Specific recruitment of corepressor complexes containing histone deacetylases (HDAC) by transcription factors is believed to play an essential role in transcriptional repression. Recent studies indicate that repression by unliganded nuclear hormone receptors and by the Mad family of repressors requires distinct HDAC-containing corepressor complexes. In this work, we show that unliganded TR specifically recruits only the closely related N-CoR and SMRT-HDAC3 complexes, whereas the Mad1 recruits only the Sin3-HDAC1/2 complex. Significantly, both the Sin3 and Mi-2/NURD complexes also exhibit constitutive association with chromatin and contribute to chromatin deacetylation in a nontargeted fashion. These results suggest that HDAC complexes can contribute to gene repression by two distinct mechanisms as follows: (1) specific targeting by repressors and (2) constitutive association with chromatin.

BACKGROUND

Histone deacetylase (HDAC) inhibition has been demonstrated to change the expression of a restricted set of cellular genes. T cells are essential in the pathogenesis of allergen-induced airway inflammation. It was recently reported that treatment with HDAC inhibitors induces a T cell-suppressive effect.

OBJECTIVE

The purpose of this study was to determine whether treatment with trichostatin A (TSA), a representative HDAC inhibitor, would reduce allergen-induced airway inflammation in a mouse asthma model.

METHODS

BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and challenged with an aerosol of OVA. TSA (1 mg/kg body weight) was injected intraperitoneally every 2 days beginning on day 1. Mouse lungs were assayed immunohistochemically for HDAC1, a major HDAC subtype, and for infiltration of CD4+ cells. The effect of TSA on airway hyper-responsiveness (AHR) was determined, and the bronchoalveolar lavage fluid (BALF) of these mice was assayed for the number and types of inflammatory cells, and for the concentrations of IL-4, IL-5, and IgE.

RESULTS

HDAC1 was localized within most airway cells and infiltrating inflammatory cells of asthmatic lungs. Treatment with TSA significantly attenuated AHR, as well as the numbers of eosinophils and lymphocytes in BALF. TSA also reduced infiltration of CD4+ and inflammatory cells and mucus occlusions in lung tissue, and decreased the concentrations of IL-4, IL-5, and IgE in BALF.

CONCLUSIONS

TSA attenuated the development of allergic airway inflammation by decreasing expression of the Th2 cytokines, IL-4 and IL-5, and IgE, which resulted from reduced T cell infiltration. Our results suggest that HDAC inhibition may attenuate the development of asthma by a T cell suppressive effect.

Hypoxia-inducible factor 1 α (HIF1α) is an essential part of the HIF-1 transcriptional complex that regulates angiogenesis, cellular metabolism, and cancer development. In von Hippel-Lindau (VHL)-null kidney cancer cell lines, we reported previously that HIF1α proteins can be acetylated and inhibited by histone deacetylase (HDAC) inhibitors or specific siRNA against HDAC4. To investigate the mechanism and biological consequence of the inhibition, we have generated stable HDAC4 knockdown via shRNA in VHL-positive normal and cancer cell lines. We report that HDAC4 regulates HIF1α protein acetylation and stability. Specifically, the HIF1α protein acetylation can be increased by HDAC4 shRNA and decreased by HDAC4 overexpression. HDAC4 shRNA inhibits HIF1α protein stability. In contrast, HDAC1 or HDAC3 shRNA has no such inhibitory effect. Mutations of the first five lysine residues (lysine 10, 11, 12, 19, and 21) to arginine within the HIF1α N terminus reduce protein acetylation but render the mutant HIF1α protein resistant to HDAC4 and HDACi-mediated inhibition. Functionally, in VHL-positive cancer cell lines, stable inhibition of HDAC4 decreases both the HIF-1 transcriptional activity and a subset of HIF-1 hypoxia target gene expression. On the cellular level, HDAC4 inhibition reduces the hypoxia-related increase of glycolysis and resistance to docetaxel chemotherapy. Taken together, the novel biological relationship between HDAC4 and HIF1α presented here suggests a potential role for the deacetylase enzyme in regulating HIF-1 cancer cell response to hypoxia and presents a more specific molecular target of inhibition.

Oct4, Sox2, Klf4, and cMyc (OSKM) reprogram somatic cells to pluripotency. To gain a mechanistic understanding of their function, we mapped OSKM-binding, stage-specific transcription factors (TFs), and chromatin states in discrete reprogramming stages and performed loss- and gain-of-function experiments. We found that OSK predominantly bind active somatic enhancers early in reprogramming and immediately initiate their inactivation genome-wide by inducing the redistribution of somatic TFs away from somatic enhancers to sites elsewhere engaged by OSK, recruiting Hdac1, and repressing the somatic TF Fra1. Pluripotency enhancer selection is a stepwise process that also begins early in reprogramming through collaborative binding of OSK at sites with high OSK-motif density. Most pluripotency enhancers are selected later in the process and require OS and other pluripotency TFs. Somatic and pluripotency TFs modulate reprogramming efficiency when overexpressed by altering OSK targeting, somatic-enhancer inactivation, and pluripotency enhancer selection. Together, our data indicate that collaborative interactions among OSK and with stage-specific TFs direct both somatic-enhancer inactivation and pluripotency-enhancer selection to drive reprogramming.

Receptor-interacting protein 140 (RIP140) encodes a histone deacetylase (HDAC) inhibitor-sensitive repressive activity. Direct interaction of RIP140 with HDAC1 and HDAC3 occurs in vitro and in vivo as demonstrated in co-immunoprecipitation and glutathione S-transferase pull-down experiments. The HDAC-interacting domain of RIP140 is mapped to its N-terminal domain, between amino acids 78 and 303 based upon glutathione S-transferase pull-down experiments. In chromatin immunoprecipitation assays, it is demonstrated that histone deacetylation occurs at the chromatin region of the Gal4 binding sites as a result of Gal4 DNA binding domain-tethered RIP expression. The immunocomplexes of RIP140 from cells transfected with RIP140 and HDAC are able to deacetylate histone proteins in vitro. This study presents the first evidence for RIP140 as a negative coregulator for nuclear receptor actions by directly recruiting histone deacetylases and categorizes RIP140 as a novel negative coregulator that is able to directly interact with HDACs.

Oncoproteins from DNA tumor viruses such as adenovirus E1a, simian virus 40 T antigen, and human papillomavirus E7 contain an LXCXE sequence, which they use to bind the retinoblastoma protein (Rb) and inhibit its function. Cellular proteins such as histone deacetylases 1 and 2 (HDAC1 and -2) also contain an LXCXE-like sequence, which they use to interact with Rb. The LXCXE binding site in Rb was mutated to assess its role in Rb function. These mutations inhibited binding to HDAC1 and -2, which each contain an LXCXE-like sequence, but had no effect on binding to HDAC3, which lacks an LXCXE-like sequence. Mutation of the LXCXE binding site inhibited active transcriptional repression by Rb and prevented it from effectively repressing the cyclin E and A gene promoters. In contrast, mutations in the LXCXE binding site did not prevent Rb from binding and inactivating E2F. Thus, the LXCXE mutations appear to separate Rb's ability to bind and inactivate E2F from its ability to efficiently recruit HDAC1 and -2 and actively repress transcription. In transient assays, several of the LXCXE binding site mutants caused an increase in the percentage of cells in G(1) by flow cytometry, suggesting that they can arrest cells. However, this effect was transient, as none of the mutants affected cell proliferation in longer-term assays examining bromodeoxyuridine incorporation or colony formation. Our results then suggest that the LXCXE binding site is important for full Rb function. Mutation of the LXCXE binding site does not inhibit binding of the BRG1 ATPase component of the SWI/SNF nucleosome remodeling complex, which has been shown previously to be important for Rb function. Indeed, overexpression of BRG1 and Rb in cells deficient for the proteins led to stable growth inhibition, suggesting a cooperative role for SWI/SNF and the LXCXE binding site in efficient Rb function.

Histone deacetylase inhibitors (HDACi) represent a new group of drugs currently being tested in a wide variety of clinical applications. They are especially effective in preclinical models of cancer where they show antiproliferative action in many different types of cancer cells. Recently, the first HDACi was approved for the treatment of cutaneous T cell lymphomas. Most HDACi currently in clinical development act by unspecifically interfering with the enzymatic activity of all class I HDACs (HDAC1, 2, 3, and 8), and it is widely believed that the development of isoform-specific HDACi could lead to better therapeutic efficacy. The contribution of the individual class I HDACs to different disease states, however, has so far not been fully elucidated. Here, we use a genetic approach to dissect the involvement of the different class I HDACs in tumor cells. We show that deletion of a single HDAC is not sufficient to induce cell death, but that HDAC1 and 2 play redundant and essential roles in tumor cell survival. Their deletion leads to nuclear bridging, nuclear fragmentation, and mitotic catastrophe, mirroring the effects of HDACi on cancer cells. These findings suggest that pharmacological inhibition of HDAC1 and 2 may be sufficient for anticancer activity, providing an experimental framework for the development of isoform-specific HDAC inhibitors.

Human cystic fibrosis transmembrane conductance regulator gene (CFTR) transcription is tightly regulated by nucleotide sequences upstream of the initiator sequences. Our studies of human CFTR transcription focus on identifying transcription factors bound to an inverted CCAAT consensus or "Y-box element." The human homeodomain CCAAT displacement protein/cut homolog (CDP/cut) can bind to the Y-box element through a cut repeat and homeobox. Analysis of stably transfected cell lines with wild-type and mutant human CFTR-directed reporter genes demonstrates that human histone acetyltransferase GCN5 and transcription factor ATF-1 can potentiate CFTR transcription through the Y-box element. We have found 1) that human CDP/cut acts as a repressor of CFTR transcription through the Y-box element by competing for the sites of transactivators hGCN5 and ATF-1; 2) that the ability of CDP/cut to repress activities of hGCN5 and ATF-1 activity is contingent on the amount of CDP/cut expression; 3) that histone acetylation may have a role in the regulation of gene transcription by altering the accessibility of the CFTR Y-box for sequence-specific transcription factors; 4) that trichostatin A, an inhibitor of histone deacetylase activity, activates transcription of CFTR through the Y-box element; 5) that the inhibition of histone deacetylase activity leads to an alteration of local chromatin structure requiring an intact Y-box sequence in CFTR; 6) that immunocomplexes of CDP/cut possess an associated histone deacetylase activity; 7) that the carboxyl region of CDP/cut, responsible for the transcriptional repressor function, interacts with the histone deacetylase, HDAC1. We propose that CFTR transcription may be regulated through interactions with factors directing the modification of chromatin and requires the conservation of the inverted CCAAT (Y-box) element of the CFTR promoter.

BACKGROUND

Epigenetic programming, dynamically regulated by histone acetylation, is a key mechanism regulating cell proliferation and survival. Little is known about the contribution of histone deacetylase (HDAC) activity to the development of pulmonary arterial hypertension, a condition characterized by profound structural remodeling of pulmonary arteries and arterioles.

RESULTS

HDAC1 and HDAC5 protein levels were elevated in lungs from human idiopathic pulmonary arterial hypertension and in lungs and right ventricles from rats exposed to hypoxia. Immunohistochemistry localized increased expression to remodeled vessels in the lung. Both valproic acid, a class I HDAC inhibitor, and suberoylanilide hydroxamic acid (vorinostat), an inhibitor of class I, II, and IV HDACs, mitigated the development of and reduced established hypoxia-induced pulmonary hypertension in the rat. Both valproic acid and suberoylanilide hydroxamic acid inhibited the imprinted highly proliferative phenotype of fibroblasts and R-cells from pulmonary hypertensive bovine vessels and platelet-derived growth factor-stimulated growth of human vascular smooth muscle cells in culture. Exposure to valproic acid and suberoylanilide hydroxamic acid was associated with increased levels of p21 and FOXO3 and reduced expression of survivin. The significantly higher levels of expression of cKIT, monocyte chemoattractant protein-1, interleukin-6, stromal-derived factor-1, platelet-derived growth factor-b, and S100A4 in R-cells were downregulated by valproic acid and suberoylanilide hydroxamic acid treatment.

CONCLUSIONS

Increased HDAC activity contributes to the vascular pathology of pulmonary hypertension. The effectiveness of HDAC inhibitors, valproic acid, and suberoylanilide hydroxamic acid, in models of pulmonary arterial hypertension supports a therapeutic strategy based on HDAC inhibition in pulmonary arterial hypertension.

Specific cell type differentiation is driven by programmed regulation of gene expression, which is the result of coordinated modulation of the transcription machinery and chromatin-remodeling factors. We present evidence here that the down-regulation of histone deacetylases is an important process during adipocyte differentiation. In 3T3-L1 cells, histone hyperacetylation was selectively induced at the promoter regions of adipogenic genes during adipocyte differentiation. Interestingly, this was accompanied by a dramatic decrease in the expression level of several histone deacetylases including HDAC1, -2, and -5 and a reduction in overall histone deacetylase enzyme activity. Inhibition of histone deacetylase activity using sodium butyrate resulted in stimulation of adipogenic gene expression and adipocyte differentiation. Consistently, HDAC1 knock-down promoted adipogenesis whereas HDAC1 overexpression attenuated adipocyte differentiation in 3T3-L1 cells. Together, these results suggest that the regulation of not only adipogenic transcription factors, but also chromatin-modifying enzymes is crucial for the execution of bona fide adipogenesis.

OBJECTIVE

The hepatitis B virus X protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanisms remain unknown. The aim of this study was to identify underlying mechanisms involved in HBx-mediated epigenetic modification.

METHODS

Interactions between HBx and DNA methyltransferase (DNMT) or histone deacetylase-1 (HDAC1) were assessed by co-immunoprecipitation. DNA methylation of gene promoters was detected by bisulfite sequencing, and HBx-mediated protein binding to gene regulatory elements was evaluated by chromatin immunoprecipitation. Target gene transcriptional activity was measured by real-time polymerase chain reaction.

RESULTS

HBx can interact directly with DNMT3A and HDAC1. HBx recruited DNMT3A to the regulatory promoters of interleukin-4 receptor and metallothionein-1F and subsequently silenced their transcription via de novo DNA methylation. By contrast, the transcription of CDH6 and IGFBP3 was triggered by HBx through the deprivation of DNMT3A from their promoters. Transcriptional levels of target genes in hepatocellular carcinoma (HCC) specimens were strongly correlated with the occurrence of HBx.

CONCLUSIONS

The interaction of HBx and DNMT3A facilitates cellular epigenetic modification (via regional hypermethylation or hypomethylation) at distinct genomic loci, providing an alternative mechanism within HBx-mediated transcriptional regulation, and a profound understanding of hepatitis and HCC pathogenesis.

The diverse function of proliferating cell nuclear antigen (PCNA) is thought to be due, in large part, to post-translational modifications. Here we show by high resolution two-dimensional PAGE analysis that there are three distinct PCNA isoforms that differ in their acetylation status. The moderately acetylated main (M) form was found in all of the subcellular compartments of cycling cells, whereas the highly acetylated acidic form was primarily found in the nucleoplasm, nuclear matrix, and chromatin. Interestingly, the deacetylated basic form was most pronounced in the nucleoplasm of cycling cells. The cells in G(0) and the cytoplasm of cycling cells contained primarily the M form only. Because p300 and histone deacetylase (HDAC1) were co-immunoprecipitated with PCNA, they are likely responsible for the acetylation and deacetylation of PCNA, respectively. We also found that deacetylation reduced the ability of PCNA to bind to DNA polymerases beta and delta. Taken together, our data support a model where the acidic and M forms participate in DNA replication, whereas the basic form is associated with the termination of DNA replication.

Transcriptional repression by sequence-specific DNA binding factors is mediated by the recruitment of a corepressor complex to the promoter region. The NK-3 homeodomain protein is a transcriptional repressor that recruits the nuclear protein kinase, homeodomain interacting protein kinase 2 (HIPK2). Here we show that HIPK2 is a component of a corepressor complex containing Groucho and a histone deacetylase complex. Groucho, like HIPK2, acts as a corepressor for NK-3 and binds to NK-3 and HIPK2. Moreover, HIPK2 appears to regulate the corepressor activity of Groucho. Transcriptional repression by NK-3 and Groucho is relieved by the histone deacetylase inhibitor trichostatin A, and both NK-3 and Groucho directly interact with the histone deacetylase HDAC1 that is associated with mSin3A in vivo. Recruitment of the histone deacetylase complex by NK-3 decreases the acetylated histones that are associated with the target gene promoter. These results indicate that NK-3 represses transcription by recruiting a complex containing Groucho and a histone deacetylase complex that leads to histone modification on chromatin and suggest that HIPK2 may play a regulatory role in the corepressor complex formation.

OBJECTIVE

Histone deacetylases (HDACs) play an important role in chromatin remodeling, gene repression and regulating cell cycle progression and differentiation. This study was designed to clarify the role of HDAC1 expression in hepatocellular carcinoma (HCC).

METHODS

The expression of HDAC1 in 47 patients with surgically resected HCC was immunohistochemically examined and analyzed in relation to their clinicopathological factors. The patients were divided into two groups according to the expression status of HDAC1: a high HDAC1 group (n = 25) with more than 20% of positively stained cells and a low HDAC1 group (n = 22) with 20% or fewer positively stained cells.

RESULTS

A high HDAC1 expression indicated a higher incidence of cancer cell invasion into the portal vein, a poorer histological differentiation, and a more advanced TNM stage. The survival rates after a surgical resection in low and high HDAC1 patients at 1, 3, 5 and 10 years were 100, 95.5, 81.8 and 60.8% and 88.0, 60.0, 40.0 and 32.0%, respectively (p = 0.008). A multivariate analysis using the Cox regression analysis showed that a high HDAC1 expression was an independent prognostic factor of HCC in patients after hepatic resection (relative risk: 10.1, p = 0.0018).

CONCLUSIONS

High HDAC1 expression might have an important role in the aggressiveness and cell dedifferentiation, and its expression status may be a useful biomarker for predicting the outcome of the patients with HCC.

We review here evidence defining a molecular pathway that includes cell cycle-related molecules and that appears to play a required role in neuron death during normal development as well as in disease and trauma. The pathway starts with inappropriate activation of cyclin dependent kinase 4 (Cdk4) in neurons which leads to hyper-phosphorylation of the pRb family member p130. This in turn results in dissociation of p130 and its associated chromatin modifiers Suv39H1 and HDAC1 from the transcription factor E2F4. Dissociation of this complex results in de-repression of genes with E2F binding sites including those encoding the transcription factors B- and C-Myb. Once elevated in neurons, B- and C-Myb proteins bind to the promoter for the pro-apoptotic BH3-only protein Bim and promote its induction. Bim then interacts with the core cellular apoptotic machinery, leading to caspase activation and apoptotic death. This pathway is supported by a variety of observations and experimental findings that implicate it as a required element for neuron loss in development and in many nervous system traumas and disorders. The components of this pathway appear to represent potential therapeutic targets for prevention of disease-associated neuron death.

Covalent modifications of histone proteins, in particular deacetylation of lysine residues, are important for the regulation of gene transcription both in normal and malignant cells. These processes are controlled by histone acetyltransferases and histone deacetylases (HDAC) and have up to now not been described in solid mesenchymal tumors. The present study shows differences in the HDAC1 and HDAC2 expression in endometrial stromal sarcomas (ESS) and a cognate cell line (ESS-1) compared with nonneoplastic endometrial stroma. We show for the first time that HDAC2 expression is consistently increased in ESS. In contrast, HDAC1 expression is generally lower than HDAC2 both in nonneoplastic stroma and in ESS, suggesting that these two proteins, although closely related, are regulated in different ways. In vitro experiments with an ESS cell line showed that valproate, an inhibitor of the class I HDACs, led to significant HDAC2 decrease and to cell differentiation. HDAC2 inhibition in ESS-1 cells caused significant changes in the cell cycle by inhibiting G1-S transition and influencing expression of p21WAF1 and cyclin D1. Moreover, in ESS-1 cells, increased expression of the p21WAF1 was associated with reduction of HDAC2 expression after transfection with small interfering RNA directed against HDAC2. Our results suggest that HDAC2 might be considered as potential drug target in the therapy of ESS and that HDAC inhibitors should be further evaluated in clinical trials in ESS.

OBJECTIVE

Hypoxia-inducible factor 1alpha (HIF-1alpha) is a transcription factor that plays an important role in tumor growth and metastasis. Inhibition of histone deacetylase shows a marked inhibition of HIF-1alpha expression; however, the association between HIF-1alpha and histone deacetylase 1 (HDAC1), metastasis-associated protein 1 (MTA1) is not fully understood.

METHODS

Hypoxia-inducible factor 1alpha, HDAC1, and MTA1 expressions were detected by immunohistochemistry in 39 pancreatic carcinoma patients. The correlations between the expression of HIF-1alpha, HDAC1, or MTA1 and clinical features and the prognosis were analyzed.

RESULTS

Hypoxia-inducible factor 1alpha, HDAC1, and MTA1 positive stainings were found in 41%, 56%, and 31%, respectively. There was no correlation between HIF-1alpha, HDAC1, or MTA1 expression levels and any clinical parameters. The survival rate for patients with HIF-1alpha and HDAC1-positive stainings were significantly lower than for patients with HIF-1alpha and HDAC1-negative stainings. The MTA1 overexpression group did not have a significantly lower prognosis than the MTA1 underexpression group. The survival rate for the HDAC1(+)/MTA1(2-3) group was significantly lower than for the other groups.

CONCLUSIONS

These results suggest that HIF-1alpha expression may be regulated through HDAC1/MTA1, which is associated with a poor prognosis for pancreatic carcinoma and indicates that HIF-1alpha and HDAC1/MTA1 are a promising therapeutic target in pancreatic carcinoma treatment.

Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors broadly used for the control of hypercholesterolemia. Recently, they are reported to have beneficial effects on certain cancers. In this study, we show that statins inhibited the histone deacetylase (HDAC) activity and increased the accumulation of acetylated histone-H3 and the expression of p21(WAF/CIP) in human cancer cells. Computational modeling showed the direct interaction of the carboxylic acid moiety of statins with the catalytic site of HDAC2. In the subsequent enzymatic assay, it was shown that lovastatin inhibited HDAC2 activity competitively with a K(i) value of 31.6 micromol/L. Sp1 but not p53 sites were found to be the statins-responsive element shown by p21 luciferase-promoter assays. DNA affinity protein binding assay and chromatin immunoprecipitation assay showed the dissociation of HDAC1/2 and association of CBP, leading to the histone-H3 acetylation on the Sp1 sites of p21 promoter. In vitro cell proliferation and in vivo tumor growth were both inhibited by statins. These results suggest a novel mechanism for statins through abrogation of the HDAC activity and promoter histone-H3 acetylation to regulate p21 expression. Therefore, statins might serve as novel HDAC inhibitors for cancer therapy and chemoprevention.

Class I histone deacetylases (HDACs) are cellular enzymes expressed in many tissues and play crucial roles in differentiation, proliferation, and cancer. HDAC1 and HDAC2 in particular are highly homologous proteins that show redundant or specific roles in different cell types or in response to different stimuli and signaling pathways. The molecular details of this dual regulation are largely unknown. HDAC1 and HDAC2 are not only protein modifiers, but are in turn regulated by post-translational modifications (PTMs): phosphorylation, acetylation, ubiquitination, SUMOylation, nitrosylation, and carbonylation. Some of these PTMs occur and crosstalk specifically on HDAC1 or HDAC2, creating a rational "code" for a differential, context-related regulation. The global comprehension of this PTM code is central for dissecting the role of single HDAC1 and HDAC2 in physiology and pathology.

BACKGROUND

The histone-modifying enzymes histone deacetylase (HDAC) and histone acetyltransferase (HAT) control gene transcriptional activation and repression in human malignancies.

OBJECTIVE

To analyse the expression of HDAC/HAT-associated molecules such as HDAC1, CREB-binding protein (CBP) and p300 in human colorectal carcinomas, and investigate the relationship between their expression levels and clinicopathological parameters.

METHODS

Expression levels of HDAC1, CBP, and p300 in human colorectal cancer were investigated by immunohistochemistry. In situ hybridisation (ISH) and reverse transcription (RT)-PCR analyses were also carried out to confirm mRNA expression levels of these genes. Immunoreactivity was evaluated semi-quantitatively using a staining index (SI). The relationships between the SIs and clinicopathological findings were analysed and survival curves were calculated using the Kaplan-Meier method and log-rank tests.

RESULTS

The mean SIs for HDAC1, CBP, and p300 in this series of tumours were much higher than those in normal colonic mucosa. The presence of HDAC1 and CBP mRNAs on colorectal carcinoma cells as well as normal epithelial cells was confirmed by ISH analysis. A marked increase in p300 mRNA levels was detected in a majority of cases by RT-PCR. Among the patients with colorectal cancer, overexpression of p300 (SI>11.9) correlated with a poor prognosis, whereas high CBP expression levels (SI>16.6) indicated long-term survival.

CONCLUSIONS

Results showed the up-regulation of these three histone-modifying molecules in this series of colorectal cancers and suggested that monitoring of CBP and p300 may assist prediction of the prognosis in patients with colorectal adenocarcinoma.

Covalent modifications of histone tails play important roles in gene transcription and silencing. We recently identified an ERG ( ets -related gene)-associated protein with a SET (suppressor of variegation, enhancer of zest and trithorax) domain (ESET) that was found to have the activity of a histone H3-specific methyltransferase. In the present study, we investigated the interaction of ESET with other chromatin remodelling factors. We show that ESET histone methyltransferase associates with histone deacetylase 1 (HDAC1) and HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A and mSin3B. Deletion analysis of ESET reveals that an N-terminal region containing a tudor domain is responsible for interaction with mSin3A/B and association with HDAC1/2, and that truncation of ESET enhances its binding to mSin3. When bound to a promoter, ESET represses the transcription of a downstream luciferase reporter gene. This repression by ESET is independent of its histone methyltransferase activity, but correlates with its binding to the mSin3 co-repressors. In addition, the repression can be partially reversed by treatment with the HDAC inhibitor trichostatin A. Taken together, these data suggest that ESET histone methyltransferase can form a large, multi-protein complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in multiple pathways of transcriptional repression.

Class I Histone deacetylases (HDACs) play a central role in controlling cell cycle regulation, cell differentiation, and tissue development. These enzymes exert their function by deacetylating histones and a growing number of non-histone proteins, thereby regulating gene expression and several other cellular processes. Class I HDACs comprise four members: HDAC1, 2, 3, and 8. Deletion and/or overexpression of these enzymes in mammalian systems has provided important insights about their functions and mechanisms of action which are reviewed here. In particular, unique as well as redundant functions have been identified in several paradigms. Studies with small molecule inhibitors of HDACs have demonstrated the medical relevance of these enzymes and their potential as therapeutic targets in cancer and other pathological conditions. Going forward, better understanding the specific role of individual HDACs in normal physiology as well as in pathological settings will be crucial to exploit this protein family as a useful therapeutic target in a range of diseases. Further dissection of the pathways they impinge on and of their targets, in chromatin or otherwise, will form important avenues of research for the future.

AML1/ETO is the chimeric protein resulting from the t(8;21) in acute myeloid leukemia. The Nervy homology 2 (NHR2) domain in ETO mediates oligomerization and AML1/ETO's interactions with ETO, MTGR1, and MTG16, and with the corepressor molecules mSin3A and HDAC1 and HDAC3. We solved the NHR2 domain structure and found it to be an alpha-helical tetramer. We show that oligomerization contributes to AML1/ETO's inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and affects AML1/ETO's activity on several endogenous genes. Oligomerization is also required for AML1/ETO's interactions with ETO, MTGR1, and MTG16, but not with other corepressor molecules.

C-terminal binding protein (CtBP) family proteins CtBP1 and CtBP2 are highly homologous transcriptional corepressors and are recruited by a large number of transcription factors to mediate sequence-specific transcriptional repression. In addition to DNA-binding repressors, the nuclear protein complex of CtBP1 consists of enzymatic constituents such as histone deacetylases (HDAC1/2), histone methyl transferases (HMTases; G9a and GLP), and the lysine-specific demethylase (LSD1). Additionally, CtBPs also recruit the components of the sumoylation machinery. The CtBPs contain two different unique structural elements, a hydrophobic cleft, with which factors that contain motifs related to the E1A PLDLS motif bind, and a surface groove that binds with factors containing motifs related to the sequence RRTGXPPXL (RRT motif). By structure-based functional dissection of CtBP1, we show that the PLDLS-binding cleft region functions as the primary recruitment center for DNA-binding factors and for the core and auxiliary enzymatic constituents of the CtBP1 corepressor complex. We identify HDAC1/2, CoREST/LSD1, and Ubc9 (E2) as the core constituents of the CtBP1 complex, and these components interact with the PLDLS cleft region through non-PLDLS interactions. Among the CtBP core constituents, HDACs contribute predominantly to the repression activity of CtBP1. The auxiliary components include an HMTase complex (G9a/Wiz/CDYL) and two SUMO E3 ligases, HPC2 and PIAS1. The interaction of auxiliary components with CtBP1 is excluded by PLDLS (E1A)-mediated interactions. Although monomeric CtBP1 is proficient in the recruiting of both core and auxiliary components, NAD(H)-dependent dimerization is required for transcriptional repression. We also provide evidence that CtBP1 functions as a platform for sumoylation of cofactors.