aed Bird 2012), The reason transcription usualy ene nan nonexclusive ways 's initiated near CpG islands is thought to involve proteins prose al transcription factors (ich 8 TBP) which are Once Bout transcription factor can bind cofactors that resent in every ce Those proteins bind tothe cea ny recruit nuclecsome-modifying proteins uch as histone sites and form a “saddle” that can recruit RNA polymmorsce ethylicansferases and acetyltransferases) that make {and position i so the polymerase can begin transcription ra grea of the genome accessbie for RNA pelymeraee (Rosteewa eta) 2000) U to bind and enabie the chromatin in that vicinity to be But RNA polymerase I! does not bind to every promoter in unwound and transcribed paseame atthe some tine Rather Risrecrutedtoandster 2. Tanserplon nt {orm bridges ooping the chroma bilizedon the promoters by DNA sequences called enhancers tin such tht the transcription factors (and thelr histone. that signal where and when a promoter can be used an! here ‘modifying enzymes) on enhancers can be brought into ich gene product to make. in other words, enhanvers conned the vicinity ofthe promoter. In the activation of ame the efficiency and rate of transcription from a sperifc promoter satian B-slobin genes, such a bridge uniting the po. {eee Ong and Cores 201), Enhancors bind specifi transcrip, ‘moter anci enhancer is formed by proteins thet bind co Aton factors, proteins that activate the gene by () recruiting transcription factors on both the enhancer and promoter (rrr crc—— Promoter Se “Transcription FIGURE 2.6 The ordge between enhancer and promoter can be made by tarscription factors. Cert tranecrpton factors bin to DNA on the pxometerfunere PNA polymerase iw inriate tra- slot), while other Yarecription factors bid to the enhancer {ebsch regulaae whan and where vanscxpton can accu). Other ‘ranserpion factors do not bie!to Uwe DNA, but rater ir the ‘tareeripfon factors that nave bound to the enfanar an promoter sequences nhis wy, the chromatin lagps to brng the enhancer tothe promoter. The example showin hare isthe mouse B-alobh gene (Aj Transcintion facore assemble onthe ennances but te ‘omaters not used until he GATAL transcription factor ids to the promoter. {6} GATAT can rect sovoral othe fecters, including Lab. which forme nk uniting the enhanice-ourd factors ta the romter bound factors. (aftr Deng eta. 2012) ‘sequences. These proteins recruit the nucleosome-modt fying enzymes and TRS that stabilize RNA polymerase TUFIGURE 2.6; Deng et al. 2012; Noordermees and Duboule 2013) THE MEDIATOR COMPLEX: LINKING ENHANCER AND PRO- MOTER In many genes, a bridge between enhancer and promoter is made by a large, multimeric complex called the Mediator, whose nearly 30 protein subunits connect RNA polymerase Il to enhancer regions that relay developmen: tal signals (Malik and Roeder 2010). This forms the pre~ initiation complex at the promoter. Theretore, the Media tor initiates a chromatin loop, bringing the enhancers to the promoter. This chramatin loop is stabilized by the protein cohesin, which becomes associated with the Mediator after the Mediator is bound by transcription factors (FIGURE 2.7), Although the Mediator may help bring the RNA poly ‘erase II ta the promoter, in ord: for transcription to take place, the connection between the Mediator and the RNA. polymerase Il has tbe broken, and RNA polymerase Il must be released from the promoter. The release of RINA poly’ ‘merase II is accomplished by a transcription elongation complex {TEC} made up of several transcription factors. This release coincides with the capping of the transcript and the phosphorylation of the polymerase, The enfiancer-bound Differential Gene Expression in Development 98. Medistor complex can presumably recruit new RNA poly- rmetases to the promoter, maintaining transcriptional activ- ity there, However, in some instances (discussed later in the chapter), the RNA polymerase Il either does not dissociate yom the Mediator, ori dissociates but only transcribes afew nucleotides before it pauses. in the latter case, a transcription ‘elongation suppressor (such as NELF) appears to prevent the transcription elongation complex from associating with the polymerase, and the RNA polymerase If is paused, held in readiness or a new developmental signal Enhancer functioning (One ofthe principal methods of identifying enhancer sequen «esis to clone DNA sequences lanking the gene of interest and fuse them to reporter ganes whose products are both read- ily kientifable and not usually made inthe organism. being studied, Researchers can insert constructs of possible enhan- crs and reporter genes into embryos and then monitor the expression ofthe zeposter gene (uch as GFP: FIGURE 2.08), If the sequence contains an enhancer, the reporter gene should become active at particular times and places. Forinstance, the E cali gene for B-gelatosidase (he lacZ gene) can be used as ‘roporter gene and fused to () 2 promoter that can be acti vated in any cell and (2) an enhancer that directs expression ofa particular gene (Myf) only in mouse muscles. When the resulting transgene is injected into a newly fertilized mouse egg and becomes incorporated int its DNA, P-galactosiéase protein reveals the expression patter ofthat muscle specific gene (FIGURE 2.88), More recently, enomic techniques such as ChIP-Seq (discussed later in the chapter) have enabled researchers to dently enhancer elements by sequencing the DNA regions bound by transcription factors Enhancers generally activate only cis-tinked promoters {ie., promoters on the same chromosome); therefore, they are sometimes called cis-regulatory elements." However, because of DNA folding, enhancers.can regulate genes at reat cistances ome as great asa million bases away) from the promoter (Visel eta. 2009). Moreover, enhancers do not need tobe on the 5! (upstream) side ofthe gene; they can be at the 3 end, and are frequently in the introns (Maniatis et at. 1987). As we will see in Chapter 24, an important enhancer for a gene involved in specifying the “pinky” of each of our *Cis-andtrane-regulatry elements are so named by analogy with onl genetics and cxgante chemistry. Therefore cs-elements ere regulatory elements that reside onthe same chrcmosome (=, “on the same side as", whereas trans- elements ae tnose that could be supplied from another ehtomesome (rans, “on the other side of"). The term es-eguatory elements now refers to those DNA Sequences that regulate a gene onthe same stctch of DNA (Le. the promoters nd enhancers). rn-rogulatory factors are solvble iolecules whose genes are focated elsewhere inthe genome and ‘Which bind to te c-tegulatory elements, They are usually ran $ctipton factors ar micro&NAs. Some evidence points tothe bi fy ofan enhancer to setivase a tans promote (Le, a promoter on ‘nother chromosome), hut these appear to be exceptional and rare events (Noordermeer etal. 2010,40 Chapter 2 FIGURE 2.7 ‘The role of he Meciator complex in forming the tanscrcton preciation complex (A) Peavy open clyomatn is composed on DONA cold ature rucieosomes. (8) Tenscrs- ton factors bind to the enhaneer and bind ucteesoma-modihing anaymes that remove >uctsosomas ftom the ares ncuaing tre errancar and promoter. (C) The tanacriton factors aso bind a large protein complex calad he Macistr, (D) The Mediators able to recrut and sabe NA polymerase Il and its cofactors (TAFS A, I, tc) at te promoter se, This is cae he pre ination complex. The cheamatin looping is further stabized by cohesin (E] Ater RNA paiymerase leavas the promoter thera are general two cu comes. Fut ig, t ean asecciate with thee Saption elongation compiex (TEC) to elongate the RNA wie tha Mediator conainuas to reerat new ANA polymerase it protein to the complex. ar nately etl, BNA polymerase I can beinstructag ‘stop elongation by a repressive venscipton faotor[NELF| hat prevents the assembly of the TEC. when given a secand developmental signa, ELF can bs omovod and transerition alonga- tion cortinusd, (Aer Mak and Roeder 2070; hisson 2010) eRe stir 2 See o Nascent Meth gro ‘wanscript blocked fae Transcript longetesa Differential Gene Expression in Development 44 ) FIGURE 28 The genetic elements regulating ssue-spectic ransciption can be idertifed by fusing reporter genes to cus ected enhancer regions of tne gen expressed in particular cl ‘p08. (A) The GFP gone ie fused to a zebrafish gene thats cive only n cortain cals ofthe retin, The rasut is expression of graan iuoreagent proton in the larval retina oetow if, socal in the re calls (below ign (6) The enhancer rajon o the gare forthe rmusele-epacitprotan MyS & fuser toa P-galactosiase reporter {mbes found in an intron of another gene, some million base pairs away from its promoter (Lettice etal. 2008). In each cel, the enhancer becomes associated with particular transcription factors, binds nucleosome regulators and the Mediator com plex, and engages with the promoter to transcribe the gere in that particular type of cel (FIGURE 2.9) ENHANCER MODULARITY The enhancer sequences on the DNA are the ssime in every cell type; what differs isthe com bination of transcription factor proteins that the enhancers ‘expesience. Once bound to enhancers, transcription actors are able toenhance orsuppress the ability of RNA polymerase Ilo initiate transcription, Enhancers can bind several transcription factors, and itis the specifi combination of transcription factors present that allows a gene to be active in a particular cell type. “That is, the same transcription factor, in conjunction with dif ant cther factors, will activate different promoters in differ ent cells. Morcover, the same gene can have several enhancers, with each enhancer binding transcript that same gene to be expressed in different cell types. ‘The mouse Paxé gene (which is expresses comea, and retina of the eye, in al tube, and in the pancreas) has severaf enhancers (FIGURE 2.98,C). The 5 reg: latory regions of the mouse Paz6 gene were discovered by taking regions from its 5 flanking sequence and introns and fusing them to a lacZ reporter gene. Each of these transgenes ‘as then microinjected into newiy fertilized mouse pronuclel, jon factors that enable In the lens, fe ‘gone and incorporated into @ mouse amioryo. When stahod f [galactose civty oar stag region, the 13 5cay mouse femlryo shows tat she reporter gave s expressed inthe muscles fae, nack, and fora and i the segerented mye ‘ie ise to the back musculature (8 trom Tekechiet purtesy ofS, Kawamura, Hamaoka and M. Takeo zatesy of A. Patapoutian and B. Wold.) and the resulting embryos were stained for f-galactosidase (FIGURE 2.90; et al 1998; Wiliams etal. 1998) Analysis of the results reveated that the enhancer farthest upstream from the promoter con‘ains the regions necessary for ‘Paxé expression in the pancreas, while @ second enhancer acti- vvates Pisé expression in surface ectoderm (lens, comea, and conjunctiva) A third enhancer resides in the leader sequence; gains the sequences that dizect Paxé expression in the neural tube. A fourth enhancer, located in an intron shortly downstream ofthe translation initiation site, determines the expression of Pax6 in the retine. The Pax6 gene illustrates the principle of enhancer modularity, wherein having multiple, sepazaie enhancers allows protein to be expressed in several different tissues while not being expressed at all in others. COMBINATORIAL ASSOCIATION While there is modularity betieen enhancers, there are cadependent units withix each ‘enhancer. Enhancers contain regions of DNA that bind tran scription factors, and itis this combination of transeription factors that activates the gene. For instance, the pancreas specific enhancer of the Pax6 gene has binding sites for the Phx and Meis transcription factors (see Figure 2.9C). Both need to be present in order for the enhancer to activate Pax in the pancreas cells (Zang etal 2006). Moreover, the product of the Paxé gene encodes a tran- scription factor that works in combinatorial partnerships ‘with other transcription factors, Figure 2.10 shows two gene42. Chapter 2 in rain and lim Genea expressed in brain Brajn-expresced transcription factors andlimb a” sinspecificenancet 4m SOWELL ES Madu Ds Be eects, L 2 mnbrain Gene A (aap) expressed ees inbrain Limb-specific enhancer (not used) se) spresed “8 38S ~ ~ transcription factors if /GeneA VA expressed \ inkimb @) Lensand Pancreas comme Neural tube enhancer enhancer enhancer a Z et Exons, o + : eet 5 recognizes the metnated oytosres of DNA, fines to the DNA aa Methyl | fae thereby abi reo Ariston deaootyaes (hic take ace youn group \| Mecea | Histone, ote Histon} or stone metnytrarsterases wich add meth vai \ fee Groupe to te hlrones}, Both modcstions prometa re statiy ofthe § lecsere and he tt pack of ONA, hereby repressing gone fexgressonin ines regions of DNA met aton. Aer Fuks 2005) seen, ba on eG St ots Sacer ee Poe @ aa sr in ety group, enone aoe Fide 9 of hrstone 13 ta DNA methylation pattern is established in a cell, it can be stably inherited by all the progeny of that cel Roinforcement between repressive chromatin and repres~ sive DNA has also been observed, Just as methylated DNA is able to attract proteins that deacetylate histones and attract 1 linker histones (both of which will stabilize nucleosomes), 0 repressed states of chromatin are able to recruit enzymes that methylate DNA. DNA methylation patterns during gametogenesis depend in part on the DNA mnethyltzansterase amb (ée novo ‘sthytrensferase) Damtt (pecpetuating methyieanslerase) s6 & x FIGURE 2.19 yo DNA methylvancferesse ar crea important In mecying DNA. Ths ‘de novo" motnytranstrasa Dnt can lace a matty group on urinated cytsines. The “pemetvat 5° metyvantarass, Dre, racogelzes mthated Os on one ‘Strand and metystes the G onthe CG pair onthe opeosite stand Drmf3L. It actualy has lost its enzymatic activity, butt can still bind avidly to the amine end of histone H3. However, if the lysine at H3K4 is methylated, it will not bind. Once ound, however, it recruits andlor activates a Dnmt3 methyl- transferase to methylate the eytosines on nearby CG pairs (Gan et al, 2007; Oot et al. 2007) “Poised chromatin Promoters can exist in three major states: an active state, a repressed state, and an intermediate, or “poised” state (see Figure 2.15). This poised chromatin state allows for a rapid response to developmental signals, and it characterizes the high CpG-content promoters (HCP) that regulate the transcription of developmental control genes, The DNA of HCPs is relatively unmethylated, and nucleosomes tend to be enriched with “activating” H3K4me3. Asa result, RNA poly ‘merase If is usually alzeady present on HCPs (Hon et al. 2009; Emnst and Kellis 2010). Indeed, there is offen a small, truncat- ed transcript of RNA already initiated (but no completed) at these promoters (see Figure 2.15), DNA methylation does not appear to play a major role in HICP regulation. Rather, HCP can be repressed by modifying the histone 3 to H3K27me3, which recruits Polycomb repressive complex 2 (Peng et al 2009; Li etal, 2010), a camplex that appeors to inbibit further RNA polymerase I binding as well as preventing elongation of the existing nA transcripts HPs become poised for activation by having nucleo somes containing both HaKame® {activating) and H3K27me3 (repressive) histones (this is sometimes called a bivalent state). Thus, the rate-limiting step of RNA transcription srom CPS isnot the initiation of transcription (as t sin the LCP), but RNA elongation, This “poised for activation” state may be predominane during early development (Muse et al. 2007; ‘Zeitlinger 2007). The genes may be put into an active state by specitic transcription factors that activate the elongation of RNA transcripts (Peterlin and Price 2006), and they may be repressed later in development (Hargreaves et al. 2008; ‘Ramirez-Carrozzi etal. 200; Rah etal. 2010) ‘These transcription factors may act on several levels to promote RNA elongation. In mammalian cells, where about ‘30% of the genes have promoters that already contain RNA polymerase Il and nascent RNA chains (Core and Lis 2008), transcription factors appear to act through the Mediator complex. Here transcription is paused because the RNA polymerase II remains tethered to TRHD, which remains bound so the promoter sequence of the gene. This tether ing is accomplished by the Mediator complex, espectally by the Mediator protein Med26, which binds the Mediator to52 Chapter 2 Genomic Imprinting and DNA Methylation least one very cuzzing phenom ‘encn, that of genomic imprinting (Ferguson-Smith 2011), is usualy ‘assumed that the genes one inherits from one’s father and the genes one Inherits ror one’s mother are equiva: lent, in fact, the basis for Mendilian ratios fand ihe Punnett square analy {586 usod to teach ther) is that it does ‘ot mattor whether the genes cama from the sperm or from the egg. But In rmammats, there are about 100 ganes for which it does matter (ternational Human Epigenome Consortium in ‘these cales, the chromosomes from the male and the female are not equivs- lent; only ha sperm-derived or only tne egg-derived allele of the gene is fexprossad, This means that @ severe or lethal concition arises ia mutant allele is derived fram one parent, but that the same mutant alee will have no. deleterious effects if nherted trom the ‘other garent. In soma of these cases, the nonfunctioning gene nas been renderec inactive by ONA methylation. (This eneans that @ mammal must nave oth @ mala parent and @ female pay: cent Uniike sea urchins, fles, and even some turkeys, mammals carinot exer ence parthenogenesis, or ‘virgin bith.) ‘The rethy’ groups ere pleced on the: DNA durng spormatogenesia and oogenesis by a series of anzyres that firs take the exssting matryl groupe otf the chromatin and then place new sex specific ones on the DNA (Ciscone et al, 2009; Gu et a. 2071, {As daseribed inthis chapter, met ated DNA is associated with stable DNA Slanaing. shor {by wtorearg rete the binding of gene-activating tranecrip ton factors or @) by reevuling rapressor proteins that stablize cucleosomes a restrictive manner along the gene. The presonce of a methyl group inthe minor groove of ONA can prevent ceriain D= ‘methyfation has sxplcined at "Alictof imprinted mouso gonas fs main tained at worweharmre ae ub/research/ genemic.imprnting/introduetion htm, (8) Bgg- derived tmaterna Enhancer H19 ‘Transcription ©) Sperm-terived (patemal) chromosome Enhancer nie No teanscrption FIGURE 2.20 Regulation of he mini gf gane in by an enhancer element it shores wih the 479 gen, The at ~ DM (CTCF insulator protein binds “oN ylted DMR ig Transcription mouse. This gone is activated ertally mestyated region {OMR} 2 sequence located batwean tha annnear and tne i gene, and is eund on bolh sperm and egg-derved chromosoras. iA} the egg-darvea crremeeeme, the OM isunmethy ted, Te CTOF insulator poten binds to the DM and blocks the enhance signal {8 Inthe sper-derved chrornceome, the DMP ie matty, Te CTCF insiator protein cans i vanscrption. {ranscription factors tom binding fo the NA, thereby preventing the gene from being activated (Wet! and Mollay 1988). For examote, during early embry- ‘onic development in mice, the gi? gene (for insulir-like growth factor Is transcribod only from the spor derived (paternal chromosome 7, The 299-derived {maternal ig/2 gene does ot function during embryonic devel- opment. Ths is because the CTOF protein is an inhibitor that can black tho promoter from getting activation signais trom enhancers, It binds 1o a ragion near the fgf2 gene in females Dacause this region is not mathyiated. Once bound, it prevents the matemaly derived igf2 gene from functioning. in the paternally derived chromosome 7, the region where CTCF would bind ic methylated. CTOF cannct tint and the gene is not inhibited trom bind to the mettyised sequence, and the signa from the enhances able functioning (FIGURE 2.20; Bartolorrel al, 1995; Ferguson-Smith et al 1999; Sei and Felsentold 2000) Ir humans, miseaguiation of 13F2 methyiation causes Beckwith-Wie- demenn growth syndrome. Athough DNA methylation is the mechanism for imprinting this gene in eath mice an humans, the machanisme responsible for the differential 72 methyation between sperm and egg appesr to be very aifferant in the two species (Far- gusan-Smith etal, 2003; Walter ang Paulsen 2003), Diferantial merhyation is one of the most important mecha- nigms of epigenetic changes and is ' reminder that an organiem cannot be explained solely by its genes, One needs knowiedga of daveloomental parameters (such as whether the gene ‘was modified by the gamete transmit ing l} as wo as genetic ones, EE ES PONENT RE EIRENEbe ey Ination ‘ery eatly elongation rome Le ansrpton ‘TFIID (FIGURE 2.21). In onierto elongate the RNA, a signal ‘must enable the multiprotein transcription elongation com- lex (TEC) to compete with TFIID for the favors of Med26. Goce the transcription elongation comple frees the RNA. polymereee Il from TFUD, RNA polymerase Hl can become Phosphorylated and travel along the DNA to transcatbe the gene (Takahashi etal. 2010. ‘Drosophila may use a slighty diferent mechanism to pause the transccption (com HCP’. In many ofthese genes, there appears to be a DNA sequence in the proximal promoter (.¢, the sequences ofthe promoter closest tothe exons that acts asa “pause button” (Hendrix eta. 2008). About 1500 genes in Drosophila embryos have RNA polymerase I already on their promoters, an these genes are primarily those actve in regulating ealy development (Muse etal. 2007; Zetlinger et al. 2007}, Itis possible that these “pause button” sequences maybe more difficul to unwind, anc the presence ofthe poly- erase may enable elongation inhibitory factors to assemble there Levine 201) ‘But hovr does the release of a single transript influence ‘he synthesis ofthat protein? It certainty takes more than one ‘uansctipt to produce significant amounts of gene product. In some cases, it appears that the transcript ofthe paused poly merase can reruis histone-actvating proteins, enabling fut ‘her transcription to occur as soon as elongation commences {Petesch and Lis 2008) Ie is also possle that paused RNA polymerase I! prevents the assemly of new nucleosomes “on the promoter, keeping the gene in an open configuration (Gitchrist et al. 2010; Nechaev etal. 2010) Thus bath high and low CpG-content promoters regulate RNA synthesis, but they do so in different manners. LCPs are usually ured off, requiring transcription factors to enable gene expression by promoting access of RNA polymerase Tl to the DNA, HCD alzeady have initiated transcription, but ‘tanserption isnot completed. Here, developmental signs allow the elongation of the nascent nRNA. Both LCPs end HCPs have repressed states that prevent transcription, as ‘wel as poised states that enable the genes tobe transcribed amumediatly when the appropriate signal is received Differential Gene Expression in Development 63 FIGURE 2.21 Model forthe rquiation of INA elongation by the Messator protein Mod25. nthe iation an early elongation chase cf trenccrnton, the Meckztor tethers RNA polymerase I 0 TFIO at the promoter trough fs Meo26 proten, The Med2® orcien can ‘se bind tothe transcription elongation complex (TEC). Tarsorip tin elongation car be reactivated by ranscrpio factors promot ing the binding of Med26 tothe TEC rather than to TRID. iter “ehahasti etal. 2011) © See WEBSITE 27 Chromatin diminution © See WEBSITE 2.8 The nuclear envelope's roles in ‘gene regulation Differential RNA Processing “The regulation of gene expression is not confined to the dif- ferential transcription of DNA. Even ifa particular RNA tren script i synthesized, there is no guaranive that it will create ‘functional protein in the cell. Te become an active protein, the nuclear RNA must be (1) processed into messenger RNA by the emaval of introns, @) translocated from: the nucleus to the cytoplasm, and (3) translated by the protein-synthesizing apparatus, In some cases, the synthesized protein is notin its mature form and must be (4) posttranslationally modified to become active. Regulation during development can occur at any of these steps ‘The essence of differentiation is the production af dif- forent sets of proteins in different types of cells. In bacte ‘a, differential gene expression can be effected at the lev- cls of transcription, translation, and protein modification In eukaryotes, however, another possible Level of regula tion exists—namely, control at the level of RNA processing land :ransport, Differential RNA processing isthe spicing of InRNA precursors into messages that specify different pro~ teins by using different combinations of potential exons. If ‘an mRNA precursor had five potential exons, one cell type might use exons 1, 2 4, and 5; a different cell type might use exons 1, 2, and 3; and yet another cell type might use al five (FIGURE 2.22). Thus, a single gene can procuce an centre family of proteins. The different proteins encoded by the same gene are called splicing isoforms of the protein. THHSEE a) mRNA 1 vec 004 i SO ea a sae ener nese canny FIGURE 2.22 Difarential INA procecaing. By convention, so= Ing pans are chow by fhe V-shaped ines. Dire spicing can process the same nucleer RNA ho ferent mBNAS by selectively using core exons.84 Chapter 2 X Chromosome Inactivatio: Noncoding RNAs in Transcriptional Gene Regulation Drosophila, nematodes, and mam- mals, females have two X chrome. ‘2omes er cell, while males Nave & singe X chromosome per cal. Unie the ¥ cnromosome. the X chromosome Contains thousands of genes that are essential for cell activity. Yet despite the female's osts naving double the ‘number of X chromosomes, male and female cols contain anproxmatoy equsl lamounts of X chroeosome-encoded {gene products. This aquaization phe- Romerion is calles dosage compen- sation, andit can be accomplished in tee ways (Migoon 2002), in Dro- sophia, the transcription rate ofthe male X chromosomes is doubiad 60 that the single mais X chromosora makes the ‘same amount of transcript asthe two female X chromosomes (Luccresi and Manning 1987}. This hyperactive ofthe male X chromosome is accor FIGURE 2.20 The ition °C" let was tho fret household pet tobe successtuly ionee using somatic nuciear transfer om "Rabon" ight), a female calco cal, However, ese ‘wo genetcay Idea! cats ara not wntical baccuge calico coloration is the ves fran dem x chromosome inactivation. In random pigment cal, the “orange” aise is nactvsted, ‘whi in char gigmont cal, the "isc" ale is randomly inactivated, [Courtesy of he plshod by enhanced wanenteie Colage of Veterinary Mecicine, Texas ARM Unuersty, elengation and by acetylation of fe ucteosomes throughout the male's X.chromosomes (Akhter et al. 2000; a pigmentation gene an the X chro. «This process is ireversibie, On Larschan et l.201%), nC. siegans, both mosome, a differant resut ie seon @ partculsr X chromasome (either Chromosomes are partaly repressed patches of ane parental color altarnata ‘the one dived from the mother (Chu ot el, 2002) so that the products with patches of the other paronta, oF the one derived from tne father of the X crromosomes are equaized coer. Calico and tertolseshell cate? has been Inactivated ina cel, the between the sexes are normally emele; their coat color ime X chromosome is inactivated In mammals, doeags compensation alleles (black and orange) are onthe X_N. af af that cel’ progeny {FIGURE ‘occurs through the inactivation of one chromosome (FIGURE 2.20: Cont 2.248,0), Because X inactivation X chromosomis in each femato col wall and Benirscks 1873) To account ‘happens relatively eary in devel. Thus, each mammatian somatic cel, for these resutts, Lyon proposed the ‘Soment, an entire region of cals whether male or female, nas only one folowing hypothesis derived fram a single coll may have functioning X chromosome. This pho. the same X chromosome inactvat- *+ Very eatly in the daveiopment of fed, Thus, al issues in tamale many female manvals, both X chromo. uw aru mais are mosaics of two coll type: nomenon is called X chromasome inactivation. The chromatin of the into heterochromatin chyomatin inactivated in each cel FIGURE S078 6 comolcated: indeed, Its & mosiotieceleydoandresenas | 2248) : love gt hough Wigeon 2007 tater than most of the other chromatin Pee eer ee The mechanisms of X chromosome ine euciromatin ofthe muceus Tig Some cols the patrnly dered x Fpesrarr aero SaTeSor was first snown by Mary Lyon (1964), Shromosome is inactivated: in other mammalian groups (Migeon 2007; iho ovceried cost colorpatems n SH, hemateray dared Xchrax Gemma Soups MgO 20 mica. fa mouse is heterozydous for mechanisms each use a noncoding an autosomal gene controing hair RNA callod Xt (Xinactvation spe Bigrentation then i esemeles one “Atough tho tre cleo and tertsise- Ge wanserpl to mena ae Gfte wo paras or has a clr intr. setae sonetnos usd prone, rena ene actia® Oe meciate between the two, meter : ony eitoca uly Xist ANA Is intaty tancersed een Sur eke and orange ony eadto cate usualy Farias esse ensle color But have white patonas that, patches wth levels in both X chomescnen tae Ya temaie mouse is heterazygous for no pigment as we oe becomes actively transcriced on only tenn rrDifferential Gene Expression in Development 55 a x t FIGURE 2.24. Xchvomosome inactivation. (A) Inactivated X chromosomes, or Barbod: ies, inthe nuoe of huren ova epthalal cote. The top cali rom a normal femal ard has single Bar bod ltrovt the lower cf, toma female with tree X com oscres, ‘ho Barr bodies ean be seen. both cases, chiy one X chromosome per oa fs tive. {B.Ol The paternal dorian X enramascrve ofthis mouse ernbny> contained a 87 tans- ‘gene, Cals i which the paternal ehvomasame is active make P-gaactosidase and stain ue, (Btn the eary blastocyst stage (day 4), ath X chromosomes ae active nat col (©) At day 6, random inactivation of one of the chromosomes occurs, Embeyonic cals in hich the maternal is active appeer pk, whie thse with an actve psterralX ae bye. in mouse (ou not human) ronhoblass, the patemaly darvad X cromosame is prefererialyinactvated, 9 th trephodlast cals are unto pink (A courtesy cI ML. ‘Bar, 8,C fom Sugimete et al, 2000, courtesy of N. Take) one X chramosome in each call—but which one's random. Although Xist is intially present only near the sito of fs transcriotion, st transcripts ‘sproad aut, evertwaly covering the ‘entra chramosome and recruiting Polycom repressive complexes (PRC: 1 and 2 to that particular X Chromosome. These repressive com- plexes modify the nucleosomes 19 pravont transcription (see Wutz 2011). “Tha PRC-Nist complexes algo recruit DNA methyttranstarases that further Siabilze the repressive state by meth YYating the gene promoters, In mice {and humans, the promater regions of humerous genes bacome methylated on the inactive X chromosome but are Lnmethyiatod on the active X chromo- ‘some (Wotf et al. 1964; Keth et al 4886; Ivigeon et si. 1991), The mem- (oy of this "x inactivation” is trans- ‘mitted to the progeny of the calls by successive DNA methylation through Gnmet (goa pp. 50-51), ‘though Xist RINA transcription ‘appears ta be the major step in X ‘chromosome inactivation, different rmammais may regulate it indifferent ways. n mice, it appears that the mac tive X chromasome is Doing selected. Both X chromosomes transcribe two factors that inhibit Xist vanscription, and the local concentration of these inhibitors determines the levels of Nat land which X chromosome becomes Inactive (Gontan et al. 2009) In pumans, nowever, the auto- somes may provide an Inhibitor of 2st, Humans with an extra X ohrorno- fsome (K trisomy) have only one active X chromosome, On the ather hand, ‘niplodd humans have an entire extra set of haploid chromosomes and often die around the time of bith. Some of these rare fetuase have an extra oO Extraembryonic tistue of placenta —~ (Crephoblast) S Embryonic els = SH = ‘eg? Extraembryonic "94 volksac - precursors X chromosome (a condition in which ‘wo X-bearing sperm enter the ©99 ‘and the embryo has 66 éutosomes ‘and 3 X chromosomes) and often have ‘wo active X chromosomes per oe! ‘Tres suggests that the Xist being constiutvely synthesized and that an ‘autosomal inhibitor of human Nit tran- seription (e., an X chromosome ecti- vvator) might exist Migeon et a. 2008, Migeon 2011). “ist is a member of a newly dis covered aroun of transeristionsl reguiatore callod tno long noncoding RINAs (IncRNAS). Those reguistors ara often used to stance genes on fone of the two chromosome. The Airs IncBINA, for instance, sinces the [gi2R promoter on the paternal (but ‘not the materna) mouse chromosome 17. (The Ig!2R protein isthe receotor {or the Igf2 insulin growth factor men: tioned earlier). The paternal promoter for Aim Is unmethylated and active in the eerly embryo (Seidl et al. 2006: Latos et al, 2012) @ See WEBSITE 2.9 Smal noncoding RNAs that repress ‘transcription r56 Chapter 2 Creating families of proteins through differential RNA splicing Alternative nRNA splicing is a means of producing a wide variety of proteins from the same gene, and most vertebrate genes make nRNAs that are alternatively spliced* (Wang et al, 2008; Nilsen and Graveley 2010). The average vertebrate ARNA consists of several relatively short exons (averaging. about 140 bases) separated by introns that are usually much Jonger. Most mammalian nRNAs contain numerous exons, By splicing together diferent sets of exons, different cells can make different types of mRNAs, and hence, different pro- teins. Recognizing a sequence of aRNA as either an exon oF an intron isa crucial step in gene cegulation. Alternative nRNA splicing is based on the determina- tion of which sequences will be spliced out as introns. This can occur in several ways. Most genes contain “consensus “Mutations can generate species-specific splicing events, and tissue-specific differences in RRNA splicing between vertebrate species occur 10-100 times moe frequently than changes in gene transcription (Barbosa-Morais etal. 2012; Merkin et al 2012). (A) Casserte exon: Type I procollagen GEES 1A: Precursor chondrocytes a a) t eet ' ‘SERED UB: Macure chondrocytes te? (©) Alternative 5 splice ste Bele FIGURE 2.25 Some examples of atemathe FINA sola, Blue ‘and ovited portions ofthe bers represent exars; gray represents introns. Aematve solling pattems ars shown with V-shaped Ines. (@)A “cassette tyalon) that can be used as exon or rerioved a8 =n non clstinguises tho type I colagen types of chondrocyte precursors and mature cnonateces (carting cal) (} Nataly sequences” at the 5° and 3° ends of the introns. These sequences are the “splice sites” of the intron, The splicing DE NRNA is mediated through complexes known as spliceo- somes that bind to the splice sites. Spliceosomes ate made up ‘of small nuclear RNAs (mRNAs) and proteins called splie- ing factors that bind to splice sites ot to the areas adjacent ‘o them, By their production of specific splicing factors, cells can gitfor in their ability to recognize a sequence as an intron, ‘That to say, a sequence that isan exo in one col type may bean intron in another (FIGURE 2.25A,8). In other instances, the factors in one cell might recognize different 5’ sites (at the beginning ofthe intron) or different 3 sites (atthe end of the intron; FIGURE 2.25¢,0) ‘The 5’ splice ste is normally recognized by small nuclear RNA Ul (U1 snRNA) and splicing factor 2 (SF2; also known as alternative splicing factor). The choice of alternative 3° splice sites is often controlled by which splice site can best bind a protein called U2AF. The spliceosome forms when the proteins that accumulate at the S' splice site contact those Proteins bound to the 3" splice site. Once the 5” and 3 ends are brought together, the intervening intron is excised and the two exons are ligated together. (B) Mutually excuse cxons FeR2 SD yh Link ectoderm 7 8 10 on ve a { SOD fh 2 Limb mesoderm eee - (D) Alternative 3 splice ste: Chorin ED Wild ype she cbs eneeesne omy 4 pee : FIGURE 2.92 _Hycothetioal made of he reguaton of -4 mANA ‘tandation byt PNAS. The in gene does not produce an "ANA, Rather, t produces small ANAS that are complementary to a rigeated socuence inthe 3° UTR of te r-14 mANA, which bina 0 ital prevent is translation. (Aer Wickers ard Takayama 1996) Proteins of the Argonaute family ace particulasly important members of this complex. Such small regulatory RNAs can bind to the 3 UTR of messages and inhibit their translation. In some cases (especially when the binding of the miRNA to the 3” UTR is perfect), the RNA is cleaved, More usually, however, several RISCs attach to sites on the 3° UTR and pre- vent the message from being translated (see Bartel 200¢; He and Hannon 2004) of ie sts con Gytoplasm —| fo fe Diver siRNA duplex SS fo» nd Asymmetric RISC ‘sembly Fong region aie ‘Translational repression FIGURE 2.83 Modal or th formation and use of miroINAS, ‘Tre mRNA gene encodes a pri-mFINA tat oten has several raion regions where the FNA Sos neartiy complementary bases wih which to pat The prk-miBNA is prooassed into eviua pre AnRNA “hapins” by te Drosra RNase, end these are exntec ‘tor the riceus. Once in the eytapiasm, another FNAase, Dice. ‘irises the non-base-parealoop, Dicar aso acts ae &neinase to Separaie the strands o the doubio-cranded miRNA. One strand itobably recognized by placement of Bice) packaged! wth totes ino the AN4nduoad siencng complex (ISG), whicn zseequenty binds fo the 2’ UTRs to efec transitional suppres sion or ceevage. depending (atleast is par} onthe trang af te complementary between the mRNA and s target. (ter He end Hannon 2004.)Lymphoid precursor call a Lymphoid precursor cll + virally activated miR187 ae 4 yoy Gg © Teall Number amber i181) i223 of Bees of Teells (no miR223} {mo miRISI) ——_enbanced reduced FIGURE 2.84 Lymotoid racursor cols can cenerate ether B cals fymohooytes thal make antibocies| or T cals (yriphocates that i val infected oa, depending on the organ which te pracurcor resides. Ths requation ofthe ineage pathway is con- ‘role! in part by levels of the miccoANA miz787. Tre ympnecyte precursor cat has ite mF. A oat has high ievo's of mE ‘whereas T calls do not appear ta have ary. Klyrpheoyle precursor cals are wel renstocied with mif?187, they preferentaly generate Balls atthe excense of T cals ‘The abundance of microRNAs and their apparent conser- vation among different groups-—inciding flies, nematodes, verterates, and even plants—soggest that such RNA regula tions previously unrecognized but potentially important seats of regulating gone expression. This hidden layer of gone regulation parallels the better known protein-level gene control mechanisms, and it may be just as important [ntepating cel fat. Indeed, computer analyses of miRNA sequences and their potontialtargels suggest that about half of our genes ace subject 0 contol by miroRNA® (Friedman tal 2009) ecent studies have shown that mica ae involved in mammalian heat and blood cell differentiation. Duting mouse heat development. the mictoRNA mi can repress the messages encoding the Hand? transition factor (hao 21 2008) The transcription factors ritalin the prolif eration of ventricle heart naecle cll, and miRd may control che balance betvreen ventricle growth and differentiation, The miR 57 miRNA is essential for committing progenitor cals to diferentate into Blymphocytes, and ectopic expres sion of iRTB7 in mice causes a preponderance of B lympho: ‘yes (FIGURE 2.36; Chen ot al 2004) MicroRas ae also used to “lean wp” and fine-tune the level of gene products. We mentioned those maternal RNAS in the oocyte that allow eary development to o¢cur, How does the embryo get td of matemnal RNAs once they have been used and the emmbryotc eels are making theit own RNAS? In zebrafish, this cleanup operation is assigned to microRNAs such as miR630, This is one ofthe first genes transcribed by the fish embryonic cell, and there are about 90 copies of the gene inthe zabratish genome. So the level of R430 goo up very reply. This microRNA has handreds of argts about 40% ofthe maternal RNA types) and when binds tthe 3° UTR ofthese target mRNA these RNAS lose theic polyA tails and are degraded (Giraldez ot al. 2006). Sight ater in development, this same microRNA is used in —S Differential Gene Expression in Development 63, the fish embryo to fine-tune the expression of Nodal mRNA (Choi et al. 2007), The consequence of this latter use of ‘miR430 is the determination of how many cells become com mitted to the endoderm and how many become committed tobe mesoderm, Although the microRNA is usually 22 bases long, it rec- ‘ognizes its target primarily through a “seed!” region of about 5 bases in the 5 end of the microRNA (usually at positions 2-7), This seed segion recognizes targets in the 3° UTR of the message. What happens, then, Ifan mRNA has a mutated 3° UTR? Such a mutation appears to have given rise to the ‘Texel sheep, abreod with a lerge and well-defined muscula- ture that is the dominant meat-producing sheep in Europe. Genetic techniques mapped the basis of the sheep's meaty phenotype to the myostatin gene. We have alveady seen that a mutation in the myostatin gene that prevents the proper splicing of the nRNA can produce a latge-muscled pheno: type (see Figure 2.27). Another way of reciucing the levels of myostatin involves a mutation in its 8'UTR sequence. In the Texel breed, there has been a G-to-A transition in the 3’ UTR of the gene for myostatin, creating a target for the rmir1 and mir266 microRNAs that are abundant in skeletal ‘muscle (Clop etal. 2006). This mutation causes the depletion cof myestatin messages and the increase in muscle mass char- acteristic of these sheep, The binding of microRNAs and their associated RISCs to the 9” UTR can regulate translation in two ways (FQURE 2.85; Filipowicz etal. 2008). Fst, this binding can block ink tiation of translation, preventing the binding of initiation fac- torsor ribosomes. The Argonaute proteins, for instance, have been found to bind dectly to the methylated guanosine cap at the 5 end of the mRNA message (Djurenovie etal. 2010, 2011}. Second, this binding can recruit endonucleases that (A) Initiation block Initiation complex (8) Endonuclease digestion (deadenyation) Coding mRNA Cop aaa Endonuclease digests polyA tail FIGURE 2.85 The miPNA complex, ictuaing numeous pro- tee tat bins to te -nFINA fmiANP), can block translaen in two male ways. (4) One way is by lacking the bredhg ofthe mRNA te ntaton factors or ribosomes. (B) The other way i by recruting fercnucieases to chew away the pay tal ofthe mRNA, thoreby causing #8 deststion. Attar Flows eta, 2008),64 Chanter 2 digest the mRNA, usually starting with the polyA tail (Guo et al. 2010), The later seems to be commonly used in mam malian cells, Control of RNA expression by cytoplasmic localization Not only isthe timing of m&NA translation regulated, but 30 is the place of RNA expression, A majority of mRNAs (about 70% in Drosopiila embryos) ate localized to specific places in the cell (Lécuyer tl. 2007), ust lke the selective repression of mRNA translation, the selective localization of messzges is often accomplished through theit 3’ UTRs, There are three ‘major mechanisins for the localization of an mRNA (see Palacios 2007); © Diffusion and local anchoring. Messenger RNAS such as numos diffuse freely in the cytoplasm. However, when they diffuse tothe posterior pole ofthe Drosophila oocyte. However, they ate trapped there by proteins tha resicie pacticulatly n these regions, These proteins also activate the mRNA, allowing it 30 be translated (FIGURE 2.368), ‘Localized protection. Messenger RNAS such as those encoding the Drosophila heat shock protein hsp83 {Which helps protect the emryos from thermal exttemes) also Eloat freely inthe cytoplasm, Like nanos mRNA, igp83 accumulates at the posterior pole but its mechanism for getting there is different. Throughout the embryo, the mRNA is degraded. However, proteins atthe posterior pole protect the hsp3 mRNA from being destroyed (FIG- URE 2.360), ‘Active transport along the cytoskeleton. This is probably _most widely used mechanism for mRNA localization. Here, the 3” UTR of the mRNA is recognized by pro teins that can bind these messages to “motor proteins” that travel along the cytoskeletan to their final destina: tion (FIGURE 2.960). These motor proteins are usually ATPases such as dynein or kinesin that spit ATP for ‘their motive force. We will see in Chapters 6 and 17 that this is very important for lacaliaing transcription factor MRNAS into different regions of the Drosepila aocyte. @ Soe WEBSITE 2.12 Stored mRNA in brain cells, FIGURE 2.56 Localization of mRNAs. (A) Difusion and local lanchoring, Nanos mRNA discos through the Drosophils 29g and is bound fn part by he Osiar protein atthe posterior end of the ‘.00/. Tis anchoring lows the aanoe mRNA to be translated. {G5 Lovalzed protecton. The mRNA tor Crosoania het shock rt tein (nso82} wl be degraded unlss it hinds toa protecter proton, (ithe 2260, algo atthe nosteror terminal of the aceyta. (2) Active transport on the cytoskeleton, causing the accumultion of RNA sta partcuar sta. Her, blocs MANA ze trensperied to the tert ofthe oneyte by den and kinesin moter proteins Mecrahi, (star MENA i brought othe posterer pole by trangpor along microtubules by nash ATPases. (ter Pezcios 2007 } (A) Diffusion and local anchoring Anterior _—~ (@== nanos mRNA being translated Q) Anchor | ramos BSA © Deadenyase = hyp mRNA G Protector protein complex ie complen (Gegrades hsp83 mRNA) eee nena ene (©) Active cansport along cytoskeleton | Gem oskarmRNA — Gee bicoid mRNA | | Boysen | ceiPosttranslational Regulation of Gene Expression ‘The story is not over when a protein is synthesized, Once a protein is made, t becomes part of a larger level of organiza tion. For instance, it may become part ofthe structural frame- work ofthe cell, or it may become involved in one ofthe many enzyenatic pathways for the synthesis or breakdown of cellular ‘metabolites. In any case, the individual protein isnow part ofa complex ecosystem that integrates it into a relationship with ‘numerous other proteins. Several changes can still take place that determine whether or not the protein will be active. Some newly synthesized proteins remain inactive until certain inhibitory sections are cleaved away. This is what happens when insulin is made from its larger protein pre- cursor. Some proteins must be “addressed” to their specific {intracellular destinations in order to funetion. Proteins are ‘often sequestered in certain regions ofthe cel, such as mem- branes, lysosomes, nuclei, or mitochondria. Some proteins need to assemble with other proteins in order to forma fune= tdonal unit. The hemoglobin protein, the microtubule, and the ‘bosome are all examples of multiple proteins joining togeth- certo forma functional unit. And some proteins are not active unless they bind an ion (such as Ca) or are modified by the Differential Gene Expression in Development 65 covalent addition of a phosphate ot acetate group. The impor- tance of this last type of protein modification will become obvious in Chapter 3, since many of the critical proteinsin embryonic cals just sit theze until some signa! activates them Coda ‘The emergence ofthe physical argenism is orchestrated by its inherited genome. Thi process does not require “decoding” that genome so much a8 interpreting it, much as an arches- tra interprets a rmusical score. [ris important to remember that all the processes we have discussed in this chapter—the interacting transcription factors, the binding of RNA poly- merase Il tothe promoter, the initiation of transcription, the elongation of the mRNAs, the kinetics of RNA splicing, and the hal ives of mRNAS in the cytoplasm—are all stochastic events. Each event depends on the different concentrations of the interacting proteins (Cacace et al. 2012; Murugan and Kreiman 2012; Costa et al. 2013; Neuert et al. 2013). Thus, each organism is a unique “performance” coordinated by interactions that tell the individual cell which genes are to be expressed in that cell and which genes are to remain silent. (Chapter 3 will detail the mechanisms by which cells tell one another to activate only 4 certain subset of the genome. ONC C NAOT EL LCC LN ONO t TILT SNAPSHOT SUMMARY: Differential Gene Expression in Development 1. Evidence from molecular biology, csi biology and somatic ‘call rucisr cloring has shown that each cel of the body (wth very fee exceptions) caries the sama nuciser gonome, 2. Differential gene expression from geneteally idorical mucia crsates diferent col types. Dilerential gene expression ‘can occur at the levels of gene transcription, nuclear ANA processing, mRNA tvansiation, and protein modification Notice that RNA procassing and export can occut vile {ne ANA is sti beng wransertoed from the gene 3. Crromatin is made of DNA and proteins. The Histone pro: {ins tor nucioosames, and tho mathy’ston and acstyie tion of specic hstone residues can activate or repress ‘gene transcration 4. Histone rrethylation is often used to sience gene expres 80n, Histones can be metylated by histone methytrans- ‘rasan and demethylated by histone demethylases. 5. Acetylated histones are often associated with active gene ‘expression, Histone acatytvansferases sco acetyl groups toristones, whle histone dscetyiasas romove ther. 8. Eukaryotic ganes contain promoter sequanees to which FRNA polymerese Ii can bid to inte transcriation. To -Sccompish this, the eukaryotic RNA po'ymerases ere bound! by a series of proteins called transcristor-assodlat 9 factors including TEID and TFS. 7. Eukaryotic genss expressed in specie cot types contain ‘enhancer sequences that regulate ther tenscristion in time and space, Enhsncers aotivate genes on the same @womosomna, Enhanear sequences can be within ions or the 3'UTR: they can even be millons of base pais away fiom the gone thay aetvate. Enhencers can ao act as once to suppress the transcription of @ gene in inap~ propriate ost types 8, Spoctc transcript factors can recognize speciic ‘sequences of DNA inthe promoter and enhancer regions. “These protains actvate or repress transcription from the gonee to which they have bound 9, Enhancer work in a combiratotial faction, The oinling of ‘Several transcrption factors can act fo promote or inhibit tranecrintion from a cata romoter In some cases, tren- ‘scription is activated oniy i both factor A and factor B are present; in othor cases, ranscription is activated if ether factor A or factor is present 410. Enhancers work ina modular fashion, A gene can contain several enhancers, ch directing the geneé expression ‘a partoular eal typo, 11. Agene encoding a transcription factor can maintain itsst In the activsted state ithe transcription facto it encodes also activates ts o#n promoter Thus, a transcription factor (gore can have cre set of enhancer sequeres to iliate fis activation and 2 second set of enancer sequences