Human maintenance DNA cytosine methyltransferase (DNMT1) regulates gene expression in a methylation-dependent and -independent manner. Anti-apoptotic survivin gene down-regulation is mediated by p53 recruitment of DNMT1 to its promoter. Survivin inhibits programmed cell death, regulates cell division, and is expressed in cancer cells. The survivin gene promoter is CG-rich containing several Sp1 canonical, Sp1-like, cell cycle-dependent element/cell cycle gene homology region, and p53-binding sites. Here we demonstrate that Sp1 transcription factor(s) play a role in transcriptional activation of the survivin promoter in Drosophila and human cells. Sp1 inhibition in vivo by mithramycin A leads to down-regulation of a luciferase reporter driven by the human survivin promoter in transfected cells. Mithramycin A or Sp1-specific short interfering RNA down-regulated the endogenous survivin gene expression, confirming Sp1 as the primary determinant for transcriptional activation. Furthermore, immobilized DNMT1 ligand bound to seven consensus amino acids corresponding to the N-terminal region of the Sp class of transcription factors in a phage display analysis. In the co-immunoprecipitation assay, the endogenous Sp1 or Sp3 pulled down DNMT1 and methyltransferase activity. Similarly, a glutathione S-transferase pulldown assay between DNMT1 and Sp1 demonstrates a direct interaction between the two proteins. Fluorescent fusions of DNMT1 and Sp1 co-localized in the mammalian nucleus, thus supporting binary complex formation between both the proteins. The kinetics of survivin promoter occupancy via chromatin immunoprecipitation following doxorubicin treatment show the presence of Sp1 and gradual accumulation of transcriptional repressors p53, DNMT1, histone methyltransferase G9a, and HDAC1 onto the promoter along with histone H3K9me2. These data suggest that the Sp1 transcription factor acts as a platform for recruitment of transcriptional repressors.

The (8;21) translocation, found in 12% of acute myeloid leukemia (AML), creates the chimeric fusion product, AML1-ETO. Previously, we demonstrated that the ETO moiety recruits a transcription repression complex that includes the histone deacetylase (HDAC1) enzyme. Here, we used inhibitors of HDAC1 to study the pathophysiology of AML1-ETO. Both the potent inhibitor, trichostatin (TSA), and the well-known but less specific inhibitor, phenylbutyrate (PB), could partially reverse ETO-mediated transcriptional repression. PB was also able to induce partial differentiation of the AML1-ETO cell line, Kasumi-1. With the intention of developing a clinically useful protocol, we combined PB with a number of other agents that induced differentiation and apoptosis of Kasumi-1 cells. In summary, transcriptional repression mediated by AML1-ETO appears to play a mechanistic role in the t(8;21) AML, and relief of repression using agents such as PB (alone or in combination) may prove to be therapeutically useful.

Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC) family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC). However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs) interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA) targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS) to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues. Here we show that E2F-Rb family complexes participate in L1 elements epigenetic regulation via nucleosomal histone modifications and recruitment of histone deacetylases (HDACs) HDAC1 and HDAC2. Our experiments demonstrated that (i) Rb and E2F interact with human and mouse L1 elements, (ii) L1 elements are deficient in both heterochromatin-associated histone marks H3 tri methyl K9 and H4 tri methyl K20 in Rb family triple knock out (Rb, p107, and p130) fibroblasts (TKO), (iii) L1 promoter exhibits increased histone H3 acetylation in the absence of HDAC1 and HDAC2 recruitment, (iv) L1 expression in TKO fibroblasts is upregulated compared to wild type counterparts, (v) L1 expression increases in the presence of the HDAC inhibitor TSA. On the basis of these findings we propose a model in which L1 sequences throughout the genome serve as centers for heterochromatin formation in an Rb family-dependent manner. As such, Rb proteins and L1 elements may play key roles in heterochromatin formation beyond pericentromeric chromosomal regions. These findings describe a novel mechanism of L1 reactivation in mammalian cells mediated by failure of corepressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation.

Epigenetic control of liver proliferation involves cooperation between transcription factors and chromatin-remodeling proteins. In this work, we found that the levels of HDAC1 (histone deacetylase 1) are increased in quiescent livers of old mice. The elevation of HDAC1 in liver is mediated by the RNA-binding protein CUGBP1. We found that the age-associated CUGBP1-eIF2 complex binds to the 5' region of HDAC1 mRNA and increases translation of HDAC1 in the liver. Further analyses showed that CUGBP1 also increases expression of HDAC1 in cultured cells, in the livers of CUGBP1 transgenic mice, and in the livers of mice injected with cyclin D3, which enhances the formation of the CUGBP1-eIF2 complex. In livers of old mice, HDAC1 interacts with the transcription factor C/EBPalpha and is recruited by this protein to E2F-dependent promoters as a component of high M(r) C/EBPalpha-Brm complexes. The recruitment of HDAC1 to c-Myc and FoxM1B promoters leads to deacetylation of histone H3 at Lys-9 on these E2F-dependent promoters. We show that HDAC1 is an important mediator of growth-inhibitory activity of C/EBPalpha and that small interfering RNA-mediated inhibition of HDAC1 reduces the ability of C/EBPalpha to inhibit cell proliferation. In addition, we have found that both elevation of HDAC1 and interaction of C/EBPalpha with HDAC1 are controlled by cyclin D3-dependent mechanisms. Treatment of old mice with growth hormone, which reduces cyclin D3 levels, leads to the reduction of the CUGBP1-eIF2 complex, normalization of HDAC1 levels, and inhibition of interactions of HDAC1 with C/EBPalpha-Brm complexes. Thus, our data demonstrate that translational elevation of HDAC1 in livers of old mice is involved in the assembly of high M(r) protein-protein complexes that inhibit liver proliferation.

Phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B (NF-kappaB) signaling pathways play a critical role in mediating survival signals. In this study we have investigated how loss of dystrophin (the primary cause of Duchenne muscular dystrophy) modulates the activation of PI3K/Akt and NF-kappaB signaling pathways in skeletal muscle in response to mechanical stimulation. Activation of Akt was significantly higher in diaphragm muscle from dystrophin-deficient mdx mice compared to normal mice at both prenecrotic and necrotic states. Higher activation of Akt was also observed in cultured dystrophin-deficient primary myotubes differentiated in vitro. Application of passive mechanical stretch ex vivo synergistically increased the activation of Akt in diaphragm of mdx mice. Stretch-induced activation of PDK-1 and PI3K were also higher in diaphragm of mdx mice compared to normal mice. Pretreatment of diaphragm with PI3K inhibitor LY294002 blocked the activation of Akt in normal and mdx mice. Higher activation of Akt was associated with increased phosphorylation of its downstream targets glycogen synthase kinase 3beta (GSK3beta), FKHR, and mammalian target of rapamycin (mTOR). Treatment of diaphragm muscle with LY294002 inhibited the stretch-induced activation of IkappaB (IkappaB) kinase (IKK) and NF-kappaB transcription factor in normal and mdx mice. Mechanical stretch also reduced the interaction of HDAC1 with RelA subunit of NF-kappaB in diaphragm muscle. Finally, cellular levels of Bcl-2, cIAP1, and integrin beta1 and activation of integrin linked kinase were higher in diaphragm muscle of mdx mice compared to normal mice. Taken together, our data suggest that loss of dystrophin and/or mechanical stretch results in the up-regulation of P13K/Akt and NF-kappaB signaling pathways in skeletal muscle.

Histone deacetylases (HDACs) are epigenetic erasers of lysine-acetyl marks. Inhibition of HDACs using small molecule inhibitors (HDACi) is a potential strategy in the treatment of various diseases and is approved for treating hematological malignancies. Harnessing the therapeutic potential of HDACi requires knowledge of HDAC-function in vivo. Here, we generated a thymocyte-specific gradient of HDAC-activity using compound conditional knockout mice for Hdac1 and Hdac2. Unexpectedly, gradual loss of HDAC-activity engendered a dosage-dependent accumulation of immature thymocytes and correlated with the incidence and latency of monoclonal lymphoblastic thymic lymphomas. Strikingly, complete ablation of Hdac1 and Hdac2 abrogated lymphomagenesis due to a block in early thymic development. Genomic, biochemical and functional analyses of pre-leukemic thymocytes and tumors revealed a critical role for Hdac1/Hdac2-governed HDAC-activity in regulating a p53-dependent barrier to constrain Myc-overexpressing thymocytes from progressing into lymphomas by regulating Myc-collaborating genes. One Myc-collaborating and p53-suppressing gene, Jdp2, was derepressed in an Hdac1/2-dependent manner and critical for the survival of Jdp2-overexpressing lymphoma cells. Although reduced HDAC-activity facilitates oncogenic transformation in normal cells, resulting tumor cells remain highly dependent on HDAC-activity, indicating that a critical level of Hdac1 and Hdac2 governed HDAC-activity is required for tumor maintenance.

Histone deacetylases (HDACs) are associated with the development and progression of cancer, but it is not known which of the HDAC isoforms play important roles in breast cancer metastasis. This study identified the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) compared to MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 cells by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.

Though the linkages between germline mutations of BRCA1 and hereditary breast cancer are well known, recent evidence suggests that altered BRCA1 transcription may also contribute to sporadic forms of breast cancer. Here we show that BRCA1 expression is controlled by a dynamic equilibrium between transcriptional coactivators and co-repressors that govern histone acetylation and DNA accessibility at the BRCA1 promoter. Eviction of the transcriptional co-repressor and metabolic sensor, C terminal-binding protein (CtBP), has a central role in this regulation. Loss of CtBP from the BRCA1 promoter through estrogen induction, depletion by RNA interference or increased NAD+/NADH ratio leads to HDAC1 dismissal, elevated histone acetylation and increased BRCA1 transcription. The active control of chromatin marks, DNA accessibility and gene expression at the BRCA1 promoter by this 'metabolic switch' provides an important molecular link between caloric intake and tumor suppressor expression in mammary cells.

CREST plays a critical role in activity-dependent development, but its mechanism of action is not well understood. Here, we show that a CREST-BRG1 complex regulates promoter activation by orchestrating a calcium-dependent release of a repressor complex and a recruitment of an activator complex. In resting neurons, transcription of the c-fos promoter is inhibited by BRG1-dependent recruitment of a phospho-Rb-HDAC repressor complex. Upon calcium influx, Rb becomes dephosphorylated at serine 795 by calcineurin, which leads to release of the repressor complex. At the same time, there is increased recruitment of CBP to the promoter by a CREST-dependent mechanism, which leads to transcriptional activation. The CREST-BRG1 also binds to the NR2B promoter, and activity-dependent induction of NR2B expression involves a release of HDAC1 and recruitment of CBP, suggesting that this mechanism may be generally involved in regulating calcium-dependent transcription of neuronal genes.

BACKGROUND

Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.

RESULTS

We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.

CONCLUSIONS

These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.

Long noncoding RNA H19 is repressed after birth, but can be induced by hypoxia. We aim to investigate the impact on and underlying mechanism of H19 induction after ischemic stroke.

Circulating H19 levels in stroke patients and mice subjected to middle cerebral artery occlusion were assessed using real-time polymerase chain reaction. H19 siRNA and histone deacetylase 1 (HDAC1) plasmid were used to knock down H19 and overexpress HDAC1, respectively. Microglial polarization and ischemic outcomes were assessed in middle cerebral artery occlusion mice and BV2 microglial cells subjected to oxygen-glucose deprivation.

Circulating H19 levels were significantly higher in stroke patients compared with healthy controls, indicating high diagnostic sensitivity and specificity. Moreover, plasma H19 levels showed a positive correlation with National Institute of Health Stroke Scale score and tumor necrosis factor-α levels. After middle cerebral artery occlusion in mice, H19 levels increased in plasma, white blood cells, and brain. Intracerebroventricular injection of H19 siRNA reduced infarct volume and brain edema, decreased tumor necrosis factor-α and interleukin-1β levels in brain tissue and plasma, and increased plasma interleukin-10 concentrations 24 hours poststroke. Additionally, H19 knockdown attenuated brain tissue loss and neurological deficits 14 days poststroke. BV2 cell-based experiments showed that H19 knockdown blocked oxygen-glucose deprivation-driven M1 microglial polarization, decreased production of tumor necrosis factor-α and CD11b, and increased the expression of Arg-1 and CD206. Furthermore, H19 knockdown reversed oxygen-glucose deprivation-induced upregulation of HDAC1 and downregulation of acetyl-histone H3 and acetyl-histone H4. In contrast, HDAC1 overexpression negated the effects of H19 knockdown.

Our findings indicate that H19 promotes neuroinflammation by driving HDAC1-dependent M1 microglial polarization, suggesting a novel H19-based diagnosis and therapy for ischemic stroke.

The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.

The ATM gene is mutated in individuals with ataxia telangiectasia, a human genetic disease characterized by extreme sensitivity to radiation. The ATM protein acts as a sensor of radiation-induced cellular damage and contributes to cell cycle regulation, signal transduction, and DNA repair; however, the mechanisms underlying these functions of ATM remain largely unknown. Binding and immunoprecipitation assays have now shown that ATM interacts with the histone deacetylase HDAC1 both in vitro and in vivo, and that the extent of this association is increased after exposure of MRC5CV1 human fibroblasts to ionizing radiation. Histone deacetylase activity was also detected in immunoprecipitates prepared from these cells with antibodies to ATM, and this activity was blocked by the histone deacetylase inhibitor trichostatin A. These results suggest a previously unanticipated role for ATM in the modification of chromatin components in response to ionizing radiation.

OBJECTIVE

Aberrant histone acetylation has been associated with malignancy and histone deacetylase (HDAC) inhibitors are currently being investigated in numerous clinical trials. So far, the malignancy most sensitive to HDAC inhibitors has been cutaneous T-cell lymphoma (CTCL). The reason for this sensitivity is unclear and studies on HDAC expression and histone acetylation in CTCL are lacking. The aim of this study was to address this issue.

RESULTS

The immunohistochemical expression of HDAC1, HDAC2, HDAC6, and acetylated H4 was examined in 73 CTCLs and the results related to histological subtypes and overall survival. HDAC1 was most abundantly expressed (P < 0.0001), followed by HDAC2; HDAC6 and H4 acetylation were equally expressed. HDAC2 (P = 0.001) and H4 acetylation (P = 0.03) were significantly more common in aggressive than indolent CTCL subtypes. In contrast, no differences were observed for HDAC1 and HDAC6. In a Cox analysis, elevated HDAC6 was the only parameter showing significant influence on survival (P = 0.04).

CONCLUSIONS

High expression of HDAC2 and acetylated H4 is more common in aggressive than indolent CTCL. HDAC6 expression is associated with a favorable outcome independent of the subtype.

Histone deacetylase 1 (HDAC1) is a major regulator of chromatin structure and gene expression. Tight control of HDAC1 expression is essential for normal cell cycle progression of mammalian cells. HDAC1 mRNA levels are regulated by growth factors and by changes in intracellular deacetylase activity levels. Stimulation of the mitogen-activated protein kinase cascade by anisomycin or growth factors, together with inhibition of deacetylases by trichostatin A (TSA), leads to stable histone H3 phosphoacetylation and strongly induced HDAC1 expression. In contrast, activation of the nucleosomal response by anisomycin alone results only in transient phosphoacetylation of histone H3 without affecting HDAC1 mRNA levels. The transcriptional induction of the HDAC1 gene by anisomycin and TSA is efficiently blocked by H89, an inhibitor of the nucleosomal response. Detailed studies of the kinetics of histone acetylation and phosphorylation show that the two modifications are synergistic and essential for induced HDAC1 transcription. Activation of the HDAC1 gene by anisomycin together with TSA or by growth factors is accompanied by phosphoacetylation of HDAC1 promoter-associated histone H3. Our results present evidence for a precise regulatory mechanism which allows induction of the HDAC1 gene in response to proliferation signals and modulation of HDAC1 expression dependent on intracellular deacetylase levels.

BACKGROUND

Epigenetic regulation of gene expression is under normal circumstances tightly controlled by the specific methylation of cytosine residues in CpG dinucleotides and coordinated by adjustments in the histone-dependent configuration of chromatin. Following our original report, providing the first description of potential tumor suppressor function associated with the histone methyltransferase SET domain containing 2 (SETD2) in breast cancer, the objective of this study was to determine the expression profiles of 16 further histone-modifier genes in a well annotated cohort of patients with primary operable breast cancer.

METHODS

Breast cancer tissues (n=127) and normal tissues (n=33) underwent RNA extraction and reverse transcription, and histone-modifier gene transcript levels were determined using real-time quantitative PCR. The histone-modifier genes included: histone acetyltransferases (cAMP response element-binding protein-binding protein (CREBBP)); class I (histone deacetylase 1 (HDAC1) and histone deacetylase 2 (HDAC2)), II (histone deacetylase 5 (HDAC5)) and III (sirtuin 1 (SIRT1)) histone deacetylases; and histone methyltransferases (SET domain containing suppressor of variegation 3-9 homolog 1 (SUV39H1) and suppressor of variegation 3-9 homolog 2 (SUV39H2)) amongst others. Expression levels were analysed against tumor size, grade, nodal involvement, histological subtype, receptor status, TNM stage, Nottingham Prognostic Index, and disease-free and overall survival over a 10-year follow-up period.

RESULTS

Expression of histone-modifier genes in breast cancer differed significantly from those in normal tissue (HDAC5, HDAC1, lysine (K)-specific demethylase 4A (KDM4A) and lysine (K)-specific demethylase 6A (KDM6A)). Differences in expression profiles were also found to exist between individual breast tumors and, in some cases, were significantly associated with conventional pathological parameters and prognostic indices: tumor grade (K (lysine) acetyltransferase 5 (KAT5), HDAC1, KDM4A, SUV39H1 and KDM6A)); TNM stage (SUV39H1, K (lysine) acetyltransferase 2B (KAT2B), lysine (K)-specific demethylase 1A (KDM1A), KDM4A, lysine (K)-specific demethylase 5C (KDM5C), K (lysine) acetyltransferase 8 (KAT8), HDAC5 and KAT5)); Nottingham Prognostic Index (KDM5C, myeloid/lymphoid or mixed-lineage leukemia (MLL), KAT8 and SET and MYND domain containing 3 (SMYD3)); receptor status (KAT5, SMYD3 and KDM1A); histological type (KAT5, KDM5C, KAT8, KDM4A and MLL); disease-free survival (SUV39H1, SMYD3, HDAC5, KDM6A, HDAC1, KDM1A, KDM4A, KAT8, KDM5C, KAT5 and MLL) and overall survival (KAT8). Significant correlations were identified between the differential expression profiles of particular histone-modifying genes.

CONCLUSIONS

Expression levels of histone-modifier genes in breast cancer differ significantly from normal tissue. Differences in expression profiles exist between breast tumors and are significantly associated with conventional pathological parameters and clinical outcomes. Further study is warranted to determine the consequences of altered expression for each specific histone-modifier gene and the biological and clinical implications of combinatorial variations in expression profiles. Histone-modifier enzymes offer utility as biomarkers and potential for targeted therapeutic strategies.

The aryl hydrocarbon receptor (AHR) is the ligand-activated transcription factor responsible for mediating the toxicological effects of dioxin and xenobiotic metabolism. However, recent evidence has implicated the AHR in additional, nonmetabolic physiological processes, including immune regulation. Certain tumor cells are largely nonresponsive to cytokine-mediated induction of the pro-survival cytokine interleukin (IL) 6. We have demonstrated that multiple nonresponsive tumor lines are able to undergo synergistic induction of IL6 following combinatorial treatment with IL1beta and the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin. Such data implicate the AHR in tumor expansion, although the mechanistic basis for the AHR-dependent synergistic induction of IL6 has not been determined. Here, we demonstrate that ligand-activated AHR is involved in priming the IL6 promoter through binding to nonconsensus dioxin response elements located upstream of the IL6 start site. Such binding appears to render the promoter more permissive to IL1beta-induced binding of NF-kappaB components. The nature of the AHR-dependent increases in IL6 promoter transcriptional potential has been shown to involve a reorganization of repressive complexes as exemplified by the presence of HDAC1 and HDAC3. Dismissal of these HDACs correlates with post-translational modifications of promoter-bound NF-kappaB components in a time-dependent manner. Thus the AHR plays a role in derepressing the IL6 promoter, leading to synergistic IL6 expression in the presence of inflammatory signals. These observations may explain the association between enhanced expression of AHR and tumor aggressiveness. It is likely that AHR-mediated priming is not restricted to the IL6 promoter and may contribute to the expression of a variety of genes, which do not have consensus dioxin response elements.

Autophagy is a process where cytoplasmic materials are degraded by lysosomal machinery. Histone deacetylase (HDAC) inhibitors induce autophagy, and HDAC6, one of class II HDAC isotypes, is directly involved in autophagic degradation in the cell. However, it is unclear if class I HDAC isotype such as HDCA1 is involved in this process. To investigate if class I HDAC isotype is involved in autophagy, a specific class I HDAC inhibitor and an siRNA of HDAC1 were used to treat HeLa cells. Autophagic markers were then investigated. Both inhibition and genetic knock-down of HDAC1 in the cells significantly induced autophagic vacuole formation and lysosome function. Moreover, disruption of HDAC1 leads to the conversion of LC3-I to LC3-II. Together, these results demonstrate that HDAC1 could play a role in autophagy and specific inhibition of HDAC1 can induce autophagy.

Histone deacetylase (HDAC) inhibitors are emerging as effective therapies in the treatment of cancer, and the role of HDACs in the regulation of promoters is rapidly expanding. GRP78/BiP is a stress inducible endoplasmic reticulum (ER) chaperone with antiapoptotic properties. We present here the mechanism for repression of the Grp78 promoter by HDAC1. Our studies reveal that HDAC inhibitors specifically induce GRP78, and the induction level is amplified by ER stress. Through mutational analysis, we have identified the minimal Grp78 promoter and specific elements responsible for HDAC-mediated repression. We show the involvement of HDAC1 in the negative regulation of the Grp78 promoter not only by its induction in the presence of the HDAC inhibitors trichostatin A and MS-275 but also by exogenous overexpression and small interfering RNA knockdown of specific HDACs. We present the results of chromatin immunoprecipitation analysis that reveals the binding of HDAC1 to the Grp78 promoter before, but not after, ER stress. Furthermore, overexpression of GRP78 confers resistance to HDAC inhibitor-induced apoptosis in cancer cells, and conversely, suppression of GRP78 sensitizes them to HDAC inhibitors. These results define HDAC inhibitors as new agents that up-regulate GRP78 without concomitantly inducing the ER or heat shock stress response, and suppression of GRP78 in tumors may provide a novel, adjunctive option to enhance anticancer therapies that use these compounds.

Preadipocyte differentiation in culture is driven by an insulin and cAMP dependant transcriptional cascade which induces the bzip transcription factors C/EBPbeta and C/EBPdelta. We have previously shown that glucocorticoid treatment, which strongly potentiates this differentiation pathway, stimulates the titration of the corepressor histone deacetylase 1 (HDAC1) from C/EBPbeta. This results in a dramatic enhancement of C/EBPbeta-dependent transcription from the C/EBPalpha promoter, concomitant with potentiation of preadipocyte differentiation. Here, we show that C/EBPbeta is acetylated by GCN5 and PCAF within a cluster of lysine residues between amino acids 98-102 and that this acetylation is strongly induced by glucocorticoid treatment. Arginine substitution of the lysine residues within the acetylation motif of C/EBPbeta prevented acetylation and blocked the ability of glucocorticoids to enhance C/EBPbeta-directed transcription and to potentiate C/EBPbeta-dependent preadipocyte differentiation. Moreover, acetylation of C/EBPbeta appeared to directly interfere with the interaction of HDAC1 with C/EBPbeta, suggesting that PCAF/GCN5-dependent acetylation of C/EBPbeta serves as an important molecular switch in determining the transcriptional regulatory potential of this transcription factor.

The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation and is implicated in carcinogenesis. It contains multiple conserved chromatin-associated domains, including three PHD fingers of unknown function. Here, we show that the first and third, but not the second, PHD fingers of KDM5B possess histone binding activities. The PHD1 finger is highly specific for unmodified histone H3 (H3K4me0), whereas the PHD3 finger shows preference for the trimethylated histone mark H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for genes involved in inflammatory responses, cell proliferation, adhesion, and migration. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with the histone deacetylase 1 (HDAC1) in gene repression. KDM5B is downregulated in triple-negative breast cancer relative to estrogen-receptor-positive breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion, and the PHD1-H3K4me0 interaction is essential for inhibiting migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a multivalent mechanism for KDM5B-mediated transcriptional regulation.

The expression of the NKG2D ligands on cancer cells leads to their recognition and elimination by host immune responses mediated by natural killer and T cells. UL16-binding proteins (ULBPs) are NKG2D ligands, which are scarcely expressed in epithelial tumours, favouring their evasion from the immune system. Herein, we investigated the epigenetic mechanisms underlying the repression of ULBPs in epithelial cancer cells. We show that ULBP1-3 expression is increased in tumour cells after exposure to the inhibitor of histone deacetylases (HDACs) trichostatin A (TSA), which enhances the natural killer cell-mediated cytotoxicity of HeLa cells. Our experiments showed that the transcription factor Sp3 is crucial in the activation of the ULBP1 promoter by TSA. Furthermore, by small interfering RNA-mediated knockdown and overexpression of HDAC1-3, we showed that HDAC3 is a repressor of ULBPs expression in epithelial cancer cells. Remarkably, TSA treatment caused the complete release of HDAC3 from the ULBP1-3 promoters. HDAC3 is recruited to the ULBP1 promoter through its interaction with Sp3 and TSA treatment interfered with this association. Together, we describe a new mechanism by which cancer cells may evade the immune response through the epigenetic modulation of the ULBPs expression and provide a model in which HDAC inhibitors may favour the elimination of transformed cells by increasing the immunogenicity of epithelial tumours.

Ataxia telangiectasia mutated (ATM)- and Rad3-related protein (ATR) is a phosphatidylinositol-kinase (PIK)-related kinase that has been implicated in the response of human cells to multiple forms of DNA damage and may play a role in the DNA replication checkpoint. The purification of an ATR complex allowed identification of chromodomain-helicase-DNA-binding protein 4 (CHD4) as an ATR-associated protein by tandem mass spectrometric sequencing. CHD4 (also called Mi-2beta) is a component of a histone-deacetylase-2 (HDAC2)-containing complex, the nucleosome remodeling and deacetylating (NRD) complex. Endogenous ATR, CHD4, and HDAC2 are shown to coimmunoprecipitate, and ATR and HDAC2 coelute through two biochemical purification steps. Other members of the NRD complex, HDAC1, MTA1, and MTA2, are also detectable in ATR immunoprecipitates. ATR's association with CHD4 and HDAC2 suggests that there may be a linkage between ATR's role in mediating checkpoints induced by DNA damage and chromatin modulation via remodeling and deacetylation.