Chromatin Marks and Machines, the Missing Nucleosome Is a Theme

Molecular Cell, Vol. 17, 323–330, February 4, 2005, Copyright ©2005 by Elsevier Inc.

DOI 10.1016/j.molcel.2005.01.013

Chromatin Marks and Machines, Meeting Review the Missing Nucleosome Is a Theme: Gene Regulation Up and Downstream Mike Carey* Department of Biological Chemistry David Geffen School of Medicine at UCLA 10833 LeConte Avenue Los Angeles, California 90095

A remarkable insight to emerge from chromatin immunoprecipitation studies is that the steps leading to chromatin remodeling and preinitiation complex (PIC) assembly differ significantly, depending upon the gene and its biological context (Cosma, 2002). However, when multiple systems are compared, the differences illuminate checkpoints and generalities that provide insights into the most salient features of mechanism. This concept dominated presentations at the 2004 Chromatin and Transcription by RNA Polymerase II meeting held at the Lake Tahoe Granlibakken Conference Center.

Players and Problems The coupling of gene biology with system-wide approaches highlighted emerging themes in the eukaryotic transcription field while reinforcing and clarifying traditional views. Some important questions covered at the meeting included: does “chromatin remodeling” involve moving or removing histones? How are modification and remodeling enzymes targeted to promoters and to coding regions? How are these processes coupled with elongation and RNA processing? How are boundaries between heterochromatin and euchromatin established? A typical nucleosome comprises 146 bp of DNA wrapped 1.65 times in a left-handed supercoil around a globular, disc-shaped octamer bearing two H2A:H2B heterodimers and an H3:H4 heterotetramer (Luger et al., 1997). The minor groove of the DNA contacts the nucleosome at 14 locations on the lateral surface via nonspecific interactions. The aggregate affinity greatly exceeds that of typical sequence-specific interactions between promoters and proteins that nucleate transcription complex assembly. Eukaryotic cells have evolved specific mechanisms to remodel nucleosomes to permit binding of the transcriptional machinery. The process of remodeling typically involves binding of one or more sequence-specific activators or repressors to an accessible region, sometimes between nucleosomes and sometimes within. The binding sites for activators and repressors can be located close to the gene as in the yeast UAS and mammalian proximal promoter. Alternatively, the activator and sometimes repressor binding sites can be positioned 100 kb away as is the case with some important enhancers in metazoans. Activators and repressors recruit the remodeling machinery, which sets in motion a series of catalytic events that in the case of gene activation, position RNA polymerase II (pol II) at the start of a gene, link it with *Correspondence: [email protected]

machines that mark its location on a chromatin template, and coordinate its action with the mRNA processing and transport machinery. Many talks at the meeting centered on how the remodeling machinery is targeted to a gene and how coupled mRNA processing is achieved. Remodeling typically involves covalent modifications of the flexible N-terminal histone tails protruding radially from the lateral surface of the nucleosome and ATPdependent nucleosome mobilization. Acetylases (HATs), kinases, methylases (HMT), ubiquitinylases, sumoylases, and ADP-ribosylases modify the tails, thereby creating signals and binding sites for factors that direct the decondensation or condensation of chromatin (Jenuwein and Allis, 2001). These factors recognize the modification patterns via specific domains such as the bromodomain (acetylated lysine) and chromodomain (methylated lysine). Several talks centered on novel aspects of histone modification and the role of domains that recognize them. The machines that modify, remodel, and transcribe chromatin are quite large compared with a nucleosomal core particle. Indeed, a typical S. cerevisiae intergenic region is but 635–940 bp long and contains only two to four 206 Kda nucleosomes. The ATP-dependent SWI/ SNF remodeling enzyme, which increases nucleosome mobility, tips the scales at ⵑ1.15 Mda, and a recent EM structure reveals a 25 ⫻ 15 nm oblate particle with a 15 ⫻ 5 nm pocket that could easily fit a 10 nm nucleosome (Smith et al., 2003). The meeting featured several new reports into how SWI/SNF and enzymes like it function both in vitro and on genes. In the case of gene activation, ATPases uncover the core promoter for assembly of a preinitiation complex (PIC) that weighs in at ⬎3.5 Mda. The Mediator coactivator complex plays a central role in PIC assembly by recruiting pol II, and the general transcription factors TFIIA, IIB, IID, IIE, IIF and IIH. TFIID, with the help of TFIIA, positions the machinery at the TATA box, TFIIB positions the polymerase at the startsite, and TFIIH mediates several important catalytic events involved in DNA melting and promoter escape (Hahn, 2004). The KIN28/CDK7 subunit of TFIIH also signals the recruitment of complexes, which facilitate transcription through chromatin (Ng et al., 2003). As yeast pol II traverses the gene, it carries along an impressive array of factors involved in chromatin modification, remodeling, and elongation, all dedicated to transcribing a typical 1.5 kb gene bearing about eight nucleosomes. An intricate signaling network involving pol II phosphorylation and histone modification coordinates different stages of mRNA processing to initiation, promoter escape, and productive elongation (Belotserkovskaya and Reinberg, 2004; Gerber and Shilatifard, 2003; Hampsey and Reinberg, 2003; Hartzog, 2003). Insights into these stages emerged during several talks and at the posters. The following review will focus first on the concept of histone removal, a major theme of the meeting. Then I will discuss talks and posters related to each of the transcriptional steps described above.

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Missing Nucleosomes and Chaperones: Mobility versus Removal The meeting highlighted an emerging paradigm shift in the field—the observation that active promoters and coding regions are depleted of nucleosomes. Historically speaking, this represents a paradigm reversal rather than a shift. The concept that nucleosomes were depleted from actively transcribed DNA was first apparent in the classic electron microscopy (EM) studies of Oscar Miller in the 1970s, where nucleosomes were positioned behind and in front of, but not within, a string of pol I molecules in the act of transcribing rRNA genes (Miller, 1981). Additionally, in the early 1980s, actively transcribed loci such as the mammalian ␤-globin and Drosophila heat shock genes were found to display increased global DNase I sensitivity within the gene locus and DNase I hypersensitivity at the promoter, suggesting the absence of nucleosomes (Elgin, 1981). Finally, in the late 1980s, Grunstein showed that histone H4 depletion in S. cerevisiae caused widespread activation of yeast genes, including PHO5 (Han and Grunstein, 1988). However, over the past decade two observations altered the view that nucleosomes are removed during transcription. One was that ATP-dependent remodeling machines did not remove nucleosomes from templates in vitro, but only set them aside or looped out the DNA to make it more accessible (Flaus and Owen-Hughes, 2004). The second observation was the fact that nucleosomes are heavily acetylated around the promoter and methylated/acetylated within the transcribed region as measured by ChIP. Hence the paradox, if the modifications are detected on the gene, then the nucleosomes must be there. The Horz and Kornberg labs recently reported a solution to this problem (Boeger et al., 2003, 2004; Reinke and Horz, 2003). Both labs analyzed the PHO5 promoter, where studies by Horz had established the presence of several positioned promoter nucleosomes, which are remodeled during gene activation by the PHO4 activator. Both groups used ChIP to show that the nucleosomes are indeed missing as originally predicted by the nuclease sensitivity and positioning assays. Horz found that nucleosomes at the UAS and TATA were first hyperacetylated and subsequently disappeared as measured by ChIP. However, the nucleosomes could have been moved to regions not detected by ChIP. Because nucleosomes constrain supercoils, Kornberg employed topological analysis of promoter minicircles excised from the genome via sites-specific recombinases to test for the presence of the nucleosomes. His group discovered that approximately two of three nucleosomes in the circles were missing in the activated state. The GCN5containing histone acetylase complex SAGA and ATPremodeling complex SWI/SNF were hypothesized to play a role in remodeling based on analysis of mutants, although the process was only delayed in SAGA and SWI2 mutants, possibly because the ISWI ATPase, INO80, can also function at the locus. The ESA1/NuA4 HAT complex is, however, essential (Nourani et al., 2004). The theme of histone depletion was reiterated in several talks and posters at the meeting. Cheol-Koo Lee (University of North Carolina) presented his ChIP study

on genome-wide nucleosome distribution of histones H3 and H4 on S. cerevisiae promoters and open reading frames (ORFs) using microarray analysis (Lee et al., 2004). By comparing histone occupancy to published studies of gene activity, Lee demonstrated an inverse relationship between histone deposition and gene activation. David Gross (Louisiana State University Health Sciences Center) also reported locus-wide loss of nucleosomes at HSP82 within a minute after heat shock. By using mutant strains, Gross found that efficient removal of histones could occur in SWI2⌬, GCN5⌬, SET1⌬, ASF1⌬, and PAF1⌬ (see below) mutants. Kevin Struhl (Harvard Medical School) showed on a GAL4-regulated promoter that H2B density decreased in proportion to the number of transcribing pol II molecules upon addition of the inducer galactose. However, upon addition of the inhibitor glucose, levels of bound pol II decreased as nucleosomes were replenished within a minute (Schwabish and Struhl, 2004), much like the case of HSP82 after temperature downshift. The authors of these studies point out that nucleosome reassembly on promoters and coding regions is a highly rapid process. Therefore, it seems unlikely that nucleosomes are completely stripped off a gene during transcription but more likely exist in a dynamic equilibrium related to the presence of activators at the promoter and pol II density in the ORF. The known biochemical activities of SAGA and SWI/ SNF cannot explain how nucleosomes are removed from a promoter. However, previous studies had shown that histone chaperones participate in histone deposition and work in concert with ATP remodeling enzymes (Haushalter and Kadonaga, 2003; Korber and Horz, 2004). Melissa Adkins (University of Colorado Health Sciences Center) reported that the Anti-silencing function 1 protein (ASF1), an H3/H4 chaperone, is involved in gene activation at PHO5 (Adkins et al., 2004) in contrast to the case of HSP82 reported by Gross. Mutations in ASF1 prevented nucleosome removal during activation, hindering recruitment of TBP to TATA. Surprisingly, the mutants did not hinder PHO4 recruitment. Two important remaining issues are the mechanism of removal and the fate of the histones. A potential mechanism for histone removal could involve modifications of the globular surface of the nucleosome. Recent proteomic data reveal that histones from mouse and chicken are heavily modified within the globular region (Zhang et al., 2003). Michael Cosgrove (Johns Hopkins University School of Medicine) modeled these modifications on the nucleosome structure and showed that, in principle, they could greatly reduce the affinity of DNA for the octamer and facilitate nucleosome mobilization (Cosgrove et al., 2004) and perhaps removal. Cosgrove mentioned that although the concept is unproven, it is supported by previous genetic data identifying switch (SWI/SNF)-independent (Sin) mutations or loss of ribosomal gene silencing (Lrs) mutations in the globular core of the S. cerevisiae nucleosome. Although biophysical experiments suggest that the Sin and Lrs mutations increase thermal mobility of nucleosomes on DNA, it is not clear how the lateral surface modifications would work. Would ATPases open the nucleosome to allow access to the modification enzymes or do the modifications recruit the ATPase? Do the modifications facilitate

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mobilization or histone removal? Lateral modifications represent a tantalizing new aspect of nucleosome metabolism, but their significance awaits a detailed proteomic analysis of yeast histones in various HAT and ATPase mutant backgrounds.

Mechanisms of Enhancer Function Metazoan enhancer sequences can function up to 100 kb from the target gene. Remarkably, little is known about how this communication is enabled. Two talks highlighted interesting new features of the mechanisms. Dale Dorsett (Saint Louis University) described genetic studies in Drosophila to identify proteins that facilitate the function of the distant enhancer for the cut gene, encoding a homeodomain protein involved in numerous developmental processes. His screen identified a protein termed Nipped-B, the fly homolog of a protein mutated in Cornelia de Lange syndrome in humans. Nipped-B is an adherin required for sister chromatid cohesion during mitosis. Nipped-B normally loads cohesin complexes onto the chromatin to establish cohesion, but cohesin inhibits cut activation. Dorsett speculated that cohesin may actually possess properties of an insulator, preventing enhancer-promoter communication, and Nipped-B removes cohesin during longrange gene activation (Dorsett, 2004). Ann Dean (National Institutes of Health) provided additional insights into how enhancers function (Zhao and Dean, 2004). The ␤-globin locus control region (LCR) contains several DNase hypersensitive (HS) sites, of which murine and human HS2 and HS3 are enhancers, whereas chicken HS4 has well-characterized insulator function and is presumed to prevent the LCR from influencing genes outside of the globin locus. Normally, the process of activation involves an extended region of histone hyperacetylation between the LCR and the promoter of the most proximal (embryonic) globin gene. Dean examined the consequences of placing the insulator between the enhancer and the target gene and found that the insulator prevented histone hyperacetylation and recruitment of p300 and CBP. The insulator trapped pol II at the enhancer and prevented its transfer to the proximal promoter. Dean also mentioned that the binding of pol II to the intervening region is associated with the presence of non-mRNA transcripts. She suggested a model whereby pol II tracks along the acetylated chromatin in a unidirectional manner until it reaches the proximal promoter. Insulators somehow block the tracking. The finding agrees with the model proposed by Talianidis for the HNF4␣ enhancer (Hatzis and Talianidis, 2002), where a wave of acetylation spreads between the enhancer and promoter during enterocyte differentiation. The enhancer binding proteins track along with this wave. The tracking model contrasts with the looping mechanism proposed by Brown, who had previously found that acetylated histones and pol II are located at both the enhancer and promoter during activation of the PSA gene but not in the intervening region (Shang et al., 2002). It will be important to determine the generality of the looping and tracking mechanisms and what parameters determine which strategy will be employed.

Targeting the Chromatin Machines to Promoters The recruitment of HATs and ATPases to promoters is a critical checkpoint subject to numerous modes of regulation. In the now-classic example of the HO promoter, the activator SWI5 binds transiently during telophase and recruits SWI/SNF, which in turn, leads to SAGA recruitment, histone modifications, and binding of the activator SBF. Long after SWI5 has left, SBF recruits the Mediator, which is followed by CDK1-dependent binding of pol II and the GTFs (Cosma et al., 2001). Jerry Workman (Stowers Institute) discussed how activators accomplish the recruitment and remodeling processes in different ways. However, the common theme is their interdependence. SAGA and SWI/SNF have a long history and appear to work in unison on many promoters. Both complexes interact tightly with numerous activators in vitro. SAGA can be recruited by acidic activators via the TRA1 subunit but until recently the mechanism of SWI/SNF recruitment by activators was unknown. Workman’s lab employed crosslinking, affinity and genetic assays to show that that SNF5 and SWI1 subunits of SWI/SNF are sites of acidic activator contact; both interaction sites must be deleted to abolish recruitment in vitro and in vivo (Prochasson et al., 2003). Previous biochemical studies had shown that activator-targeted SWI/SNF could be retained on chromatin acetylated by SAGA after removal of activator. However, the retention required an intact bromodomain. Workman addressed the generality of this mechanism by microarray analysis in mutant backgrounds. His group showed that genes requiring the SWI/SNF activator interaction domains generally required the bromodomain of SWI2. Thus, the concept of activator- and acetylation-dependent targeting of SWI/SNF appears widespread. Because both the GCN5 subunit of SAGA and the SWI2 subunit of SWI/SNF contain bromodomains, the effects may be self-propagating within a gene locus. The theme of SWI/SNF and HAT recruitment was reiterated in numerous talks, which emphasized the diverse ways this mechanism can be exploited for combinatorial gene regulation. Pier Lorenzo Puri (Burnham Institute and DTI) showed that activation of the myogenic program in mammals requires the combined action of human SWI/SNF and the HATs p300 and PCAF. The MKK6/ p38 MAPK pathway leads to phosphorylation of the BAF60 subunit of human SWI/SNF and its subsequent recruitment to the promoter (Simone et al., 2004), whereas phosphorylation of p300 by AKT causes recruitment of the HATs. The use of kinase inhibitors revealed that SWI/SNF recruitment couldn’t occur via promoter acetylation alone, reiterating the theme discussed by Workman. Barbara Graves (University of Utah) showed that MAPK-mediated recruitment of p300 to ETS-1/-2 responsive promoters can be achieved by ERK-2 phosphorylation of the unstructured ETS N-terminal tail located at a conserved distance from the Pointed domain (Foulds et al., 2004). Remarkably, as shown previously by Berk and colleagues, ERK also phosphorylates ELK-1, another member of the ETS family. In this case, phosphorylation leads to recruitment of Mediator via interaction with MED23 (Cantin et al., 2003). The selective recruitment of different complexes by different ETS

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family members is one mechanism to rationalize the wide diversity of family members found in any given cell. However, as discussed by Graves, it is still unclear what recruits the different ETS proteins to unique genes given their near-identical DNA binding specificities (Hollenhorst et al., 2004). Stimulation of the RAS-MAPK pathway by EGF activates many immediate-early response genes by promoting H3S10 phosphorylation (Nowak and Corces, 2004). H3S10 phosphorylation has been shown to facilitate GCN5 binding via direct interactions, providing another mechanism of targeting a HAT to a promoter. Jim Davie (Manitoba Institute of Cell Biology) showed that MAPK also promotes H3S28 phosphorylation in cells with MAPK-activated H3 kinase MSK1 (Drobic et al., 2004). In Ras-transformed cells, MSK1 activity, but not protein level, was increased, leading to elevated levels of phosphorylated H3. The H3S10 and S28 events appear to be distinct residing in chromatin domains with differing steady-state levels of acetylated H3, but both are related to growth factor gene activation. The proteins recruited by H3S28 phosphorylation and their relationship to early gene control are unknown. Numerous talks centered on the role of MAPK in activating transcription, but little is known of how MAPK activity is specifically targeted to the nucleus or to a specific gene. One insight comes from analysis of the helix-loop-helix protein TFII-I. Ananda Roy (Tufts University) discussed how upon EGF stimulation, tyrosine phosphorylated TFII-I⌬ interacts with ERK1/2, translocates to the nucleus, and is recruited to the c-fos promoter. It will be interesting to determine whether TFII-I is a loading factor that localizes MAPK to specific upstream promoters of other early response genes. Although many studies have focused on the cooperation of SWI/SNF and SAGA, SWI/SNF clearly exists in other complexes in vivo. Wei Xu (Salk Institute) showed that SWI/SNF is associated with the arginine methylase CARM1, which methylates H3R17 on nuclear receptor (NR)-responsive genes (Xu et al., 2004). CARM1 is recruited to NR-regulated genes via interaction with p160 factors that bind the ligand-responsive AF2 activation domain. In vitro SWI/SNF stimulates CARM1’s ability to methylate histone tails in the context of chromatin, whereas CARM1 conversely stimulates the ATPase activity of BRG1, one of two SWI2-like proteins present in metazoan SWI/SNF. Xu discussed how CARM1 and BRG1 are corecruited to ER-responsive genes in vivo and how the interdependence of the CARM1 and SWI/ SNF catalytic activities is conceptually similar to the relationship between SAGA and SWI/SNF. The mechanisms regulating methylation by CARM1 are unknown. Tony Kouzarides (University of Cambridge) discussed how peptidyl arginine deiminase 4 (PADI4) can deiminate arginine at several positions including H3R17 to generate citrulline. As such, PADI4 can block histone methylation by CARM1 (Cuthbert et al., 2004). PADI4 is bound to inactive ER-responsive genes in vivo suggesting that arginine deimination and methylation by CARM1 are opposing mechanisms. The mechanism for SWI/SNF has undergone numerous revisions since the initial discoveries of the yeast and human enzymes. Two models have been proposed for how SWI/SNF and other ATPases work. In one model,

a DNA twist is propagated throughout the nucleosome, changing the translational position of the nucleosome on DNA. In the other, the enzyme creates a bulge, which diffuses throughout the nucleosome. Blaine Bartholomew (Southern Illinois University) discussed his recent studies comparing the mechanisms of SWI/SNF and ISWI. Both enzymes interact 20 bp away from the nucleosome dyad. Introduction of DNA gaps would, in principle, prevent changes in twist from being transmitted to other parts of the nucleosome and can prevent translocation of the enzyme along DNA. Indeed, such gaps near the binding sites prevented remodeling from occurring, whereas gaps farther away did not until they were slid closer to the binding site. A new model was proposed that involved highly localized twist diffusion resulting in the formation and propagation of a bulge on the nucleosome surface. Recruitment of the Mediator and PIC Assembly Chromatin remodeling at the promoter is an important transition to the next stage in transcription, recruitment of coactivators that assemble the GTFs and pol II over the core promoter. The major coactivator complexes include the Mediator and TFIID. Many subunits of yeast and mammalian Mediator and TFIID interact with activators, whereas others interact with pol II and GTFs. EM reconstructions suggest that yeast and mammalian Mediators share a similar shape and interact with the heptapeptide repeat constituting the carboxyl terminal domain (CTD) of pol II. Yeast and human Mediator stimulate basal transcription and are necessary for activated transcription. There has been much confusion over the precise subunit composition of the mammalian Mediator isolated by different groups and in the similarity between the yeast and mammalian Mediators (Bourbon et al., 2004). Two forms of Mediator had been proposed, one containing a module bearing CDK8, CyclinC, MED12, and MED13. The other lacks the CDK8 module but contains a metazoan specific subunit termed MED26. To resolve the issue of subunit variations Joan and Ron Conaway (Stowers Institute) prepared HeLa cell lines expressing FLAG-tagged subunits of several different Mediator subunits and analyzed the purified complexes by multidimensional protein identification technology (MuDPIT)(Sato et al., 2004). Joan Conaway, in the Plenary Lecture, described how all forms of the Mediator contain a consensus set of subunits with some minor exceptions. First, in addition to CDK8, a highly related protein was found, termed CDK11. CDK8 and 11 are found in mutually exclusive complexes Second, the FLAG-MED26 Mediator was enriched in pol II but relatively depleted in CDK8 and 11, although these differences were in relative amounts, not absolute differences as originally suggested. The association of various subunits appears quite dynamic and may signal functional differences in the complexes. Finally, the Conaways identified a number of new human homologs of yeast subunits and several metazoan-specific subunits but none that corresponded to the yeast MED2, 3 and 5. The lack of yeast MED5 is conspicuous because it has been previously reported to contain HAT activity. The recruitment of Mediator by activators involves

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specific activator-Mediator subunit interactions. For example, nuclear receptor AF-2 domains interact with MED1, and phosphorylated ELK-1 interacts with MED23. However, the possible conserved mechanisms by which activators recruit Mediator have been difficult to discern because of the sequence divergence of Mediator subunits between yeast and mammalian cells. Anders Na¨a¨r (Harvard Medical School/MGH Cancer Center) showed that the mammalian activator SREBP interacts with a domain of human MED15 that is structurally highly similar to the KIX domain found in the activator binding sites of the metazoan-specific HATs p300 and CBP. Remarkably, the GAL11 Mediator subunit of S. cerevisiae also contains a KIX domain and Na¨a¨r showed that PDR1, an activator involved in the multidrug-resistance pathway, physically and genetically interacts with it. Much remains to be discovered about the 32 subunit human Mediator. Which subunits contact the GTFs? Is the CDK8 module associated with repression in vivo and what pathways does it intersect? Do Mediator subunits interact with chromatin machines to link remodeling with PIC assembly? Importantly, Mediator stimulates the CTD kinase activity of the KIN28/CDK7 subunit of TFIIH, which is necessary for recruitment of the PAF1 elongation complex and in linking 5⬘ mRNA capping with transcription initiation. The central role of Mediator in this transition suggests it is an important nexus for regulatory decisions. Transcription Elongation The themes of targeted histone modifications, remodeling, and chaperones were extended in the talks on pol II elongation. Pol II must typically pass through eight to ten nucleosomes on the average yeast gene. Elongin, ELL, and TFIIF help accelerate pol II on naked DNA. However, a complex series of interdependent machines are dedicated to transcription on chromatin. These machines include the PAF1 complex (PAF1, CTR9, CDC73, RTF1, and LEO1), FACT (SPT16 and POB1), SPT6, DSIF (SPT4 and 5), P-TEFb, SET2, and COMPASS (complex of proteins associated with SET1). The machines, in addition to promoting transcription on chromatin, are coupled to CTD phosphorylation and communicate the location of pol II to RNA processing enzymes and the transport machinery, thereby ensuring the fidelity of mRNA synthesis (Gerber and Shilatifard, 2003; Hampsey and Reinberg, 2003; Shilatifard, 2004). Don Luse (Cleveland Clinic) showed that pol II promoter escape requires a critical minimum size for both the transcriptional bubble and for the transcript. The helicase subunits of TFIIH help form the open complex and stimulate promoter escape. Promoter escape marks the end of the requirement for the TFIIH helicase. The KIN28/CDK7 subunit of TFIIH phosphorylates Ser5 of the CTD. Ser5 phosphorylation recruits the PAF complex, COMPASS, and the mRNA capping machinery (Ng et al., 2003). Adam Wood (St. Louis University) described how the PAF complex interacts with COMPASS, which also directly contacts pol II (Krogan et al., 2003a). The SET1 subunit of COMPASS methylates H3K4 at and near the promoter. The activity of SET1 depends upon ubiquitinylation of H2B by the RAD6/BRE1 complex (Dover et al., 2002; Wood et al., 2003).

DSIF and NELF cause pol II to pause further downstream. Grant Hartzog (University of California, Santa Cruz) described how the SPT5 subunit of yeast DSIF is in a complex with numerous proteins including the mRNA capping enzymes CEG1 and CET1 (Lindstrom et al., 2003). Previous studies by Buratowski had shown that binding of these enzymes requires Ser5 phosphorylation by KIN28 (Rodriguez et al., 2000). The pause is believed to permit time for the capping reaction to occur. The close proximity of the CTD to the RNA exit channel in pol II suggests that capping occurs shortly after the RNA has begun to protrude from the enzyme. In mammalian cells, the transition from the pause to productive elongation is catalyzed by P-TEFb. Yuki Yamaguchi (Tokyo Institute of Technology) described how phosphorylation of the Thr4 residue in the C-terminal repeat domain of the SPT5 subunit of DSIF by the P-TEFb CDK9-CyclinT kinase somehow transitions pol II into the elongation mode. P-TEFb also phosphorylates the CTD of pol II. CTK1 is a yeast kinase similar in some respects to P-TEFb. As pol II begins productive elongation, the chromatin becomes methylated at H3K36 by SET2 in a reaction dependent upon CTD phosphorylation at Ser2 by CTK1 (Hampsey and Reinberg, 2003). Ser2 phosphorylation is also linked to proper mRNA polyadenylation and 3⬘ end formation (Ahn et al., 2004). The distinct catalytic events associated with transitions in elongation may be necessary to ensure the proper coupling of RNA processing with elongation. Indeed, as discussed by Hartzog, the role of DSIF in these coupling processes is emphasized by association of SPT5 with numerous proteins involved in both chromatin modification (PAF1) and remodeling (CHD1), and mRNA capping, polyadenylation, and transport (THO/TREX complex) (Lindstrom et al., 2003). However, the specific roles, if any, of the nucleosomes remains unclear. Does the promoter proximal nucleosome assist in pol II pausing? Does phosphorylation of SPT5 influence the transition from H3K4 to H3K36 methylation and Ser5 to Ser2 phosphorylation? Do the chromatin events influence mRNA processing or do they simply represent a “memory” of recent transcription? A possible answer to this final question was revealed in microarray experiments described by Hartzog, where mutations in subunits of the PAF1 complex lead to splicing defects. It is unclear how pol II passes through nucleosomes during elongation. In vitro pol II can transcribe nucleosomal templates without accessory factors. Maria Kireeva (National Cancer Institute) showed that pure pol II transcribes into a nucleosome in vitro unless it stops on a naturally occurring sequence-dependent pause site. The histone-DNA contacts that are reestablished downstream of the paused pol II promote backtracking and stabilize the inactive state of the elongation complex. If the backtracking is blocked, however, the efficiency of transcription through the nucleosome significantly increases. In vivo, the extensive modification of nucleosomes within a gene and the observation that heavily transcribed regions are depleted of nucleosomes suggests the participation of ATP-dependent remodelers and histone chaperones. FACT associates with the PAF complex and DSIF as discussed by Hartzog and seems to have intrinsic H2A/H2B chaperone activity, which pro-

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motes pol II passage through nucleosomes in vitro, whereas SPT6 possesses H3 chaperone abilities in vitro (Belotserkovskaya and Reinberg, 2004). It is unclear whether the chaperone activity would be necessary for removing nucleosomes in vivo or simply redepositing them behind the polymerase or both. As for the ATPases, in vitro studies have shown that SWI/SNF has the capability of remodeling nucleosomes during elongation. However, in vivo ISWI ATPases and CHD1 are associated with the coding regions of active genes. Some data suggest that ISW1 binds H3K4-methylated chromatin. However, Hartzog discussed how CHD1, a chromodomain-containing ATPase that associates with the PAF1 complex, DSIF and FACT, may also play a role in elongation (Hartzog, 2003; Simic et al., 2003). CHD1 could target methylated histones for remodeling via its chromodomains, but the exact role and mechanism of CHD1 in elongation remain to be established. The close linkage of transcription and mRNA processing raises the possibility that some so-called elongation factors may simply be involved in coupling. Struhl described how he fused the GAL1 promoter to the 8 kb YLR454 ORF and measured pol II density by ChIP at multiple sites throughout the gene. By turning off transcription with glucose he could examine how various elongation factor mutants affected the rate of elongation (nucleotide additions per minute) and the efficiency by which elongating pol II completed transcription of the gene (processivity). He found that mutations in the RPB2 subunit of pol II affected elongation rate and processivity but mutations in THO complex subunits, PAF1 complex subunits, SET1 and 2, CHD1, Elongin A, TFIIS, and BRE1, among others, had no effect on elongation rate. These intriguing observations are certain to stimulate discussion of the role played by elongation factors in vivo. Complexes involved in elongation also participate in other processes. Joel Eissenberg (Saint Louis University) reported domain-mapping studies of Drosophila ELL (Eissenberg et al., 2002). The N-terminal domain allows ELL recruitment by pol II, whereas the central region is involved in the redistribution of ELL to heat shock genes at elevated temperatures. However, N-terminal-deleted ELL seems to complement recessive lethal mutations in ELL suggesting a developmental role for the protein that is distinct from its elongation function. Distinguishing between On and Off: Silencers and Boundary Elements Silenced chromatin or heterochromatin occurs at the HMR and HML loci and near the telomeres in yeast. At HMR and HML, two silencers direct the recruitment of SIR2, an NAD-dependent histone deacetylase, and SIR 3 and 4, which spread and coat the deacetylated chromatin. Transcriptionally active genes are thought to use several strategies for preventing heterochromatin formation. Histone variants such as H3.3 and H2AZ are associated with active genes and may be inserted cotranscriptionally by chaperones in concert with ATPases such as SWR1. Additionally, as discussed by Wood, DOT1 associates with the PAF complex and methylates H3K79 throughout the active locus. Collectively these alterations may promote transcription while hindering heterochromatin assembly. However, the mechanisms

are not well established, and their relationship to the barriers that delimit active from inactive regions is unknown. The proteins found at natural barriers typically include HATs and ATPases. Ro Kamakaka (National Cancer Institute) described his group’s efforts to identify the mechanisms by which barriers function (Oki et al., 2004). The HMR-E silencer normally inactivates the downstream MAT-␣ gene. Kamakaka used a mating screen to identify GAL4 fusion proteins that would block silencer function and permit mating when positioned between HMR-E and MAT-␣. Of 5500 GAL4-ORF fusions tested, a majority of the selected proteins were found to be involved in chromatin remodeling, much like proteins identified at native barriers. The screen detected SAGA and SAS1 subunits as well as many transcription factors and TFIID subunits. Changes in acetylation and chromatin structure at the boundary differed among different blocking proteins like SNF6 and SAS2. This finding suggested that although the specific consequences may vary, general alterations in chromatin are sufficient to create a barrier. Kamakaka proposed that the formation of barriers involves a junction of opposing activities. Formation of Mammalian Heterochromatin The mechanism of mammalian heterochromatin formation appears to be somewhat more complicated than in yeast. A variety of mechanisms lead to H3K9 and H3K27 methylation, causing recruitment of chromodomaincontaining repressors such as the Polycomb group Repressive Complex (PRC1) and Heterochromatin Protein 1 (HP1) isoforms. The Polycomb proteins were originally identified in Drosophila and fall into several groups that form multiprotein complexes. PRC2 and 3 contain the Ezh2 protein, which methylates H3K27 in vitro thereby recruiting the PRC1 complex that prevents access to remodeling enzymes like SWI/SNF. The specific DNA binding proteins that target PRC2/3 in Drosophila are known and include GAGA and PHO. Several important questions regarding PRC2/3 in mammalian cells include: what recruits PRC2/3? Does the complex methylate histones in vivo and what are its target genes? Peggy Farnham (University of California, Davis) described her group’s use of gene expression microarrays to identify target genes of PRC2/3 in colon cancer cells, where the PRC2/3 Suz12 subunit is upregulated (Kirmizis et al., 2004). By using RNAi approaches, Farnham identified a series of genes that responded to depletion of Suz12. This strategy was coupled with ChIP analysis of Suz12 on CpG island microarrays. She identified a number of Suz12 binding sites and showed that they also bound Ezh2 and Eed, two other PRC2/3 subunits. Furthermore, the binding correlated with methylation of H3K27, but not H3K9. The binding patterns of Suz12 mapped closely with the corepressor CtBP2, originally identified as an E1A-interacting protein that binds sequence-specific repressor proteins. Little is known of the mechanism of heterochromatin formation and how boundaries are established in mammalian cells. The identification of target genes will provide important tools to address this problem. New Discoveries There were many talks where the subject matter did not fit neatly into the sections on gene regulation. Jacques

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Coˆte´ (Laval University) described multiple HAT/HDAC complexes emphasizing structural conservation between yeast and man, and the key role played by the ING subunits, implicated as tumor suppressors and regulators of p53, to control acetylation in eukaryotes. Coˆte´ also proposed how the human Tip60 complex is the structural equivalent to a combination of the yeast NuA4 HAT and SWR1 complexes responsible for H2AZ dimer exhange (Doyon et al., 2004; Kobor et al., 2004; Krogan et al., 2003b; Mizuguchi et al., 2004). Jerry Workman recent work showed that the TIP60 histone acetylase works in concert with the Domino ATPase (a SWR1 homolog) in a complex to acetylate phosphorylated H2Av in Drosophila (human H2AX) and exchange it with unmodified H2Av at double stranded DNA breaks during repair (Kusch et al., 2004). These processes and others like it provides example for histone exchange processes associated with transcription and other metabolic events. Tony Kouzarides (University of Cambridge) identified SET9 as an H4 methylase that participates in DNA repair and recruits CRB2, the adaptor for the checkpoint kinase CHK1. New transcriptional roles were found for long and short interspersed repetitive elements (LINES and SINES) found in mammals. These elements, long thought to constitute junk DNA, are emerging as important regulatory sequences. Jef Boeke (Johns Hopkins University) described how LINE-1 retrotransposon elements can act as transcriptional elongation pause sites and discussed how LINE-1 is generally associated with introns or UTRs of poorly transcribed genes and may fine tune transcription levels throughout the genome (Han et al., 2004). Jim Goodrich (University of Colorado) showed that the RNA encoded by the B2 SINE is upregulated during heat shock and can bind and inhibit RNA polymerase II initiation (Allen et al., 2004; Espinoza et al., 2004). Ramin Shiekhattar (Wistar institute) described his purification of the DROSHA complex that functions with Dicer to process microRNAs. He has successfully recreated this processing reaction in vitro (Gregory et al., 2004). One of the most remarkable insights of the meeting was concept that the size and composition of transcriptional coactivators and chromatin machines don’t change significantly from yeast to man; the key subunits and machines are largely conserved, as are important aspects of mechanism. Perhaps we shouldn’t be surprised given that man has only four times more genes than yeast. Yet how the machinery is used in mammalian cells seems much more complex. The more effective combinatorial usage of activators and signaling pathways, coupled with differential splicing, seems to make a world of difference. If indeed combinatorial usage is the key, then we can look forward to many novel discoveries illustrating how different developmental and extracellular signaling pathways intersect the transcriptional machinery. References Adkins, M.W., Howar, S.R., and Tyler, J.K. (2004). Chromatin disassembly mediated by the histone chaperone Asf1 is essential for transcriptional activation of the yeast PHO5 and PHO8 genes. Mol. Cell 14, 657–666.

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