BACKGROUND

The status of estrogen receptor-α (ERα) is critical to the clinical prognosis and therapeutic approach in breast cancer. ERα-negative breast cancer is clinically aggressive and has a poor prognosis because of the lack of hormone target-directed therapies. Previous studies have shown that epigenetic regulation plays a major role in ERα silencing in human breast cancer cells. Dietary green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), is believed to be an anticancer agent in part through its regulation of epigenetic processes.

RESULTS

In our current studies, we found that EGCG can reactivate ERα expression in ERα-negative MDA-MB-231 breast cancer cells. Combination studies using EGCG with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), revealed a synergistic effect of reactivation of ERα expression in ERα-negative breast cancer cells. Reactivation of ERα expression by EGCG and TSA treatment was found to sensitize ERα-dependent cellular responses to activator 17β-estradiol (E2) and antagonist tamoxifen in ERα-negative breast cancer cells. We also found that EGCG can lead to remodeling of the chromatin structure of the ERα promoter by altering histone acetylation and methylation status thereby resulting in ERα reactivation. A decreased binding of the transcription repressor complex, Rb/p130-E2F4/5-HDAC1-SUV39H1-DNMT1, in the regulatory region of the ERα promoter also contributes to ERα transcriptional activation through treatment with EGCG and/or TSA.

CONCLUSIONS

Collectively, these studies show that green tea EGCG can restore ERα expression by regulating epigenetic mechanisms, and this effect is enhanced when combined with an HDAC inhibitor. This study will facilitate more effective uses of combination approaches in breast cancer therapy and will help to explore more effective chemotherapeutic strategies toward hormone-resistant breast cancer.

Chemical manipulations performed on aroyl-pyrrolyl-hydroxyamides (APHAs) led to (aryloxopropenyl)pyrrolyl hydroxamates 2a-w, and their inhibition against maize HDACs and their class I or class II HDAC selectivity were determined. In particular, from these studies some benzene meta-substituted compounds emerged as highly class II (IIa)-selective HDAC inhibitors, the most selective being the 3-chloro- and 3-fluoro-substituted compounds 2c (SI = 71.4) and2f (SI = 176.4). The replacement of benzene with a 1-naphthyl ring afforded 2s, highly active against the class II homologue HD1-A (IC(50) = 10 nM) but less class II-selective than 2c,f. When tested against human HDAC1 and HDAC4, 2f showed no inhibitory activity against HDAC1 but was able to inhibit HDAC4. Moreover, in human U937 acute myeloid leukaemia cells 2f did not produce any effect on apoptosis, granulocytic differentiation, and the cell cycle, whereas 2s (that retain class I HDAC inhibitory activity) was 2-fold less potent than SAHA used as reference.

Altered expression of histone deacetylases (HDACs) is a common feature in several human malignancies and may represent an interesting target for cancer treatment, including haematological malignancies. We evaluated the mRNA gene expression profile of 12 HDAC genes by quantitative real-time polymerase chain reaction in 94 consecutive childhood acute lymphoblastic leukaemia (ALL) samples and its association with clinical/biological features and survival. ALL samples showed higher expression levels of HDAC2, HDAC3, HDAC8, HDAC6 and HDAC7 when compared to normal bone marrow samples. HDAC1 and HDAC4 showed high expression in T-ALL and HDAC5 was highly expressed in B-lineage ALL. Higher than median expression levels of HDAC3 were associated with a significantly lower 5-year event-free survival (EFS) in the overall group of patients (P = 0·03) and in T-ALL patients (P = 0.01). HDAC7 and HADC9 expression levels higher than median were associated with a lower 5-year EFS in the overall group (P = 0.04 and P = 0.003, respectively) and in B-lineage CD10-positive patients (P = 0.009 and P = 0·005, respectively). Our data suggest that higher expression of HDAC7 and HDAC9 is associated with poor prognosis in childhood ALL and could be promising therapeutic targets for the treatment of refractory childhood ALL.

Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum-infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease.

Bortezomib is now widely used for the treatment of multiple myeloma (MM); however, its action mechanisms are not fully understood. Despite the initial results, recent investigations have indicated that bortezomib does not inactivate nuclear factor-kappaB activity in MM cells, suggesting the presence of other critical pathways leading to cytotoxicity. In this study, we show that histone deacetylases (HDACs) are critical targets of bortezomib, which specifically down-regulated the expression of class I HDACs (HDAC1, HDAC2, and HDAC3) in MM cell lines and primary MM cells at the transcriptional level, accompanied by reciprocal histone hyperacetylation. Transcriptional repression of HDACs was mediated by caspase-8-dependent degradation of Sp1 protein, the most potent transactivator of class I HDAC genes. Short-interfering RNA-mediated knockdown of HDAC1 enhanced bortezomib-induced apoptosis and histone hyperacetylation, whereas HDAC1 overexpression inhibited them. HDAC1 overexpression conferred resistance to bortezomib in MM cells, and administration of the HDAC inhibitor romidepsin restored sensitivity to bortezomib in HDAC1-overexpressing cells both in vitro and in vivo. These results suggest that bortezomib targets HDACs via distinct mechanisms from conventional HDAC inhibitors. Our findings provide a novel molecular basis and rationale for the use of bortezomib in MM treatment.

TAL1 is a critical transcription factor required for hematopoiesis. However, perturbation of its activity often leads to T cell leukemia. Whether and how its transcriptional activities are regulated during hematopoiesis remains to be addressed. Here, we show that TAL1 is associated with histone demethylase complexes containing lysine-specific demethylase 1 (LSD1), RE1 silencing transcription factor corepressor (CoREST), histone deacetylase 1 (HDAC1), and histone deacetylase 2 in erythroleukemia and T cell leukemia cells. The enzymatic domain of LSD1 plays an important role in repressing the TAL1-directed transcription of GAL4 reporter linked to a thymidine kniase minimal promoter. Furthermore, we demonstrate that the TAL1-associated LSD1, HDAC1, and their enzymatic activities are coordinately down-regulated during the early phases of erythroid differentiation. Consistent with the rapid changes of TAL1-corepressor complex during differentiation, TAL1 recruits LSD1 to the silenced p4.2 promoter in undifferentiated, but not in differentiated, murine erythroleukemia (MEL) cells. Finally, shRNA-mediated knockdown of LSD1 in MEL cells resulted in derepression of the TAL1 target gene accompanied by increasing dimeH3K4 at the promoter region. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs.

The loss of E-cadherin gene expression can cause the dysfunction of the cell-cell junction to trigger tumor metastasis. Members of the Snail family of transcription factors are repressors of the expression of the E-cadherin gene. In this study, we showed that the activated androgen receptor (AR) is a novel repressor of E-cadherin gene expression and can promote metastasis. Our results demonstrated that the activated AR could bind to the E-cadherin promoter in vitro and in vivo. The activated AR and HDAC1 had synergistic effects in downregulating E-cadherin gene expression. Treating cells with the AR ligand, dihydrotestosterone (DHT), triggered the reduction of E-cadherin expression and induced changes in cell morphology from an epithelial-like to a mesenchymal-like appearance. When nonmetastatic breast cancer cells expressing cytoplasmic AR were transplanted into mice and the mice were treated with DHT, tumors were detected at metastatic sites, whereas no tumors were detected in transplanted mice without DHT treatment. Furthermore, clinical data from breast cancer patients with invasive ductal carcinomas showed high levels of AR expression in the nuclei and low levels of E-cadherin expression. These results suggest that, similarly to Snail and Twist, the activated AR can downregulate E-cadherin expression to promote the activation of epithelial-mesenchymal transition and tumor metastasis.

The pocket domain of pRB is required for pRB to arrest the cell cycle. This domain was originally defined as the region of the protein that is necessary and sufficient for pRB's interaction with adenovirus E1A and simian virus s40 large T antigen. These oncoproteins, and other pRB-binding proteins that are encoded by a variety of plant and animal viruses, use a conserved LXCXE motif to interact with pRB. Similar sequences have been identified in multiple cellular pRB-binding proteins, suggesting that the viruses have evolved to target a highly conserved binding site of pRB that is critical for its function. Here we have constructed a panel of pRB mutants in which conserved amino acids that are predicted to make close contacts with an LXCXE peptide were altered. Despite the conservation of the LXCXE binding site throughout evolution, pRB mutants that lack this site are able to induce a cell cycle arrest in a pRB-deficient tumor cell line. This G(1) arrest is overcome by cyclin D-cdk4 complexes but is resistant to inactivation by E7. Consequently, mutants lacking the LXCXE binding site were able to induce a G(1) arrest in HeLa cells despite the expression of HPV-18 E7. pRB mutants lacking the LXCXE binding site are defective in binding to adenovirus E1A and human papillomavirus type 16 E7 protein but exhibit wild-type binding to E2F or DP, and they retain the ability to interact with CtIP and HDAC1, two transcriptional corepressors that contain LXCXE-like sequences. Consistent with these observations, the pRB mutants are able to actively repress transcription. These observations suggest that viral oncoproteins depend on the LXCXE-binding site of pRB for interaction to a far greater extent than cellular proteins that are critical for cell cycle arrest or transcriptional repression. Mutation of this binding site allows pRB to function as a cell cycle regulator while being resistant to inactivation by viral oncoproteins.

The de novo DNA methyltransferase Dnmt3a is one of three mammalian DNA methyltransferases that has been shown to play crucial roles in embryonic development, genomic imprinting and transcriptional silencing. Despite its importance, very little is known about how the enzymatic activity and transcriptional repression functions of Dnmt3a are regulated. Here we show that Dnmt3a interacts with multiple components of the sumoylation machinery, namely the E2 sumo conjugating enzyme Ubc9 and the E3 sumo ligases PIAS1 and PIASxalpha, all of which are involved in conjugating the small ubiquitin-like modifier polypeptide, SUMO-1, to its target proteins. Dnmt3a is modified by SUMO-1 in vivo and in vitro and the region of Dnmt3a responsible for interaction maps to the N-terminal regulatory domain. Functionally, sumoylation of Dnmt3a disrupts its ability to interact with histone deacetylases (HDAC1/2), but not with another interaction partner, Dnmt3b. Conditions that enhance the sumoylation of Dnmt3a in vivo abolish its capacity to repress transcription. These studies reveal a new level of regulation governing Dnmt3a whereby a post-translational modification can dramatically regulate its interaction with specific protein partners and alter its ability to repress transcription.

The epigenetic down-regulation of genes is emerging as a possible underlying mechanism of the GABAergic neuron dysfunction in schizophrenia. For example, evidence has been presented to show that the promoters associated with reelin and GAD67 are down-regulated as a consequence of DNA methyltransferase (DNMT)-mediated hypermethylation. Using neuronal progenitor cells to study this regulation, we have previously demonstrated that DNMT inhibitors coordinately increase reelin and GAD67 mRNAs. Here, we report that another group of epigenetic drugs, histone deacetylase (HDAC) inhibitors, activate these two genes with dose and time dependence comparable with that of DNMT inhibitors. In parallel, both groups of drugs decrease DNMT1, DNMT3A, and DNMT3B protein levels and reduce DNMT enzyme activity. Furthermore, induction of the reelin and GAD67 mRNAs is accompanied by the dissociation of repressor complexes containing all three DNMTs, MeCP2, and HDAC1 from the corresponding promoters and by increased local histone acetylation. Our data imply that drug-induced promoter demethylation is relevant for maximal activation of reelin and GAD67 transcription. The results suggest that HDAC and DNMT inhibitors activate reelin and GAD67 expression through similar mechanisms. Both classes of drugs attenuate, directly or indirectly, the enzymatic and transcriptional repressor activities of DNMTs and HDACs. These data provide a mechanistic rationale for the use of epigenetic drugs, individually or in combination, as a potential novel therapeutic strategy to alleviate deficits associated with schizophrenia.

The NF-kappaB pathway plays a pivotal role in proliferation, differentiation, apoptosis, and immune responses in mammals. The NF-kappaB inhibitor, IkappaB, has classically been characterized for its ability to sequester NF-kappaB transcription factors in the cytoplasm. Nevertheless, a nuclear fraction of IkappaBalpha has consistently been detected and associated with repression of nuclear NF-kappaB. Now we show that IkappaBalpha physically associates with different repression elements such as nuclear corepressors and histone acetyltransferases and deacetylases (HDACs). More remarkably, chromatin immunoprecipitation experiments demonstrate that IkappaBalpha is recruited to the promoter regions of the Notch-target gene, hes1, together with HDAC1 and -5, whereas we did not detect IkappaBalpha associated with classical NF-kappaB target genes such as IL6 and RANTES. TNF-alpha treatment results in a temporary release of IkappaBalpha from the hes1 promoter that correlates with increased histone acetylation and transcriptional activation. In addition, we demonstrate that both IkappaB kinase-alpha and -beta are simultaneously recruited to the hes1 promoter in response to TNF-alpha, coinciding with a maximum of IkappaBalpha release and gene activation. Moreover, TNF-alpha-dependent histone H3 acetylation, release of IkappaBalpha from the hes1 promoter, and hes1 mRNA synthesis are affected in IKK-alpha(-/-) mouse embryonic fibroblasts. We propose that IkappaBalpha plays a previously undescribed role in regulating the recruitment of repression elements to specific promoters. Recruitment of IKKs to the nucleus in response to TNF-alpha may induce chromatin-associated IkappaBalpha release and gene activation. These findings provide additional insight in the cross-talk between NF-kappaB and other signaling pathways.

Beta interferon (IFN-beta) gene expression in response to virus infection relies on the dynamic assembly of a multiprotein enhanceosome complex that is initiated by the activation of two inducible transcription factors, interferon regulatory factor 3 (IRF3) and NF-kappaB. Virus or double-stranded RNA-induced activation of IFN-beta gene expression is prevented by the addition of protein deacetylase inhibitors. The isolated IRF-responsive positive regulatory domain was found to require deacetylation for its activity, but IRF3 protein activation leading to its nuclear translocation and DNA binding was not impaired by deacetylase inhibition. In contrast, NF-kappaB activity was not affected by deacetylase inhibitors. RNA interference indicated that several deacetylase enzymes, including histone deacetylase 1 (HDAC1), HDAC8, and HDAC6, influence IFN-beta gene expression with opposing effects. While HDAC1 and HDAC8 repress IFN-beta expression, HDAC6 acts as a coactivator essential for enhancer activity. Virus replication is enhanced in HDAC6-depleted cells, demonstrating HDAC6 is an essential component of innate antiviral immunity.

NF-kappaB activation is required for TNF-alpha-induced transformation of JB6 mouse epidermal cells. Deficient activation of p65 contributes to the lack of NF-kappaB activation in transformation-resistant (P-) cells. We hypothesized that the differential NF-kappaB activation involves differential p65 phosphorylation arising from enzyme activity differences. Here we show that TNF-alpha induces greater ERK-dependent p65 phosphorylation at S536 in transformation sensitive (P+) cells than in P- cells. Our results establish that limited ERK content contributes to a low IkappaB kinase (IKKbeta) level, in turn resulting in insufficient p65 phosphorylation at S536 upon TNF-alpha stimulation in P- cells. Phosphorylation of p65 at S536 appears to play a role in TNF-alpha-induced p65 DNA binding and recruitment of p300 to the p65 complex as well as in release of p65 bound to HDAC1 and 3. Blocking p65 phosphorylation at S536, but not at S276 or S529, abolishes p65 transactivational activity. Over-expression of p65 but not p65 phosphorylation mutant (S536A) in transformation-resistant P- cells renders these cells sensitive to TNF-alpha-induced transformation. Over-expression of p65 phosphorylation mimics p65-S536D or p65-S536E in P- cells and also rescues the transformation response. These findings provide direct evidence that phosphorylation of p65 at S536 is required for TNF-alpha-induced NF-kappaB activation in the JB6 transformation model. The lack of NF-kappaB activation seen in P- cells can be attributed to an insufficient level of p65 phosphorylation on S536 that arises from insufficient IKKbeta that in turn arises from insufficient ERK. Thus, p65 phosphorylation at S536 offers a potential molecular target for cancer prevention.

RUNX3 is a tumor suppressor that is silenced in cancer following hypermethylation of its promoter. The effects of hypoxia in tumor suppressor gene (TSG) transcription are largely unknown. Here, we investigated hypoxia-induced silencing mechanisms of RUNX3. The expression of RUNX3 was downregulated in response to hypoxia in human gastric cancer cells at the transcriptional level. This downregulation was abolished following treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and cytosine methylation inhibitor 5-aza-2-deoxycytidine (5-Aza), suggesting that an epigenetic regulatory mechanism may be involved in RUNX3 silencing by hypoxia. DNA methylation PCR and bisulfite-sequencing data revealed that hypoxia did not affect the methylation of RUNX3 promoter. A chromatin immunoprecipitation (ChIP) assay revealed increased histone H3-lysine 9 dimethylation and decreased H3 acetylation in the RUNX3 promoter following hypoxia. Hypoxia resulted in the upregulation of G9a histone methyltransferase (HMT) and HDAC1; additionally, overexpression of G9a and HDAC1 attenuated RUNX3 expression. The overexpression of G9a and HDAC1, but not their mutants, inhibited the nuclear localization and expression of RUNX3. Diminished mRNA expression and nuclear localization of RUNX3 during hypoxia was abolished by siRNA-mediated knockdown of G9a and HDAC1. This study suggests that hypoxia silences RUNX3 by epigenetic histone regulation during the progression of gastric cancer.

Modification of histones can have a dramatic impact on chromatin structure and function. Acetylation of lysines within the N-terminal tail of the histone octamer marks transcriptionally active regions of the genome whereas deacetylation seems to play a role in transcriptional silencing. Recently, the methylation of the histone tails has also been shown to be important for transcriptional regulation and chromosome structure. Here we show by immunoaffinity purification that two activities important for chromatin-mediated gene silencing, the histone methyltransferase SU(VAR)3-9 and the histone deacetylase HDAC1, associate in vivo. The two activities cooperate to methylate pre-acetylated histones. Both enzymes are modifiers of position effect variegation and interact genetically in flies. We suggest a model in which the concerted histone deacetylation and methylation by a SU(VAR)3-9/HDAC1-containing complex leads to a permanent silencing of transcription in particular areas of the genome.

A subgroup of genes induced by IFN-gamma requires both STAT1 and IRF1 for transcriptional activation. Using WT, stat1(-/-), or irf1(-/-) cells, we analyzed the changes induced by IFN-gamma in gbp2 promoter chromatin. STAT1 associated with the promoter independently of IRF1 and played an essential role in the ordered recruitment of the coactivator/histone acetyl transferase CREB-binding protein (CBP) and the histone deacetylase HDAC1. Hyperacetylation of histone 4 also required STAT1. Phosphorylation at S727 in the transactivating domain increased transcriptional activity of STAT1. In cells expressing a STAT1S727A-mutant CBP recruitment, histone 4 hyperacetylation and RNA polymerase II association with the gbp2 promoter were strongly reduced. IRF1 association with the gbp2 promoter followed that of STAT1, but STAT1 association with DNA or histone hyperacetylation were not necessary for IRF1 binding. RNA polymerase II association with the gbp2 promoter required both STAT1 and IRF1, suggesting that both proteins mediate essential steps in transcriptional activation. IRF1, but not STAT1, was found to coimmunoprecipitate with RNA polymerase II. Together, the data support the assumption that the main role of STAT1 in activating gbp2 transcription is to provide transcriptionally competent chromatin, whereas the function of IRF1 may lie in directly contacting RNA polymerase II-containing transcriptional complexes.

Cells use redox signaling to adapt to oxidative stress. For instance, certain transcription factors exist in a latent state that may be disrupted by oxidative modifications that activate their transcription potential. We hypothesized that DNA-binding sites (response elements) for redox-sensitive transcription factors may also exist in a latent state, maintained by co-repressor complexes containing class I histone deacetylase (HDAC) enzymes, and that HDAC inactivation by oxidative stress may antagonize deacetylase activity and unmask electrophile-response elements, thus activating transcription. Electrophiles suitable to test this hypothesis include reactive carbonyl species, often derived from peroxidation of arachidonic acid. We report that alpha,beta-unsaturated carbonyl compounds, e.g. the cyclopentenone prostaglandin, 15-deoxy-Delta12,14-PGJ(2) (15d-PGJ(2)), and 4-hydroxy-2-nonenal (4HNE), alkylate (carbonylate), a subset of class I HDACs including HDAC1, -2, and -3, but not HDAC8. Covalent modification at two conserved cysteine residues, corresponding to Cys(261) and Cys(273) in HDAC1, coincided with attenuation of histone deacetylase activity, changes in histone H3 and H4 acetylation patterns, derepression of a LEF1.beta-catenin model system, and transcription of HDAC-repressed genes, e.g. heme oxygenase-1 (HO-1), Gadd45, and HSP70. Identification of particular class I HDACs as components of the redox/electrophile-responsive proteome offers a basis for understanding how cells stratify their responses to varying degrees of pathophysiological oxidative stress associated with inflammation, cancer, and metabolic syndrome.

Synaptic scaling is a form of homeostatic synaptic plasticity characterized by cell-wide changes in synaptic strength in response to changes in overall levels of neuronal activity. Here we report that bicuculline-induced increase in neuronal activity leads to a decrease in mEPSC amplitude and a decrease in expression of the AMPA receptor subunit GluR2 in rat hippocampal cultures. Bicuculline treatment also leads to an increase in the levels of the transcriptional repressor MeCP2, which binds to the GluR2 promoter along with the corepressors HDAC1 and mSin3A. Downregulation of MeCP2 by shRNA expression or genetic deletion blocks the bicuculline-induced decrease in GluR2 expression and mEPSC amplitude. These observations indicate that MeCP2 mediates activity-dependent synaptic scaling, and suggest that the pathophysiology of Rett syndrome, which is caused by mutations in MeCP2, may involve defects in activity-dependent regulation of synaptic currents.

Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

The class-I histone deacetylases (HDACs) HDAC1 and HDAC2 belong to a family of 11 zinc-dependent human HDACs and are overexpressed in many cancers. Inhibitors of these HDACs now in clinical trials show activity against several types of cancers. This review is focused on recent advances in both clinical and preclinical efforts to understand the basis for the actions of HDACis, with emphasis on implications for rational combinations with conventional or other targeted agents. We will address new perspectives on the molecular mechanisms by which HDACs act and how these actions relate to cancer. We will also review new evidence showing that HDACs are direct intracellular targets of the potent sphingolipid mediator S1P, the first identified endogenous nuclear regulator of these enzymes, linking sphingolipid metabolism in the nucleus to remodeling of chromatin and epigenetic regulation of gene expression. Understanding how endogenous molecules regulate HDAC activity in vivo may facilitate the search for safer and more effective anticancer drugs capable of interfering with HDAC functions in a highly specific manner.

Expression level of metastasis-associated protein 1 (MTA1) is closely related to tumor growth and metastasis in various cancers. Although increased expression level of MTA1 was observed in hepatocellular carcinoma (HCC), role of MTA1 complex containing histone deacetylase (HDAC) in hepatitis B virus (HBV)-associated hepatocarcinogenesis has not been studied. Here, we demonstrated that HBx strongly induced the expression of MTA1 and HDAC1 genes at transcription level. MTA1 and HDAC1/2 physically associated with hypoxia-inducible factor-1 alpha (HIF-1 alpha) in vivo in the presence of HBx, which was abolished by knockdown of MTA1 by short interfering RNA (siRNA). HBx induced deacetylation of the oxygen-dependent degradation domain of HIF-1 alpha, which was accompanied with dissociation of prolyl hydroxylases and von Hippel-Lindau tumor suppressor from HIF-1 alpha. These results indicate that HBx-induced deacetylation is important for proteasomal degradation of HIF-1 alpha. Further, we observed that protein levels of MTA1 and HDAC1 were increased in the liver of HBx-transgenic mice. Also, there was a higher expression of HDAC1 in HCC than in the adjacent non-tumorous cirrhotic nodules in 10 out of 12 human HBV-associated HCC specimens. Together, our data indicate a positive cross talk between HBx and the MTA1/HDAC complex in stabilizing HIF-1 alpha, which may play a critical role in angiogenesis and metastasis of HBV-associated HCC.

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4(+) T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells.

The SNF2h-containing chromatin-remodeling complex NoRC is responsible for silencing a fraction of mammalian rRNA genes (rDNA). NoRC silences transcription by establishing heterochromatic features-including DNA methylation, hypoacetylation of histone H4, and methylation of H3K9-at the rDNA promoter []. We have investigated the mechanism of NoRC-mediated rDNA silencing and show that binding of the bromodomain of TIP5, the large subunit of NoRC, to acetylated nucleosomes is a prerequisite for NoRC function. A point mutation within the bromodomain impairs the association of NoRC with chromatin, prevents heterochromatin formation, and abolishes transcriptional repression. Moreover, the association of NoRC with chromatin requires acetylation of histone H4 at lysine 16 (acH4K16), and binding to acH4K16 is required for subsequent deacetylation of H4K5, H4K8, and H4K12, indicating that acetylation of H4K16 plays an active role in NoRC-mediated heterochromatin formation. The bromodomain cooperates with an adjacent PHD finger to recruit HDAC1, DNMT1, DNMT3, and SNF2h to rDNA. If specifically targeted to the rDNA promoter, the PHD finger/bromodomain is capable of establishing heterochromatic features and rDNA silencing. Thus, the PHD finger/bromodomain represents an autonomous unit that binds to acH4K16 and coordinates the chain of events that establish the repressed state of rDNA.

The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. The AR acetylation site point mutants showed wild-type trans repression of NF-kappa B, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.