JOURNAL OF CELLULAR PHYSIOLOGY 184:116 (2000)

REVIEW ARTICLE

Histone Deacetylases, Transcriptional Control, and Cancer

W. DOUGLAS CRESS AND EDWARD SETO* Molecular Oncology Program, H. Lee Moftt Cancer Center and Research Institute, University of South Florida, Tampa, Florida A key event in the regulation of eukaryotic gene expression is the posttranslational modication of nucleosomal histones, which converts regions of chromosomes into transcriptionally active or inactive chromatin. The most well studied posttranslational modication of histones is the acetylation of -amino groups on conserved lysine residues in the histones amino-terminal tail domains. Signicant advances have been made in the past few years toward the identication of histone acetyltransferases and histone deacetylases. Currently, there are over a dozen cloned histone acetyltransferases and at least eight cloned human histone deacetylases. Interestingly, many histone deacetylases can function as transcriptional corepressors and, often, they are present in multi-subunit complexes. More intriguing, at least some histone deacetylases are associated with chromatinremodeling machines. In addition, several studies have pointed to the possible involvement of histone deacetylases in human cancer. The availability of the cloned histone deacetylase genes has provided swift progress in the understanding of the mechanisms of deacetylases, their role in transcription, and their possible role in health and disease. J. Cell. Physiol. 184:116, 2000.
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For many years, studies aimed at elucidating the mechanisms of transcriptional activation sought to determine how DNA-bound transcription factors could affect transcription initiation and elongation by RNA polymerase II. A major leap forward in this endeavor was the discovery that some DNA-bound transcriptional activation domains function, at least in part, by binding so-called coactivator complexes that possess histone acetyltransferase activity. Almost in parallel, investigators seeking to decipher the mechanisms of transcriptional repression identied corepressor complexes that possess histone deacetylase activity. The physiological signicance of these ndings is underscored by the fact that some human cancers have been associated with malfunctions of coactivator or corepressor components. The histone deacetylases, their roles in transcriptional repression, and their involvement in human cancer are the central topics of this review. DNA PACKAGING AND HISTONE-MODIFYING ENZYMES Eukaryotic DNA is packaged into chromatin. It is becoming increasingly clear that the transcription of these tightly packaged genes is regulated, at least in part, by chromatin-remodeling events, which can render the DNA either more or less accessible to transcription factors. The most basic element of DNA packaging is the nucleosome, which consists of DNA wrapped twice around a histone octamer, which contains two copies each of four different histone proteins (H2A, H2B, H3, and H4). Nucleosomes form on DNA at approximately every 200 base pairs, with 146 base pairs
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tightly wound around the histone octamer and the remaining 54 base pairs intervening. This arrangement forms what appears, under electron microscopy, to be a series of beads on a string. The nucleosomes themselves can interact with one another to form a spiral that has been termed the solenoid structure. This structure is 30 nm in diameter, contains six nucleosomes per turn, and, in some eukaryotic cells, requires one copy of the histone H1 protein to interact with each nucleosome. It is thought that DNA packaged into this compact structure is transcriptionally inert. Histone acetyltransferases Crystallography studies revealed that the N-terminal tails of histone proteins protrude from the nucleosome complex (Luger et al., 1997). These extensions appear to be involved in higher-order packaging by mediating contacts between adjacent nucleosomes. Histone tails contain highly conserved lysine residues that can be acetylated on their -amino groups. Each acetylation event eliminates another positive charge and potentially weakens the electrostatic interactions that tether the octamer tails to the DNA phosphate backbone. Histone acetylation may affect chromatin strucContract grant sponsor: National Institutes of Health. *Correspondence to: Edward Seto, Molecular Oncology Program, H. Lee Moftt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612. E-mail: setoe@moftt.usf.edu Received 10 February 2000; Accepted 21 February 2000

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TABLE 1. Classification of histone deacetylases Species Yeast RPD3 Class I: RPD3-like

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Class II: HDA1-like HDA1 HOS1 HOS2 HOS3

Class III: HD2-like

Reference Vidal and Gaber, 1991; Rundlett et al., 1996

C. elegans Drosophila Xenopus Chicken Mouse Human

HDA1 HDA2 HDA3 dHDAC1 (dRPD3, RPD3) dHDAC2 (DmHDAC2) dHDAC3 (DmHDAC3) HDm (RPD3) HDAC1 (HDAC1A) HDAC2 (HD2) HDAC3 (HD3) HDAC1 (HD1) HDAC2 (mRPD3) HDAC3 HDAC1 (HD1) HDAC2 (hRPD3) HDAC3 RPD3

Shi and Mello, 1998 dHDA2 DeRubertis et al., 1996; Johnson et al., 1998 Ladomery et al., 1997 Takami et al., 1999 mHDA1 mHDA2 HDAC4 (HDAC-A) HDAC5 (HDAC-B) HDAC6 HDAC7 HDAC8 HD2 Yang et al., 1996; Bartl et al., 1997; Mahlknecht et al., 1999; Verdel and Khochbin, 1999 Taunton et al., 1996; Yang et al., 1996, 1997; Dangond et al., 1998; Emiliani et al., 1998; Fischle et al., 1999; Grozinger et al., 1999; Miska et al., 1999 Lusser et al., 1997; Aravind and Koonin, 1998

Maize

ture by at least two different mechanisms. First, the net reduction in positive charge could lead to destabilization and consequent dissociation of nucleosomes, thus allowing access of transcription factors and RNA polymerase to the DNA. Second, histone acetylation may inhibit the stacking of nucleosomes into the solenoid structure and thus the formation of higher-order structure. With these phenomena in mind, it is easy to envision a model in which the transcription of genes in any given region is essentially controlled by accessibility. The disruption of higher-order structures by acetylation would be expected to stimulate transcription; conversely, the facilitation of higher-order structures by deacetylation would inhibit transcription. This concept seems to work well in providing a theoretical platform from which to approach numerous model systems, but is actually an oversimplication. Indeed, it is quite possible that acetylation may have either positive or negative effects, depending on the particular gene involved (Sun and Hampsey, 1999). For example, a third mechanism by which histone acetylation could regulate transcription is by affecting the binding of regulatory proteins to the histones themselves. Furthermore, it is possible that some histone acetyltransferases and deacetylases, in fact, act on other transcription factors instead of, or in addition to, the histones (Gu and Roeder, 1997; Gu et al., 1997; Boyes et al., 1998; Zhang and Bieker, 1998; Sartorelli et al., 1999). The rst eukaryotic transcription factor recognized to encode an acetyltransferase was the protein GCN5. Yeast GCN5 was initially identied as a global transcriptional coactivator via genetic techniques (Georgakopoulos and Thireos, 1992; Marcus et al., 1994); however, its biological function was not apparent until the momentous cloning of Tetrahymena histone acetyltransferase A (Brownell et al., 1996). The deduced amino acid sequence of the Tetrahymena enzyme revealed obvious sequence homology with yeast GCN5 and established, for the rst time, a direct link between transcriptional coactivators and histone acetylation.

This link was rmly reinforced by the demonstration that GCN5 possesses acetyltransferase activity, and that this activity is essential for transcriptional activation in vivo (Candau et al., 1997). It is now clear that numerous yeast and mammalian transcriptional coactivators are, in fact, histone acetyltransferases (for recent reviews see Hampsey, 1997; Struhl, 1998). Initially described as a transcriptional coactivator for a number of enhancer-binding proteins (Janknecht and Hunter, 1996), p300/CBP (CREB-binding protein) is one of the best understood examples of human histone acetyltransferases (Bannister and Kouzarides, 1996; Ogryzko et al., 1996b). The p300/ CBP protein forms complexes with P/CAF (p300/CBPassociated factor), which is also a well-characterized histone acetyltransferase (Yang et al., 1996b). One transcription factor that utilizes the p300/PCAF complex as a coactivator is the retinoic acid receptor alpha (RAR ). RAR forms a DNA-binding heterodimer with a retinoid-X receptor (RXR). The RAR/RXR dimer represses transcription in the absence of the hormone ligand retinoic acid. Upon hormone binding, the dimer releases the transcriptional inhibitory complex and subsequently binds to the p300/PCAF complex. This complex can activate the transcription of a number of developmentally regulated genes, such as those involved in hematopoiesis (Lenny et al., 1997). Histone deacetylases Broadly speaking, histone deacetylase proteins from various species can be divided into three categories: (1) the class I RPD3-like proteins; (2) the class II HDA1like proteins; and (3) the class III maize HD2 protein (Table 1). Class I. The rst breakthrough in the identication of the histone deacetylases came with the cloning of HDAC1 (initially termed HD1). HDAC1 and an associated protein, RbAp48 (Rb-associated protein 48), were copuried by afnity chromatography using a modied form of the HDAC inhibitor trapoxin as an afnity

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reagent (Taunton et al., 1996). Sequence analyses revealed that HDAC1 was very similar to the yeast RPD3 protein (reduced potassium dependency), a known yeast transcriptional regulator (Vidal et al., 1996). These ndings provided the rst direct experimental evidence linking histone deacetylation and transcriptional control. Almost concurrently, a second HDAC (HDAC2) was identied as a protein that binds to the transcription factor YY1 via use of the yeast two-hybrid system (Yang et al., 1996a). Human HDAC1 and HDAC2 are 75% identical in DNA sequence and 85% identical in protein sequence, respectively. Three groups have independently cloned an additional member of the class I human histone deacetylase family (Yang et al., 1997; Dangond et al., 1998; Emiliani et al., 1998). Analysis of the predicted amino acid sequence of this protein, HDAC3, revealed an open reading frame of 428 amino acids with a predicted molecular mass of 49 kDa. HDAC3 and the cloned human HDAC1 share 50% sequence identity at the DNA level, and 53% identity at the protein level. Comparisons of the HDAC3 protein and DNA sequences with those from human HDAC2 yielded similar results, with 51% identity in DNA sequence and 52% identity in protein sequence. Class II. Yeast cells have at least two distinguishable histone deacetylase activities catalyzed by different enzymatic complexes (Carmen et al., 1996; Rundlett et al., 1996). The active component of the histone deacetylase A complex is the HDA1 catalytic subunit. The histone deacetylase A complex has a native molecular mass of approximately 350 kDa. The catalytic subunit of the 600-kDa histone deacetylase B complex is RPD3 or the RPD3-related proteins HOS1, HOS2, or HOS3 (where HOS stands for HDA one similar) (Rundlett et al., 1996). Mammalian deacetylases are found in much larger complexes (2,000 kDa) that contain an estimated 20 proteins, many of which have not yet been identied. RPD3 and HDA1 share significant sequence homology as well as homology with prokaryotic proteins known to interact with acetylated substrates. A highly conserved region termed the RPD3 homology domain is clearly involved in catalysis. Deacetylase activity can be abolished by the introduction of mutations within this domain. The class II HDA1-like protein has at least ve homologs in vertebrates; mHDA1 and mHDA2 in mouse; and HDAC4, HDAC5, HDAC6, HDAC7, and HDAC8 in human (Fischle et al., 1999; Grozinger et al., 1999; Miska et al., 1999; Verdel and Khochbin, 1999; Wang et al., 1999). Mouse HDA2 (human HDAC6) is particularly interesting in that it contains two yeast HDA1 homology domains. Class III. The rst two classes of histone deacetylases have considerable homology with each other; for example, RPD3 and HDA1 are 25% identical and 50% similar over a region of 490 amino acids. In contrast, the only Class III histone deacetylase, maize HD2, does not share obvious or extended regions of sequence similarities with the Class I or Class II enzymes (Lusser et al., 1997). Maize HD2, however, does share regions of sequence homology with acidic nucleolar phosphoproteins (e.g., UBF1, UBF2, nucleolin, and B23), and the immunophilin FK506-binding proteins. Whether the nucleolar phosphoproteins or the FK506-binding pro-

teins can function as histone deacetylases remains to be determined. Within each class of deacetylase, homologs from different species share relatively high sequence similarities. For example, human HDAC1 and yeast RPD3 share 60% sequence identity and 80% sequence similarity over a region of 450 amino acids. Many additional members of each deacetylase class have recently been uncovered by searching EST databases for homologous sequence elements, and have not yet been extensively characterized. HISTONE DEACETYLASES AND TRANSCRIPTIONAL COREPRESSORS A major breakthrough in our understanding of transcriptional repression came from the recent discoveries that indicated that transcriptional repression, by at least some proteins, is directly linked to the recruitment of multiprotein complexes containing histone deacetylases. We discuss three specic examples of transcriptional repressors that have served as paradigms: YY1, Mad/Max, and the nuclear hormone receptors. YY1 As its name suggests, the transcription factor YY1 (Yin Yang 1) can act as either a transcriptional activator or a transcriptional repressor. This evolutionarily conserved protein is ubiquitously expressed in many cells. A multitude of promoters and enhancers contain potential YY1-binding sites and many of the genes containing these elements have been shown to be regulated by YY1 (recently reviewed by Thomas and Seto, 1999). In an effort to identify YY1-binding proteins (and thus better understand its mechanisms of action) a mouse RPD3 homolog, now known as HDAC2, was identied using a yeast two-hybrid screen (Yang et al., 1996a). A GAL4 DNA-binding domain HDAC2-fusion protein strongly repressed transcription from a promoter containing GAL4-binding sites, suggesting that YY1 negatively regulates transcription by tethering HDAC2 to DNA as a corepressor, and that this transcriptional mechanism is highly conserved from yeast to human. This was the rst demonstration that a mammalian histone deacetylase can have a direct effect on transcriptional control. Subsequently, it was found that HDAC1 and HDAC3 can also bind YY1 and repress transcription when targeted to promoters (Yang et al., 1997). In addition to HDAC1, HDAC2, and HDAC3, YY1 also interacts with the nucleolar phosphoprotein B23 (Inouye and Seto, 1994). Because the maize deacetylase HD2 is closely homologous to nucleolar proteins UBF1, UBF2, nucleolin, and, most interestingly, B23, it is tempting to speculate that YY1 recruits B23 as part of the deacetylase enzymatic activities. The nding that YY1 interacts with three human HDACs closely related to yeast RPD3 does not exclude the possibility that additional mammalian histone deacetylases, perhaps with limited sequence homology to yeast RPD3, may also be recruited by YY1 to repress transcription. Ribosomal RNA genes are localized distinctly in the nucleolus and have their own specic subset of transcription factors for transcription. Given that a subpopulation of YY1 is also localized within the

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nucleolus (Guo et al., 1995), it seems reasonable to speculate that YY1 may direct a distinct family of deacetylases devoted to the regulation of rRNA synthesis. Of the many other proteins that have been reported to interact with YY1, an exceptionally interesting case is the closely related coactivators CBP and p300 (Lee et al., 1995). Both of these coactivators are HAT enzymes. It is, therefore, quite appealing to speculate that YY1 activates transcription by the recruitment of HAT activities. Perhaps it is the selective binding of either HAT or HDAC that determines whether YY1 will mediate the activation or repression of transcription of a given gene. Further studies aimed at uncovering the signals that favor the recruitment of HDAC over HAT may shed some light on the mechanisms of YY1-mediated repression. Although a number of different assays have been used to conrm that YY1 interacts with class I HDACs, both in vitro and in vivo, it is noteworthy that YY1 has not yet been copuried with any of the HDAC complexes. It is conceivable that the YY1-HDAC interaction is direct and occurs in the absence of other intermediate proteins. In this regard, YY1 may be similar to the Drosophila corepressor Groucho, which also interacts directly with the Drosophila RPD3 protein (Chen et al., 1999). Alternatively, it is possible that YY1 is present in a substoichiometric quantity compared with that of other proteins in an HDAC complex. In any case, further studies will be required to determine how the YY1-HDAC interaction is regulated and to identify additional candidate components of the YY1-HDAC complex. Mad-Max and the Sin3/HDAC complex In growing cells, Myc-Max heterodimers activate transcription of growth stimulatory genes, such as E2F-2, that are regulated by E-box elements in their promoters (Sears et al., 1997; McArthur et al., 1998). However, upon cellular differentiation Myc is replaced by Mad and the Mad/Max heterodimer represses transcription of growth stimulatory genes (McArthur et al., 1998). Using the yeast two-hybrid system, the transcriptional repression domain of Mad was shown to bind to two mouse proteins, mSin3A and mSin3B, both of which exhibit signicant sequence homology with the yeast SIN3 general transcriptional repressor (Ayer et al., 1995; Schreiber-Agus et al., 1995). The obvious question of what does Sin3 do? was quickly answered with the discoveries that mSin3A can be coimmunoprecipitated with HDAC1 and HDAC2, as well as a nuclear hormone transcriptional corepressor called NCoR (nuclear corepressor) (Alland et al., 1997; Hassig et al., 1997; Heinzel et al., 1997; Zhang et al., 1997). These observations tied the transcriptional repressor Mad to a histone deacetylase as a potential mechanism for repression, reminiscent of the case with YY1 (Heinzel et al., 1997; Laherty et al., 1997). However, unlike YY1, mSin3A can repress transcription in the absence of the HDAC interaction region, suggesting that mSin3 contains an alternative repression domain that is active in the absence of bound HDAC. Subsequent work demonstrated that the Sin3A/ HDAC complex contains additional components, including RbAp46 and RbAp48 (Qian et al., 1993; Qian

and Lee, 1995), as well as SAP18 and SAP30 (Zhang et al., 1997; Laherty et al., 1998; Zhang et al., 1998b). RbAp46 and RbAp48 are related histone-binding proteins; they are thought to function at least in part by facilitating interactions of the complex with the histone substrate (Parthun et al., 1996; Verreault et al., 1996, 1998). The roles of SAP18 and SAP30 are not well understood. GST-SAP18 binds mSin3 and also interacts weakly with HDAC1 (Zhang et al., 1997). GSTSAP30 binds in vitro translated mSin3A, mSin3B, and NCoR, but does not bind directly to HDAC1, HDAC2, or RbAp48. SAP30 has a structural and functional homolog in yeast. Its deletion in yeast confers a phenotype very similar to those obtained subsequent to deletion of RPD3 or SIN3. This result suggests that SAP30 is a critical component of a transcriptional regulatory pathway in yeast. In mammalian cells, microinjection of anti-SAP30 antibodies demonstrates that SAP30 is required for repression by the estrogen receptor, but not by thyroid or retinoic acid receptors, suggesting that it may have a gene-specic role in corepressor function (Laherty et al., 1998). Nuclear hormone receptors The thyroid hormone (TH) and retinoic-acid receptors (RARs) are ligand-dependent transcription factors that control cell development and homeostasis as the result of both transcriptional repression and activation (Chambon, 1994). These factors activate transcription in the presence of ligand by associating with the histone acetyltransferases p300/PCAF, whereas in the absence of ligand these receptors repress transcription. The ligand-binding domains of these receptors are capable of transferring active repression to the GAL4 DNA-binding domain (Glass et al., 1988; Baniahmad et al., 1992). The purication of a 240-kDa protein that bound to this modular repression domain identied NCoR (Horlein et al., 1995; Kurokawa et al., 1995; Zamir et al., 1996) and also a related protein, SMRT (Chen and Evans, 1995). Receptor mutations that abolish NCoR binding also block transcriptional repression, but do not block activation, suggesting that NCoR mediates transcription by the hormone receptors. Subsequent experiments using NCoR antibodies demonstrated that Sin3 and histone deacetylase are also components of this complex (Alland et al., 1997; Heinzel et al., 1997). It was suggested that the repressor complex involved in Mad-mediated transcriptional repression is identical to the complex used by the nuclear hormone receptors and that this complex contains Sin3, HDAC1/2, RbAp46/48, SAP30, and SAP18. The emerging model for transcriptional regulation by the hormone receptors is that, in the absence of ligand, the RAR/RXR dimer binds what many refer to as the Sin3 complex; this complex represses transcription. In the presence of ligand, the repressor complex is dissociated. The receptor is then free to form a complex with the histone acetyltransferase, which activates transcription. HISTONE DEACETYLASE-INTERACTING PROTEINS In addition to their roles in the Sin3 complex, HDAC1 and HDAC2 also exist in a separate composite known as the Mi2 complex. In addition to two proteins

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teract with other members of the HDAC family. For example, the myocyte enhancer factor MEF2A associates exclusively with HDAC4 (Miska et al., 1999). Binding of HDAC4 to MEF2A results in the repression of MEF2A transcriptional activation, a function that requires the deacetylase domain of HDAC4. Naturally, many more examples of proteins that interact with HDACs will be expected to emerge if a local alteration in chromatin structure is a general means of regulating gene expression. Perhaps future studies will even reveal that most transcriptional repressors function, in part, by recruiting histone deacetylases; those devoid of this recruiting activity may turn out to be the interesting exceptions. HISTONE DEACETYLASES AND CANCER GUILT BY ASSOCIATION During the past few years, results from basic research studies of histone deacetylases have added greatly to our general understanding of the regulation of transcription in eukaryotic cells. Increasing importance is now being given to assess the relevance of deacetylases in health and disease. The interaction of Mad and Max is important in tumor suppression, whereas the thyroid hormone and retinoic acid receptors are indispensable in the control of development and homeostasis. The nding that histone deacetylases are key components in the regulation of gene expression by Mad and by hormone receptors underscores the important implications of deacetylases in human disease. No direct alterations in the histone deacetylase genes have yet been demonstrated in human oncogenesis. However, it is now known that histone deacetylases associate with a number of well-characterized cellular oncogenes and tumor-suppressor genes. Thus, histone deacetylases may represent candidate targets for anticancer drugs and therapies. In the following sections, we review the potential roles that HDACs play in human cancer. In general, there are three separate known levels that are dependent on histone deacetylases and are likely linked to tumorigenesis. On the rst level are cell-cyclerestraining transcriptional repressors including Mad and Rb. On a second level are transcriptional repressors that normally block the process of differentiation in certain cellular lineages, for example, the nuclear hormone receptors such as RAR in hematopoietic differentiation. A nal level of cell biology controlled by histone deacetylase containing complexes is the strong correlation between genomic methylation and the transcriptional silencing of various tumor-suppressor genes, such as p21WAF/CIP1. Disruption of the Mad/Sin3/HDAC by v-Ski The roles of the Mad/Sin3A/HDAC complex in growth control and differentiation were discussed in an earlier section. The most commonly known alteration of this pathway in human cancer is the overexpression of the alternative Max binding partner Myc, which occurs as a result of translocations, gene amplications, or activating point mutations (see Dang, 1999 and references therein). When Myc is overexpressed, Mad/Max heterodimers are excluded and Mad-mediated transcriptional repression is blocked. However, in this event it is not clear whether transformation occurs

Fig. 1. HDAC-associated proteins. Proteins that interact with HDAC1/2 through the Sin3 complex: MeCP2 (Jones et al., 1998; Nan et al., 1998; Wade et al., 1998a), Ikaros (Koipally et al., 1999), UME6 (Kadosh and Struhl, 1997), Ski (Nomura et al., 1999), p53 (Murphy et al., 1999), NCoR/SMRT (Alland et al., 1997; Heinzel et al., 1997), MAD (Ayer et al., 1995; Schreiber-Agus et al., 1995); and through the Mi2 complex: Hunchback (Kehle et al., 1998), PcG (van der Vlag and Otte, 1999), Ikaros (Kim et al., 1999b), HPV E7 (Brehm et al., 1999b), MBD2 (Ng et al., 1999; Zhang et al., 1999b) are displayed on the upper left and upper center panels, respectively. All other HDAC-interacting proteins bind HDACs either directly or through unknown mechanisms: YY1 (Yang et al., 1996a, 1997), Groucho (Chen et al., 1999), p107/p130/pRB (Brehm et al., 1998; Ferreira et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998a,b; Stiegler et al., 1998; Lai et al., 1999), TR 1 (Sasaki et al., 1999), LIM (Bach et al., 1999), REST (Huang et al., 1999), CBF (Hsieh et al., 1999), menin (Gobl et al., 1999), MBP-1 (Ghosh et al., 1999), Sp1 (Sowa et al., 1997; Doetzlhofer et al., 1999; Sowa et al., 1999; Xiao et al., 1999), LAZ3 (BCL-6) (Dhordain et al., 1998), Net (Criqui-Filipe et al., 1999), PLZF (PLZFRAR ) (Grignani et al., 1998; He et al., 1998; Lin et al., 1998), BRCA1 (Yarden and Brody, 1999), ATM (Kim et al., 1999a), TGIF (Wotton et al., 1999), Rbp1 (Lai et al., 1999). References for HDAC3 interactions include: HDAC4 and HDAC5 (Grozinger et al., 1999), YY1 (Yang et al., 1997), NCoR/SMRT (Kao et al., 2000), Rbp1 (Lai et al., 1999). References for HDAC4 interactions include: HDAC3 (Grozinger et al., 1999), MEF2A (Miska et al., 1999; Wang et al., 1999), NCoR/SMRT (Huang et al., 2000; Kao et al., 2000). References for HDAC5 interactions are: HDAC3 (Grozinger et al., 1999), NCoR/SMRT (Huang et al., 2000; Kao et al., 2000). Reference for HDAC7: Kao et al., 2000.

that are also present in the Sin3 complex (RbAp46 and RbAp48), the Mi2 complex contains Mi2, MTA2, and MBD3. Proteins such as NCoR and Mad interact with HDAC1/2 through the Sin3 complex, whereas some repressors interact with HDAC1/2 through the Mi2 complex. In addition, many other proteins interact with HDAC1/2 either directly or through yet unidentied proteins (summarized in Fig. 1). Although nearly all HDAC-interacting proteins discovered to date are known to interact with HDAC1/2, there are clearly other cellular proteins that could in-

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as a result of a blockage of repression per se, or if additional transcriptional activation by Myc/Max is also required. Recent studies of the cellular proto-oncogene c-Ski may address this issue. Over 10 years ago, the oncogenic form of Ski, v-Ski, was found to transform chicken embryo broblasts (Li et al., 1986). However, the mechanism of this transformation has remained unclear. A recent study has demonstrated that the cellular Ski protein is a component of the Sin3/NCoR/HDAC complex (Nomura et al., 1999). Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that Ski may interact simultaneously with both NCoR and Sin3A. Domain-mapping experiments determined that c-Ski binds to NCoR via its N-terminal region, and Sin3A through its Cterminal domain. It therefore seems possible that c-Ski may function, at least in part, as a bridging protein that tethers Sin3A and NCoR. It has been shown that mutations within the N-terminal region of v-Ski (which lacks the Sin3A-binding C-terminus) blocks transformation. These observations suggested that v-Ski exerts its transforming effects by blocking the function or assembly of the Sin3/HDAC/NCoR/c-Ski complex. To test this hypothesis, mutants of c-Ski lacking the Cterminal Sin3A-interacting domain were examined for the ability to block transcriptional repression mediated by Mad. As anticipated, v-Ski and C-terminally truncated forms of c-Ski blocked repression by Mad in a dominant-negative fashion. The observations discussed earlier demonstrate that v-Ski is oncogenic, at least in part, because of its ability to block Mad-mediated repression in the absence of Myc overexpression. Furthermore, they suggest a broad potential for disruptions of the Sin3A/NCoR/ HDAC complex in tumorigenesis. The Sin3A/NCoR/ HDAC complex is likely involved in other regulatory pathways that may also contribute to v-Ski transformation. For instance, it has been shown that c-Ski may also be involved in Rb-mediated transcriptional repression (Tokitou et al., 1999), and other work has demonstrated that c-Ski can modulate RAR -mediated transcriptional repression (Dahl et al., 1998). Is c-Ski a universal component of the complex, or is it necessary only in a subset of Sin3A/NCoR/HDAC-regulated pathways? Future work is required to characterize the biological roles of c-Ski. Histone deacetylases interact with the Rb tumor-suppressor protein Although disruptions of the Myc/Mad pathway contribute signicantly to human cancer, they are not present in all tumors. In contrast, the Rb/E2F transcriptional regulatory pathway is disrupted in virtually every human tumor, making it a nearly universal target for anticancer drug and therapy development (Sellers and Kaelin, 1997). The 105-kDa Rb protein is known to interact with numerous other proteins. Its best-understood binding partner is the dimeric transcription factor E2F, which is central in the control of cell-cycle progression (for reviews see Sellers and Kaelin, 1997; Dyson, 1998; Johnson and Schneider-Broussard, 1998; Nevins, 1998; Brehm et al., 1999a). E2Fregulated promoters are involved in the expression of two classes of activities: (1) enzymes required for DNA synthesis, such as dihydrofolate reductase and thymi-

Fig. 2. Model for cell-cyclespecic transcriptional regulation by E2F and Rb. In quiescent cells, Rb interacts with DNA-bound E2F. Rb represses transcription of E2F-regulated promoters by recruiting HDAC1, HDAC2, or HDAC3. Rb may interact directly with HDACs, or may require the bridging factor Rbp1. Growth stimulation activates G1 cyclin-dependent kinases to phosphorylate Rb and dissociate it from E2F and thus the promoter. Recent evidence suggests that E2F may then associate with TRRAP, which likely possesses HAT activity (McMahon et al., 1998; Vassilev et al., 1998). Basal transcription factors and RNA polymerase may then associate with the promoter.

dine kinase; and (2) cell-cycle regulatory proteins, such as cyclin A and cyclin E. The current Rb/E2F paradigm is highlighted in Fig. 2. In this model, an E2F family member binds DNA as a heterodimer with a DP family partner (Helin et al., 1993). In quiescent cells, this heterodimeric complex is bound by a member of the Rb family, which converts this potential transcriptional activator to a transcriptional repressor (Adnane et al., 1995; Weintraub et al., 1995; Luo et al., 1998). This Rb-mediated transcriptional repression is critical for the active downregulation of many E2F/Rb-regulated promoters (Lam and Watson, 1993; Johnson et al., 1994b; Lam et al., 1995; Sellers et al., 1995; Zwicker et al., 1996). Upon growth stimulation, the Rb protein is phosphorylated by the cyclin-dependent kinases (cdks); and the hyperphosphorylated Rb then releases the E2F heterodimer, which goes on to activate transcription. The mechanism of E2F/DP-mediated transcription activation is not fully understood, but is thought to involve the recruitment of HAT activity. Specically, it has been demonstrated that the E2F-1 activation domain associates with an ATM-related protein, TRRAP (McMahon et al., 1998; McMahon et al., 2000), and that TRRAP associates with HAT activity (Vassilev et al., 1998). Rbs phosphorylation state, and thus its growth inhibitory activity, is tightly regulated by the activities of the cdks. The cdks are in turn regulated by cyclindependent kinase inhibitor proteins such as p21WAF/CIP1. Both active transcriptional repression and transcrip-

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tional activation via E2F are likely to be critical for proper cell-cycle regulation (Dobrowolski et al., 1994; Sellers et al., 1995; Ishizaki et al., 1996; Wu et al., 1996; Zhang et al., 1999a). Evidence that the G0 Rb/ E2F complex serves as a critical transcriptional repressor involved in cell-cycle control includes the following: 1. Rb overexpression in Rb / cells blocks the transcription of E2F-regulated genes (Hiebert et al., 1992) and can, in some Rb-negative cell lines, lead to growth arrest (Qin et al., 1992). 2. In some Rb-regulated promoters, such as B-myb, an E2F binding site has been shown to be occupied only during G0 (when the promoter is off) and not in S phase, implying that the E2F site functions exclusively as a repressor in this context (Zwicker et al., 1996). 3. Numerous transfection/reporter experiments have demonstrated that E2F binding sites can serve as Rb-dependent repressing elements in cis for both articial and natural promoters (Weintraub et al., 1992; Lam and Watson, 1993; Johnson et al., 1994b). 4. When fused to the DNA-binding domain of GAL4, the Rb protein represses transcription of articial promoters containing GAL4 binding sites (Adnane et al., 1995; Weintraub et al., 1995). 5. When fused to the DNA-binding domain of E2F-1, the Rb protein blocks cell-cycle progression (Sellers et al., 1995). 6. Transcriptional repression by Rb is reversed by its phosphorylation (Weintraub et al., 1995). 7. In serum-starved broblasts from Rb knockout animals, certain E2F-regulated promoters are constitutively activated (Hurford et al., 1997). As mentioned earlier, the Rb protein binds to a host of cellular proteins (Taya, 1997). One of the proteins found to bind to Rb was RbAp48 (Rb-associated protein 48) (Huang et al., 1991; Qian et al., 1993). Both of these proteins are ubiquitously expressed in the nucleus and they form complexes with Rb both in vitro and in vivo. RbAp48 shares sequence homology with MSI1, a negative regulator of the yeast Ras-cyclic AMP pathway (Qian et al., 1993; Qian and Lee, 1995). The early observation that RbAp48 was also present in complexes containing HDAC1 (Taunton et al., 1996) led several groups to specically test whether the Rb protein associates with histone deacetylases in vivo. Not surprisingly, several laboratories have provided evidence that Rb associates with HDACs in vivo (Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998). However, it is now known that Rbs interaction with HDACs is not dependent on the binding to RbAp46 or RbAp48, but rather, is mediated through the bridging protein Rbp1 (Lai et al., 1999). The following evidence from three initial studies (Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998) rmly established the existence of a bona de interaction between Rb and HDAC1/2: 1. A GST-Rb fusion protein was demonstrated to specically associate with HDAC1 and HDAC2. Furthermore, the association of GST-Rb with histone deacetylases was dependent on Rb domains known

2. 3.

4. 5. 6. 7.

8.

to be critical for cell-growth control and transcriptional repression. Rb antibodies were used to coimmunoprecipitate histone deacetylase activity from crude extracts. Viral oncoproteins known to cause the dissociation of Rb complexes had similar effects on the Rb/ HDAC interaction. As expected, inactive mutants of these oncoproteins did not result in the dissociation of Rb/HDAC. An E2F/Rb/histone deacetylase complex could be formed in vitro from recombinant proteins. GST-E2F associated with histone deacetylase from crude extracts when Rb protein was provided as a tether. Transfection of HDAC1 together with Rb resulted in collaborative repression of an E2F-regulated promoter. The histone deacetylase inhibitor trichostatin A (TSA) activated the transcription of several E2Fregulated promoters, including thymidine kinase and dihydrofolate reductase in Rb-expressing cells, but not in cells lacking Rb. Recruitment of Rb to a promoter resulted in decreased histone acetylation of nucleosomes bound to that promoter.

HDAC1 and HDAC2 contain an IXCXE sequence, similar to the LXCXE motif, through which several viral and cellular proteins interact with the A/B pocket of Rb (Nevins, 1994). This domain may be sufcient for interaction with Rb since its deletion abolished the HDAC1/Rb interaction in vitro. Although there is potential for a direct interaction between Rb and HDAC1 and HDAC2, recent evidence suggests that HDAC3 also associates with Rb in vivo (Lai et al., 1999). Since HDAC3 lacks an LXCXE-like motif, it seems likely that it may be tethered to Rb via interaction with a bridging protein. A prime bridging protein candidate is Rbp1, which contains a consensus LXCXE motif and is known to interact with HDAC1, -2, and -3 (Lai et al., 1999). Rbp1 possesses two transcriptional repression domains, one that binds HDACs and a second that represses transcription in an HDAC-independent manner. It is not yet known whether other HDACassociated proteins such as Sin3A, NCoR, RbAp46, and RbAp48 are components of the Rb/HDAC/Rbp1 complex. In one study, immunoprecipitations were utilized to demonstrate that Rb associates with c-Ski, a related protein Sno, and Sin3A. However, interactions between Rb and NCoR were not detected in this effort. In another study, the expression of v-Ski was correlated with diminished interaction between Rb and HDACs, as well as lower levels of Rb-mediated transcriptional repression (Tokitou et al., 1999). Taken together, these results suggest a complex scenario in which Rb associates with HDACs through a variety of mechanisms. Additional work will be required for a more complete understanding of the regulation and functions of the Rb family of proteins. The molecular mechanisms underlying the ne-tuning of cell growth control will undoubtedly be the subject of future studies for years to come.

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Alterations of the Rb/HDAC interaction in human cancer There are numerous genetic alterations that result in loss of Rb/HDAC interactions in cancer. The most obvious genetic alteration is simply the loss of a functional Rb gene, by deletions and point mutations, which occurs in many solid tumors in both sporadic and heritable patterns. A second common mechanism of Rb inactivation occurs in cervical cancers caused by highrisk strains of the human papilloma virus. These viruses express the E7 oncoprotein, which binds Rb and blocks its interaction with E2F and with HDACs (Phelps et al., 1991; Brehm et al., 1998). In a third mechanism, numerous genetic alterations not involving the Rb gene directly lead to constitutive Rb phosphorylation (inactivation). For example, the loss of cdk inhibitor proteins (most commonly p16) or the overexpression of cyclin D1 or cdk4 can each result in constitutive Rb phosphorylation and enhanced tumor growth (Hall and Peters, 1996). Adding further to the complexity of the Rb/E2F pathway is the fact that Rb, E2F, and DP are not single polypeptides but are each a member of protein families. Rb has two close protein relatives, p107 (Ewen et al., 1991) and p130 (originally named pRb2/p130) (Hannon et al., 1993; Li et al., 1993; Mayol et al., 1993). Collectively, these proteins are referred to as the pocket proteins, since each shares a large conserved structural domain (the pocket) required for interaction with E2F and with viral oncogenes such as E7 (Kaelin et al., 1991). Both p107 and p130 possess the ability to repress transcription and to block cell growth (Zhu et al., 1993; Claudio et al., 1994; Zhu et al., 1995). Furthermore, recent work has shown that both p107 and p130 bind to HDACs (Ferreira et al., 1998; Stiegler et al., 1998; Iavarone and Massague, 1999). Although they share many structural and functional similarities, the members of the Rb family clearly have different functional roles. Rb is the only member of its family that is known to be commonly mutated in human cancer; it is also the only protein of its class that has been shown to be required for mouse viability (Lee et al., 1992; Cobrinik et al., 1996; Lee et al., 1996; Helin et al., 1997). However, p130 is also a bone de tumorsuppressor gene, since it has been found to be mutated in primary nasopharyngeal carcinoma (Claudio et al., 2000a) and in lung tumors (Claudio et al., 2000b), as well as in cell lines derived from small cell lung carcinoma (Helin et al., 1997). Replacement of the wild-type p130 allele by retrovirus leads to suppression of tumor growth. It is not yet clear why Rb has such a special role. All members of the Rb family interact with members of the E2F family; however, there is some specicity, in that p107 and p130 do not physiologically interact with E2F-1, E2F-2, or E2F-3. In contrast, Rb interacts with the ve known E2F proteins (Beijersbergen et al., 1994; Ginsberg et al., 1994; Hijmans et al., 1995; Sardet et al., 1995). Perhaps the unique role of Rb lies in its ability to interact with all members of the E2F family. Alternatively, Rb-family knockout mice reveal that Rb, p107, and p130 regulate slightly different subsets of E2F-driven promoters (Hurford et al., 1997). Specically, Rb appears to be uniquely able to regulate the cyclin E and p107 promoters. Thus, Rbs unique roles may be a function of its ability to regulate

a relatively small number of critical cell-cycle regulatory factors. The E2F family itself is theoretically a target for mutations that would activate cell growth. For example, a mutation that would increase E2F expression or make E2F unable to interact with Rb (and thus unable to tether Rb and associated HDACs to promoters) would be predicted to be oncogenic. This principle has been demonstrated in numerous experiments designed to overexpress E2F or to express altered Rb-insensitive forms of E2F (Beijersbergen et al., 1994; Johnson et al., 1994a; Singh et al., 1994; Jooss et al., 1995; Wang et al., 1995; Xu et al., 1995; Guy et al., 1996; Pierce et al., 1998a,b). There is also limited evidence that alterations in E2F occur in human tumors. The E2F-1 gene has been shown to be amplied in a human erythroleukemia cell line (Saito et al., 1995) and E2F-4 is frequently altered within a serine repeat motif near its C-terminus (Yoshitaka et al., 1996; Souza et al., 1997). Although there is little evidence that known members of the E2F family themselves are commonly altered in human oncogenesis, it should be noted that the eld is still relatively young, and the list of known E2F variants involved in oncogenesis may very well remain to be discovered. Paradoxical observations In the cases of both Mad and Rb, transcriptional repression is critical in evoking growth arrest. Because the capacity of these proteins to repress transcription is dependent on HDAC association, one would predict that histone deacetylase inhibitors would prevent cellcycle arrest. Although histone deacetylase inhibitors activate certain Rb/E2F-regulated promoters, they do not promote cell growth, but rather lead to growth arrest. These observations seem paradoxical, especially considering the central role that Rb/E2F is thought to play in growth control. The most general way to reconcile these observations is to point out that histone deacetylase inhibitors may affect various transcriptional pathways in addition to Mad and Rb. The precise identity of the critical pathway or pathways is not known, but one would speculate that HDAC inhibitors would affect events upstream of Myc and E2F in the G1 phase of the cell cycle. A putative target would be the tumor-suppressor protein p21WAF/CIP1, which has recently been shown to be strongly stimulated at the level of transcription by the addition of histone deacetylase inhibitors (Archer et al., 1998); p21WAF/CIP1 is a cyclindependent kinase inhibitor, whose expression leads to hypophosphorylated (growth-suppressing) Rb. Normally, p21WAF/CIP1 is induced by p53 in response to DNA damage and, thus, it is also known as WAF1 or CIP1 (wild-type p53-activated factor or cdk inhibitor protein-1) (Di Leonardo et al., 1994). In this model, addition of histone deacetylase inhibitor induces p21WAF/CIP1, which in turn leads to the activation of Rb and growth arrest. Although the mechanism by which HDAC inhibitors activate p21 is not clear, it is clearly p53 independent (Xiao et al., 1997) and is dependent on Sp1-binding sites within the promoter (Sowa et al., 1997, 1999). An obvious difculty with this model is that the HDAC inhibitor might block Rbs transcriptional repression function and, thus, p21WAF/CIP1 induction

HISTONE DEACETYLASES AND CANCER

would be pointless. However, there are several examples of promoters that are transcriptionally repressed by Rb/E2F independent of histone deacetylase. For example, among genes controlled by well-characterized E2F-regulated promoters, TSA induces p107 and DHFR transcription, but does not affect the transcription of ribonucleotide reductase, thymidine kinase, or PCNA (Luo et al., 1998). Thus, it is clear that Rb has transcriptional-repressing properties that are not blocked by histone deacetylase inhibitors. This may be expected since Rb binds to many proteins, which potentially may function as HDAC-independent transcriptional repressors, such as Rbp1 (Lai et al., 1999). Furthermore, it has been known for some time that Rb can inhibit transcriptional activation by E2F in vitro in the absence of chromatin (Dynlacht et al., 1994). Using highly puried components, it was shown that Rb directly blocks the interaction between E2F and the basal transcription factors TFIID and TFIIA, thus preventing the formation of a preinitiation complex in vitro (Ross et al., 1999). These ndings strongly suggest that Rb utilizes a number of mechanisms to repress transcription. The exact mechanism used may be either promoter dependent or contingent on the mechanisms stimulating growth arrest. Future work is necessary to characterize the mechanisms underlying Rbs growth-restraining properties. Histone deacetylases and interaction with fusion proteins in leukemia Acute leukemia results when the differentiation of immature hematopoietic cells is blocked and the cells proliferate without restraint. Acute promyelocytic leukemia (APL) is most often associated with chromosomal translocation t(15;17), which fuses a large portion of the retinoic acid receptor (RAR ) to the coding sequence of a second gene, PML (promyelocytic leukemia). In a smaller percentage of APL cases, a similar translocation t(11;17) occurs in which the same coding region of RAR is fused to a different protein, PLZF (promyelocytic leukemia zinc nger). PLZF is a Krupple-like DNA-binding protein containing nine zinc-nger motifs. It normally plays a role in central nervous system development and in hematopoiesis. RAR also plays a role in hematopoiesis. Specically, RAR binds to DNA as a heterodimer with an RXR (retinoid-X receptor) protein. In the absence of retinoic acid, which normally induces differentiation of promyelocytic cells, the RAR /RXR heterodimer binds to a transcriptional corepressor that contains NCoR, Sin3A, and histone deacetylase (Grignani et al., 1998). In the presence of retinoic acid, the corepressor complex is displaced and is replaced by a coactivator complex that contains the histone acetyltransferases p300/ CBP and PCAF. However, when the RAR protein is present in either of these fusion proteins it is no longer responsive to physiological levels of retinoic acid. Thus, under these conditions, RAR becomes a constitutive transcriptional repressor, which blocks normal differentiation and leads to leukemia. Although both of the RAR fusion proteins discussed earlier are insensitive to physiological levels of retinoic acid, the PMLRAR fusion (and thus patients with this translocation) will respond to pharmacological doses of all-trans-retinoic acid (ATRA). In contrast, the

PLZFRAR fusion protein is completely insensitive to retinoic acid; patients with this translocation do not benet from retinoic acid treatment. This difference results from the fact that the PLZF protein itself contains an interaction domain that can bind to the NCoR/ Sin3A/HDAC complex. Thus, the fusion protein interacts with corepressors through two domains, one of which is not sensitive to retinoic acid. Consistent with this model, the histone deacetylase inhibitor trichostatin A restores RA sensitivity to PLZFRAR and allows these leukemic cells to respond to ATRA (Grignani et al., 1998; He et al., 1998; Lin et al., 1998). These observations clearly demonstrate a role for the histone deacetylases in oncogenesis and suggest that histone deacetylase inhibitors may be valuable in treating certain forms of leukemia. AML The AML1 gene is disrupted by the t(8;21) translocation in acute myeloid leukemia (for review see Fenrick and Hiebert, 1998). Normally, AML1 functions as the DNA-binding component of CBF (the enhancer core-binding factor). This complex apparently activates expression of genes required for myeloid differentiation (Lenny et al., 1997). In the DNA-binding complex, AML1 forms a heterodimer with a second protein CBF ; this complex appears to function as a transcriptional activator via interaction with the p300/PCAF histone acetyltransferase complex (Kitabayashi et al., 1998). In the t(8;21) translocation, the DNA-binding domain of AML1 is fused to a second protein referred to as ETO (8;21 or eight, twenty-one). Numerous AML translocations in addition to t(8;21) occur in acute myeloid leukemia. The common element of these fusion proteins is that they all fuse the DNAbinding domain of AML1 with a second protein that interferes with AML-dependent transcriptional activation (Fenrick and Hiebert, 1998). The t(12;21) translocation fuses most of the AML protein to a second transcription factor TEL (translocation, ets, leukemia). The t(16;21) translocation fuses AML1 to MTG16 (myeloid tumor gene 16), a protein very similar in structure to ETO (Gamou et al., 1998). Finally, the t(3;21) translocation fuses AML1 to Evi I, a known transcriptional repressor (Nucifora and Rowley, 1995). The inv(16) inversion fuses the CBF protein to a smooth muscle myosin heavy-chain gene. Although this does not affect the AML1 gene directly, the inv(16) retains its ability to interact with AML1 and, thus, is thought to induce leukemia through the disruption of AML1-mediated transcriptional activation. Each of the fusion proteins mentioned previously has been shown to inhibit AML1-dependent transcription (Fenrick and Hiebert, 1998). So far, the histone deacetylases have been directly implicated in only the AMLETO translocation. However, recent evidence suggests that ETO, like RAR and PLZF, binds to NCoR and to mSin3A and recruits histone deacetylases (Lutterbach et al., 1998). Because of its similarity in structure to ETO, it is predicted that the AMLMTG16 fusion will also bind constitutively to the NCoR, mSin3A, and histone deacetylase complex; however, this has yet to be demonstrated experimentally. If histone deacetylases are commonly involved in AML fu-

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sions, then histone deacetylase inhibitors will no doubt have value in AML therapy. Another link between HDAC and AML1 came recently with the discovery that the Drosophila corepressor Groucho interacts functionally with the Drosophilia HDAC protein (Chen et al., 1999). HDAC potentiates repression by Groucho, and histone deacetylase inhibitors were used to show that deacetylase activity is required for efcient Groucho-mediated repression. Mutations in Groucho and HDAC genes have synergistic effects on embryonic lethality and pattern formation in ies. Given that the human homolog of Groucho is known to bind to AML1, it is tempting to speculate that it plays roles in hematopoiesis and may even be involved in leukemia. Chromatin remodeling, histone deacetylases, and methylation: potential roles in cancer Recent work has demonstrated the existence of cellular complexes with both histone deacetylase and ATP-dependent nucleosome-remodeling activity (Tong et al., 1998; Wade et al., 1998b; Zhang et al., 1998a, 1999b). One of these complexes contains at least seven subunits and has been named NRD or NuRD (nucleosome-remodeling histone deacetylase complex; identical to the Mi2 complex). The Mi2 complex does not contain Sin3A or SAP30 and is thus distinct from the Sin3A/HDAC complex. Four of the NuRD subunits, the histone deacetylases HDAC1/2 and the histone-binding proteins RbAp48 and RbAp46 are common to both the Mi2 complex and the Sin3A/HDAC complex. The three remaining subunits have apparent molecular masses of 230, 70, and 32 kDa, respectively, and appear to be unique to the Mi2 complex. Sequence analyses revealed that the largest of these subunits is identical to the dermatomyositis-specic autoantigen Mi2 (which is identical to the protein CHD3) (Targoff and Reichlin, 1985). CHD3 is closely related to another polypeptide known as CHD4 (Woodage et al., 1997). The CHD3/4 subunit of the Mi2 complex contains a helicase/ATPase domain and possesses nucleosome-remodeling activity. Peptide sequence analyses of the 70-kDa subunit of the Mi2 complex revealed that it is related to the MTA1 (metastasis-associated protein 1); accordingly, it was termed MTA2. The MTA2 subunit is essential for high levels of histone deacetylase activity. The overexpression of MTA1 correlates with the metastatic potential of numerous cancer cells; it will therefore be of great interest to determine whether MTA1 overexpression alters Mi2 complex function. The 32-kDa subunit corresponds to MBD3, a member of a family of proteins containing methyl-CpG binding domains (Zhang et al., 1999b). MBD3 is required for association of MTA2 with the core histone deacetylase complex. Despite possessing a methyl-CpG-binding domain, the MBD3 subunit is apparently unable to bind methylated DNA. Rather, the Mi2 complex can be tethered to methylated DNA via an interaction with an eighth protein, MBD2, which is highly homologous to MBD3. Although able to tether NuRD to methylated DNA, MBD2 was not found to be stably associated with either HDAC1 or HDAC2. These observations suggest that some chromatin remodeling is mediated by cellular complexes with histone deacetylase activity. It is speculated that the coupling of remodeling to deacetylation may be required to

allow access of the RbAp46 and RbAp48 subunits to the core histones. Furthermore, this complex may bind directly to methylated genomic DNA. The methylation of promoter CpG dinucleotides has been tightly correlated with transcriptional repression (Siegfried and Cedar, 1997; Siegfried et al., 1999). Results from recent studies suggest that methylation-mediated transcriptional silencing of tumor-suppressor genes may be a critical event in the formation of certain cancers. Although various mechanisms may underlie this repression (dependent on the promoter), recent work has demonstrated that one mechanism mediating this repression may involve the recruitment of histone deacetylases to methylated CpG dinucleotides. MeCP2 is the best-characterized member of a family of methylCpG binding proteins (Meehan et al., 1989; Lewis et al., 1992; Cross et al., 1997; Hendrich and Bird, 1998). It has recently been demonstrated that MeCP2 is a transcriptional repressor that recruits histone deacetylase via the Sin3 complex (Nan et al., 1997, 1998; Jones et al., 1998). Four proteins with homology to the MeCP2 methyl-CpG binding domain, MBD1MBD4 (Hendrich and Bird, 1998), have been identied through searches of EST databases. Of these four, MBD2 appears to be a demethylase (Bhattacharya et al., 1999) and MBD4 is an endonuclease likely involved in DNA repair (Bellacosa et al., 1999). The roles of methyl-CpG binding/HDAC complexes in human cancer are not yet fully understood. One possibility is the strong correlation between methylation and transcriptional silencing of tumor-suppressor genes. Aberrant CpG methylation is observed in a number of tumor-suppressor gene promoters including Rb, the cdk inhibitors p15 and p16, and the DNA repair gene MLH1 (reviewed in Jones and Laird, 1999). Rb is the most extensively characterized example to date. In a number of tumor types, one Rb allele is abolished by a structural mutation, and the otherwise normal allele is transcriptionally silenced by aberrant methylation (Stirzaker et al., 1997). The methylation of the silenced Rb promoter is extensive, and encompasses a pair of critical E2F-binding elements. Recent work has demonstrated that methylation of these E2F sites in the Rb promoter abolishes E2F binding (and thus E2F-mediated activation of the promoter) and creates a binding site for MeCP2, thus silencing the allele (Di Fiore et al., 1999). An important question that certainly needs to be addressed is: what events lead to the hypermethylation of the Rb-promoter? Recent evidence suggests that fos may transform cells by a mechanism involving activation of DNA 5-methylcytosine transferase (Bakin and Curran, 1999). Perhaps activation of CpG-methylation, whether promoter-specic or not, will be found to be an important oncogenic activity. Equally important, blocking this activity pharmacologically may prove to be an effective anticancer therapy in the future. HISTONE DEACETYLASE INHIBITORS AS CANCER CHEMOTHERAPEUTIC AND CHEMOPREVENTIVE AGENTS The initial cloning of histone deacetylases and the subsequent rapid advances in understanding the mechanisms of histone deacetylases and transcriptional repression have been greatly attributed to the

HISTONE DEACETYLASES AND CANCER

11

availability of histone deacetylase inhibitors. Early on, it was clear that in addition to alterations in deacetylation activity and transcription, many histone deacetylase inhibitors alter cellular functions. Treatment of mammalian cell cultures with deacetylase inhibitors causes cell-cycle arrest at either stage G1 or stage G2, consistent with the fact that histone deacetylation is tightly linked to cell-cycle control (Ogryzko et al., 1996a). Furthermore, a large number of studies have shown that histone deacetylase inhibitors can effectively arrest and revert transformation of some cells (e.g, Yoshida et al., 1990; Yoshida and Sugita, 1992; Kijima et al., 1993; Futamura et al., 1995; Richon et al., 1996; McBain et al., 1997; Richon et al., 1998) and can block the formation of tumors in rodent models (Cohen et al., 1998; Desai et al., 1999). These observations suggest that histone deacetylase inhibitors may be effective chemotherapeutic agents in human cancer. The histone deacetylase inhibitor butyrate has been tested in clinical trials for -thalassemia (Collins et al., 1995) and for prostate and brain cancers (Samid et al., 1997). HDAC inhibitors target critical cell-cycle regulatory pathways and may induce the expression of various silenced tumor-suppressor genes. Thus, these agents may have a broad application in chemotherapy and may hold special promise for the treatment of acute leukemias. As discussed in detail previously, fusion proteins that utilize HDACs have been implicated in many acute leukemias (Fenrick and Hiebert, 1998). These fusion proteins block the differentiation of immature hematopoietic cells, resulting in unstrained growth. Histone deacetylase inhibitors block the ability of these fusion proteins to repress transcription of genes required for differentiation. This repression can often be relieved by addition of high levels of all-transretinoic acid, which allows these cells to differentiate (thus the term differentiation therapy). However, in other cases these fusion proteins interact with HDACs via retinoic acidinsensitive domains (Guidez et al., 1998). In these cases, HDAC inhibitors are capable of restoring RA responsiveness, suggesting they may serve to augment retinoic acid differentiation therapy. This principle has been demonstrated in a mouse model in which trichostatin A was shown to be an effective treatment for leukemias induced by transgenic RAR -fusion proteins (He et al., 1998). Furthermore, because of the central role of HDACs in hematopoiesis, it is likely that HDAC inhibitors will be of therapeutic value, regardless of the exact nature of the fusion causing the leukemia. Butyrate and colon cancer High-ber diets are associated with a signicant decrease in the incidence of colon cancer (Trock et al., 1990a,b). Butyrate, a four-carbon fatty acid, which is produced in millimolar quantities by the bacterial fermentation of ber, is thought to mediate a substantial portion of dietary bers chemopreventive activity (McIntyre et al., 1991). The growth-inhibiting properties of butyrate have been demonstrated both in vitro, using colorectal cancer cell lines (Barnard and Warwick, 1993; Whitehead et al., 1986), and in vivo, using chemically induced colorectal tumors in rat (McIntyre et al., 1993).

Although the mechanism of butyrates action is not yet fully understood, early work demonstrated that one result of exposure to butyrate was the induction of histone hyperacetylation (Riggs et al., 1977). This effect can now be explained from the understanding that butyrate is a noncompetitive inhibitor of histone deacetylases (Sealy and Chalkley, 1978). HDAC5 was initially described as an antigen in human colon cancer and named NY-CO-9 (Scanlan et al., 1998). Moreover, recent work may provide a direct connection between butyrate exposure and stimulation of Rb tumor-suppressor gene function (Archer et al., 1998). Specically, expression of the cyclin-dependent kinase inhibitor p21WAF/CIP1 mRNA is sharply upregulated following butyrate treatment. This induction occurs within 2 h of treatment, even in the presence of cycloheximide. An experiment comparing p21 ( / ) human colon carcinoma cells with isogenic p21 ( / ) cells demonstrated that p21 is absolutely required for butyrate-induced growth arrest. It is likely that this therapeutic effort would be irrelevant to tumor cells that have already lost p21 or Rb function. However, these experiments suggest that butyrate and other more specic HDAC inhibitors may have a potent antitumor effect if administered at appropriate doses over the lifetime of susceptible individuals. Such preventative treatment may be broadly applicable to various types of cancer, assuming that future clinical trials do not reveal broad side effects or toxicity problems. CONCLUSIONS The purication, cloning, and partial characterization of histone deacetylases in the past few years have provided important insights into the transcriptional regulation of chromatin. Undoubtedly, these lines of study will continue to hone our understanding of enzymatic machines in gene regulation. As with the discovery of any molecules related to cancer, the initial link between HDACs and cancer is greeted with great excitement and overwhelming expectations. The future hope and challenge will be to convert this tenuous link into a substantiated, rm connection, and to discover ways to destroy cancer through inhibitors of HDACs. ACKNOWLEDGMENTS We thank Tanya Butler, Rhonda Croxton, Nancy Olashaw, Matt Thomas, Bill Tsai, Wen-Ming Yang, and Alice Yao for their helpful suggestions regarding the manuscript. We apologize to the many colleagues whose work could not be referenced because of space limitations. LITERATURE CITED
Adnane J, Shao Z, Robbins PD. 1995. The retinoblastoma susceptibility gene product represses transcription when directly bound to the promoter. J Biol Chem 270:8837 8843. Alland L, Muhle R, Hou H Jr, Potes J, Chin L, Schreiber-Agus N, DePinho RA. 1997. Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387:49 55. Aravind L, Koonin EV. 1998. Second family of histone deacetylases. Science 280:1167. Archer SY, Meng S, Shei A, Hodin RA. 1998. p21(WAF1) is required for butyrate-mediated growth inhibition of human colon cancer cells. Proc Natl Acad Sci USA 95:6791 6796. Ayer DE, Lawrence QA, Eisenman RN. 1995. Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3. Cell 80:767776.

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