R E V I E W S
`
` M O D E S O F T R A N S C R I P T I O N A L R E G U L AT I O N
`
`Inducible gene expression:
`diverse regulatory mechanisms
`
`Vikki M. Weake and Jerry L. Workman
`
`Abstract | The rapid activation of gene expression in response to stimuli occurs largely
`through the regulation of RNA polymerase II-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
`147 base pairs of DNA are
`wrapped around an octamer of
`histone 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
`polymerase II and the general
`transcription factors. Many
`co-activators also catalyse
`chromatin modifications that
`assist the kinetics of
`recruitment of the general
`transcription machinery.
`
`Stowers Institute for Medical
`Research, 1000 East 50th
`Street, Kansas City,
`Missouri 64110, USA.
`Correspondence to J.L.W.
`e-mail: jlw@stowers.org
`doi:10.1038/nrg2781
`Published online
`27 April 2010
`
`Cells must be able to rapidly respond to changes in
`their external environment — such as temperature or
`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-
`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-
`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-
`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 genes1–6.
`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 inducible gene expression that were once
`thought to function exclusively in the cytoplasm such
`as mitogen-activated protein kinases (MAPKs) are
`recruited to chromatin and are integral components of
`transcription complexes7.
`We use three well-characterized examples of induc-
`ible gene expression to illustrate some of the key mecha-
`nisms involved in transcription activation in response
`to stimuli: Gal gene induction in response to galactose
`in Saccharomyces cerevisiae, heat-shock gene induction in
`
`Drosophila melanogaster and osmostress regulation r
`in S. cerevisiae. We first discuss the initial steps of the
`transcription cycle: activator-dependent recruitment of
`the transcriptional machinery and the role of co-activators
`and nucleosome-remodelling complexes in facilitat-
`ing this recruitment. We then examine the events that
`occur following recruitment of the general transcription
`machinery, including promoter clearance and release
`of paused Pol II into productive transcription elonga-
`tion. Finally, we examine the role of signalling kinases
`that seem to play an integral part in multiple aspects of
`
`426 | JUNE 2010 | VOLUME 11
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`
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`
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`R E V I E W S
`
`Figure 1 | Early steps in the transcription cycle.
`a | Promoter selection is determined by the interaction
`of one or more transcriptional activator(s) with specific
`DNA sequences (recognition sites) near target genes.
`Activators then recruit components of the transcription
`machinery to these genes through protein–protein
`interactions. b | Activation of gene expression is induced
`by the sequential recruitment of large multi-subunit
`protein co-activator complexes (shown in purple and
`pink) through binding to activators. Activators also
`recruit ATP-dependent nucleosome-remodelling
`complexes, which move or displace histones at the
`promoter, facilitating the rapid recruitment and
`assembly of co-activators and the general transcription
`machinery. c | Together, co-activators and nucleosome
`remodellers facilitate the rapid recruitment of RNA
`polymerase II (Pol II) and the general transcription
`factors (GTFs) TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH
`to form the pre-initiation complex (PIC) on the core
`promoter9. These first three steps (a–c) constitute acti-
`vator-dependent recruitment. d | After PIC assembly,
`CDK7 in human TFIIH (Kin28 in yeast) phosphorylates
`the serine 5 (S5) position of the Pol II carboxy-terminal
`domain (CTD). At the same time, another subunit of
`TFIIH, the DNA helicase XPB (Rad25 in yeast), remodels
`the PIC, and 11–15 bases of DNA at the transcription
`start site (TSS) is unwound to introduce a
`single-stranded DNA template into the active site
`of Pol II83. Pol II then dissociates from some of the GTFs
`and transitions into an early elongation stage of
`transcription83. This step is often referred to as promoter
`escape or clearance but is not sufficient for efficient
`passage of Pol II into the remainder of the gene.
`e | Following promoter clearance, Pol II transcribes
`20 – 40 nucleotides into the gene and halts at the
`promoter-proximal pause site. Efficient elongation by
`Pol II requires a second phosphorylation event at the S2
`position of the Pol II CTD by CDK9,a subunit of human
`P-TEFb (Ctk1 in yeast)8,104. Phosphorylation of the CTD
`creates binding sites for proteins that are important for
`mRNA processing and transcription through chromatin
`such as the histone H3 lysine 36 (H3K36) methylase
`SET2 (REF. 104). Nucleosome remodellers also facilitate
`passage of Pol II during the elongation phase of
`transcription. The transcription cycle continues with
`elongation of the transcript by Pol II, followed by
`termination and re-initiation of a new round of
`transcription (not shown).
`
`Activator-dependent recruitment
`
`Promoter clearance
`
`proximal pausing
`Release from promoter-
`
`Target gene
`
`Target gene
`
`Activator
`
`Recognition site
`
`TSS
`
`Co-activator
`
`Activator
`
`Nucleosome-
`remodelling complex
`
`a
`
`b
`
`c
`
`Co-activator
`
`GTFs
`
`PIC
`
`Target gene
`
`RNA polymerase II
`
`Activator
`
`Nucleosome-
`remodelling complex
`
`d
`
`GTFs
`CDK7
`
`Target gene
`
`S5
`P
`
`RNA polymerase II
`
`Nucleosome-
`remodelling complex
`
`XPB
`
`GTFs
`
`e
`
`P-TEFb
`
`Target gene
`
`H3
`
`H4
`
`H2A
`
`H2B
`
`S5
`P
`
`S2
`P
`
`RNA polymerase II
`
`Nucleosome-
`remodelling complex
`
`General transcription
`machinery
`RNA polymerase II together
`with the general transcription
`factors TFIIA, TFIIB, TFIID,
`TFIIE, TFIIF and TFIIH.
`
`Transcriptional activator
`A sequence-specific
`DNA-binding protein that
`increases the rate of
`transcription by recruiting RNA
`polymerase II, either directly
`in prokaryotes or through
`co-activators in eukaryotes.
`
`these 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 provide a broad over-
`view of the principles involved in inducible activation
`of transcription.
`
`Activator-dependent recruitment
`Gene activation involves a multistep recruitment
`process that consists of several potential rate-limiting
`steps during the initial stages of the transcription cycle
`(reviewed in REF. 8) (FIG. 1). During the initial steps of
`gene induction, transcriptional activators bind to specific
`DNA sequences near target genes and recruit transcrip-
`tional co-activators and components of the transcription
`
`machinery to these genes through protein–protein
`interactions. These steps result in formation of the
`pre-initiation complex (PIC) on the promoter 9,10. For
`the purposes of this Review, these first three steps
`can be regarded as a single rate-determining process,
`which we refer to as activator-dependent recruitment
`(FIG. 1a–c). An additional level of regulation is required
`for polymerase to proceed to productive transcription
`elongation (FIG. 1d,e). Although all of the steps in the
`transcription cycle are subject to regulation11, we focus
`in this Review on those steps that are most important
`for inducible gene expression: activator-dependent
`recruitment resulting in PIC formation; activation of the
`PIC and transcription initiation; and release of paused
`polymerase into productive elongation.
`
`NATURE REVIEWS | GENETICS
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`

`R E V I E W S
`
`a
`
`b
`
`c
`
`d
`
`e
`
`Gal4
`
`Gal80
`
`UASGAL
`
`TSS
`
`Gal4
`
`UASGAL
`
`Tra1
`
`Gal4
`
`UASGAL
`
`SAGA
`
`SAGA
`
`Tra1
`
`Gcn5
`
`Gal4
`
`SWI/SNF
`
`Mediator
`
`H3Ac
`
`Cytoplasm
`
`Gal3
`
`– Galactose
`GAL1
`
`+ Galactose
`
`GAL1
`
`Gal3
`
`Gal80
`
`Activator-dependent SAGA recruitment
`
`GAL1
`
`Activator-dependent Mediator recruitment
`Nucleosome acetylation by SAGA
`stimulates nucleosome remodelling by
`SWI/SNF, which is also recruited by Gal4
`
`GAL1
`
`Pre-initiation complex formation
`
`SAGA
`
`GTFs
`
`PIC
`
`Spt3
`
`RNA polymerase II
`
`GAL1
`
`Gal4
`
`TBP
`
`Mediator
`
`SWI/SNF
`
`Figure 2 | 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 medium causes an inducer
`protein, Gal3, to bind and sequester Gal80 in the cytoplasm, releasing it from Gal4 (b).
`Gal4 binds target UASGAL (upstream activating sequence) sites in the promoters
`of Gal genes such as GAL1 and sequentially recruits co-activators, such as the
`acetyltransferase SAGA (c) and Mediator (d). Gal4 also recruits ATP-dependent
`nucleosome-remodelling complexes such as SWI/SNF that remove nucleosomes at
`the promoter and are stimulated 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). Nucleosome
`removal, catalysed by SWI/SNF, aids in the kinetics of Mediator and GTF recruitment,
`thereby facilitating rapid PIC formation and initiation of transcription at Gal genes.
`H3Ac, histone H3 acetylation; TBP, TATA-binding protein; TSS, transcription start site.
`
`Gal4-mediated Gal gene induction in yeast.The expression
`of Gal genes, which encode products that are required
`for the import and metabolism of galactose, is rapidly
`induced when galactose is added to the growth medium
`of S. cerevisiae (reviewed in REF. 12). Activation of the
`Gal genes is regulated primarily through activator-
`dependent recruitment. Expression is initiated by
`the transcriptional activator Gal4, which binds to an
`upstream activating sequence (UASUU GAL) in the promot-
`ers of Gal genes (FIG. 2). The affinity of Gal4 binding var-
`ies among the Gal genes, thereby leading to differential
`levels of activation12.
`The initiation of the entire response of the Gal reg-
`ulon to galactose is dependent on this transcriptional
`activator, Gal4. How then is Gal4 itself regulated? The
`regions of Gal4 that contain the DNA-binding and
`transcription-activation activities are separable13. In
`the absence of galactose, the acidic activation 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 complex14,15. When galac-
`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
`to the Gal genes16–21. The activation function of Gal4
`is further regulated by post-translational mechanisms
`that include phosphorylation and ubiquitin-mediated
`degradation12.
`
`Gal gene induction requires co-activators. In Gal gene
`induction and many other examples of inducible
`gene expression, recruitment of co-activators and the
`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-
`activator to bind to Gal promoters following a shift to
`galactose-containing medium is SAGA, which is directly
`recruited by Gal4 (REFS 22–24). A few minutes follow-
`ing this, Mediator is recruited to Gal promoters through
`direct contact with Gal4 (REFS 23,25–28). Finally, Pol II
`and components of the general transcription machinery,
`including TBP, TFIIH, TFIIE and TFIIF, are recruited
`to Gal promoters23. None of these final components,
`including Pol II, is recruited in the absence of SAGA,
`which indicates that Gal4 alone is not sufficient to acti-
`vate transcription23,24. Rather, a combination of SAGA
`and Mediator activities is required for the recruitment of
`TBP, Pol II and the remainder of the GTFs23,24,29,30.
`Although this response to galactose might seem
`specific to yeast and other closely related fungi, the
`mechanisms by which Gal4 activates gene expression
`must be widely conserved because Gal4 can activate
`UASUU GAL-specific transcription in organisms that range
`from D. melanogaster to humans31,32. Furthermore,
`SAGA, Mediator and the GTFs are highly conserved
`from yeast to humans (Supplementary information S1
`(figure and tables)). The presence of cis-regulatory ele-
`ments that have varying affinities for Gal4 at many dif-ff
`ferent galactose-inducible genes provides a mechanism
`
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`R E V I E W S
`
`to coordinate both the timing and relative levels of
`expression of the Gal genes. In higher eukaryotes, genes
`that are co-regulated often share common cis-regulatory
`elements. These regulatory elements can be bound by
`individual activators or by combinations of transcrip-
`tional activators that have varying affinities. For exam-
`ple, the Forkhead and Ets transcriptional activators bind
`together to the same DNA motif that is present upstream
`of a set of co-regulated genes to synergistically acti-
`vate the transcription of these genes in the developing
`vascular endothelium33.
`
`Co-activators facilitate gene activation
`In prokaryotes, transcriptional activators directly contact
`RNA polymerase34, so why are co-activators required in
`eukaryotes? Many studies have shown that, in contrast
`to prokaryotes, most genomic DNA in a eukaryotic cell
`is compacted into chromatin and therefore is not directly
`accessible to components of the general transcription
`machinery (reviewed in REF. 35). Although some tran-
`scriptional activators such as the human glucocorticoid
`receptor can bind their target DNA sequence in a
`nucleosomal context36, PIC formation and subsequent
`transcription are inhibited by nucleosomes in vitro37–39.
`Furthermore, studies in yeast suggest that inducible
`genes tend to have a higher density of nucleosomes cov-
`ering their promoters than constitutively active genes,
`which have more open, nucleosome-depleted promot-
`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 nucleosomal barrier to transcrip-
`tion initiation requires complexes such as SAGA and
`Mediator. Intriguingly, complexes involved in tran-
`scription activation often possess enzymatic activities
`directed towards the amino-terminal tails of histone pro-
`teins in the nucleosome. SAGA, for instance, contains
`the histone acetyltransferase (HAT) Gcn5 (REF. 41). So,
`are histone-modifying activities required for recruitment
`of Pol II and the general transcription machinery?
`Although nucleosome acetylation by activator-
`recruited SAGA stimulates transcription in vitro42,43,
`surprisingly, the acetyltransferase activity of SAGA is
`not directly required for Pol II recruitment and PIC
`formation at Gal genes22. However, mutations in other
`SAGA components such as Spt3 that only modestly
`reduce SAGA recruitment substantially decrease PIC
`formation22,24. Therefore, importantly, the co-activator
`function of SAGA requires more than its enzymatic
`activity. Rather, both SAGA and Mediator have struc-
`tural roles during inducible gene expression, forming
`a scaffold on which components of the general tran-
`scription machinery and Pol II can assemble. Mediator
`interacts directly with the unphosphorylated form of
`the carboxy-terminal heptapeptide repeat sequences
`(carboxy-terminal domain; CTD) of the RBP1 subunit
`of Pol II (reviewed in REF. 44). Therefore, in the case of
`Gal gene induction, Mediator links Gal4 and the general
`transcription machinery.
`
`A HAT-independent role for other co-activators such
`as p300 has also been reported. Mutation of the KIX tran-
`scription factor-binding domain of mouse p300 results
`in severe defects in haematopoiesis but mutation of the
`HAT domain has little effect45. Furthermore, the role of
`human PCAF (also known as KAT2B) as a co-activator
`in activation of human T cell leukaemia virus type 1
`long terminal repeat transcription is also independent
`of its HAT activity46. Although these examples show that
`some HATs can have histone acetylation-independent
`functions in gene activation, at some genes, histone
`acetylation is required for PIC formation. For example,
`in yeast, mutation of the HAT Gcn5 in SAGA decreases
`the levels of TBP and Pol II recruitment at some genes47.
`However, the findings discussed above show that, in the
`early stages of the transcription cycle, the structural role
`of co-activators is of at least equal importance to their
`histone-modifying activities.
`
`Chromatin remodelling and transcription
`To understand why histone acetylation is required for
`the activation of some genes but not others, we need to
`consider another class of transcription regulators known
`as ATP-dependent nucleosome-remodelling complexes.
`Nucleosome-remodelling complexes use the energy from
`ATP hydrolysis to move histones or displace them from one
`piece of DNA onto another or onto a histone-binding
`protein, known as a histone chaperone (reviewed in
`REF.FF 48). Following galactose induction, Gal4 recruits the
`nucleosome-remodelling complex SWI/SNF to Gal gene
`promoters at which it rapidly removes promoter nucleo-
`somes28,49,50. Intriguingly, a recent study has shown that
`in the absence of SWI/SNF, these promoter nucleosomes
`are still removed and transcription is initiated, albeit
`much more slowly49. Therefore, nucleosome removal
`facilitated by nucleosome-remodelling complexes is
`important for the swift induction of gene expression but
`is not necessarily essential for overall levels of induc-
`tion at every inducible gene. This implies that at some
`inducible genes, given enough time, recruitment of co-
`activators and the general transcription machinery by
`an activator alone is sufficient to remove nucleosomes
`at the promoter. However, nucleosome remodelling is
`crucial for inducible gene expression as it facilitates the
`rapid activation of transcription. Furthermore, at some
`promoters such as at the human α1 antitrypsin gene,
`recruitment of nucleosome-remodelling proteins like
`brahma is rate-limiting for activation51.
`Is histone acetylation important for chromatin
`remodelling? At least in the example of the Gal genes,
`SWI/SNF recruitment does not depend on SAGA28.
`However, in vitro studies suggest that histone acetylation
`is important for the binding of SWI/SNF to nucleosomes
`and that acetylated histones are preferentially displaced
`by SWI/SNF52–54. Nucleosome-remodelling complexes
`such as SWI/SNF or RSC are also important for assist-
`ing Pol II transcription of a nucleosomal template55.
`Intriguingly, HAT complexes such as NuA4 and
`SAGA increase RSC-stimulated transcription by Pol II
`in vitro55. These findings support a model in which
`the acetylation activity of SAGA promotes SWI/SNF
`
`Pre-initiation complex
`A 44 polypeptide complex,
`which consists of RNA
`polymerase II and the general
`transcription factors TFIIA,
`TFIIB, TFIID, TFIIE, TFIIF
`and TFIIH. Its formation
`at TATA-containing core
`promoters is nucleated by
`binding of TATA-binding protein
`(a component of TFIID) to the
`TATA element in the promoter
`DNA sequence.
`
`Acidic activation domain
`The type of transactivation
`domain found in transcriptional
`activators such as Gal4. This
`domain contains a stretch of
`acidic amino acids and is
`required for interactions
`with co-activators.
`
`ATP-dependent nucleo-
`some-remodelling complex
`A transcriptional regulatory
`complex that uses the energy
`obtained from ATP hydrolysis
`to move or displace histone
`octamers from DNA.
`
`NATURE REVIEWS | GENETICS
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`R E V I E W S
`
`activity, therefore facilitating the rapid removal of
`promoter nucleosomes after galactose induction, leading
`to the swift activation of Gal transcription. In addition to
`this role in the early stages of transcription initiation,
`HAT complexes and nucleosome remodellers assist
`polymerase elongation through the coding region of
`Gal genes.
`The interplay between SAGA and SWI/SNF at Gal
`genes is similar to that observed at the yeast PHO5 gene
`under phosphate starvation conditions56,57. SAGA and
`SWI/SNF are not essential for PHO5 activation but are
`important for the kinetics of activation. Therefore, the
`functions of a transcriptional co-activator complex
`such as SAGA can be separated into HAT-independent
`activities that are essential for PIC formation and HAT-
`dependent activities that are required for chromatin
`remodelling and the kinetics of gene induction. At some
`inducible genes these functions overlap, for example,
`the Gcn4-dependent genes in yeast46, as HAT activity
`is also required for nucleosome remodelling before
`PIC formation.
`The role of co-activators in stimulating nucleosome
`remodelling is not limited to HAT complexes. At some
`genes, Mediator is also required for the recruitment of
`SWI/SNF or for its chromatin remodelling activity28,58.
`However, at other genes such as yeast CHA1, chromatin
`remodelling is independent of Mediator59. Therefore,
`nucleosome remodelling can occur before or subsequent
`to the activities of Mediator and SAGA and, in some
`cases, is even required for their co-activator functions.
`
`Is nucleosome loss sufficient to activate transcription?
`In some examples in yeast, nucleosome loss alone is
`sufficient to induce PIC formation and activate gene
`expression, even in the absence of an inducing sig-
`nal60,61. Moreover, when yeast are shifted to growth
`media containing glucose, transcription of the Gal genes
`is repressed and nucleosomes rapidly reform at Gal
`promoters62,63. Therefore, at many genes, nucleosome
`loss correlates positively with transcription. However,
`several lines of evidence across different species show
`that a nucleosome-free region on its own is not neces-
`sarily sufficient to activate transcription. Furthermore,
`repression is possible in the absence of nucleosomes.
`In yeast, expression of the Gal genes is repressed in the
`presence of a combination of glucose and galactose,
`although Gal4 still recruits SWI/SNF and maintains a
`nucleosome-free promoter region49. Another example
`of this — heat shock induction in D. melanogaster — isr
`
`discussed below,
`
`PARP-dependent nucleosome loss at D. melanogaster
`Hsp70. Heat shock induces a rapid loss of nucleo-
`somes across the entire Hsp70 locus in D. melanogaster
`that precedes and is independent of transcription64–66
`(FIG. 3a). This nucleosome loss corresponds at least
`partially to the diffuse appearance or puffing of the
`heat-shock loci on polytene chromosome spreads and
`is important for optimal levels of Hsp70 expression64
`(reviewed in REF. 67). Although nucleosome loss is
`required for optimal transcription of the heat-shock
`
`▶
`
`Figure 3 | Heat shock induces nucleosome loss and
`release of paused RNA polymerase II. a | At the top is a
`schematic of the Drosophila melanogaster 87A heat-shock
`
`r
`locus; the example of Hsp70Ab is used below. Equivalent
`events occur at Hsp70Aa. The arrows at the Hsp70Aa
`and Hsp70Ab promoters indicate the direction of
`transcription through the gene. RNA polymerase II (Pol II,
`shown in orange) and GAGA factor (GAF or Trithorax-like)
`are bound at the promoters of the Hsp70 genes before heat
`shock. After heat shock, the transcriptional activator
`heat-shock factor (HSF) forms a stable trimeric complex
`that binds the Hsp70 promoter105,106. Heat shock
`stimulates nucleosome loss at the Hsp70 locus.
`Nucleosome loss is dependent on HSF, GAF and
`poly(ADP)-ribose polymerase (PARP)64. PARP localizes
`at many sites along polytene chromosomes but only
`catalyses formation of ADP-ribose polymers from donor
`NAD+ at the heat-shock loci after induction by heat
`shock107. Nucleosome loss proceeds outwards from the
`5(cid:96) end of the Hsp70 genes ahead of Pol II, extending as far
`as the SCS and SCS(cid:96) boundary elements (region of loss is
`grey in top panel). The CG31211 and CG3281 genes are
`not transcribed (as shown by blunt-headed arrows at their
`promoters) although Pol II is bound at their promoters and
`nucleosomes are lost. Ba | Before heat-shock activation
`at Hsp70, GAF recruits co-activators, the GTFs and
`ATP-dependent nucleosome-remodelling complexes,
`thereby facilitating pre-initiation complex (PIC) formation
`at the promoter. At Hsp70, however, PIC formation is
`not sufficient to activate productive transcription
`elongation89,108–110. The CDK7 subunit of TFIIH
`phosphorylates serine 5 (S5) of the carboxy-terminal
`domain (CTD) and Pol II initiates transcription into the first
`20–40 bases of the gene, at which it pauses at the
`promoter-rr proximal pause site. Pol II is held here by the
`negative elongation factor (NELF) and DRB sensitivity-
`inducing factor (DSIF), which is composed of SPT4 and 5
`(REFS 111,112). Bb | Heat shock induces binding of HSF,
`which recruits additional co-activators and nucleosome-
`remodelling activities. HSF is required but is not sufficient
`to recruit the pause release factor P-TEFb to Hsp70
`(REF.FF 85). Recruitment of Mediator by HSF, which occurs
`independently of PIC formation, might contribute to
`P-TEFb binding113. Recruited P-TEFb phosphorylates S2 of
`the CTD, SPT5 and NELF. These phosphorylations cause
`NELF to dissociate from Pol II, releasing polymerase into
`productive transcription elongation. Although Pol II still
`pauses briefly at the promoter-r proximal pause site under
`heat-shock conditions, the duration of these pauses are
`much shorter than at normal temperatures.
`
`genes at Hsp70, it is not sufficient to activate expression
`of other genes that lie in the region of nucleosome dis-
`ruption64. Furthermore, chemicals that induce puffing
`and nucleosome loss at heat-shock loci such as sodium
`salicylate do not activate expression of the heat-shock
`genes68. Therefore, although nucleosome loss is impor-
`tant for rapid activation of gene expression and is some-
`times required for optimal levels of gene expression,
`these studies show that nucleosome loss alone is not
`necessarily sufficient to induce activated transcription.
`Rather, nucleosome remodelling can be an important
`step in providing access to transcriptional activators and
`the general transcription machinery.
`
`430 | JUNE 2010 | VOLUME 11
`
` www.nature.com/reviews/genetics
`
`
`
`© 20 Macmillan Publishers Limited. All rights reserved10
`
`LCY Biotechnology Holding, Inc.
`Ex. 1012
`Page 5 of 12
`
`

`

`SCS
`
`CG31211
`
`Hsp70Aa
`
`Hsp70Ab
`
`CG3281
`
`SCS(cid:1)
`
`A 87A heat-shock locus
`
`NELF
`
`Hsp70Ab
`
`GAF GAF
`
`RNA polymerase II
`
`GAF
`
`GAF
`
`HSF
`
`GAF
`
`GAF
`
`HSF
`
`Ba Pre-heat shock
`
`Bb Post-heat shock
`
`Heat shock
`
`PARP
`
`RNA polymerase II
`
`Hsp70Ab
`
`Hsp70Ab
`
`RNA polymerase II
`
`GTFsCDK7
`
`PIC
`
`RNA polymerase II
`
`S5
`P
`Co-activators
`
`GAF
`
`GAF
`
`Nucleosome-
`remodelling complexes
`
`GTFs
`
`DSIF
`
`SPT4 SPT5
`RNA polymerase II
`
`NELF
`
`S5
`P
`
`Co-activators
`
`GAF
`
`GAF
`
`Nucleosome-
`remodelling complexes
`
`Promoter-proximal
`paused Pol II
`
`S2
`P
`Co-activators
`
`S5
`P
`
`HSF
`
`GAF
`
`GAF
`
`HSF
`
`P-TEFb
`
`Mediator
`
`Nucleosome-
`remodelling complexes
`
`P
`SPT4 SPT5
`
`P
`NELF
`
`PARP
`
`Release of paused polymerase
`P
`NELF
`
`P
`SPT4 SPT5
`
`S2
`P
`
`S5
`P
`
`RNA polymerase II
`
`Co-activators
`Co-activators
`
`HSF
`
`GAF
`GAF
`
`GAF
`GAF
`
`HSF
`
`Mediator
`
`Nucleosome-
`remodelling complexes
`
`R E V I E W S
`
`These findings might seem at first to contrast with
`results from older studies in which histone H4 was
`depleted in yeast using a genetic approach69. In the
`absence of H4, the yeast PHO5 promoter was activated
`independently of its UAS. However, although nucleoUU
`-
`
`some depletion resulted in activation of PHO5, the
`level of activation was significantly lower than that
`observed under physiological induction conditions. A
`similar result is observed for GAL1 in which nucleo-
`some loss activates a low level of transcription that is
`independent of UASUU GAL. In both these examples, the
`level of transcription observed after nucleosome loss is
`significantly lower than that observed under inducing
`conditions when nucleosomes are present. Therefore,
`although some transcription may occur in the absence
`of nucleosomes, full activation of inducible gene tran-
`scription generally requires additional factors such as
`transcriptional activators.
`
`Post-recruitment initiation of transcription
`Together, the activator-dependent recruitment of
`the general transcription machinery and chromatin
`remodelling at the promoter facilitate formation of the
`PIC. However, once Pol II and the GTFs are present
`in the PIC, a series of sequential chromatin modifica-
`tion events are required for Pol II to clear the promoter
`and initiate efficient transcription elongation (FIG. 4).
`Although loss of some histone modifications can
`impair overall levels of transcription activation at some
`inducible genes (for example, loss of acetylation by
`the HAT Gcn5 (REF. 70) or loss of H2B ubiquitylation
`by Rad6 (REF. 71)), these histone modifications are often
`not essential for co-activator-dependent transcription
`from a chromatin template in vitro72. It is likely that
`the in vitro experiments do not completely recapitu-
`late in vivo regulation. In addition, it seems that many
`of these histone modifications are important for later
`stages of the transcription cycle. Therefore, it is useful to
`describe the chromatin modification events that occur
`during the early steps of PIC formation and promoter
`clearance to provide an overview of the role of these
`chromatin marks in activating efficient transcription
`elongation by Pol II.
`
`Histon