Hat Hdac and Histone Acetylationa More Complicated Picture

Although it is generally thought that histone deacetylation causes gene repression, several studies now reveal a much more complex picture. For example, in a genome-wide CHIP analysis in yeast, it was revealed that the recruitment of the histone deacetylase HOS2 to the coding region of specific genes is correlated with gene activation, rather than repression (Wang et al., 2002). Since there is concurrent deacetylation of histone H3 and H4, these observations demonstrate that histone deacetylation by a deacetylase does not necessarily cause gene repression. How HOS2-catalyzed deacetylation causes transcriptional activation is not yet clear. Wang et al. propose that deacetylation by HOS2 might be required to restore a permissive transcription state after one round of acetylation associated with active transcription (Wang et al., 2002). It is of interest to note that although Rpd3 is also present in the genomic regions occupied by HOS2, Rpd3 mutation gives rise to the opposite phenotype (Robyr et al., 2002). On the other hand, Rpd3 is also able to behave as a transcriptional activator in conditions of osmotic stress (De Nadal et al., 2004). Upon activation of osmotic stress signaling, Rpd3 is required for the deacetylation of promoters from stress response genes, including the heat shock factor HSP12 among others. It was concluded that Rpd3 responds to various stimuli, including osmo-stress and heat shock, by positively regulating target gene expression. These results clearly illustrate the complexity of HDAC function. HOS2 was found to associate with SET3, which belongs to a family of histone methyltransferases, although such an activity has not been demonstrated for SET3 (Pijnappel et al., 2001). One interesting possibility is that deacetylation of specific histone residues in conjunction with specific methylation might confer gene activation instead of repression. Thus, although all HDAC family members possess histone deacetylase activity, their functions could be vastly different depending on many other factors.

It was initially thought that HATs and HDACs are recruited to specific promoter regions and affect histone acetylation spanning as little as two nucleosomes (Kadosh and Struhl, 1998; Wu et al., 2001). However, genome-wide surveys by chromatin-immunoprecipitation (ChIP) in yeast also reveal non-targeted and broad distributions of both HATs and HDACs throughout the yeast genome. This configuration is proposed to allow for rapid reversal of the hyper- or hypo-acetylation of chromatin induced by the targeted recruitment of HAT and HDAC, thereby restoring the chromatin to a "ground" state (Vogelauer et al., 2000; Katan-Khaykovich and Struhl, 2002). This model is somewhat analogous to that proposed for HOS2-dependent gene activation.

The last important issue concerns the complexity of histone acetylation. Different HATs and HDACs appear to have distinct preferences toward different lysine residues in histones. For example, in yeast cells, Rpd3 is capable of deacetylating all lysines examined in the core histones, whereas Hdal specifically deacetylates histone H2B and H3, and HOS2 deacetylates histone H3 and H4 (for review, see Kurdistani and Grunstein, 2003). The specificity of HDAC family members in higher eukaryotes is less well defined; however, it is likely it exists there as well. HATs also show specificity. GCN5 acetylates mostly H3 and H2B while CBP and p300 appear to acetylate all histones (for review, see Sterner and Berger, 2000). Whether this apparent specificity toward different histones is important for the chromatin remodeling activity of these enzymes remains to be established.

Acetylation of Non-histone Proteins

Histones are the prototypical target of acetylation, but they are far from the only ones. It has become clear in recent years that acetylation has a much wider range than simply histones. As already mentioned in previous sections, a growing number of non-histone acetylated proteins have been identified (reviewed in Cohen and Yao, 2004; Yang, 2004b). These include several different types of proteins. First, other non-histone chromatin proteins can be acetylated, such as several members of the High Mobility Group proteins (HMG1, HMG2, HMG14, and HMG17) and cohesin subunits (Sterner et al., 1978; Sterner et al., 1981; Ivanov et al., 2002). Secondly, a number of transcription factors can be acetylated, including such well-known proteins as p53, E2F1, CREB, MyoD, and more than 30 other activators (For a list of acetylated TFs, see (Yang, 2004b)). Several general transcription factors are also acetylated, including TFIIB, E, and F (Choi et al., 2003; Imhof et al., 1997). Acetylation of proteins involved in DNA replication and repair, such as PCNA, and the nuclear transport factor importin-a have also been reported (Naryzhny and Lee, 2004; Bannister et al., 2000). In the cytoplasm, a-tubulin has long been known to be acetylated (LHernault and Rosenbaum, 1985). A recent study also demonstrates that the molecular chaperone Hsp90 is also regulated by acetylation (Yu et al., 2002). The recent explosion of acetylated proteins suggests that acetylation is a very common modification, and that many more can and will be identified in the future. Furthermore, these observations strongly support the proposition that reversible protein acetylation plays a broad biological role beyond histone modification.

Given the wide array of targets, it is not surprising that acetylation can have diverse functional consequences. Since many acetylated substrates are DNA-binding proteins, a common effect of acetylation is to alter the DNA-binding ability of the protein. Acetylation can stimulate DNA binding, as shown for GATA1 (Boyes et al., 1998), or it can disrupt it, as is the case with histones and HMGI(Y) (Munshi et al., 1998). Acetylation can affect protein transport. The deacetylation of RelA subunit of NF-kB by HDAC3 leads to NF-kB nuclear export, attenuating its transcriptional activity (Chen et al., 2001). Acetylation has also been reported to affect protein stability, possibly by interfering with protein ubiquitination. In fact, acetylation, ubiquitination, sumoylation, methylation, and neddylation all target lysine residues. An emerging concept is that acetylation can compete with other modifications for a specific lysine residue and block its effects. If acetylation blocks ubiquitin binding of a lysine, for example, it may prevent its degradation by the proteasome, suggesting a mechanism for the stability increase mentioned above. It appears p53 acetylation can function in this manner (Ito et al., 2002). Importantly, these acetylation events appear to be controlled by the same enzymes that were initially thought to be dedicated to histone modification. Thus, the classically defined HAT and HDAC are likely to have functions other than chromatin-dependent processes. In fact in at least one case, HDAC6, it does not appear to function as a histone deacetylase at all (see above), further splitting the "H" from the "AT" and "DAC".

HDAC Inhibitors in Diseases and Therapy

HDAC inhibitors are currently being examined for clinical use in several different areas. So far, these approaches focus on class I and class II inhibitors, although class III HDACs may soon be considered as therapeutic targets for ageing or metabolic disorders (see above). Generally, the inhibitors lack specificity, inhibiting all of class I and II, although several do not inhibit HDAC6 or 10 very efficiently (Furumai et al., 2001). One major area of research is the use of HDAC inhibitors as anti-cancer chemotherapeutic agents. Sodium butyrate is one inhibitor that has been approved for clinical use (Thiagalingam et al., 2003). It is able to reduce proliferation of tumor cells, induce apoptosis, and increase differentiation (Sawa et al., 2001; Chopin et al., 2002). It has, however, effects on several different enzymes besides HDACs. Trichostatin A (TSA), originally discovered as an antifungal agent, is another widely used HDAC inhibitor (Tsuji et al., 1976). It causes cell cycle arrest and apoptosis in a number of cell lines (Kim et al., 2000b; Sawa et al., 2001; Herold et al., 2002). TSA is a reversible inhibitor that binds to the active site of HDACs (Finnin et al., 1999). In contrast, trapoxin is an irreversible inhibitor that was originally identified as a compound capable of reversing transformation of NIH3T3 cells (Itazaki et al., 1990). It was subsequently shown to be an HDAC inhibitor. Unfortunately TSA and trapoxin do not have high antitumor activity in vivo, probably due to stability issues (Yoshida et al., 2001). On the other hand, suberoylanilide hydroxamic acid (SAHA), which is similar to TSA, appears to be more effective (Richon et al, 1998). Like other inhibitors, it stimulates apoptosis and differentiation while inhibiting growth of cancer cell lines (Butler et al., 2002; Munster et al., 2001), and it also shows substantial antitumor activity in animal models (Cohen et al., 2002). It reduced both tumor number and size in rats with mammary carcinomas as well as inhibiting tumor growth in at least two different mouse models (Cohen et al., 2002; Marks et al., 2000). SAHA is currently undergoing clinical trials for both solid tumors and hematological malignancies (Kelly et al., 2003). Several other HDAC inhibitors have also been identified, such as FK228, MS-275, and apicidin, which have also shown promise as chemotherapy drugs (Nakajima et al., 1998; Jaboin et al., 2002; Kim et al., 2000a).

Although HDAC inhibitors are effective against many cell lines and tumors, surprisingly, they appear to have the opposite effect in a different context. Another major area of study for clinical use of HDAC inhibitors is neurodegenerative diseases. HDAC inhibitors were first shown to reduce neurodegeneration in Drosophila in models of polyglutamine disease (Steffan et al., 2001). Since then, SAHA and sodium butyrate have both been shown to ameliorate symptoms in mouse models of Huntington's disease (Ferrante et al., 2003; Hockly et al., 2003). Sodium butyrate has also been shown to ameliorate the phenotypes of another polyQ disease, spinal and bulbar muscular atrophy (SBMA) (Minamiyama et al., 2004). As mentioned above, polyQ proteins can sequester CBP, thus reducing HAT activity and leading to histone hypoacetylation (Taylor et al., 2003). HDAC inhibition may be a way of restoring the proper balance between HATs and HDACs in these diseases (Rouaux et al., 2004). However, the fact that HDAC inhibition does work in these animal models suggests that HDACs do play an active role in this system. This is supported by the high level of expression of several HDACs in the brain (Grozinger et al., 1999), and the observation that HDAC5 can induce neuronal cell death when overexpressed (Linseman et al., 2003). The advantage of developing inhibitors specific for individual HDAC members is apparent, though, given the important role for HDAC5 and 9 in preventing cardiac hypertrophy (see above).

The reasons for the difference in response to HDAC inhibitors between tumor and neurodegenerative models are not clear. It may be due to the relative expression levels of various HDACs in the tissues or cell lines. There are also likely to be differences in associated factors and targets in different tissues and even in the same tissues under different conditions. This may cause completely different responses to the same HDAC inhibitors. For instance, HDAC inhibitors such as TSA have been shown to arrest the cell cycle (Kim et al., 2000b). However, neurons are largely postmitotic in the adult brain, so they might be insensitive to effects caused by the arrest. These recent studies underline the potential therapeutic utility of HDAC inhibitors and further highlight the importance of protein acetylation in human disease.

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