Reduced Histone Acetylation Research Articles

Survival factor NFIL3 restricts FOXO-induced gene expression in cancer

Depending on the circumstance, FOXO (Forkhead O) (FOXO1, FOXO3, and FOXO4) transcription factors activate the expression of markedly different sets of genes to produce different phenotypic effects. For example, distinct FOXO-regulated transcriptional programs stimulate cell death or enhance organism life span. To gain insight into how FOXOs select specific genes for regulation, we performed a screen for genes that modify FOXO activation of TRAIL, a death receptor ligand capable of inducing extrinsic apoptosis. We discovered that the bZIP transcriptional repressor NFIL3 (nuclear factor interleukin 3-regulated) hindered FOXO transcription factor access to chromatin at the TRAIL promoter by binding to nearby DNA and recruiting histone deacetylase-2 (HDAC2) to reduce histone acetylation. In the same manner, NFIL3 repressed expression of certain FOXO targets--e.g., FAS, GADD45α (growth arrest and DNA damage-inducible, α), and GADD45β--but not others. NFIL3, which we found to be overexpressed in different cancers, supported tumor cell survival largely through repression of TRAIL and antagonized hydrogen peroxide-induced cell death. Moreover, its expression in cancer was associated with lower patient survival. Therefore, NFIL3 alters cancer cell behavior and FOXO function by acting on chromatin to restrict the menu of FOXO target genes. Targeting of NFIL3 could be of therapeutic benefit for cancer patients.

Identification of a Novel Jasmonate-Responsive Element in the AtJMT Promoter and Its Binding Protein for AtJMT Repression

Jasmonates (JAs) are important regulators of plant biotic and abiotic stress responses and development. AtJMT in Arabidopsis thaliana and BcNTR1 in Brassica campestris encode jasmonic acid carboxyl methyltransferases, which catalyze methyl jasmonate (MeJA) biosynthesis and are involved in JA signaling. Their expression is induced by MeJA application. To understand its regulatory mechanism, here we define a novel JA-responsive cis-element (JARE), G(C)TCCTGA, in the AtJMT and BcNTR1 promoters, by promoter deletion analysis and Yeast 1-Hybrid (Y1H) assays; the JARE is distinct from other JA-responsive cis-elements previously reported. We also used Y1H screening to identify a trans-acting factor, AtBBD1, which binds to the JARE and interacts with AtJAZ1 and AtJAZ4. Knockout and overexpression analyses showed that AtBBD1 and its close homologue AtBBD2 are functionally redundant and act as negative regulators of AtJMT expression. However, AtBBD1 positively regulated the JA-responsive expression of JR2. Chromatin immunoprecipitation from knockout and overexpression plants revealed that repression of AtJMT is associated with reduced histone acetylation in the promoter region containing the JARE. These results show that AtBBD1 interacts with JAZ proteins, binds to the JARE and represses AtJMT expression.

Insights into Negative Regulation by the Glucocorticoid Receptor from Genome-wide Profiling of Inflammatory Cistromes

How the glucocorticoid receptor (GR) activates some genes while potently repressing others remains an open question. There are three current models for suppression: transrepression via GR tethering to AP-1/NF-κB sites, direct GR association with inhibitory elements (nGREs), and GR recruitment of the corepressor GRIP1. To gain insights into GR suppression, we used genomic analyses and genome-wide profiling of GR, p65, and c-Jun in LPS-stimulated macrophages. We show that GR mediates both activation and repression at tethered sites, GREs, and GRIP1-bound elements, indicating that motif classification is insufficient to predict regulatory polarity of GR binding. Interestingly, sites of GR repression utilize GRIP1's corepressor function and display reduced histone acetylation. Together, these findings suggest that while GR occupancy confers hormone responsiveness, the receptor itself may not participate in the regulatory effects. Furthermore, transcriptional outcome is not established bysequence but is influenced by epigenetic regulators, context, and other unrecognized regulatory determinants.

Pulmonary artery smooth muscle cell proliferation and migration in fetal lambs acclimatized to high-altitude long-term hypoxia: role of histone acetylation

High-altitude long-term hypoxia (LTH) is known to induce pulmonary arterial smooth muscle cell (PASMC) proliferation in the fetus, leading to pulmonary arterial remodeling and pulmonary hypertension of the newborn. The mechanisms underlying these conditions remain enigmatic however. We hypothesized that epigenetic alterations in fetal PASMC induced by high-altitude LTH may play an important role in modulating their proliferation during pulmonary arterial remodeling. To test this hypothesis, we have analyzed epigenetic alterations in the pulmonary vasculature of fetal lambs exposed to high-altitude LTH [pregnant ewes were kept at 3,801 m altitude from ~40 to 145 days gestation] or to sea level atmosphere. Intrapulmonary arteries were isolated, and fetal PASMC were cultured from both control and LTH fetuses. Compared with controls, in LTH fetus pulmonary arteries measurements of histone acetylation and global DNA methylation demonstrated reduced levels of global histone 4 acetylation and DNA methylation, accompanied by the loss of the cyclin-dependent kinase inhibitor p21. Treatment of LTH fetal PASMCs with histone deacetylase (HDAC) inhibitor trichostatin A decreased their proliferation rate, in part because of altered expression of p21 at both RNA and protein level. In PASMC of LTH fetuses, HDAC inhibition also decreased PDGF-induced cell migration and ERK1/2 activation and modulated global DNA methylation. On the basis of these observations, we propose that epigenetic alterations (reduced histone acetylation and DNA methylation) caused by chronic hypoxia leads to fetal PASMC proliferation and vessel remodeling associated with vascular proliferative disease and that this process is regulated by p21.

Epigenetic imprinting by commensal probiotics inhibits the IL-23/IL-17 axis in an in vitro model of the intestinal mucosal immune system

The pathophysiology of IBD is characterized by a complex interaction between genes and the environment. Genetic and environmental differences are attributed to the heterogeneity of the disease pathway and to the epigenetic modifications that lead to altered gene expression in the diseased tissues. The epigenetic machinery consists of short interfering RNA, histone modifications, and DNA methylation. We evaluated the effects of Bifidobacterium breve (DSMZ 20213) and LGG (ATCC 53103), as representatives of commensal probiotics on the expression of IL-17 and IL-23, which play an important role in IBD, and on the epigenetic machinery in a 3D coculture model composed of human intestinal HT-29/B6 or T84 cells and PBMCs. The cells were treated with LPS in the presence or absence of bacteria for 48 h, and the expression of IL-17, IL-23, and CD40 at the mRNA and protein levels was assessed using TaqMan qRT-PCR and ELISA, respectively. Western blotting was used to assess the expression of the MyD88, the degradation of IRAK-1 and IκBα, the expression of the NF-κB p50/p65 subunits, the p-p38 MAPK and p-MEK1, as well as histone modifications. NF-κB activity was assessed by NF-κB-dependent luciferase reporter gene assays. The accumulation of Ac-H4 and DNA methylation was quantitatively assessed using colorimetric assays. B. breve and LGG diminished the LPS-induced expression of IL-17, IL-23, CD40, and histone acetylation, while slightly enhancing DNA methylation. These effects were paralleled by a decrease in the nuclear translocation of NF-κB, as demonstrated by a decrease in the expression of MyD88, degradation of IRAK-1 and IκBα expression of the nuclear NF-κB p50/p65 subunits, p-p38 MAPK and p-MEK1, and NF-κB-dependent luciferase reporter gene activity in LPS-stimulated cells. B. breve and LGG may exert their anti-inflammatory effects in the gut by down-regulating the expression of the IBD-causing factors (IL-23/IL-17/CD40) associated with epigenetic processes involving the inhibition of histone acetylation and the optimal enhancement of DNA methylation, reflected in the limited access of NF-κB to gene promoters and reduced NF-κB-mediated transcriptional activation. We describe a new regulatory mechanism in which commensal probiotics inhibit the NF-κB-mediated transcriptional activation of IBD-causing factors (IL-23/IL-17/CD40), thereby simultaneously reducing histone acetylation and enhancing DNA methylation.

LANP mediates neuritic pathology in Spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease that results from a pathogenic glutamine-repeat expansion in the protein ataxin-1 (ATXN1). Although the functions of ATXN1 are still largely unknown, there is evidence to suggest that ATXN1 plays a role in regulating gene expression, the earliest process known to go awry in SCA1 mouse models. In this study, we show that ATXN1 reduces histone acetylation, a post-translational modification of histones associated with enhanced transcription, and represses histone acetyl transferase-mediated transcription. In addition, we find that depleting the Leucine-rich Acidic Nuclear Protein (LANP)—an ATXN1 binding inhibitor of histone acetylation—reverses aspects of SCA1 neuritic pathology.

Cooperative Binding of KLF4, pELK-1, and HDAC2 to a G/C Repressor Element in the SM22α Promoter Mediates Transcriptional Silencing During SMC Phenotypic Switching In Vivo

We previously identified conserved G/C Repressor elements in the promoters of most smooth muscle cell (SMC) marker genes and demonstrated that mutation of this element within the SM22α promoter nearly abrogated repression of this transgene after vascular wire injury or within lesions of ApoE-/- mice. However, the mechanisms regulating the activity of the G/C Repressor are unknown, although we have previously shown that phenotypic switching of cultured SMC is dependent on Krupple-like factor (KLF)4. The goals of the present studies were to ascertain if (1) injury-induced repression of SM22α gene after vascular injury is mediated through KLF4 binding to the G/C Repressor element and (2) the transcriptional repressor activity of KLF4 on SMC marker genes is dependent on cooperative binding with pELK-1 (downstream activator of the mitogen-activated protein kinase pathway) and subsequent recruitment of histone de-acetylase 2 (HDAC2), which mediates epigenetic gene silencing. Chromatin immunoprecipitation (ChIP) assays were performed on chromatin derived from carotid arteries of mice having either a wild-type or G/C Repressor mutant SM22α promoter-LacZ transgene. KLF4 and pELK-1 binding to the SM22α promoter was markedly increased after vascular injury and was G/C Repressor dependent. Sequential ChIP assays and proximity ligation analyses in cultured SMC treated with platelet-derived growth factor BB or oxidized phospholipids showed formation of a KLF4, pELK-1, and HDAC2 multiprotein complex dependent on the SM22α G/C Repressor element. Silencing of SMC marker genes during phenotypic switching is partially mediated by sequential binding of pELK-1 and KLF4 to G/C Repressor elements. The pELK-1-KLF4 complex in turn recruits HDAC2, leading to reduced histone acetylation and epigenetic silencing.

CPTH6, a Thiazole Derivative, Induces Histone Hypoacetylation and Apoptosis in Human Leukemia Cells

We previously identified novel thiazole derivatives able to reduce histone acetylation and histone acetyltransferase (HAT) activity in yeast. Among these compounds, 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) has been selected and used throughout this study. The effect of CPTH6 on histone acetylation, cell viability and differentiation, cell-cycle distribution, and apoptosis in a panel of acute myeloid leukemia and solid tumor cell lines has been evaluated. Here, we showed that CPTH6 leads to an inhibition of Gcn5 and pCAF HAT activity. Moreover, it inhibits H3/H4 histones and α-tubulin acetylation of a panel of leukemia cell lines. Concentration- and time-dependent inhibition of cell viability, paralleled by accumulation of cells in the G(0)/G(1) phase and depletion from the S/G(2)M phases, was observed. The role of mitochondrial pathway on CPTH6-induced apoptosis was shown, being a decrease of mitochondrial membrane potential and the release of cytochrome c, from mitochondria to cytosol, induced by CPTH6. Also the involvement of Bcl-2 and Bcl-xL on CPTH6-induced apoptosis was found after overexpression of the two proteins in leukemia cells. Solid tumor cell lines from several origins were shown to be differently sensitive to CPTH6 treatment in terms of cell viability, and a correlation between the inhibitory efficacy on H3/H4 histones acetylation and cytotoxicity was found. Differentiating effect on leukemia and neuroblastoma cell lines was also induced by CPTH6. These results make CPTH6 a suitable tool for discovery of molecular targets of HAT and, potentially, for the development of new anticancer therapies, which warrants further investigations.

Stress-Induced C/EBP Homology Protein (CHOP) Represses MyoD Transcription to Delay Myoblast Differentiation

When mouse myoblasts or satellite cells differentiate in culture, the expression of myogenic regulatory factor, MyoD, is downregulated in a subset of cells that do not differentiate. The mechanism involved in the repression of MyoD expression remains largely unknown. Here we report that a stress-response pathway repressing MyoD transcription is transiently activated in mouse-derived C2C12 myoblasts growing under differentiation-promoting conditions. We show that phosphorylation of the α subunit of the translation initiation factor 2 (eIF2α) is followed by expression of C/EBP homology protein (CHOP) in some myoblasts. ShRNA-driven knockdown of CHOP expression caused earlier and more robust differentiation, whereas its constitutive expression delayed differentiation relative to wild type myoblasts. Cells expressing CHOP did not express the myogenic regulatory factors MyoD and myogenin. These results indicated that CHOP directly repressed the transcription of the MyoD gene. In support of this view, CHOP associated with upstream regulatory region of the MyoD gene and its activity reduced histone acetylation at the enhancer region of MyoD. CHOP interacted with histone deacetylase 1 (HDAC1) in cells. This protein complex may reduce histone acetylation when bound to MyoD regulatory regions. Overall, our results suggest that the activation of a stress pathway in myoblasts transiently downregulate the myogenic program.

Epigenetic Enhancement of BDNF Signaling Rescues Synaptic Plasticity in Aging

Aging-related cognitive declines are well documented in humans and animal models. Yet the synaptic and molecular mechanisms responsible for cognitive aging are not well understood. Here we demonstrated age-dependent deficits in long-term synaptic plasticity and loss of dendritic spines in the hippocampus of aged Fisher 344 rats, which were closely associated with reduced histone acetylation, upregulation of histone deacetylase (HDAC) 2, and decreased expression of a histone acetyltransferase. Further analysis showed that one of the key genes affected by such changes was the brain-derived neurotrophic factor (Bdnf) gene. Age-dependent reductions in H3 and H4 acetylation were detected within multiple promoter regions of the Bdnf gene, leading to a significant decrease in BDNF expression and impairment of downstream signaling in the aged hippocampus. These synaptic and signaling deficits could be rescued by enhancing BDNF and trkB expression via HDAC inhibition or by directly activating trkB receptors with 7,8-dihydroxyflavone, a newly identified, selective agonist for trkB. Together, our findings suggest that age-dependent declines in chromatin histone acetylation and the resulting changes in BDNF expression and signaling are key mechanisms underlying the deterioration of synaptic function and structure in the aging brain. Furthermore, epigenetic or pharmacological enhancement of BDNF-trkB signaling could be a promising strategy for reversing cognitive aging.

The Eleven-Nineteen Lysine-rich Leukemia Gene (ELL2) Influences the Histone H3 Protein Modifications Accompanying the Shift to Secretory Immunoglobulin Heavy Chain mRNA Production

In plasma cells, immunoglobulin heavy chain (IgH) secretory-specific mRNA is made in high abundance as a result of both increased promoter proximal poly(A) site choice and weak splice-site skipping. Ell2, the eleven-nineteen lysine rich leukemia gene, is a transcription elongation factor that is induced ∼6-fold in plasma cells and has been shown to drive secretory-specific mRNA production. Reducing ELL2 by siRNA, which reduced processing to the secretion-specific poly(A) site, also influenced the methylations of histone H3K4 and H3K79 on the IgH gene and impacted positive transcription factor b (pTEFb), Ser-2 carboxyl-terminal phosphorylation, and polyadenylation factor additions to RNA polymerase II. The multiple lineage leukemia gene (MLL) and Dot1L associations with the IgH gene were also impaired in the absence of ELL2. To investigate the link between histone modifications, transcription elongation, and alternative RNA processing in IgH mRNA production, we performed chromatin immunoprecipitation on cultured mouse B and plasma cells bearing the identical IgH γ2a gene. In the plasma cells, as compared with the B cells, the H3K4 and H3K79 methylations extended farther downstream, past the IgH enhancer to the end of the transcribed region. Thus the downstream H3K4 and H3K79 methylation of the IgH associated chromatin in plasma cells is associated with increased polyadenylation and exon skipping, resulting from the actions of ELL2 transcription elongation factor.

Post-training intrahippocampal inhibition of class I histone deacetylases enhances long-term object-location memory

Long-term memory formation involves covalent modification of the histone proteins that package DNA. Reducing histone acetylation by mutating histone acetyltransferases impairs long-term memory, and enhancing histone acetylation by inhibiting histone deacetylases (HDACs) improves long-term memory. Previous studies using HDAC inhibitors to enhance long-term memory have focused on the fear-conditioning task using broad-spectrum HDAC inhibitors. We have found that post-training intrahippocampal administration of the broad-spectrum HDAC inhibitor trichostatin A (TSA) or the class I HDAC-selective inhibitor MS275 enhances long-term object-location memory, supporting a role for class I HDACs in the enhancement of hippocampus-dependent memory induced by HDAC inhibition.

Abstract 3929: ADA3, a component of ATAC complex regulates DNA damage response

Abstract Our long term research goal is to understand the biochemical mechanism of immortalization of human epithelial cells that represents early step in cancer progression. Over the years, we have generated several epithelial cell immortalization models, using viral oncogenes, mutated cancer genes, as well as radiation treatment of cells. In one of such immortalization model, we identified alteration/deficiency in activation (ADA3) protein as a novel human papilloma virus E6 oncoprotein binding protein, and as a regulator of p53 function. We and others have shown that human ADA3 protein is an essential component of various Histone acetyl transferase complexes (HATs) and thus controls transactivation of various transcriptional factors. To gain insight into the physiological importance of ADA3, we established a conditional knockout mouse model and showed that loss of ADA3 led to embryonic lethality at an early stage of development. Using ADA3lox/lox mouse embryonic fibroblast (MEF) cell lines, we further showed that ADA3 plays an important role in DNA damage and repair pathway. Adenovirus cre-mediated deletion of ADA3 led to a global reduction of histone acetylation, severe G2/M cell cycle arrest, various aberration in nuclear structures, and massive chromosome aberration. Significantly, cells lacking ADA3 showed defects in recruitment of DNA repair associated proteins, BRCA1 and 53BP1 to the DNA damage foci. These results implicate ADA3 as a major cell cycle and DNA repair response regulating protein. These findings in the context of oncogenesis and chemotherapy will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2011-3929

Valproic acid improves outcome after rodent spinal cord injury: Potential roles of histone deacetylase inhibition

Histone deacetylases (HDAC) inhibitors including valproic acid (VPA) have emerged as a promising therapeutic intervention in neurological disorders. We investigated the levels of acetylated histone and the therapeutic potential of VPA in a rat model of spinal cord injury (SCI). At different time points (12 h, 1 day, 3 days, 1 week and 2 weeks) after SCI or sham surgery, the spinal cords were collected to evaluate the levels of acetylated histone H3 (Ac-H3) and H4 (Ac-H4). VPA or vehicle was injected for 1 week starting immediately after SCI and histone acetylation, apoptosis, as well as neurobehavior were observed to test the effect of VPA. The levels of Ac-H3 and Ac-H4 in the injured spinal cord started to significantly decrease as early as day 1, and remained below those in uninjured controls for at least 2 weeks after SCI. Injection of VPA markedly prevented the reductions of Ac-H3 and Ac-H4, upregulated the expressions of Hsp70 and Bcl-2, reduced apoptosis and finally promoted locomotion recovery. Our data demonstrated that SCI led to marked reduction in histone acetylation; VPA was neuroprotective in the SCI model, and the mechanism may involve HDAC inhibition and protective proteins induction.

The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1

The dynamics of histone methylation have emerged as an important issue since the identification of histone demethylases. We studied the regulatory function of Rph1/KDM4 (lysine demethylase), a histone H3K36 demethylase, on transcription in Saccharomyces cerevisiae. Overexpression of Rph1 reduced the expression of PHR1 and increased UV sensitivity. The catalytically deficient mutant (H235A) of Rph1 diminished the repressive transcriptional effect on PHR1 expression, which indicates that histone demethylase activity contributes to transcriptional repression. Chromatin immunoprecipitation analysis demonstrated that Rph1 was associated at the upstream repression sequence of PHR1 through zinc-finger domains and was dissociated after UV irradiation. Notably, overexpression of Rph1 and H3K36A mutant reduced histone acetylation at the URS, which implies a crosstalk between histone demethylation and acetylation at the PHR1 promoter. In addition, the crucial checkpoint protein Rad53 acted as an upstream regulator of Rph1 and dominated the phosphorylation of Rph1 that was required for efficient PHR1 expression and the dissociation of Rph1. The release of Rph1 from chromatin also required the phosphorylation at S652. Our study demonstrates that the histone demethylase Rph1 is associated with a specific chromatin locus and modulates histone modifications to repress a DNA damage responsive gene under control of damage checkpoint signaling.

Genome-Wide Analysis of Transcriptional Reprogramming in Mouse Models of Acute Myeloid Leukaemia

Acute leukaemias are commonly caused by mutations that corrupt the transcriptional circuitry of haematopoietic stem/progenitor cells. However, the mechanisms underlying large-scale transcriptional reprogramming remain largely unknown. Here we investigated transcriptional reprogramming at genome-scale in mouse retroviral transplant models of acute myeloid leukaemia (AML) using both gene-expression profiling and ChIP-sequencing. We identified several thousand candidate regulatory regions with altered levels of histone acetylation that were characterised by differential distribution of consensus motifs for key haematopoietic transcription factors including Gata2, Gfi1 and Sfpi1/Pu.1. In particular, downregulation of Gata2 expression was mirrored by abundant GATA motifs in regions of reduced histone acetylation suggesting an important role in leukaemogenic transcriptional reprogramming. Forced re-expression of Gata2 was not compatible with sustained growth of leukaemic cells thus suggesting a previously unrecognised role for Gata2 in downregulation during the development of AML. Additionally, large scale human AML datasets revealed significantly higher expression of GATA2 in CD34+ cells from healthy controls compared with AML blast cells. The integrated genome-scale analysis applied in this study represents a valuable and widely applicable approach to study the transcriptional control of both normal and aberrant haematopoiesis and to identify critical factors responsible for transcriptional reprogramming in human cancer.

ATP citrate lyase is required for normal sexual and asexual development in Gibberella zeae

Adenosine triphosphate (ATP) citrate lyase (ACL) is a key enzyme in the production of cytosolic acetyl-CoA, which is crucial for de novo lipid synthesis and histone acetylation in mammalian cells. In this study, we characterized the mechanistic roles of ACL in the homothallic ascomycete fungus Gibberella zeae, which causes Fusarium head blight in major cereal crops. Deletion of ACL in the fungus resulted in a complete loss of self and female fertility as well as a reduction in asexual reproduction, virulence, and trichothecene production. When the wild-type strain was spermatized with the ACL deletion mutants, they produced viable ascospores, however ascospore delimitation was not properly regulated. Although lipid synthesis was not affected by ACL deletion, histone acetylation was dramatically reduced in the ACL deletion mutants during sexual development, suggesting that the defects in sexual reproduction were caused by the reduction in histone acetylation. This study is the first report demonstrating a link between sexual development and ACL-mediated histone acetylation in fungi.

Inhibition of p300-HAT results in a reduced histone acetylation and down-regulation of gene expression in cardiac myocytes

Aims Histone acetylation plays an important role in cardiogenesis, but the underlying mechanism is unclear. In this study, we investigated the relationship between histone hypo-acetylation and the expression of cardiac-specific genes to explore the underlying mechanisms. Main methods Cardiac-specific genes that physically interacted with p300 protein in mouse hearts were analyzed using chromatin immunoprecipitation (ChIP) assays. The cultured mouse neonatal cardiac myocytes were treated with curcumin with different concentrations and durations. The changes of histone acetyltransferase (HAT) activities, histone acetylation, cardiac-specific genes expression, and structure of chromatin were assessed by ELISA, Western blotting, quantitative RT-PCR, and ChIP assays, respectively. Key findings Results from the ChIP assay showed that GATA4, Nkx2.5, and Mef2c physically interacted with p300 protein. After treatment with 30 μM curcumin for 24 h, the HAT activities of cardiac myocytes were inhibited significantly. And the acetylation of whole histone H3 was reduced by 0.3983-fold compared to control groups (P < 0.05). Accordingly, the expression of cardiac-specific genes, GATA4, Nkx2.5, and Mef2c, were significantly down-regulated. Acetylation of histone H3 bound with promoter regions of these genes was significantly reduced. Significance p300 interacts with cardiac-specific genes, GATA4, Nkx2.5 and Mef2c, and inhibition of p300-HAT by curcumin down-regulates their expression through the inhibition of histone H3 acetylation in the promoter regions. This finding indicates that p300-HAT mediated histone H3 acetylation plays an important role in the regulation of cardiac gene expression, which is a novel mechanism of epigenetic regulation in the heart during the development and in case of some congenital heart diseases.

TGF-β enforces senescence in Myc-transformed hematopoietic tumor cells through induction of Mad1 and repression of Myc activity

Inhibition of tumor growth factor (TGF)-β-mediated cell cycle exit is considered an important tumorigenic function of Myc oncoproteins. Here we found that TGF-β1 enforced G 1 cell cycle arrest and cellular senescence in human U-937 myeloid tumor cells ectopically expressing v-Myc, which contains a stabilizing mutation frequently found in lymphomas. This correlated with induced expression of the Myc antagonist Mad1, resulting in replacement of Myc for Mad1 at target promoters, reduced histone acetylation and strong repression of Myc-driven transcription. The latter was partially reversed by histone deacetylase (HDAC) inhibitors, consistent with involvement of Mad1. Importantly, knockdown of MAD1 expression prevented TGF-β1-induced senescence, underscoring that Mad1 is a crucial component of this process. Enforced Mad1 expression sensitized U-937-myc cells to TGF-β and restored phorbol ester-induced cell cycle exit, but could not alone induce G 1 arrest, suggesting that Mad1 is required but not sufficient for cellular senescence. Our results thus demonstrate that TGF-β can override Myc activity despite a stabilizing cancer mutation and induce senescence in myeloid tumor cells, at least in part by induction of Mad1. TGF-β-induced senescence, or signals mimicking this pathway, could therefore potentially be explored as a therapeutic principle for treating hematopoietic and other tumors with deregulated MYC expression.

AtMBD9 modulates Arabidopsis development through the dual epigenetic pathways of DNA methylation and histone acetylation

Mutations within the Arabidopsis METHYL-CpG BINDING DOMAIN 9 gene (AtMBD9) cause pleotropic phenotypes including early flowering and multiple lateral branches. Early flowering was previously attributed to the repression of flowering locus C (FLC) due to a reduction in histone acetylation. However, the reasons for other phenotypic variations remained obscure. Recent studies suggest an important functional correlation between DNA methylation and histone modifications. By investigating this relationship, we found that the global genomic DNA of atmbd9 was over-methylated, including the FLC gene region. Recombinant AtMBD9 does not have detectable DNA demethylation activity in vitro, but instead has histone acetylation activity. Ectopic over-expression of AtMBD9 and transient DNA demethylation promotes flowering and causes partial recovery of the normal branching phenotype. Co-immunoprecipitation assays suggest that AtMBD9 interacts in vivo with some regions of the FLC gene and binds to histone 4 (H4). Gene expression profile analysis revealed earlier up-regulation of some flower-specific transcriptional factors and alteration of potential hormonal and signal transducer axillary branching regulatory genes. In accordance with this result, AtMBD9 itself was found to be localized in the nucleus and expressed in the flower and axillary buds. Together, these results suggest that AtMBD9 controls flowering time and axillary branching by modulating gene expression through DNA methylation and histone acetylation, and reveal another component of the epigenetic mechanism controlling gene expression.