The Translation Post Vol.3 Issue 1

Review Article - Histone Deacetylases: Regulators of Genomic Free Speech

Histone Acetylation: The Shot Heard ™round the Nucleosome

Though Nobel laureate Alberech Kossel first recognized DNA-coiling histone proteins in 1884, it was nearly a century before scientists began to grasp the breadth of histone function beyond its mechanical role packaging eukaryotic DNA. Since the early 1990s, the dynamic regulatory capabilities of histones have been profiled in tens of thousands of publications, implicating histones in gene expression and silencing, transcription regulation, and regulation of cellular developmental processes. The wide variety of histone activities is due in no small part to the modifications that occur on the "tail" of the histone [Figure 1], a protruding amino-terminal of ~25-40 residues [1]. These modifications are thought to occur in an orderly manner to affect gene expression by affecting the arrangement of DNA in the nucleosome. Thus, the pattern of modifications, or "histone code," greatly increases DNA™s potential to affect cell function by controlling access to transcription factors [2]. This code is also thought to be involved in epigenetic, or heritable and potentially reversible, changes in chromatin structure [3]. Understanding the underlying patterns of these modifications may hold the key to many types of cellular dysfunction, such as tumorigenesis and myelodysplasia.
 

Figure 1: Diagram of histone tails and the sites at which they may be modified [4].

Of the observed histone modifications, including methylation, phosphorylation, ubiquitination and ADP-ribosylation, the best characterized is acetylation, which is correlated with increased transcription and may directly facilitate transcription by loosening the DNA surrounding the histone, as well as recruit specific transcriptional facilitators, such as the bromo-, chromo- and SANT-domains [5, 6]. Acetylation of histones is primarily controlled by precise interactions between histone acetylases (HATs) and their antagonists, histone deacetylases (HDACs). In cancer, tumor-suppressor genes are sometimes repressed by HDACs, consequently, compounds that inhibit HDAC activity have been identified as potential anti-cancer drugs by a number of pharmaceutical companies. HDAC inhibitors are being tested in a number of different clinical trials against a wide variety of diseases, including leukemia, AIDS, breast cancer, and spinal muscular atrophy.

HDAC Function: United We Stand

Histone deacetylases are a venerable family of enzymes that may be found in animals, plants, fungi, and bacteria. These enzymes predate their most abundant eukaryotic substrates, leading some to speculate that primary HDAC targets are not histones but rather other acetylated proteins [7, 6]. The first HDAC gene was cloned after purifying the protein target of HDAC inhibitor trapoxin A [9]. The predicted protein was a human orthologue of the yeast transcriptional regulator Rpd3p, and has since been named HDAC1. Human HDACs are classified by homology to their yeast counterparts and largely function in multisubunit protein complexes.

Class I contains HDACs 1, 2, 3, and 8, which primarily localize to the nucleus, though HDAC3 carries a nuclear import signal in addition to a nuclear localization signal, which indicates that it may also localize to the cytoplasm [9]. By contrast, Class II HDACs move freely across the nuclear membrane in a highly regulated response to cellular stimuli [10]. Class III contains orthologues of yeast silent information regulator 2 (Sir2), which participate in gene silencing and DNA repair, as well as deacetylate important nonhistone targets, including p53 [11]. HDAC11 occupies its own class because it is the only human HDAC that has no homologue in fungi and does not function in any known HDAC complexes [12]. In addition to being present in nearly all non-human organisms, HDACs are expressed in all human tissues, indicating the extensive role that HDACs and HDAC complexes play in cellular processes.

Histone acetylation, which takes place on the ε-amino group of core histone lysine residues, neutralizes the positively charged histone so that it interacts more weakly with negatively charged DNA. When histones are deacetylated, the chromatin condenses and the genes exposed in the acetylated state are silenced. Acetylation and deacetylation also work in concert with surrounding methylated DNA to express and silence genes. Methylation occurs on the CpG base pair, and methylated DNA is often targeted by HDAC-containing complexes such as mSin3A and NuRD for purposes of gene silencing [Figure 2]. Methylation may also occur on histones on the same lysines where acetylation occurs, though the resultant activity depends on the position of the lysine residue. Transcriptionally active chromatin (euchromatin) is generally unmethylated , since acetylation occurs on the same lysines that are methylated in inactive chromatin (heterochromatin) [13]. Although inhibiting HDAC activity produces large numbers of hyperacetylated histones, transcription does not occur unless chromatin is also demethylated, thus indicating that the two modifications work in concert to control gene transcription [14].
 

Figure 2: Class I HDACs complexes have been implicated in gene silencing through the recruitment of methylated DNA [15].

HDACs in Disease: Divided We Fall

Given the ubiquity of HDACs in higher life forms, it follows logically that abnormal HDAC activity would have debilitating consequences. Indeed, few pathogenic mutations of HDACs have ever been observed, and successful mammalian knockouts have been difficult to analyze because the effect, particularly in Class I HDACs, is lethal in the early stages of development. Of the Class I HDACs, HDAC1 is the only member to have been successfully knocked out, which resulted in death due to cessation of embryonic cell proliferation [6]. Finding an effective way to knock down HDAC in vivo is critical to the study of Class I HDAC absence in fully developed organisms. Because Class II HDACs are less broadly expressed, knockouts develop normally but are susceptible to hypertrophic cardiac growth when cardiac stress is introduced, whether surgically or as a product of old age [16].

However, disease states are more likely to arise from abnormal expression or recruitment of HDACs rather than from a complete absence of the enzymes. For example, mutant retinoic acid receptor-α (RARα) loses its ability to displace gene-silencing HDACs on histone tails, which causes leukemia by creating blood cells that never differentiate. Furthermore, HDACs are implicated in cancer because of HDAC ability to silence genes that are critical for regular cell activity, such as tumor-suppressing genes. Overexpression of Class II HDACs in solid malignancies, acute promyelocytic leukemia and acute myelocytic leukemia is caused by mutated oncoproteins.

The important role HDACs play in gene silencing also makes them prime targets for opportunistic viruses, whose survival depends on their ability to commandeer the cell™s transcription machinery. Malignancy-related viruses, including the human papillomavirus and Epstein-Barr, recruit HDACs to induce cell growth and immortalize B-cells, respectively [17]. The human immunodeficiency virus (HIV), which causes AIDS, uses HDACs to hide its genetic material inside inactive CD4 T-cells by burying it deep within heterochromatin. Because the T-cells do not actively produce HIV, they are not affected by antiretroviral treatment and collect in lymph nodes, ready to produce more virus at any time. These stable reservoirs of latent infected cells have thus far thwarted efforts to rid patients completely of the virus and are a major impediment to finding a cure for AIDS.

Phase I if by Land, Phase II if by Sea

Because so many disease states are dependent on inappropriate expression and recruitment of HDACs, HDAC inhibitors have become attractive as potential drug targets. Currently, major pharmaceutical companies such as Abbott Laboratories (Abbott Park, IL), Merck (Whitehouse Station, NJ), Novartis (Basel, Switzerland), and AstraZeneca (London, UK) are testing known and new HDAC inhibitors in clinical trials against diseases ranging from leukemia to HIV/AIDS.

Currently, valproic acid (VPA), which has been approved to prevent migraines under the brand name Depakote ™ (Abbott Laboratories, Abbott Park, IL), is in Phase II clinical trials to see if the drug is effective in ridding AIDS patients of latent HIV-infected CD4 T-cells. VPA is a short-chain fatty acid HDAC1 inhibitor that has been used for years to treat seizures and other brain disorders. Preliminary studies indicate that by inhibiting HDAC1, VPA forces dormant HIV genes hidden within resting T-cells to be expressed, thus making them susceptible to anti-retroviral treatments [18]. In addition to HIV/AIDS therapy, VPA is also being tested for efficacy against melanoma, and acute myelogenous leukemia. Similarly, Merck™s (Whitehouse Station, NJ) Zolinza™ (vorinostat, or SAHA), a hydroxamate derivative that has already been approved to treat cutaneous T-cell lymphoma, is being tested for other types of cancer. Another pre-existing HDAC inhibitor that is being explored to cure disease is trichostatin A (TSA), an antifungual antibiotic derived from Streptomyces platensis. In addition to its antibiotic properties, TSA is also a highly specific and reversible HDAC inhibitor that has been shown to reduce malignancy in breast cancer and inhibit lung cancer growth by promoting apoptotic gene expression [19, 20].

In addition to pre-existing drugs, entirely new HDAC inhibitors have also been created for the purpose of fighting different cancers. MethylGene, partnered with Pharmion and Taiho Pharmaceutical, is testing its isotype-specific small molecule HDAC inhibitor MGCD0103 against aggressive non-Hodgkin's lymphoma and leukemia in Phase I and Phase II clinical trials. Interim results of Phase II clinical trials with FK228, a bicyclic desipeptide isolated from Chromobacterium violaceum by Gloucester Pharmaceuticals, indicate that the drug is effective and well-tolerated in metastatic prostate cancer patients. Furthermore, prostate cancers that are resistant to hormone therapy are the target of Novartis HDAC inhibitor LBH589, which is currently in Phase I/II clinical trials, having already been shown to induce apoptosis of cancerous cells in acute myelocytic leukemia. In addition to a current Phase I clinical trial against chronic myelomonocytic leukemia, Merck™s benzamide HDAC inhibitor MS-275, has also been shown to be effective in the brain, thus raising the possibility of using it to treat psychiatric disorders [21]. This finding also highlights the tantalizing possibility that these new HDAC inhibitors may turn out to be even more important in other treatments than the ones for which they were designed.
 

A report in the October 2006 issue of Nature Medicine highlights a trend that will likely become more common as new HDAC inhibitors gain regulatory approval. Trichostatin A (TSA), an HDAC inhibitor whose cancer-fighting potential is the subject of several clinical trials, has been shown to slow the spread of muscular dystrophy (MD), a debilitating inherited disease characterized by muscle deterioration, in MDX mice [22]. Though the drug does not cure the underlying genetic abnormality that causes the disease, TSA has been shown to regrow muscle by upregulating follistatin, a molecule shown to be involved with repairing muscle damage in healthy tissue. TSA is the first drug shown to have positive effects in mouse MD, and gives hope that TSA may be used to relieve symptoms for the thousands of patients who are waiting for a cure for the disease.

Concluding Remarks

Histone acetylation, though already the best-characterized posttranslational modification on histone tails, continues to be the focus of researchers in a rich variety of fields, ranging from pathology to molecular chemistry. Because the modification occurs in every human tissue, the importance of the enzymes that control the process cannot be overstated. The search for an underlying epigenetic code of histone modifications continues, and whether or not the search ultimately proves to be fruitful, human health has already begun to benefit from renewed focus on histones and the structures that participate in their modification. Given the number and variety of HDAC inhibitors currently being investigated to treat life-threatening disease, continued research on HDACs and their roles in multprotein complexes will serve to open the door for new drugs and new uses for old drugs, as well as clarify molecular processes whose precise mechanisms have only been hypothesized by researchers to date.

Abgent and Acetylation

Abgent offers a comprehensive collection of antibodies against histone deacetylases and other acetylation targets, including HDACs 1 to 11, SIRT1 to 7, NCOR1 and HRX. In addition to our catalog of acetylation-related products, Abgent also carries a wide variety of other modification targets for phosphorylation, methylation, ubiquitination, glycosylation, and sumoylation.

References

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Other sources of information:
http://www.clinicaltrials.gov
http:///www.merck.com
http://www.methylgene.com/
http://www.abbott.com/