Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic components of corepressor complexes that modulate gene expression. In most cell types, deletion of both Hdac1 and Hdac2 is required to generate a discernible phenotype, suggesting their activity is largely redundant. We have therefore generated an ES cell line in which Hdac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon cell cycle progression, because differentiated, nonproliferating cells retain their viability. Furthermore, we observe increased mitotic defects, chromatin bridges, and micronuclei, suggesting HDAC1/2 are necessary for accurate chromosome segregation. Consistent with a critical role in the regulation of gene expression, microarray analysis of Hdac1/2-deleted cells reveals 1,708 differentially expressed genes. Significantly for the maintenance of stem cell self-renewal, we detected a reduction in the expression of the pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is regulated through binding of an inositol tetraphosphate molecule (IP4) sandwiched between the HDAC and its cognate corepressor. This raises the important question of whether IP4 regulates the activity of the complex in cells. By rescuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. Our data indicate that HDAC1/2 have essential and pleiotropic roles in cellular proliferation and regulate stem cell self-renewal by maintaining expression of key pluripotent transcription factors.

The roles of long non-coding RNAs (lncRNAs) in regulating cancer and stem cells are being increasingly appreciated. Its diverse mechanisms provide the regulatory network with a bigger repertoire to increase complexity. Here we report a novel LncRNA, Lnc34a, that is enriched in colon cancer stem cells (CCSCs) and initiates asymmetric division by directly targeting the microRNA miR-34a to cause its spatial imbalance. Lnc34a recruits Dnmt3a via PHB2 and HDAC1 to methylate and deacetylate the miR-34a promoter simultaneously, hence epigenetically silencing miR-34a expression independent of its upstream regulator, p53. Lnc34a levels affect CCSC self-renewal and colorectal cancer (CRC) growth in xenograft models. Lnc34a is upregulated in late-stage CRCs, contributing to epigenetic miR-34a silencing and CRC proliferation. The fact that lncRNA targets microRNA highlights the regulatory complexity of non-coding RNAs (ncRNAs), which occupy the bulk of the genome.

Histone modifications in chromatin regulate gene expression. A transcriptional co-repressor complex containing LSD1-CoREST-HDAC1 (termed LCH hereafter for simplicity) represses transcription by coordinately removing histone modifications associated with transcriptional activation. RE1-silencing transcription factor (REST) recruits LCH to the promoters of neuron-specific genes, thereby silencing their transcription in non-neuronal tissues. ZNF198 is a member of a family of MYM-type zinc finger proteins that associate with LCH. Here, we show that ZNF198-like proteins are required for the repression of E-cadherin (a gene known to be repressed by LSD1), but not REST-responsive genes. ZNF198 binds preferentially to the intact LCH ternary complex, but not its individual subunits. ZNF198- and REST-binding to the LCH complex are mutually exclusive. ZNF198 associates with chromatin independently of LCH. Furthermore, modification of HDAC1 by small ubiquitin-like modifier (SUMO) in vitro weakens its interaction with CoREST whereas sumoylation of HDAC1 stimulates its binding to ZNF198. Finally, we mapped the LCH- and HDAC1-SUMO-binding domains of ZNF198 to tandem repeats of MYM-type zinc fingers. Therefore, our results suggest that ZNF198, through its multiple protein-protein interaction interfaces, helps to maintain the intact LCH complex on specific, non-REST-responsive promoters and may also prevent SUMO-dependent dissociation of HDAC1.

This study explored the effect of MS-275, a novel histone deacetylase inhibitor (HDACI), against a variety of human leukemia cells with defined genetic alterations. MS-275 profoundly induced growth arrest of acute myelogenous leukemia (AML) MOLM13 and biphenotypic leukemia MV4-11 cells, which possess internal tandem duplication mutation in the fms-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD), with IC50s less than 1 microM, as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay on day two of culture. Exposure of these cells to MS-275 decreased levels of total, as well as, phosphorylated forms of FLT3, resulting in inactivation of its downstream signal pathways, including Akt, ERK, and STAT5. Further studies found that MS-275 induced acetylation of heat shock protein 90 (HSP90) in conjunction with ubiquitination of FLT3, leading to degradation of FLT3 proteins in these cells. This was blunted by treatment with the proteasome inhibitor bortezomib, confirming that FLT was degraded via ubiquitin/proteasome pathway. Moreover, we found that further inhibition of MEK/ERK signaling potentiated the action of MS-275 in leukemia cells. Taken together, MS-275 may be useful for treatment of individuals with leukemia possessing activating mutation of FLT3 gene.

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.

Epigenetic regulation mediated by lysine- and arginine-specific enzymes plays an essential role in tumorigenesis, and enhanced expression of the type II protein arginine methyltransferase PRMT5 as well as the polycomb repressor complex PRC2 has been associated with increased cell proliferation and survival. Here, we show that PRMT5 is overexpressed in three different types of non-Hodgkin lymphoma cell lines and clinical samples as well as in mouse primary lymphoma cells and that it up-regulates PRC2 expression through inactivation of the retinoblastoma proteins RB1 and RBL2. Although PRMT5 epigenetically controls RBL2 expression, it indirectly promotes RB1 phosphorylation through enhanced cyclin D1 expression. Furthermore, we demonstrate that PRMT5 knockdown in non-Hodgkin lymphoma cell lines and mouse primary lymphoma cells leads to RBL2 derepression and RB1 reactivation, which in turn inhibit PRC2 expression and trigger derepression of its CASP10, DAP1, HOXA5, and HRK pro-apoptotic target genes. We also show that reduced PRMT5 expression leads to cyclin D1 transcriptional repression via loss of TP53K372 methylation, which results in decreased BCL3 expression and enhanced recruitment of NF-κB p52-HDAC1 repressor complexes to the cyclin D1 promoter. These findings indicate that PRMT5 is a master epigenetic regulator that governs expression of its own target genes and those regulated by PRC2 and that its inhibition could offer a promising therapeutic strategy for lymphoma patients.

Expression of the NMDA receptor 2B (NR2B) gene is upregulated following chronic intermittent ethanol (CIE) treatment and withdrawal, which underlies behavioral alterations in addiction. The goal of this study was to characterize the changes of histone modifications induced by CIE treatment and its subsequent removal associated to the upregulation of NR2B gene transcription. To investigate the involvement of histone acetylation in the effect of ethanol on the NR2B gene, we examined the influence of CIE on histone acetylation in the 5' regulatory region of NR2B using a qChIP assay. CIE treatment and its subsequent removal produced a remarkable and selected increase in histone H3K9 acetylation. Interestingly, the majority of the increased H3K9 acetylation occurred after ethanol removal, which was coincident with a decrease in H3K9 methylation in the same time duration. Further examination of the mechanisms of ethanol-induced alterations on the histone modifications revealed that CIE-induced acetylation of H3K9 was not due to the changes in global enzyme activities or the expression of histone acetyltransferases (HATs) and deacetylase (HDACs). Instead, we found a significant downregulation in some histone methyltransferases (HMTs) at both the global level and the local chromatin of the NR2B gene following CIE treatment. Moreover, our experiments also indicated a decrease of G9a, Suv39 h1 and HDAC1-3 in the chromatin of the NR2B gene promoter, which may be responsible for the altered H3K9 modifications. Taken together, the findings suggest a mechanism where the changes in H3K9 modifications in the local chromatin of the NR2B gene underlie alcohol-induced neuroadaptation.

DEC1/STRA13 is a bHLH type transcriptional regulator involved with immune regulation, hypoxia response and carcinogenesis. We recently demonstrated that STRA13 interacts with STAT3 in the transcriptional activation of STAT-dependent promoters. Here, we pursue STRA13 involvement in the JAK/STAT pathway by studying its role in STAT1 expression. First, we showed that VHL deficiency or HIF-1 activation resulted in the repression of endogenous STAT1 mediated by STRA13. We then characterized the STAT1 proximal promoter to assess its response to STRA13 by transient coexpression in a luciferase reporter assay. Using sequential truncation and site-directed mutagenesis of the STAT1 promoter with STRA13 deletion constructs, we showed that the STRA13 C-terminal trans-activation domain, which is known to bind HDAC1, mostly determines the repressive activity. Involvement of HDAC activity in STAT1 regulation was validated by TSA inhibition and chromatin immunoprecipitation (ChIP) assay. Thus, we demonstrate that STRA13-mediated repression of STAT1 transcription utilizes an HDAC1-dependent mechanism. Furthermore, we show that targets of unphosphorylated STAT1, such as antigen presenting genes and CASP1, are also repressed by hypoxia possibly through the same STRA13-mediated mechanism. Thus, the newly discovered link between HIF-1 and STAT1 reveals a previously unknown role of STRA13 in hypoxia and carcinogenesis.

The translocation t(15;17) generates the chimeric PML-RARalpha transcription factor that is the initiating event of acute promyelocytic leukemia. A global view of PML-RARalpha transcriptional functions was obtained by genome-wide binding and chromatin modification analyses combined with genome-wide expression data. Chromatin immunoprecipitation (ChIP)-chip experiments identified 372 direct genomic PML-RARalpha targets. A subset of these was confirmed in primary acute promyelocytic leukemia. Direct PML-RARalpha targets include regulators of global transcriptional programs as well as critical regulatory genes for basic cellular functions such as cell-cycle control and apoptosis. PML-RARalpha binding universally led to HDAC1 recruitment, loss of histone H3 acetylation, increased tri-methylation of histone H3 lysine 9, and unexpectedly increased trimethylation of histone H3 lysine 4. The binding of PML-RARalpha to target promoters and the resulting histone modifications resulted in mRNA repression of functionally relevant genes. Taken together, our results reveal that the transcription factor PML-RARalpha regulates key cancer-related genes and pathways by inducing a repressed chromatin formation on its direct genomic target genes.

From work done largely on derived cell lines, it has been suggested that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might be a therapeutic target for many forms of malignancy. However, use of primary tumor cells, including chronic lymphocytic leukemic (CLL) cells, has shown inherent resistance to TRAIL. Although the molecular basis for this resistance remains unknown, treatment with histone deacetylase inhibitors (HDACi) often sensitizes resistant cells to TRAIL-induced apoptosis. We used structurally diverse HDACi to ascertain which HDAC needs to be inhibited for the sensitization. Inhibition of HDAC class I but not class II is required for sensitization to TRAIL-induced apoptosis of CLL cells and various cell lines. Using different HDACi together with small interfering RNA for HDAC1, HDAC2, HDAC3, and HDAC6, we report that inhibition of HDAC1 and HDAC2 but not HDAC3, HDAC6, and HDAC8 are primarily responsible for sensitization to TRAIL-induced apoptosis. Based on these data and our previous studies, we propose that a clinical trial in CLL is warranted using a combination of a selective HDACi that inhibits HDAC1 and/or HDAC2 together with a form of TRAIL that signals through TRAIL receptor 1.

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes is regulated by the interplay between extrinsic signals and intrinsic epigenetic determinants. In this study, we analyze the effect that the extracellular ligands sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4), have on histone acetylation and gene expression in cultured OPCs. Shh treatment favored the progression toward oligodendrocytes by decreasing histone acetylation and inducing peripheral chromatin condensation. BMP4 treatment, in contrast, inhibited the progression toward oligodendrocytes and favored astrogliogenesis by favoring global histone acetylation and retaining euchromatin. Pharmacological treatment or silencing of histone deacetylase 1 (Hdac1) or histone deacetylase 2 (Hdac2) in OPCs did not affect BMP4-dependent astrogliogenesis, while it prevented Shh-induced oligodendrocyte differentiation and favored the expression of astrocytic genes. Transcriptional profiling of treated OPCs, revealed that BMP4-inhibition of oligodendrocyte differentiation was accompanied by increased levels of Wnt (Tbx3) and Notch-target genes (Jag1, Hes1, Hes5, Hey1, and Hey2), decreased recruitment of Hdac and increased histone acetylation at these loci. Similar upregulation of Notch-target genes and increased histone acetylation were observed in the corpus callosum of mice infused with BMP4 during cuprizone-induced demyelination. We conclude that Shh and Bmp4 differentially regulate histone acetylation and chromatin structure in OPCs and that BMP4 acts as a potent inducer of gene expression, including Notch and Wnt target genes, thereby enhancing the crosstalk among signaling pathways that are known to inhibit myelination and repair.

One hallmark of human immunodeficiency virus type 1 (HIV-1) infection is the dysregulation of cytokine gene expression in T cells. Transfection of T cells with human T-cell leukemia type 1 or 2 transactivator results in the induction of the T-cell-restricted cytokine interleukin-2 (IL-2) and its receptor (IL-2Ralpha). However, no T-cell-specific factor(s) has been directly linked with the regulation of IL-2 and IL-2Ralpha transcription by influencing the promoter activity. Thymocytes from SATB1 (special AT-rich sequence binding protein 1) knockout mice have been shown to ectopically express IL-2Ralpha, suggesting involvement of SATB1 in its negative regulation. Here we show that SATB1, a T-cell-specific global gene regulator, binds to the promoters of human IL-2 and IL-2Ralpha and recruits histone deacetylase 1 (HDAC1) in vivo. SATB1 also interacts with Tat in HIV-1-infected T cells. The functional interaction between HIV-1 Tat and SATB1 requires its PDZ-like domain, and the binding of the HDAC1 corepressor occurs through the same. Furthermore, Tat competitively displaces HDAC1 that is bound to SATB1, leading to increased acetylation of the promoters in vivo. Transduction with SATB1 interaction-deficient soluble Tat (Tat 40-72) and reporter assays using a transactivation-negative mutant (C22G) of Tat unequivocally demonstrated that the displacement of HDAC1 itself is sufficient for derepression of these promoters in vivo. These results suggest a novel mechanism by which HIV-1 Tat might overcome SATB1-mediated repression in T cells.

Vertebrate gastrulation involves the coordinated movements of populations of cells. These movements include cellular rearrangements in which cells polarize along their medio-lateral axes leading to cell intercalations that result in elongation of the body axis. Molecular analysis of this process has implicated the non-canonical Wnt/Frizzled signaling pathway that is similar to the planar cell polarity pathway (PCP) in Drosophila. Here we describe a zebrafish mutant, colgate (col), which displays defects in the extension of the body axis and the migration of branchiomotor neurons. Activation of the non-canonical Wnt/PCP pathway in these mutant embryos by overexpressing DeltaNdishevelled, rho kinase2 and van gogh-like protein 2 (vangl2) rescues the extension defects suggesting that col acts as a positive regulator of the non-canonical Wnt/PCP pathway. Further, we show that col normally regulates the caudal migration of nVII facial hindbrain branchiomotor neurons and that the mutant phenotype can be rescued by misexpression of vangl2 independent of the Wnt/PCP pathway. We cloned the col locus and found that it encodes histone deacetylase1 (hdac1). Our previous results and studies by others have implicated hdac1 in repressing the canonical Wnt pathway. Here, we demonstrate novel roles for zebrafish hdac1 in activating non-canonical Wnt/PCP signaling underlying axial extension and in promoting Wnt-independent caudal migration of a subset of hindbrain branchiomotor neurons.

Extracellular factors control the angiogenic switch in endothelial cells (ECs) via competing survival and apoptotic pathways. Previously, we showed that proangiogenic and antiangiogenic factors target the same signaling molecules, which thereby become pivots of angiogenic balance. Here we show that in remodeling endothelium (ECs and EC precursors) natural angiogenic inhibitors enhance nuclear factor-kappaB (NF-kappaB) DNA binding, which is critical for antiangiogenesis, and that blocking the NF-kappaB pathway abolishes multiple antiangiogenic events in vitro and in vivo. NF-kappaB induction by antiangiogenic molecules has a dual effect on transcription. NF-kappaB acts as an activator of proapoptotic FasL and as a repressor of prosurvival cFLIP. On the FasL promoter, NF-kappaB increases the recruitment of HAT p300 and acetylated histones H3 and H4. Conversely, on cFLIP promoter, NF-kappaB increases histone deacetylase 1 (HDAC1), decreases p300 and histone acetylation, and reduces the recruitment of NFAT, a transcription factor critical for cFLIP expression. Finally, we found a biphasic effect, when HDAC inhibitors (HDACi) were used to test the dependence of pigment epithelial-derived factor activity on histone acetylation. The cooperative effect seen at low doses switches to antagonistic as the concentrations increase. Our study defines an interactive transcriptional network underlying angiogenic balance and points to HDACi as tools to manipulate the angiogenic switch.

Mesenchymal stem cells (MSCs) show high potential for the therapy of several human diseases; however, the effectiveness of MSC transplantation has been hampered by the relatively poor migratory capacity of these cells toward disease target sites. This study investigated whether treatment of MSCs with two mood stabilizers-valproic acid (VPA) and lithium-would enhance cell migration and, if so, to explore the mechanisms underlying their effects. Short-term (3 h) exposure of MSCs to a relatively high concentration (2.5 mM) of VPA markedly increased the transcript and protein levels of CXC chemokine receptor 4 (CXCR4). VPA-induced CXCR4 expression required inhibition of histone deacetylases (HDACs), including the HDAC1 isoform, and involved histone hyperacetylation at the promoter region of the CXCR4 gene. Notably, VPA treatment enhanced stromal cell-derived factor-1α (SDF-1α)-mediated MSC migration, which was completely blocked by AMD3100, a CXCR4 antagonist. Treatment of MSCs with lithium (2.5 mM for 1 day) selectively elevated the transcript and protein levels of matrix metalloproteinase-9 (MMP-9) and its enzymatic activity; these effects were mimicked by inhibition or gene silencing of glycogen synthase kinase-3β (GSK-3β). Lithium treatment also potentiated SDF-1α-dependent MSC migration across the extracellular matrix, which was suppressed by two MMP-9 inhibitors, doxycycline and GM6001. Combining VPA and lithium treatment further increased MSC migration. Overall, VPA and lithium stimulated MSC migration through distinct targets and mediators: HDAC-CXCR4 and GSK-3β-MMP-9, respectively.

Cyclooxygenase-2 (COX-2) is considered to be a target for anticancer therapy. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity, but the mechanisms of action are incompletely understood. We investigated whether HDAC inhibitors blocked AP-1-mediated activation of COX-2 transcription. Trichostatin A and suberoylanilide hydroxamic acid, two structurally related inhibitors of HDAC activity, blocked AP-1-mediated induction of COX-2 expression and prostaglandin E2 biosynthesis. Chromatin immunoprecipitation assays indicated that HDAC inhibitors suppressed c-Jun binding to the COX-2 promoter and thereby blocked transcription. The observed reduction in binding reflected reduced levels of c-Jun. HDAC inhibitors suppressed the induction of c-jun transcription by blocking the recruitment of the preinitiation complex (RNA polymerase II and TFIIB) to the c-jun promoter. HDAC3 but not HDAC1 or HDAC2 was required for AP-1-mediated stimulation of c-jun expression. Because HDAC inhibitors suppressed the induction of c-jun gene expression, resulting in reduced COX-2 transcription, it was important to determine whether other known AP-1 target genes were also modulated. Cyclin D1 and collagenase-1 are AP-1-dependent genes that have been implicated in carcinogenesis. HDAC inhibitors suppressed the induction of both cyclin D1 and collagenase-1 transcription by inhibiting the binding of c-Jun to the respective promoters. Taken together, these results suggest that HDAC inhibitors block the induction of c-jun transcription by inhibiting the recruitment of the preinitiation complex to the c-jun promoter. This led, in turn, to reduced expression of several activator protein-1-dependent genes (COX-2, cyclin D1, collagenase-1). These findings provide new insights into the mechanisms underlying the antitumor activity of HDAC inhibitors.

BACKGROUND

Trim28 appears up-regulated in many cancers.

RESULTS

In early stage lung tumors high Trim28 correlates with increased overall survival and Trim28 reduces cell proliferation in model lung cancer cell lines through E2F interactions.

CONCLUSIONS

Trim28 may have a tumor suppressing role in the early stages of lung cancer.

CONCLUSIONS

These results suggest a complex role for Trim28 in lung cancer. Trim28 is a poorly understood transcriptional co-factor with pleiotropic biological activities. Although Trim28 mRNA is found in many studies to be up-regulated in both lung and breast cancer tissues relative to normal adjacent tissue, we found that within a panel of early-stage lung adenocarcinomas high levels of Trim28 protein correlate with better overall survival. This surprising observation suggests that Trim 28 may have anti-proliferative activity within tumors. To test this hypothesis, we used shRNAi to generate Trim28-knockdown breast and lung cancer cell lines and found that Trim28 depletion led to increased cell proliferation. Likewise, overexpression of Trim28 led to decreased cell proliferation. Confocal microscopy indicated co-localization of E2F3 and E2F4 with Trim28 within the cell nucleus, and co-immunoprecipitation assays demonstrated that Trim28 can bind both E2F3 and E2F4. Trim28 overexpression inhibited the transcriptional activity of E2F3 and E2F4, whereas Trim28 deficiency enhanced their activity. Co-immunoprecipitations further indicated that Trim28 bridges an interaction between E2Fs 3 and 4 and HDAC1. Promoter-reporter assays demonstrated that the ability of HDAC1 to repress E2F3 and E2F4-driven transcription is dependent on Trim28. Trim28 depletion increased E2F3 and E2F4 DNA binding activity, as measured by chromatin-immunoprecipitation (ChIP) assays while simultaneously reducing HDAC1 binding. Finally, ChIP-ReChIP experiments demonstrated that Trim/E2F complexes exist on several E2F-regulated promoters. Taken together, these results suggest that Trim28 has anti-proliferative activity in lung cancers via repression of members of the E2F family that are critical for cell proliferation.

The Rpd3-like members of the class I lysine deacetylase family are important regulators of chromatin structure and gene expression and have pivotal functions in the control of proliferation, differentiation and development. The highly related class I deacetylases HDAC1 and HDAC2 have partially overlapping but also isoform-specific roles in diverse biological processes, whereas HDAC3 and HDAC8 have unique functions. This review describes the role of class I KDACs in the regulation of transcription as well as their non-transcriptional functions, in particular their contributions to splicing, mitosis/meiosis, replication and DNA repair. During the past years, a number of mouse loss-of-function studies provided new insights into the individual roles of class I deacetylases in cell cycle control, differentiation and tumorigenesis. Simultaneous ablation of HDAC1 and HDAC2 or single deletion of Hdac3 severely impairs cell cycle progression in all proliferating cell types indicating that these class I deacetylases are promising targets for small molecule inhibitors as anti-tumor drugs.

Increased consumption of cruciferous vegetables is associated with a reduced risk of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) are phytochemicals derived from cruciferous vegetables that have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in human prostate cancer cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells. I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of HDAC activity was detected in PC-3 cells. In contrast, DIM significantly inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC activity correlated with increased expression of p21, a known target of HDAC inhibitors. DIM treatment caused a significant decrease in the expression of HDAC2 protein in both cancer cell lines but no significant change in the protein levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these results show that inhibition of HDAC activity by DIM may contribute to the phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification of carcinogens, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis.

Alveolar development comprises the transition of lung architecture from saccules to gas-exchange units during late gestation and early postnatal development. Exposure to hyperoxia disrupts developmental signaling pathways and causes alveolar hypoplasia as seen in bronchopulmonary dysplasia affecting preterm human newborns. Expanding literature suggests that epigenetic changes caused by environmental triggers during development may lead to heritable changes in gene expression. Given recent data on altered histone deacetylase (HDAC) activity in lungs of humans and animal models with airspace enlargement/emphysema, we hypothesized that alveolar hypoplasia from hyperoxia exposure in neonatal mice is a consequence of cell cycle arrest and reduced HDAC activity and up-regulation of the cyclin-dependent kinase inhibitor, p21. We exposed newborn mice to hyperoxia and compared lung morphologic and epigenetic changes to room air controls. Furthermore, we pretreated a subgroup of animals with the macrolide antibiotic azithromycin (AZM), known to possess antiinflammatory properties. Our results showed that hyperoxia exposure resulted in alveolar hypoplasia and was associated with decreased HDAC1 and HDAC2 and increased p53 and p21 expression. Furthermore, AZM did not confer protection against hyperoxia-induced alveolar changes. These findings suggest that alveolar hypoplasia caused by hyperoxia is mediated by epigenetic changes affecting cell cycle regulation/senescence during lung development.

The SWI/SNF complex participates as a co-activator in the transcriptional regulation of certain genes. Conversely, we and others have recently established that Brg1 and Brm, the central components of SWI/SNF, act instead as co-repressors for E2F-mediated transcriptional repression, and for the transcription of certain other promoters. We report here that Brg-1 and Brm can switch their mode of function at same promoter between activation and repression by ligand-directed differential coordination with BAF155, BAF170, HDAC1, p300 and prohibitin. This ligand and context-dependent reprogramming of the SWI/SNF complex allows it to differentially serve as either a co-repressor or a co-activator of transcription at the same promoter.

Mutations in PHF6 are the cause of Börjeson-Forssman-Lehman syndrome (BFLS), an X-linked intellectual disability (XLID) disorder, and both T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). The PHF6 gene encodes a protein with two plant homeodomain (PHD)-like zinc finger domains. As many PHD-like domains function to target chromatin remodelers to post-translationally modified histones, this suggests a role for PHF6 in chromatin regulation. However, PHD domains are usually found in association with a catalytic domain, a feature that is lacking in PHF6. This distinct domain structure and the minimal information on its cellular function prompted us to perform a proteomic screen to identify PHF6 binding partners. We expressed recombinant Flag-tagged PHF6 in HEK 293T cells for coimmunoprecipitation, and analyzed the purified products by mass spectrometry. We identified proteins involved in ribosome biogenesis, RNA splicing, and chromatin regulation, consistent with PHF6 localization to both the nucleoplasm and nucleolus. Notably, PHF6 copurified with multiple constituents of the nucleosome remodeling and deacetylation (NuRD) complex, including CHD4, HDAC1, and RBBP4. We demonstrate that this PHF6-NuRD complex is not present in the nucleolus but is restricted to the nucleoplasm. The association with NuRD represents the first known interaction for PHF6 and implicates it in chromatin regulation.

Runt domain transcription factor 3 (RUNX3) is a tumor suppressor that is silenced in cancer via hypermethylation of its promoter. This study investigated the mechanisms involved in reactive oxygen species (ROS)-induced silencing of RUNX3 in terms of epigenetic alteration since the effects of oxidative stress in tumor suppressor gene transcription are largely unknown. RUNX3 mRNA and protein expressions were down-regulated in response to hydrogen peroxide (H(2)O(2)) in the human colorectal cancer cell line SNU-407. This down-regulation was abolished with pretreatment of the ROS scavenger, N-acetylcysteine (NAC). Moreover, methylation-specific PCR data revealed that H(2)O(2) treatment increased RUNX3 promoter methylation; however, NAC and the cytosine methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-dC), decreased it, suggesting that an epigenetic regulatory mechanism by ROS-induced methylation may be involved in RUNX3 silencing. H(2)O(2) treatment resulted in DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) up-regulation with increased expression and activity, increased binding of DNMT1 to HADC1, and increased DNMT1 binding to the RUNX3 promoter. In addition, 5-Aza-dC treatment prevented the decrease in RUNX3 mRNA and protein levels by H(2)O(2) treatment. Additionally, H(2)O(2) treatment inhibited the nuclear localization and expression of RUNX3, which was abolished by NAC treatment. Furthermore, the down-regulation of RUNX3 expression by H(2)O(2) also influenced cell proliferation. Taken together, the data suggested that ROS silenced the tumor suppressor, RUNX3, by epigenetic regulation and may therefore be associated with the progression of colorectal cancer.

Tat-interactive protein, 60kDa (Tip60) is a histone acetyltransferase with specificity toward lysine 5 of histone H2A (H2AK5) and plays multiple roles in chromatin remodeling processes. Co-immunoprecipitation experiments performed on Jurkat cells, showed that Tip60 is present in the same macro-molecular complex as UHRF1 (Ubiquitin-like containing PHD and RING domain 1), DNMT1 (DNA methyltransferase 1), and HDAC1 (histone deacetylase 1). Furthermore, immunocytochemistry experiments confirmed that Tip60 co-localizes with the UHRF1/DNMT1 complex. Although down-regulation of UHRF1 by RNA interference enhanced Tip60 expression, a significant decrease of the level of acetylated H2AK5 was observed. Consistently, we have observed that down-regulation of Tip60 and DNMT1 by RNA interference, dramatically reduced the levels of acetylated H2AK5. Altogether, these results suggest that Tip60 is a novel partner of the epigenetic integration platform interplayed by UHRF1, DNMT1 and HDAC1 involved in the epigenetic code replication.