ature '
`!\VIEWS
`
`~,o,o,orne U oo S
`1' 2
`ture com/reviews
`··
`,.,,,na
`
`Nature reviews. Genetics.
`rh11
`QH426 .N38
`v. 11 , no. 6 (2010 June)
`IIIIIIIIIIIIIIIIIIIIIJllllll/lllllllllllllllllllll/11111/111 c-Ser
`2001 -229989
`
`GENETICS
`
`I
`
`'
`
`.
`
`j ,I
`Pl REACTIONS
`115Cription factors and chromatin
`. ~
`set, or inducible gene expression
`
`The drugs don't work
`Evolutionary dynamics of bacterial
`antibiotic resistance
`
`LCY Biotechnology Holding, Inc.
`Ex. 1061
`Page 1 of 16
`
`

`

`I
`
`Hunting rare variants p41 5
`
`REVIEWS
`
`39 1
`
`405
`
`Condensin and cohesin complexity:
`the expanding repertoire of functions
`Andrew J. Wood, Aaron F. Severson and
`Barbara/. Meyer
`Cohesin a nd condensin are best known for the ir
`roles in mitosis, but these complexes achieve
`remarkable functional diversity and specificity,
`Recent s tudies have demonstra ted their involvement
`in genome organization, gene expression, organismal
`development c1nd meiosis.
`
`The population genetics of antibiotic
`resistance: integrating molecular
`mechanisms and treatment contexts
`R. Craig Maclean. Alex R. Hall. Gabriel C. Perron
`and Angus Buckling
`The authors discuss t he evo lutiona ry dyna mics of
`antibiotic resist ance in bacteria in rel.at io n t o the
`co mplex int e rplay between population ge netic
`factors a nd the spatial and temporal pattern of
`antibiot ic use.
`
`4 15
`<m>
`
`426
`
`fEA'TURED
`ARTICLE
`
`Uncovering the roles of rare variants
`in common disease through
`whole-genome sequencing
`Elizabeth T. Cirulli und David B. Goldstein
`Genome-wid e association studies have explained
`only a smaU frac tion of t he genetic basis of complex
`diseases. This Review argues that rare variants could
`have a substant ial effect o n genetic pre d is position to
`common disease, a nd t he authors outline discovery
`strategies based o n whole-genome seque ncing for
`ident ifying these genetic risk fac tors.
`
`Inducible gene expression:
`diverse regulatory mechanisms
`Vikki M. Weake and Jerry L Workman
`The rapid ind uct ion of specific sets of genes is required
`for cells to respond to external cues. Transcription of
`eukaryotic inducible genes is controlled at multiple
`steps , including activator recruitme nt and polymerase
`pausing, and is influe nced by chrom atin remodelling
`and signal transduction.
`
`On the web www.nature.com1reviews19enetiu
`Advance online public ation
`il.."Pet"at -. an aJvance o n line puhlication (J\OP• sc-rvicr lo.- .:iuth<irs ,1nc1
`ft~'!> ro..-iew the lare n artidc~ published on!i~ ,lhc,1d of print
`forihcoming articles
`~)'J)e inipt1\ation for genome-wide .inocia tion studies
`11\an Mr1rc-M11i and Uryan Howie
`I.
`t~~~ene, aUon genon1ic1,: an Integrative approach
`wwrd llowklm. GaryC. Hon and 81119 Ren
`1ionary mkrobial ge nomics: iruigh u into b.-.c.,~ial host a daptation
`~
`'""'1111,tu To/tand SivG, LAnder:sson
`
`Links to further information
`The fuH te:itt o Fart icle~ inclt.Kies:
`• Auth-O r biogr~ies
`• Lirlli:stoi:jlossaryterms
`w Link~ to~eMSil!ld p,rote.tl~ in ddt dbasessuchas
`Entrez., flyBase, OM IM and UniPn;,tKB.
`At a glance provides a bullet-pointe dsumm.1ryof
`the m-,i11 topics covered ln each c1rt1de.
`
`More article s like this for fUfthe, reading.
`Ea.ch Rev~cOJl(;[udes with a 'Mo.-e .-..r1iclei like tlus'
`box cont <1ining Iris t o rr.la1c d content 011 NPC,
`Featured article can be browsed onlin~ hy
`rc,gi~tcred IT)(!mbers of nalufe.com.
`
`PowerPoint figures
`F'tgurasC:llf1 t>edownloadedasa PowerPoint file for
`l1se in ~ese11tatioos and for 1e-,d1ing
`E-alert table of contents
`Get m onthlye-rooi! ~lm1:s to the conteo1t of ltiis
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`Online c orresponde nce
`Comments from readers i'bour our .1rtk:lr-~.
`Al! cor, espondence will bol hi~li9htcd on t i~ t,,bl,I)
`of co11tents ar1d on o3--mail all'rts,,
`
`LCY Biotechnology Holding, Inc.
`Ex. 1061
`Page 2 of 16
`
`

`

`CONTENTS
`
`FROM THE EDITORS
`3 8 S An editorial commentary and introduction to this month's issue
`
`RESEARCH HIGHLIGHTS
`3 8 6 Selected highlights from the recent research literature
`
`4 3 9
`
`AN INTERVIEW WITH ...
`3 9 0 Shinya Yamanaka
`PERSPECTIVES
`OPINION
`Insulators and promoters: closer than we think
`Jesse R. Raab and Rohinton T. Kamakaka
`Increasing evidence suggests functional similarities between
`promoters and insulators. The authors propose that these findings
`unify existing models of insulator function, provide new directions
`for understanding how insulators work and suggest t hat insulators
`have evolved from promoters.
`
`VIEWPOINT
`4 46 Missing heritability and strategies for finding the underlying
`causes of complex disease
`Evan E. Eich/er.Jonathan Flint, Greg Gibson, Augustine Kong,
`Suzanne M. Leal.Jason H. Moore and Joseph H. Nadeau
`Seven leading geneticists express their views about where the
`unidentified components of the heritability forcomplexhuman dis.eases
`might lie and how this could affect the underlying genetic architecture,
`as well as offering suggestions of how genomic research could be
`targeted to address this key issue.
`
`SERIES
`
`APPLICATIONS OF
`NEXT-GENERATION
`SEQUENCING
`http://www.nature.com/nr 1
`series/nextgeneration
`g
`
`SERIES
`MODES OF
`TRANSCRIPTIONAL
`REGULATIO['J
`The articles in this series consider
`the range of levels at which
`transcription is controlled. the
`molecules involved and how
`the modes of regulation are
`adapted to particular type, of
`gene or developmental contexts.
`http:/ /www.nature.com/nrg/
`series/transcriptionalregulation
`
`nature
`REVIEWS
`
`GENETICS
`
`DISCOUNT SU!ISCRIPTIONS
`SllJOENTS,ID,d)'Q\lkl'IOWtMt.-.ont.~
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`Tcl:•12111269H4:Fd>'.: • \J1)Jlf.\Nlll
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`C
`
`LCY Biotechnology Holding, Inc.
`Ex. 1061
`Page 3 of 16
`
`

`

`0acure
`
`R£VIEWS
`~
`
`, c;OVll! 'Solnething N111' by fl.trick Mo..gan
`~ by l l'Mt il:eo,iew on p416.
`
`TAMACA!Cl
`
`l!AArMi.lU S
`
`MH RA SWAMI
`
`FROM THE EDITORS
`
`- - -
`
`- - - --- --- ---------- --- ---
`
`search in PubMed for papers with 'transcription' in the title or
`
`abstract published during the last year and you will get a staggering
`number of hits - more than 20,00 0 studies -
`that reflects the
`importance of this first step in gene expression. In this issue
`of Natwe Reviews Genetics we launch a series of articles on M odes of
`Transcri ptional Regulation that highlights the continuing interest in this topic.
`The first article in our series, the Review on p426 by Weake and
`Workman, examines how inducible gene regulation occurs at the level
`of transcription in eukaryotes. Classical models such as the Gal4 system of
`budding yeast and the heat-shock genes of Drosophila melanogaster
`are still widely used and cont inue to provid e new insights. Recent
`examples include the importance of RNA polymerase II pausing in
`inducible expression at many genes and the direct involvement of signal
`transduction proteins in transcript ional activation.
`Interest is growing not only in the proteins that regulate transcription,
`but also in the DNA elements to which these proteins bind. The molecular
`genetic basis of variation in transcription levels in humans has recently
`become accessible to study, thanks to genome-wide maps of nucleotide
`polymorphisms combined with technological advances. A Research Highlight
`on p388 describes a new statistical approach that extends these studies to
`understanding the coordination of transcriptional regulation across human
`tissues. Meanwhile, a particular class of regulatory element -
`insulators -
`are the topic of the Opinion article on p439. Here, Raab and Kamakaka
`present the evidence t hat insulators are closely related to promoters - a
`theory that could help to bring together conflicting models of insulator
`function. The signs are that transcriptional regulation will be the topic of
`many more studies for years to come.
`
`EDITO .. IAL ASIISTANrs: J,xq,Mn Smit,
`l.Ao~acoms
`Wl!:B l"ROOUCTIOH MANAOU t :
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`LCY Biotechnology Holding, Inc.
`Ex. 1061
`Page 4 of 16
`
`

`

`REVIEWS
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`(@ MODES OF TRANSCRIPTIONAL REGULATION
`
`Inducible gene expression:
`diverse regulatory mechanisms
`
`Vikki M. Weake and Jerry L. Workman
`Abstract I The rapid activation of gene expression in response to stimuli occurs largely
`through the regulation of RNA polymerase I I-dependent transcription. In this Review, we
`discuss events that occur during the transcription cycle in eukaryotes that are important
`for the rapid and specific activation of gene expression in response to external stimuli.
`In addition to regulated recruitment of the transcription machinery to the promoter, it has
`now been shown that control steps can include chromatin remodelling and the release of
`paused polymerase. Recent work suggests that some components of signal transduction
`cascades also play an integral part in activating transcription at target genes.
`
`Chromatin
`A nucleoprotein structure
`formed of repeating
`nucleosomal units in which
`I 4 7 base pairs of DNA are
`wrapped around an octamer of
`hi stone proteins consisting
`of an H3-H4 tetramer flanked
`by two H2A-H2B dimers
`
`Co-activator
`A protein that is recruited to
`promoters through interactions
`with transcriptional activators,
`and facilitates transcriptional
`activation through the
`recruitment of RNA
`polymera se II and the general
`transcription factors Many
`co-activators also catalyse
`chromatin modifications that
`assist the kinetics of
`recruitment of tl1e general
`transcription machinery
`
`Stowers Institute for Medical
`Research, 1000 East 50th
`Street, Kansos City,
`Missouri 64 110, USA.
`Correspondence to J_L W
`e-mail: jlw@stowers.org
`doi: 10 1038/nrg2781
`Published online
`27 April 20 I 0
`
`Cells must be able to rapidly respond to changes in
`such as temperature or
`their external environment -
`nutrient availability -
`to exploit and survive in new
`conditions. Even cells in a multicellular organism need
`to respond to developmental cues such as signalling
`molecules to determine when to divide, migrate or
`die. The production of new proteins in response to
`external stimuli results largely from rapid activation
`of gene transcription -
`this is known as inducible
`gene expression.
`Inducible gene expression has several features
`that distinguish it from the expression of genes that
`are constitutively active (for example, housekeep(cid:173)
`ing genes). Inducible genes are highly regulated and
`must be able to be rapidly and specifically activated
`in response to stimuli. Once the stimulus is removed,
`an inducible gene must quickly return to its basal,
`inactive state. Furthermore, multiple genes must
`often be synchronously activated in response to the
`same stimulus, such that the proteins required to
`respond to the stimulus are produced simultaneously
`at the appropriate relative levels. Similarly, multiple
`cells in an organism must respond to developmental
`cues in a coordinated fashion so that the appropriate
`morphogenetic process occurs over a broad region
`of cells.
`Here, we discuss mechanisms of inducible gene
`expression used by eukaryotic cells. Although proc(cid:173)
`esses that occur following transcription such as protein
`translation are also regulated as part of inducible gene
`expression, we do not discuss them in this Review.
`We focus on the events that are important for recruit(cid:173)
`ment of the transcription machinery and initiation of
`
`RNA polymerase II (Pol II)-dependent transcription.
`Although a traditional model of activator-dependent
`recruitment of Pol II and the general transcription
`factors (GTFs) holds true for many inducible genes,
`recent studies suggest that Pol II is already present and
`poised for transcription at many inducible genes 1
`6
`-
`•
`Therefore, it is becoming increasingly apparent that
`there is an additional level of regulation that occurs
`during the initial stages of transcription elongation
`before Pol II is released into a productive transcription
`cycle. In addition, several recent studies suggest that
`some components of signal transduction cascades
`that lead to inducib le gene expressi.Oll Lh.ni-we rc once
`tholtght to function exclusively ln the cytopla m such
`as mit o gen -actival cd protein kinases (MAPK) ru·~
`rec nlited to cllromaLin aud aJ'e Integral c:omponcnls or
`transcription complexes7
`•
`We use three well-characterized examples of induc(cid:173)
`ible gene expression to illustrate some of the key mecha(cid:173)
`nisms involved in transcription a tLvation in response
`to stimuli: al gene Jnduction m res pons
`to galactose
`in 'acclu1romyces cerevisiae, heat-shock gene inductioi: ill
`Drosophila melanognster and osmos lress regulation
`in S. cerevi.siae. We first discuss the initial. teps I f lh:
`transcription yde: activator-dependent recruitment ot·
`the transcriptionalmachinery and the role of co, acuvators
`and nucieo ome-remodelling complexe in facilitat(cid:173)
`ing thi · recruil.meut. Vve then exami1Je the even t lbal
`occur ~ llowlng recruitment of the gen .ral transalfJ!tOn
`machinery, including promoter clearan ce nnd release
`oi pau edPol II into productive transcription eJ~n~~(cid:173)
`Lioo. FinallJr, we examin e the role of ignalliJ1g kin, se;
`that seem to play an integral partin multiple a pe 1 0
`
`426 I JUNE 2010 I VOLUME 11
`
`LCY Biotechnology Holding, Inc.
`Ex. 1061
`Page 5 of 16
`
`

`

`Target gene
`
`Fig ure 1 I Early steps in the transcription cycle.
`a I Promoter select io n is determined by the lnteract ion
`of one or more transcriptional act ivator(s) with specific
`DNA sequences (recognition sites) near target genes.
`Activators then recruit components of t he transc ription
`machinory to these genes t hrough protei n- protein
`interactio ns.. b I Activation of gene expression is induced
`by the sequential recruitment of la rge mult i-subun it
`protein co~activator compte-xes. (show n In purp le and
`pin k) through b inding to activators. Activators also
`re cr11it ATP- dependent nucleo some-remodelling
`complexes. which move or d isplace hi stones at the
`promoter, focilitatiriy the rapid recru itment and
`a ssem bly of co-~ctivator~ a nd the genera l transcription
`machinery. c I Together, co-activators a nd nucleosome
`rernodellers facilitate t he rapid recru;tment of RNA
`polymerase II (Pol II) and t he gen~rill transcript ion
`factors (GTFs) TFIIA, TFIIB, TFIID, TFIIE, TFIIF a n d TFIIH
`to form t he p re-initiation complex (PlC) o n the c o re
`p romoter•. Thc~e firs t three steps{a-c) constitute act i(cid:173)
`vator-dependent recruitment. d I After PIC assembly,
`CD K7 in human TFIIH (Kin28 in yeast) phosp horylate,
`the serine 5 {55) posit io n of the Pol II ca rboxy-terminal
`domain (CTD). At t he same t ime, another subun it of
`TFIIH, the DNA helicase XPB (Rad 25 in yeast), remodels
`the PIC, and 11-15 bases of ONA at the t ranscript ion
`start site (TSS) is unwound to introduce a
`single -stranded DNA temp\ate into t he active site
`of Pol 11°. Pol II then dissociates from some of the GTFs
`and transitions into an early e longation stag~ of
`rrans.criptio n~J_ Thi:s ~tep is often referred to as promoter
`escape or clearance but is not sufficient for efficient
`passage of Pol U into the rema inde r of the gene.
`e I Following p romoter clearance, Pol II transc ribes
`20 -40 nucleo tides into t he ge ne and halts at t he
`p romoter-proximal p ause site. Efficient e longation by
`Pol II re q u ires a seco ri<l pho:sphorylation event at the S2
`position of the Po l II CTD by CDK9.a subunit of human
`P-TEFb (Ctkl in yeast)11
`11u_ Phosphorylation o f the CTD
`•
`c reates binding site s for proteins t hat a re impo rtant for
`mRNA processing and t ranscription t hrough ch ro matin
`~uc h as t he histone H3 lysine 36 (H3 K36~ methylase
`5ET2 (RC:F. I 04). Nucleosome remodellers also facilitate
`pass.:ige of Pol Ii du ring t he elongation phase o f
`transcription. The transcription cycle continues with
`elongation o f t he tn:rnsc, ipt by Po l IL followed by
`termination a nd re-init iatiOn of a new round o f
`trc1nscript ion (not shown).
`
`d
`
`Nucleosorne(cid:173)
`remodelHng complex
`
`H2A
`
`H2B
`
`Ce-neraJ transcriptlon
`machinery
`RNA polyrne-ra~ II togeth-er
`''iith the general transcripliun
`facto,s THIA. TFIIB. TFIID,
`l FIJE, TF11F and n-nH
`
`Transcriptional activator
`A.~ueoce-spccific
`DNA-bindirl!! protein that
`in-c,eaSt-s the rate of
`lra~criptiun hy N'!Cruitin,e RNA
`~~nl€rasc II. Cilher directly
`in Drok.'=lryotes Of through
`CO-attivators in eukaryotes
`
`t hese initial stages of the transcription cycle. Although
`the mechanisms involved in the chosen examples may
`not always be observed in all other cases of inducible
`gene expression, we hope to p rovide a broad o ver(cid:173)
`view of the principles involved in indu cib le activation
`of transcription.
`
`Activator-dependent recruitment
`Gene a c tivation in volves a multistcp recruitment
`process that co nsists of several potential rate-limiting
`steps during the in itial stages of the lranscription cyclr
`(reviewed in REF. 8) [FIG I J. During the initial steps of
`gene induction, transcriptional act[vators bind Lo specific
`DNA sequences ntar target genes and recruit tr-.an s crip (cid:173)
`tional co-activators and components of the transcription
`
`machinery to th e.sl.' gen.es through protein-protein
`interactio ns. These s teps resu lt in formation of the
`pre-initiatfori ( o m1Jlex (PIC) on t he promoter9•1-0_ For
`the purposes of this Review, these first three steps
`can be regarded as a single rate- determining process.
`which we refer Lo as activator-dependent recruitment
`(FIG. I a-cJ. An additional level o f regulation is required
`for polymerase to proceed to productive transcription
`elongation (FIG Id.el. Although all of the steps in the
`t ranscription cycle are subject to regulation 11, we focus
`in this Review on those steps that are mo~t impor tant
`for inducible gene expression: activator-dependen t
`recruitment resulting in PIC formation; activation of the
`PIC and transcription initiation; a n d release of paused
`polymerase into productive elongation.
`
`NATURE REVIEWS I GENETICS
`L
`
`VOL UME 11 I JUNE 20 1-0 I (cid:141) 27
`
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`

`REVIEWS
`
`b
`
`d
`
`e
`
`! • Galactose
`
`! Act;vato,-dependent SAGA recru;rment
`
`j Activator-depend. ent Mediator recruitment
`
`Nudeosome acetylation by SAGA
`stimulates nucleosome remodelling by
`SWI/SNF. whfch 1s also recruited by Ga\4
`
`Figure 2 I Gal4-mediated induction of Gal gene expression requires co-activators.
`In yeast, in the absence of galactose, the acidic activator Gal4 is bound by its
`repressor Gal80 (a). Addition of galactose to the growth mediurn causes an inducer
`protein, Ga 13, to bind and sequester Gal80 in the cytoplasm, releasing it from Gal4 {b).
`Gal4 binds target UASGAI. {upstream activating sequence) sites. in the promoters
`of G<1l genes such as GAL1 and s.equenrially recruits co-activators, such as the
`acetyltrans.ferase SAGA (c) an.d Mediator {d). Gal4 also recruits ATP-dependent
`nucleosome-remodelling complexes such as SWI/SNF that remove nudeosomes at
`rhe promoter and are srimulated by SAGA-catalysed histone acetylation.Together,
`SAGA and Mediator recruit RNA polymerase II and the general transcription factors
`(GTFs). leading to formation of the pre-initiation complex (PIC) (e}. Nudeosome
`removal. cat<1lysed by SWI/SNF, aids in the kinetics of Mediator and GTF recruitment,
`thereby facilitating rapid PIC formarion and initiation of transcription at Gal genes.
`H3Ac, histone H3 acetylatfon; TBP, TATA-binding protein: TSS. transcription start site.
`
`Gaf4-mediated Gal gene induction in yeast, TI1eexprcssion
`o f Gal genes, wh ich en code _prod ucts th at arc required
`for the import and m etabohsm o f galactosc, is rapid! ,
`induced when galactose is added to the growth mediu,)
`o f S. cenwisiae (reviewed in REF 12). Activation orut
`Gal genes is regula ted pri m ar ily through a,tivaic,r~
`dep e ndent rec ru it menl. Ex pr ession is in itiated b
`the transcriptio naJ activator Gal4, wh ich b inds to a~
`upstream acth1ating sequence (UASGAi) in the promot(cid:173)
`ers of Gal genes (FIG 2). The affinity of Gal4 binding var(cid:173)
`ies among lhe Gal genes, thereby leading to differential
`2
`levels of activation 1
`•
`The Jnilia ti o n of the entire response of the Gal reg(cid:173)
`ulon to galaet"ose is dependent on this transcriptional
`activator, Gal4. How then is Gal4 itself regulated? The
`regions of Gal4 Lhat contain the DNA-binding and
`transcription-activation activities are separahle1'. In
`the absence of galactose, the acidic aclivdtlon domain of
`Gal4 is bound tightly by an inhibitor protein Gal80.
`This prevents the interaction of this domain with
`co-activators, such as TATA-binding protein (TBP) or
`the SAGA acetyltransferase complex" 15• When galac(cid:173)
`tose is added to the growth medium, an inducer protein
`Gal3 sequesters Gal80) alleviating repression of Gal4
`and allowing it to interact with and recruit co-activators
`11
`to the Gal ge11esi6
`• Th~ activation function of Ga14
`-
`is further regulated. by posl-lranslalional mechanisms
`that include phosphorylation and ubiquitin-mediatcd
`degradation 11,
`
`Gal gene induction requires co-activators. In Gal gene
`induction and many other examples of inducible
`gene expression> recruitment of co-activators and lhe
`transcription machinery to promoter regions is the key
`initial step in activating transcription. Recruitment of
`co-activators to Gal genes occurs in a sequential but
`not necessarily interdependent manner. The first co(cid:173)
`activator to bind to Gal promoters following a shift to
`galactusc-containing medium is SAGA, which is directly
`recruited by Gal4 JREFS 22-24). A few minutes follow(cid:173)
`ing this, Mediator is recruited to Gal promoters through
`d irecl contact with Gal4 (REFS ,3,,S- 28). Finally, Pol II
`and components of the general transcription ,nachinery,
`including TBP, TFIIH, Tfl!E and 'l'FIIF, are rccrui1ed
`to Gal promoters2·1• None of these fin al com ponents,
`including Pol ll, is recruited in the absence of SAGA,
`which indicates that Gal4 alone is not suflJ.cicnl to acti(cid:173)
`vate Lranscriptionz-u,_ Rat her, a combination of SAGA
`and Mediator aclivities is required for the recruitment of
`T IJP, Pol TI and the remainder of the GTFs" ·"""·~-
`Allhough 1his respo nse to galactosc m ight seem
`specific to yeast and other closely related fungi, '.he
`m echanisms by which Gal4 activates gene expression
`must be widely conserved because Gal4 can ~1ctivate
`UAS -.
`-specific transcriptio n in o rganisms 1ha1 range
`front\D. mefa11ogaster to huma ns.11,J1 • Furtherrnore,
`
`SAGA, Mediator and the GT Ps are highly conserv~•:
`from yeast to human s {Supplementary infonnntion
`1
`(figure and tables)). The presence of cis-regulatory e _~(cid:173)
`ments that have varying affinities for Gal4 at many ch ·
`ferent galactose-ind ucible genes p rovides a mecha.nisOl
`
`-t2s"fTuNt-: 20m I \'CH,U,\-IF. 11
`
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`REVIEWS
`
`to co(lrdinate both the t iming and relative le,;els nf
`expression of the Gal gene.,. In higher eukaryotes, genes
`that are co-regulated often share coinmon cis-regulatory
`elements. T hese regulatory elements can be bound by
`individual activators or by combinations of transcrip(cid:173)
`tional activalor.'i that have varying affinities. J-.or exam(cid:173)
`ple, the Forkhead and Ets transcriptional activators bind
`together to the same DNA motif that is present upstream
`o f a set of co-regulated genes to synergistically acti(cid:173)
`vate the transcription of the~ genes in the developing
`vascular cndothelium1-'.
`
`Co-activators facilitate gene activation
`In prokaryotc.i;, transcriptional activator~ J ircctly contact
`, so why are co-activators required in
`RNA polymerascl4
`eukaryotes? Many .-.tudies have shown that. in contrast
`to prokaryotes, mo..i;t genomic DNA in a eukaryotic cell
`is compacted into chromatin and therefore is not directly
`acce~sible to components of the general transcription
`machinery (reviewed in RE~. lo), Although some tran(cid:173)
`scriptional activators such as the- human glucocorticoi<l
`receptor can bind t heir target DNA sequence in a
`nucleosmnal context", PIC formation and subsequent
`transcription are inhibited by nudeosomes in vitro'1 >'I,
`Furthermore, studies in yeast suggest that inducible
`genes tend to have a higher density of m,cleosomes cov(cid:173)
`ering their promoters than constitutively active genes,
`which have more open, nucleosome-deplete<l promot(cid:173)
`ers (reviewed in REF 40). In eukaryotes, co-activators
`and nucleosome-remodelling complexes act together to
`facilitate gene activation in a nucleosomal context.
`
`Co-activators are required for PIC formation .
`Overcoming the nucleosornal ba11 ic:r to transcrip(cid:173)
`tion initiation requires complexes ~uch as SAGA and
`Mediator. lnlr iguing1y, complexes involved in tran(cid:173)
`scriptio n activation often possess enzymatic activities
`directed towards the amino-terminal rails ofhistunc pro•
`tci ns in the nucleosome. SAGA, for instance, contains
`the histone acetyltransferasc (H AT) Gens (REF 41 J. So,
`are histone-modifying activities 1cquired for recruitment
`of Pol JI and the general transcription machinery?
`Although nucleosome acetylatiun by activator(cid:173)
`recruited SAGA stimulates tr.1nscription i11 vitro~1••',
`surprisingly, the acetyltransferase activity of SAGA is
`n ot directly required for Po l II recruitment and P IC
`formation at Gal genes". However, mutations in other
`SAGA components such as Spt3 that only modestly
`reduce SAGA recruitment substantially decrease PIC
`formation 22-2(cid:141) . Therefore, importantly, the co-activator
`function of SAGA requires more than its enzymatic
`activity, Rother, both SAGA and Mediator have struc(cid:173)
`tural roles during inducible gene expression, forming
`a scaffold on which components of the general tran(cid:173)
`scription machinery and l'ol II con assemble. Mediator
`interact., directly with the ,mphosphorylated form of
`the carboxy-terminal heptapeptide repeat sequences
`(carboxy-terminal domain; CTD) of the RBPl subunil
`of Pol II (reviewed in REF. 44). Therefore, in the case o f
`Gal gene induction, Mediator links Gal4 and the general
`transcription machinery.
`
`A HAT-independent role for othl.-r co•activators such
`as p300 bas also lx.'Cn reported. Mutation of the KIX tran(cid:173)
`scription factor-binding domain of mouse p300 results
`in severe defects in haenrn.topoiesis but mutation of the
`HAT dumain has little effect". Furthermore, the role uf
`human PCAF (also known as KAT211) as a co-activator
`in activation of human Tcell leukacm in virus type J
`long terminal repeat transcription is al."iO independent
`ofits HAT activity ... Although these example.show that
`some HATs can have h istone acetylation-indcpendent
`functions in gene activation, at some genes, hi~tone
`acetylation is required for PIC formation. For example,
`in yeast, mutation of Lhe HAT GcnS in SAGA decreases
`Lhe levels ofTBP and Pol JI recruitment nt some genes47
`•
`However, the findings discusse<l above show that, in the
`early stagl's of the transcription cycle, the strucrural role
`of co-activators is of at least equal importance to their
`histone-modifying activilies.
`
`Chromatin remodelling and tran,criptlon
`To understand why histone acetylation is required for
`the activation of some genes but not others, we need to
`consider another clas.i; of transcription regulators known
`as ATP-dependent nucleo-.ome remodelling complt~xPs.
`Nudcosome-remodelling cnmplCJ<es use the energy from
`Al'P hydrolysis to move histom:sur dis-place them from one
`piece of DNA onto another or onto a h istone-binding
`protein, known as a histone chaperone (reviewed in
`REF' 48). Followinggalactosei.nduclion, Gal4 recruits the
`nudeosome-remudelling c