Báo cáo Y học: Nuclear receptor-dependent transcription with chromatin Is it all about enzymes? pot

MINIREVIEW Nuclear receptor-dependent transcription with chromatin Is it all about enzymes? W Lee Kraus 1,2 and Jiemin Wong 3 1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA; 2 Department of Pharmacology, Weill Medical College of Cornell University, New York, USA; 3 Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza Houston, TX, USA Nuclear receptors (NRs) are ligand-regulated, DNA-bind- ing transcription factors that function in the chromatin environment of the nucleus to alter the expression of subsets of hormone-responsive genes. It is clear that chromatin, rather than being a passive player, has a profound effect on both transcriptional repression and activation mediated by NRs. NRs act in conjunction with at least three general classes of cofactors to regulate transcription in the context of chromatin: (a) chromatin remodelers; (b) corepressors; and (c) coactivators, many of which have distinct enzymatic activities that remodel nucleosomes or covalently modify histones (e.g. acetylases, deacetylases, methyltransferases, and kinases). In this paper, we will present a brief overview of these enzymes, their activities, and how they assist NRs in the repression or activation of transcription in the context of chromatin. Keywords: chromatin; chromatin remodeling; coactivators; corepressors; histone acetyltransferase; histone deacetylase; histone kinase; histone methyltransferase; nuclear receptor; transcription. INTRODUCTION Nuclear receptors (NRs) comprise a large superfamily of DNA-binding transcriptional regulatory proteins that con- trol the expression of distinct subsets of genes in the chromatin environment of the nucleus [1–3]. In many cases, the activities of the receptors are modulated by the binding of hormonal ligands (e.g. steroids, retinoids, thyroid hormone, and vitamin D 3 ), which function as key regulators in numerous physiological processes (e.g. growth, develop- ment, metabolism, homeostasis, and reproduction) [1,2]. Most nuclear receptors share a conserved structural and functional organization, including a highly conserved DNA-binding domain, a C-terminal ligand-binding domain, and two transcriptional activation functions (an N-terminal AF-1 and a C-terminal AF-2) (Fig. 1) [1,2]. The two most widely studied classes of NRs can be categorized based on their dimerization and DNA binding properties: (a) class I contains the steroid hormone receptors, which function primarily as homodimers, and (b) class II contains the vitamin and thyroid hormone receptors, which function primarily as heterodimers with RXR [2]. The structural organization of the receptors makes them ideally suited for the transduction of hormonal signals into gene-regulatory transcriptional responses. During the regulation of hormone-responsive genes, NRs must gain access to their cognate receptor binding sites (hormone response elements, or HREs) in promoter DNA that is assembled into chromatin, the physiological template for transcription [3,4]. The packaging of genomic DNA into nucleosomes (protein–DNA structures which are the repeat- ing units of chromatin) restricts the access of the transcrip- tional machinery to the promoters of hormone-regulated genes, thereby reducing the transcription of those genes [3–5]. Although chromatin was at one time largely overlooked or considered a passive player in NR-dependent transcription, it is now clear that it plays a critical role. The importance of chromatin in achieving a proper ligand-regulated, NR-dependent transcriptional response (i.e. on/off switch- ing with/without hormone) has been demonstrated experi- mentally using both in vitro and cell-based assays [6,7]. NRs make use of chromatin to apply an exquisite level of transcriptional control to the genes that they regulate (i.e. repress or activate), but they do not carry out this alone. NRs act in conjunction with at least three general classes of cofactors to regulate transcription in the context of chro- matin, namely: (a) chromatin remodelers; (b) corepressors; and (c) coactivators, many of which have distinct enzymatic activities [4,8,9]. For example, chromatin remodeling Correspondence to W. L. Kraus, Department of Molecular Biology and Genetics, Cornell University, 465 Biotechnology Building, Ithaca, NY 14853, USA. Fax: + 1 607 255 6249, Tel.: + 1 607 255 6087, E-mail: wlk5@cornell.edu, Abbreviations: CARM, coactivator associated arginine methyltrans- ferase; CBP, CREB-binding protein; HAT, histone acetyltransferase; HDAC, histone deacetylase; HMT, histone methyltransferase; NCoR, nuclear receptor corepressor; NR, nuclear receptor; PCAF, p300/ CBP-associated factor; PRMT, protein arginine methyltransferase; SMRT, silencing mediator for retinoid and thyroid hormone recep- tors; SRC, steroid receptor coactivator. Note: a homepage for W. L. Kraus can be found at http://www.mbg.cornell.edu/kraus/kraus.html. The Cornell University Department of MBG homepage can be found at http://www.mbg.cornell.edu. Dedication: This Minireview Series is dedicated to Dr Alan Wolffe, deceased 26 May 2001. (Received 8 October 2001, accepted 7 December 2001) Eur. J. Biochem. 269, 2275–2283 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02889.x complexes contain ATPase subunits [10]. Likewise, corepressor and coactivator complexes contain histone modifying enzymes (e.g. acetylases, deacetylases, methyl- transferases, and kinases) that covalently modify specific lysine, arginine, or serine residues in the N-terminal tails of the core histones [4,11–14]. One consequence of these post- translational modifications may be to specify a Ôhistone codeÕ that directs the binding of various regulatory factors, via specific chromatin-binding domains (e.g. bromodomains and chromodomains), to the histone tails [13]. The various enzymatic activities listed above are recruited to hormone- responsive promoters via direct or indirect interactions with NRs and subsequently modify their chromatin substrates to regulate transcription by RNA polymerase II (RNA pol II) (Fig. 2) [3,4,8]. In this paper, we will present a brief overview of these enzymes, their activities, and how they assist NRs in the repression or activation of transcription in the context of chromatin. Although the methods used to generate this information will not be presented in detail, many of them have recently been reviewed elsewhere [15]. CHROMATIN MODIFYING ENZYMES AND THE REPRESSION OF TRANSCRIPTION BY NRs Histone deacetylases (HDACs) Many class II NRs, including thyroid hormone receptor and retinoic acid receptor, have the capacity to actively repress the transcription of their target genes in the absence of their cognate ligands [3,4,9,16]. Early competition experiments indicated that NR-dependent transcriptional repression requires cellular accessory proteins, termed corepressors [17,18]. Two closely related corepressors, SMRT and NCoR, were subsequently identified in yeast two-hybrid screens based on their ability to interact with unliganded NRs [19,20]. The requirement of corepressors for repression by unliganded NRs is clearly illustrated by a lack of NR-dependent repression in isolated NCoR –/– mouse embryo fibroblasts [21]. Although transcriptional repression can be mediated via direct targeting of the basal transcription machinery by unliganded NRs and/or core- pressors [22,23], strong evidence indicates that chromatin structure plays a pivotal role in repression by unliganded NRs [6,24,25]. The importance of chromatin in NR- dependent repression was demonstrated in studies using Xenopus oocytes showing that repression of the TRbA promoter by unliganded TR/RXR heterodimers requires the proper assembly of the promoter into chromatin [6]. Following the identification of SMRT and NCoR as corepressors for NRs, a number of studies were published implicating histone deacetylase (HDAC) enzymes in SMRT/NCoR-dependent transcriptional repression by unliganded NRs (reviewed in [4,9,11,12,16]). The identifi- cation of HDACs as components of NR corepressor complexes, in conjunction with a well documented correla- tion between hypoacetylated core histones and transcrip- tionally inactive chromatin [13,26], has led to a major effort to clarify the roles of HDACs in repression by unliganded NRs. Currently, three major classes of HDACs have been identified. Class I includes HDACs 1, 2, 3, and 8, which are related to the yeast transcriptional regulator Rpd3p, and class II includes HDACs 4, 5, 6, and 7, which are related to yeast Hda1p [27]. The third class of HDACs are the NAD + -dependent Sir2 family proteins [28]. While it is clear that HDAC activity is essential for repression by unliganded nuclear receptors [24,25], questions remain regarding which HDACs are involved and how they are recruited by unliganded NRs. Class I HDACs, specifically HDACs 1 and 2, were the first to be implicated in NR-dependent transcriptional repression, as they were found to interact with mammalian Sin3 proteins (Sin3A and Sin3B), which in turn were found to interact with both SMRT and NCoR [29,30]. The observed interactions between Sin3 and the corepressors led to a model where unliganded NRs could repress tran- scription, at least in part, through the recruitment of Sin3-HDAC1/2 complexes by SMRT or NCoR [29,30]. However, the role of Sin3 proteins in repression mediated by SMRT and NCoR is not as straightforward as the model suggests. Some reports indicate that purified native SMRT– NCoR complexes contain Sin3 [31,32], while others indicate that the purified repressor complexes contain none [31,33– 35]. Whether this discrepancy reflects differences in the purification protocols used, the existence of distinct or heterogeneous SMRT–NCoR complexes, or variations in the strength of association of Sin3 complexes with different corepressor complexes under different cellular conditions is presently unclear. Further studies will be required to sort out these possibilities. Class II HDACs, including HDACs 4, 5, 6, and 7, have been shown to interact directly with SMRT and NCoR, thus representing a second possible mechanism by which HDAC activities can be recruited to unliganded NRs [36,37]. However, the extent to which class II HDACs contribute to repression by unliganded NRs is an open question, as native SMRT and NCoR complexes purified from human cells or Xenopus oocytes have so far only been found to contain class I HDACs (primarily HDAC3, but also HDACs 1 and 2) [31–33,35]. Thus, the relative importance of class I and class II HDACs in NR-dependent transcriptional repression remains undetermined. Interest- ingly, the class I HDAC3 has also recently been shown to interact directly with SMRT and NCoR, leading to a stimulation of HDAC activity [38]. Such interactions that alter enzyme activity could provide an additional level of regulatory control. AthirdmechanismbywhichclassIorIIHDAC complexes might contribute to NR-dependent transcrip- tional repression is through a nontargeting mechanism. In this scenario, interactions with histone-binding proteins such as RbAp46/48, which are found in HDAC com- plexes [11,12], could direct the HDACs to the chromatin template rather than HDAC-corepressor–NR interactions. Whether this occurs in vivo has not been determined. Fig. 1. NRs share a conserved structural and functional organization, including a highly conserved DNA-binding domain (DBD), a C-terminal ligand-binding domain (LBD), and two transcriptional activation func- tions (AF-1 and AF-2). 2276 W. L. Kraus and J. Wong (Eur. J. Biochem. 269) Ó FEBS 2002 Regardless of how the HDACs are brought to the chromatin template, it is clear that they play an important role in transcriptional repression by unliganded NRs in the context of chromatin. Although HDACs have also been shown to deacetylate nonhistone substrates, inclu- ding acetylated p53 and NF-jB [39–41], a role for factor deacetylase activity in NR-dependent transcription has not been demonstrated. HDAC complexes containing chromatin remodelers At least two types of HDAC complexes containing ATP- dependent chromatin remodeling activities have been iden- tified. The first includes variations of the Mi-2/NURD complex [42–48], which in its most complete form contains HDACs 1 and 2, the histone-binding proteins RbAp46/48, the Snf 2-related ATPase Mi-2, and methyl-DNA binding proteins such as MBD2 and MeCP1 [49]. The second is the NCoR-1 complex, which contains NCoR, HDAC3, and several subunits of the SWI/SNF complex, including the Snf2-related ATPase Brg1 [32]. Of the two types of complexes, only the Mi-2/NURD complex has been shown to repress transcription in biochemical assays, which it does through remodeling and histone deacetylation of nucleo- somes assembled from methylated DNA [49]. The role of HDAC/chromatin remodeler complexes in NR-dependent transcriptional repression has not been investigated exten- sively. However, one study has shown that microinjection of neutralizing antibodies against Mi-2 (also known as CHD4) partially relieves TR-dependent transcriptional repression in Xenopus oocytes [46]. These results suggest a role for HDAC–chromatin remodeler complexes in NR-dependent repression. However, further studies in this area are clearly needed. Interestingly, the expression of genes encoding a number of NRs (including estrogen receptor and retinoic acid receptor b) has been shown to be inhibited through CpG methylation of the NR gene promoter DNA in some cancer cell lines [50]. Thus, methylation-targeted Mi-2/NURD complexes might play a role in regulating NR activity by altering NR expression in some pathological states. Histone methyltransferases (HMTs) In addition to acetylation, core histones, especially H3 and H4, are also targets for methylation. A number of histone methyltransferases (HMTs) have been identified, including: (a) the H3 lysine 9 (H3-K9)-specific HMTs Suv39H1 and G9a, which are involved in transcriptional repression or silencing [51,52]; (b) the H3 lysine 4 (H3-K4)-specific HMT Set 9 (also known as Set7), which is involved in transcrip- tional activation [53,54]; and (c) members of the protein arginine methyltransferase (PRMT) family, such as PRMT1 and CARM1, which are also involved in transcriptional activation [55–58]. While no specific methyltransferase has been reported to participate in transcriptional repression by unliganded NRs, it is worth mentioning these enzymes because they may indirectly reduce the transcriptional activity of NRs. For example, Suv39H1 is a heterochroma- tin-associated, SET domain-containing protein with intrin- sic H3 lysine 9-specific HMT activity [51]. The methylation of H3 lysine 9 in nucleosomes generates a binding motif for the chromodomain of the heterochromatin-associated pro- tein HP1, which can promote the formation of higher order chromatin structures that are repressive to transcription [59,60]. Previous studies have shown that the incorporation of linker histones into chromatin, which also promotes the formation of higher order chromatin structures, reduces NR-dependent transcription [61–63]. Thus, it is possible that a similar effect will be observed with HP1. Whether unliganded NRs use lysine-specific HMTs to actively repress transcription, however, remains to be determined. CHROMATIN MODIFYING ENZYMES AND THE ACTIVATION OF TRANSCRIPTION BY NRs ATP-dependent chromatin remodelers As mentioned above, the packaging of genomic DNA into nucleosomes restricts the receptor-dependent assembly of transcription complexes at the promoters of hormone- regulated genes. Unlike many DNA-binding transcriptional regulators, NRs bind stably and with reasonably high affinity to DNA even when their cognate HREs are assembled into chromatin [3]. Thus, the relevant issue seems to be how the receptors promote the formation of an open chromatin architecture at the promoter. One way is through the ligand-dependent recruitment of chromatin remodeling complexes, which are multipolypeptide enzymes categorized by the type of ATPase subunit that they contain, including yeast Snf2-like (e.g. SWI/SNF) or Drosophila ISWI-like (e.g. RSF, CHRAC, ACF) [10]. Human SWI/SNF (hSWI/ SNF) represents a family of related complexes usually containing eight or nine subunits, with either hBrg1 or hBrm as the ySnf2-related ATPase subunit; however, the exact composition of the complexes can vary from one cell type to the next [10]. Chromatin remodeling complexes use the energy stored in ATP to mobilize or structurally alter nucleosomes, allowing greater access of the transcriptional machinery to promoter DNA, thus facilitating transcrip- tional activation [3,4,8,10,64]. The involvement of SWI–SNF complexes in NR- dependent transcription was originally suggested by studies in yeast and mammalian cells showing a stimulatory effect Fig. 2. Multiple proteins with chromatin remodeling or histone modify- ing activities facilitate transcriptional regulation (repression and activa- tion) by NRs. See the text for abbreviations and details. Ó FEBS 2002 Nuclear receptors and chromatin (Eur. J. Biochem. 269) 2277 of SWI–SNF components on NR-dependent activity [65– 68]. Since then, additional cell-based approaches have supported these results, including experiments showing a requirement for hBrg1-receptor interactions in estrogen receptor and glucocorticoid receptor gene regulatory activ- ity [69,70] and chromatin immunoprecipitation (ChIP) experiments showing the recruitment of hBrg1 to an estrogen-regulated promoter upon hormonal stimulation [70]. Recently, a direct demonstration of the requirement for the hSWI/SNF complex in receptor-dependent transcrip- tion was made using the purified complex and an in vitro chromatin assembly and transcription system with retinoic acid receptor/RXR heterodimers [71]. Additional in vitro transcription experiments have shown that recombinant ISWI can support progesterone receptor-dependent trans- cription with chromatin templates [72]. These in vitro studies, in conjunction with previous cell-based studies, make the important point that although ATP-dependent chromatin remodeling is required for NR-dependent trans- cription, it is not sufficient [71,73,74]. Chromatin remode- ling may set the stage for subsequent actions by coactivators with histone modifying activities, such as histone acetyl- transferases [8,71,74,75]. Histone acetyltransferases (HATs) Numerous studies in yeast and higher eukaryotic organisms have demonstrated a link between the acetylation of specific lysine residues in the N-terminal tails of core histones (e.g. histone H3 lysine 14) and the activation of transcription [26,76]. An intriguing connection between NRs and chro- matin was made when some nuclear receptor coactivators, including p300 and CBP (two closely related factors commonly referred to collectively as p300/CBP), as well as PCAF (p300/CBP-associated factor), were found to possess intrinsic nucleosomal HAT activity [77–79]. Although initial studies suggested that both p300/CBP and PCAF bind directly to NRs [80,81], more recent results indicate that the interaction of p300/CBP with many NRs is mainly indirect, and mediated by the SRC (steroid receptor coactivator) family of bridging factors [82–85]. Interestingly, some reports [86,87], but not others [84,88], have suggested that members of the SRC family also possess a weak intrinsic HAT activity. Recent biochemical experiments indicate that a critical function of ligand-activated, DNA-bound NRs is to serve as nucleation sites for the recruitment of HAT enzymes to promoters in chromatin [83], a conclusion consistent with ChIP assays showing ligand-dependent recruitment of HATs to hormone-regulated promoters in vivo [89,90]. The link between histone acetylation and transcriptional activation is, by now, well-established, yet the exact mechanism of how histone acetylation leads to enhanced activation is not clear. Although histone acetylation was originally thought to facilitate chromatin remodeling by ÔlooseningÕ the association of the histone octamer with DNA through the neutralization of positive charges in the histone tails, more recent results suggest that histone acetylation may require prior chromatin remodeling or may occur at a post-remodeling step [8,71,74,75]. The results of one study suggest that post-remodeling histone acetylation by p300 may direct the transfer of histone H2A–H2B dimers from nucleosomes to a histone chaperone [75]. Such an effect may help to establish and maintain an open chromatin confi- guration conducive to transcription. The differences observed in the order of action of chromatin remodelers and HATs in different experimental systems have not been adequately explained, but may represent promoter-specific types of regulation [8]. Recent results suggest another role for histone acetylation, namely to create binding sites on the amino-terminal tails of core histones for acetylated lysine binding domains, such as the bromodomain (reviewed in [91]), similar to the way that methylated H3-K9 serves as a binding site for chromodomain-containing proteins (des- cribed above). A mechanism like this may allow for the recruitment of bromodomain-containing factors (e.g. the HAT TAF II 250) to promoters that have nucleosomal histones with specific patterns of acetylation [91]. Another question related to histone acetylation is why a number of different HATs are required by NRs to activate the transcription of genes in chromatin. The fact that p300/ CBP and PCAF have different histones [77–79,92] and nonhistones (see, for example [93–95]), substrate specificities may provide the answer, as each can acetylate a distinct set of targets, possibly directing a distinct set of outcomes. Although HAT activity is critical for NR-dependent transcription, it is important to note that coactivators such as PCAF (which is found in a large multipolypep- tide complex with a number of other transcription-related factors [96]) and p300/CBP contribute other activities to the transcription process. For example, p300/CBP inter- acts with RNA pol II complexes [97] and possesses a glutamine-rich C-terminal region similar to the gluta- mine-rich activation domains found in some transcrip- tional activators, suggesting that p300/CBP may also function as ÔclassicalÕ coactivator by interacting with RNA pol II [98]. Furthermore, both PCAF and p300/ CBP can acetylate nonhistone, transcription-related fac- tors,whichinmanycaseshasbeenshowntoalterthe activity of those factors (reviewed in [99]). For example, the acetylation of SRC3 (also known as ACTR, an SRC family member) by p300 was shown to cause a disruption of receptor–coactivator complexes, leading to a decrease in receptor-mediated gene activation [90]. Estrogen receptor alpha has been shown to be a target for p300-mediated acetylation, which may alter the transcriptional activity of the receptor [100]. Thus some HATs, such as p300/CBP and PCAF, serve as multi- functional coactivators for NR-dependent transcription, contributing multiple activities to the process. HMTs Although some HMTs, such as Suv39H1 described above, are involved in gene silencing, other HMTs are involved in gene activation. In a recent collection of experiments, two PRMT (protein arginine methyltransferase) family mem- bers, CARM1 (coactivator-associated arginine methyl- transferase) and PRMT1, were shown to interact with SRC2 (also known as GRIP1, an SRC family member) and enhance the activity of a variety of nuclear receptors in mammalian cell-based reporter gene assays [55,56], as well as Xenopus microinjection experiments [57]. The stimulation of receptor activity by CARM1 and PRMT1 was shown to be dependent on the presence of the SRC protein and require the intrinsic methyltransferase activities of CARM1 2278 W. L. Kraus and J. Wong (Eur. J. Biochem. 269) Ó FEBS 2002 and PRMT1 [55–57]. Both CARM1 and PRMT1 can methylate histones in vitro [56,57], and recent studies suggest that both do so in vivo as well [57,58,101]. Interestingly, CARM1 and PRMT1 exhibit different HMT specificities; CARM1 primarily methylates arginines 17 and 26 of histone H3 (H3-R17 and R26) [102], whereas PRMT1 methylates arginine 3 of histone H4 (H4-R3) [57]. The fact that these two methyltransferases have distinct, rather than overlapping, HMT specificities may underlie the synergism that has been observed between them during the stimulation of NR activity [55]. Like the HATs mentioned above, CARM1 and PRMT have also been shown to methylate nonhistone substrates (e.g. p300/CBP by CARM1; STAT1 by PRMT1), thereby regulating the transcriptional activity of the target proteins [103,104]. However, the role of factor methylation in NR-dependent transcription has not yet been explored in detail. Interestingly, cooperative functional interactions between HMTs and HATs have been observed during the stimula- tion of NR activity (e.g. synergism between CARM1 and p300 with estrogen receptor) [105]. The interplay between HMTs and HATs can be observed at the enzymatic level, as well. For example, methylation of H4-R3 by PRMT1 was shown to increase acetylation of H4-K8 and K12 by p300 [57]. In contrast, preacetylation of H4 reduced subsequent H4-R3 methylation by PRMT1 [57]. Although not demon- strated in the context of NR-dependent transcription, H3-K4 methylation by Set9 (also known as Set7) has been shown to antagonize H3-K9 methylation by Suv39H1, and vice versa [53,54]. Furthermore, pre-methylation by Set9 and Suv39H1 of H3 in a core histone mixture had different effects on the subsequent acetylation of H3 and H4 by p300, with Set9 stimulating and Suv39H1 inhibiting the p300- mediated acetylation [54]. These results illustrate the com- plex interactions that can occur between different factors with histone modifying activities, generating interplay at the level of the nucleosome that results in a Ôcombinatorial histone codeÕ [91]. Furthermore, they suggest that dissecting the individual contributions of particular HMTs and HATs in the context of a large, NR-dependent transcriptional regulatory complex may be difficult, as each effect may be subtle and may influence the activity of other factors in the complex. This will be an important area of continued investigation. Linker and core histone kinases Linker histones, such as histone H1, bind to the linker DNA flanking the nucleosome core. In doing so, they facilitate the compaction of chromatin into higher order chromatin structures, which can lead to the repression of transcription with a variety of DNA-binding activators, including NRs [61,106,107]. Interestingly, the repression of NR activity by histone H1 can be relieved by the ligand- dependent phosphorylation of H1 by cdk2 (and possibly other kinases, as well), which leads to the removal of H1 from the promoter region [62,108,109]. Thus, post-trans- lational modification of linker histones must be considered when evaluating the transcriptional activity of NRs with chromatin. Interestingly, recent studies suggest that another post-translational modification of H1, namely ubiquitination, may also play a role in regulating the repressive effects of H1 [110]. However, this has not yet been shown to play a role in modulating NR transcrip- tional activity. In addition to the phosphorylation of linker histones, the phosphorylation of the core histone H3, specifically at serine 10, has also been shown to enhance the transcription of genes in chromatin (reviewed in [13]). The phosphorylation of H3-S10 appears to be tied to intracellular protein kinase signaling pathways [111,112] and can enhance the subse- quent acetylation of nearby lysine residues [111,113,114]. Thus, as was observed for methylation and acetylation of H4 [57], phosphorylation and acetylation of H3 are functionally linked. Although H3 phosphorylation has not yet been demonstrated to play a role in NR-dependent transcription with chromatin, it seems likely to play a role. In this regard, it is interesting to note that some of the same signaling pathways that enhance H3-S10 phosphorylation (e.g. MAP kinase) [111,112] have been shown to enhance NR transcriptional activity [115,116]. CONCLUSIONS In closing, we should ask, ÔIs NR-dependent transcription with chromatin really all about enzymes?Õ Given the fact that NRs rely on coactivators that target the basal transcriptional machinery (e.g. the mediator complexes) [117] in addition to enzymes involved in chromatin remodeling and histone modification, the answer is no. Nonetheless, it is clear that the enzymes described herein play critical roles in the process of NR-stimulated tran- scription by RNA pol II, which is itself a complex and fascinating enzyme. 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