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|Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. |
Tang, Jiangbo, et al.
Nat. Struct. Mol. Biol., 20: 317-25 (2013) 2013
The pathogenic sequelae of BRCA1 mutation in human and mouse cells are mitigated by concomitant deletion of 53BP1, which binds histone H4 dimethylated at Lys20 (H4K20me2) to promote nonhomologous end joining, suggesting that a balance between BRCA1 and 53BP1 regulates DNA double strand-break (DSB) repair mechanism choice. Here we document that acetylation is a key determinant of this balance. TIP60 acetyltransferase deficiency reduced BRCA1 at DSB chromatin with commensurate increases in 53BP1, whereas HDAC inhibition yielded the opposite effect. TIP60-dependent H4 acetylation diminished 53BP1 binding to H4K20me2 in part through disruption of a salt bridge between H4K16 and Glu1551 in the 53BP1 Tudor domain. Moreover, TIP60 deficiency impaired homologous recombination and conferred sensitivity to PARP inhibition in a 53BP1-dependent manner. These findings demonstrate that acetylation in cis to H4K20me2 regulates relative BRCA1 and 53BP1 DSB chromatin occupancy to direct DNA repair mechanism.
|The CpG island shore of the GLT-1 gene acts as a methylation-sensitive enhancer. |
Tatjana Perisic,Florian Holsboer,Theo Rein,Jürgen Zschocke
Glia 60 2012
Astrocytic lineage commitment and brain region-dependent specialization of glia are partly ascribed to epigenetic processes. Clearance of glutamate is an essential task, which astrocytes assume in a temporal-spatial fashion by distinct glutamate transporter expression. Glutamate transporter subtype 1 (GLT-1) is predominant in cortex (CTX), while it plays an inferior role in cerebellum (CER). Here, we set out to identify regulatory elements that could account for the differences in brain region-specific activity as well as response to dexamethasone (DEX) or epigenetic factors. We found a distal promoter element at the shore of the CpG island exhibiting enhancer function in response to DEX in reporter gene assays. This shore region showed slight enrichment in repressive trimethyl-histone H3 (Lys27) and under-representation of acetyl-histone H4 (H4ac) marks in DEX nonresponsive CER astrocytes as determined by chromatin immunoprecipitation. In addition, CpG sites of the shore region displayed higher methylation in CER than in CTX cells. Targeted in vitro methylation of CpG sites within the shore abrogated the stimulatory effects of DEX. Interestingly, the shore was characterized by a pronounced epigenetic plasticity in CTX cells since DEX exposure elicited an increase of H4ac in CTX in comparison to DEX nonresponsive CER. The transcriptional activity of this region was also affected by histone deacetylase inhibitors in a methylation- and brain region-dependent manner. Together, our study highlights the impact of an epigenetically adaptive DNA element of the GLT-1 promoter being decisive for brain region-specific activity and reactivity. © 2012 Wiley Periodicals, Inc.
|Valproate alters dopamine signaling in association with induction of par-4 protein expression. |
Saebom Lee,Jaehoon Jeong,Young-Un Park,Yongdo Kwak,Seol Ae Lee,Haeryun Lee,Hyeon Son,Sang Ki Park
PloS one 7 2012
Chromatin remodeling through histone modifications has emerged as a key mechanism in the pathophysiology of psychiatric disorders. Valproate (VPA), a first-line medication for bipolar disorder, is known to have histone deacetylase (HDAC) inhibitor activity, but the relationship between its efficacy as a mood stabilizer and HDAC inhibitory activity is unclear. Here we provide evidence that prostate apoptosis response-4 (Par-4), an intracellular binding partner of dopamine D2 receptors (DRD2), plays a role in mediating the effectiveness of VPA. We found that chronic VPA treatment enhanced the expression of Par-4 in cultured neurons and adult mouse brains. This Par-4 induction phenomenon occurred at the transcriptional level and was correlated with an increase in histone H3 and H4 acetylation of the Par-4 promoter regions. Furthermore, chronic VPA treatment potentiated the suppression of the cAMP signaling cascade upon dopamine stimulation, which was blocked by sulpiride treatment. These results indicate that VPA potentiates DRD2 activity by enhancing Par-4 expression via a chromatin remodeling mechanism.
|Local microenvironment provides important cues for cell differentiation in lingual epithelia. |
Feng Li,Mingliang Zhou
PloS one 7 2012
Transgenic Keratin14-rtTA-PTR mice specifically express Keratin14 (K14) in the tongue epithelia, as well as co-express EGFP and the dominant negative ?Tgfbr2 genes upon treatment with Doxycycline (Dox). As TGF-? signaling negatively regulates the stem cell cycle and proliferation, its disruption by Dox induction in these transgenic mice shortens the cell cycle and allows observation of the final fate of those mutated cell lineages within a short period of time. Here, we used inducible transgenic mice to track the K14+ cells through the cell migration stream by immunohistochemical an immunofluorescent imaging. We showed that these cells have different development patterns from the tip to posterior of the tongue, achieved presumably by integrating positional information from the microenvironment. The expression of the K14 gene was variable, depending on the location of the tongue and papillae. Disruption of TGF-? signaling in K14+ progenitor cells resulted in proliferation of stem cell pools.
|NFκB and AP-1 drive human myometrial IL8 expression. |
Shirin Khanjani,Vasso Terzidou,Mark R Johnson,Phillip R Bennett
Mediators of inflammation 2012 2012
The uterine expression of the chemokine IL8 increases dramatically with the onset of labour both at term and preterm. The IL8 promoter contains binding sites for the transcription factors nuclear factor-kappa B (NFκB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein (CEBP). In this study we investigated the roles of these transcription factors in IL1B regulation of the IL8 gene in human myometrium. Using chromatin immune precipitation (ChIP) assay, we showed that each of NFκB, CEBP, and AP-1 binds to the IL8 promoter upon IL1B stimulation. To examine the relative importance of each site in IL8 gene expression, site-directed mutagenesis of each of these sites was performed. We found that the NFκB site was essential for basal and IL1B-stimulated gene expression. Mutation of the AP-1 site reduced both basal and IL1B-stimulated expression but to a lesser extent. Mutation of the CEBP site had no effect upon basal expression but eliminated the IL1B response. Small interfering RNA (siRNA) silencing of NFκB abolished the IL8 response to IL1B significantly; siRNA against AP-1 reduced it to a lesser extent whilst knockdown of CEBP enhanced the response. Our data confirms a central and essential role for NFκB in regulation of IL8 in human myometrium.
|HIF2α-Sp1 interaction mediates a deacetylation-dependent FVII-gene activation under hypoxic conditions in ovarian cancer cells. |
Shiro Koizume,Shin Ito,Etsuko Miyagi,Fumiki Hirahara,Yoshiyasu Nakamura,Yuji Sakuma,Hitoshi Osaka,Yasuo Takano,Wolfram Ruf,Yohei Miyagi
Nucleic acids research 40 2012
Hypoxia-inducible factors (HIF)-1α and HIF2α are major transcription factors required for adaptive responses to hypoxia. HIFs form a complex with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to the regulatory regions of target genes. The acetylation of histones by histone acetyltransferases (HATs) is one of the epigenetic marks associated with active chromatin. Indeed, HIFs recruit p300 HAT to hypoxia response elements (HREs) within gene regulatory regions. Here, we report an unusual HIF-mediated transcriptional activation in ovarian clear cell carcinoma (CCC). While characterizing coagulation factor VII (FVII) gene induction during hypoxic conditions, we observed that the interaction of HIF2α with Sp1, but not with ARNT, could induce transcription of FVII in a HRE-independent manner. Unexpectedly, this gene activation is associated with histone deacetylation. We found that a class II HDAC, HDAC4, is recruited with HIF2α to the FVII promoter as a co-activator, while p300 HAT negatively regulated this process. Furthermore, this mechanism can be synergistically enhanced via a deacetylation-dependent pathway when cells are simultaneously exposed to hypoxic and serum-free conditions. These results suggest the presence of a stress-responsive transcription mediated by the HIF2α/Sp1/HDAC4 network and explain how CCC shed their procoagulant activity under hypoxia.
|miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells. |
Kyle L Macquarrie,Zizhen Yao,Janet M Young,Yi Cao,Stephen J Tapscott
Skeletal muscle 2 2012
|Late-onset Increases in Oxidative Stress and Other Tumorigenic Activities and Tumors With a Ha-ras Mutation in the Liver of Adult Male C3H Mice Gestationally Exposed to Arsenic. |
Keiko Nohara,Yukiyo Tateishi,Takehiro Suzuki,Kazuyuki Okamura,Hikari Murai,Shota Takumi,Fumihiko Maekawa,Noriko Nishimura,Masuko Kobori,Takaaki Ito
Toxicological sciences : an official journal of the Society of Toxicology 129 2012
Tumorigenesis is a complex process involving genetic, epigenetic, and metabolic alterations. Gestational arsenic exposure has been shown to increase hepatic tumors in adult male offspring of C3H mice, which spontaneously develop hepatic tumors often harboring activating Ha-ras mutation. We explored tumor-promoting changes by gestational arsenic exposure with a focus on Ha-ras mutation and gene expression changes. The results of this study demonstrated that gestational arsenic exposure particularly increased hepatic tumors with a C61A Ha-ras mutation. Real-time PCR analyses on the adult normal livers showed that two genes (Creld2, Slc25a30), whose expression are induced by endoplasmic reticulum stress and cellular oxidative stress, respectively, were significantly upregulated and two genes (Fabp4, Ell3), whose products are involved in lipid efflux and apoptosis, respectively, were significantly downregulated more than twofold by gestational arsenic exposure compared with control mice. The expression changes in the four genes were shown to be late-onset events and to some extent to be associated with corresponding histone modifications, and not with DNA methylation changes. The gene expression changes suggested alterations in lipid metabolism and associated oxidative stress augmentation. Consistently, expression of an oxidative-stress-inducible gene heme oxygenase-1 (HO-1) was upregulated in the livers of the arsenic group. We also found increased expression of retrotransposon L1 mRNA in the tumor-bearing livers of the arsenic group in comparison with control mice. These results suggested that gestational arsenic exposure induces tumor-augmenting changes, including oxidative stress and L1 activation, in a late-onset manner, which would particularly promote tumorigenic expansion of cells with a C61A Ha-ras mutation.
|Chromatin Modulation at the FLO11 Promoter of Saccharomyces cerevisiae by HDAC and Swi/Snf Complexes. |
Ramón R Barrales,Philipp Korber,Juan Jimenez,José I Ibeas
Genetics 191 2012
Cell adhesion and biofilm formation are critical processes in the pathogenicity of fungi and are mediated through a family of adhesin proteins conserved throughout yeasts and fungi. In Saccharomyces cerevisiae, Flo11 is the main adhesin involved in cell adhesion and biofilm formation, making the study of its function and regulation in this nonpathogenic budding yeast highly relevant. The S. cerevisiae FLO11 gene is driven by a TATA-box-containing promoter that is regulated through one of the longest regulatory upstream regions (3 kb) in yeast. We reported recently that two chromatin cofactor complexes, the Rpd3L deacetylase and the Swi/Snf chromatin-remodeling complexes, contribute significantly to the regulation of FLO11. Here, we analyze directly how these complexes impact on FLO11 promoter chromatin structure and dissect further the interplay between histone deacetylases, chromatin remodeling, and the transcriptional repressor Sfl1. We show that the regulation of chromatin structure represents an important layer of control in the highly complex regulation of the FLO11 promoter.
|Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression. |
Roberta Zini,Ruggiero Norfo,Francesco Ferrari,Elisa Bianchi,Simona Salati,Valentina Pennucci,Giorgia Sacchi,Chiara Carboni,Giovanni Battista Ceccherelli,Enrico Tagliafico,Sergio Ferrari,Rossella Manfredini,
Experimental hematology 40 2012
Histone deacetylase inhibitors represent a family of targeted anticancer compounds that are widely used against hematological malignancies. So far little is known about their effects on normal myelopoiesis. Therefore, in order to investigate the effect of histone deacetylase inhibitors on the myeloid commitment of hematopoietic stem/progenitor cells, we treated CD34(+) cells with valproic acid (VPA). Our results demonstrate that VPA treatment induces H4 histone acetylation and hampers cell cycle progression in CD34(+) cells sustaining high levels of CD34 protein expression. In addition, our data show that VPA treatment promotes erythrocyte and megakaryocyte differentiation. In fact, we demonstrate that VPA treatment is able to induce the expression of growth factor-independent protein 1B (GFI1B) and of mixed-lineage leukemia translocated to chromosome 3 protein (MLLT3), which are crucial regulators of erythrocyte and megakaryocyte differentiation, and that the up-regulation of these genes is mediated by the histone hyperacetylation at their promoter sites. Finally, we show that GFI1B inhibition impairs erythroid and megakaryocyte differentiation induced by VPA, while MLLT3 silencing inhibits megakaryocyte commitment only. As a whole, our data suggest that VPA sustains the expression of stemness-related markers in hematopoietic stem/progenitor cells and is able to interfere with hematopoietic lineage commitment by enhancing erythrocyte and megakaryocyte differentiation and by inhibiting the granulocyte and mono-macrophage maturation.
|Nuclear epidermal growth factor receptor interacts with transcriptional intermediary factor 2 to activate cyclin D1 gene expression triggered by the oncoprotein latent membrane protein 1. |
Yin Shi,Yongguang Tao,Yiqun Jiang,Yang Xu,Bin Yan,Xue Chen,Lanbo Xiao,Ya Cao
Carcinogenesis 33 2012
The epidermal growth factor receptor (EGFR), a ubiquitously expressed receptor tyrosine kinase, is an important factor in carcinogenesis. Transcriptional intermediary factor 2 (TIF2), a member of the p160 nuclear receptor co-activator gene family, is linked to the proliferation of cancer cells. However, the direct interplay between the EGFR and the nuclear receptors remains unclear. Our previous study demonstrated that nuclear EGFR could directly bind to the cyclin D1 promoter under the regulation of the oncoprotein latent membrane protein 1 (LMP1), but it also indicated that other factors are involved in the activation of target genes. In this study, we found that LMP1 upregulated the expression of TIF2 and promoted the interaction of EGFR with TIF2 in nasopharyngeal carcinoma. Furthermore, we demonstrated that the intact complex was linked with cyclin D1 promoter activity in an LMP1-dependent manner. The physiological functions of the intact complex were associated with cell proliferation and cell cycle progression. These findings suggest that TIF2 is a novel binding partner for nuclear EGFR and is involved in regulating its target gene expression.
|Role of PU.1 in MHC class II expression through transcriptional regulation of class II transactivator pI in dendritic cells. |
Nao Kitamura,Hokuto Yokoyama,Takuya Yashiro,Nobuhiro Nakano,Makoto Nishiyama,Shunsuke Kanada,Tatsuo Fukai,Mutsuko Hara,Shigaku Ikeda,Hideoki Ogawa,Ko Okumura,Chiharu Nishiyama
The Journal of allergy and clinical immunology 129 2012
PU.1 is a hematopoietic cell-specific transcription factor belonging to the Ets family. We hypothesized that PU.1 is involved in MHC class II expression in dendritic cells (DCs).
|The novel function of HINFP as a co-activator in sterol-regulated transcription of PCSK9 in HepG2 cells. |
Hai Li,Jingwen Liu
The Biochemical journal 443 2012
PCSK9 (proprotein convertase subtilisin/kexin type 9) plays an important role in control of plasma LDL (low-density lipoprotein) cholesterol metabolism by modulating the degradation of hepatic LDL receptor. Previous studies demonstrated that PCSK9 is a target gene of the SREBP2 [SRE (sterol-regulatory element)-binding protein 2] that activates PCSK9 gene transcription through an SRE motif of the promoter. In addition to SREBP2, HNF1α (hepatic nuclear factor 1α) positively regulates PCSK9 gene transcription in hepatic cells through a binding site located 28 bp upstream from SRE. In the present study, we have identified a novel HINFP (histone nuclear factor P) recognition motif residing between the HNF1 motif and SRE that is essential for basal and sterol-regulated transcriptions of the PCSK9 promoter. Mutation of this motif lowers the basal promoter activity and abolishes the sterol-mediated repression as well as the SREBP2-induced activation of the PCSK9 promoter. We show further that the activity of SREBP2 in stimulating PCSK9 promoter activity is greatly enhanced by HINFP. Additional experiments suggest that HINFP and its cofactor NPAT (nuclear protein of the ataxia telangectasia mutated locus) form a functional complex, and NPAT may subsequently recruit the HAT (histone acetyltransferase) cofactor TRRAP (transformation/transactivation domain-associated protein) to facilitate the histone H4 acetylation of the PCSK9 promoter. Knockdown of HINFP, NPAT or TRRAP each markedly reduces the amount of acetylated histone H4 on the PCSK9 promoter region and lowers PCSK9 protein levels. Importantly, by utilizing co-immunoprecipitation assays, we have demonstrated a direct interaction between SREBP2 and HINFP and its cofactors NPAT/TRRAP. Taken together, these new findings identify HINFP as a co-activator in SREBP-mediated transactivation of PCSK9 gene expression.
|Influence of epigenetic modifications of the interleukin-10 promoter on IL10 gene expression. |
Lena Larsson,Sara Thorbert-Mros,Lars Rymo,Tord Berglundh
European journal of oral sciences 120 2012
Epigenetic modifications of DNA and its associated proteins influence gene expression. The -1087 interleukin-10 (IL10) gene polymorphism is associated with differences in IL10 expression. The objectives of this study were to analyze the effect of DNA methylation and histone modifications on IL10 gene expression, the differences in epigenetic modifications between GG and AA genotypes of the -1087 IL10 gene polymorphism, and the methylation pattern in the region close to the -1087 position. Using B cells obtained from subjects with GG and AA genotypes we demonstrated that treatment with histone deacetylase inhibitors and 5-aza-2-deoxycytidine resulted in an increase in the production of IL10 mRNA. The chromatin immunoprecipitation assay revealed that stimulation with lipopolysaccharide resulted in a higher fold increase in the acetylation of histone H4 and in the methylation of histone H3 for GG genotype cells than for AA genotype cells. The increase in acetylation of histone H3 was larger for AA genotype cells than for GG genotype cells. For unstimulated cells the acetylation and methylation of histone H3 were higher for GG genotype cells than for AA genotype cells, while AA genotype cells had a higher increase in acetylation of histone H4. DNA methylation assays revealed that the three CpG sites distal to the -1087 site were methylated in blood cells and gingival tissues.
|Amyloid precursor protein is a biomarker for transformed human pluripotent stem cells. |
Venkataramani, Vivek, et al.
Am. J. Pathol., 180: 1636-52 (2012) 2012
Increasing evidence suggests an important function of the β-amyloid precursor protein (APP) in malignant disease in humans; however, the biological basis for this evidence is not well understood at present. To understand the role of APP in transformed pluripotent stem cells, we studied its expression levels in human testicular germ cell tumors using patient tissues, model cell lines, and an established xenograft mouse model. In the present study, we demonstrate the cooperative expression of APP with prominent pluripotency-related genes such as Sox2, NANOG, and POU5F1 (Oct3/4). The closest homologue family member, APLP2, showed no correlation to these stem cell factors. In addition, treatment with histone deacetylase (HDAC) inhibitors suppressed the levels of APP and stem cell markers. Loss of pluripotency, either spontaneously or as a consequence of treatment with an HDAC inhibitor, was accompanied by decreased APP protein levels both in vitro and in vivo. These observations suggest that APP represents a novel and specific biomarker in human transformed pluripotent stem cells that can be selectively modulated by HDAC inhibitors.
|Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast. |
Chhabi K Govind,Daniel Ginsburg,Alan G Hinnebusch
Methods in molecular biology (Clifton, N.J.) 833 2012
Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.
|IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome. |
Laura Belloni,Lena Allweiss,Francesca Guerrieri,Natalia Pediconi,Tassilo Volz,Teresa Pollicino,Joerg Petersen,Giovanni Raimondo,Maura Dandri,Massimo Levrero
The Journal of clinical investigation 122 2012
HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.
|Biochemical inhibition of the acetyltransferases ATase1 and ATase2 reduces β-secretase (BACE1) levels and Aβ generation. |
Yun Ding,Mi Hee Ko,Mariana Pehar,Frank Kotch,Noel R Peters,Yun Luo,Shahriar M Salamat,Luigi Puglielli
The Journal of biological chemistry 287 2012
The cellular levels of β-site APP cleaving enzyme 1 (BACE1), the rate-limiting enzyme for the generation of the Alzheimer disease (AD) amyloid β-peptide (Aβ), are tightly regulated by two ER-based acetyl-CoA:lysine acetyltransferases, ATase1 and ATase2. Here we report that both acetyltransferases are expressed in neurons and glial cells, and are up-regulated in the brain of AD patients. We also report the identification of first and second generation compounds that inhibit ATase1/ATase2 and down-regulate the expression levels as well as activity of BACE1. The mechanism of action involves competitive and non-competitive inhibition as well as generation of unstable intermediates of the ATases that undergo degradation.
|Silencing of Wnt5a during colon cancer metastasis involves histone modifications. |
Li, Qian and Chen, Hong
Epigenetics, 7: 551-8 (2012) 2012
Colorectal cancer (CRC) is the third most common cancer in the United States. Approximately 90% of colon cancer deaths arise from the metastasis of primary tumors. Aberrant expression of Wnt5a, one of the WNT signaling factors, has been reported during colon cancer development and progression. We found that both mRNA and protein expression of Wnt5a were decreased in the highly metastatic human colon cancer cell line SW620 compared with the non-metastatic human colon cancer cell SW480. This study tested the hypothesis that the silencing of Wnt5a in metastatic human colon cancer cells is related to altered epigenetic modifications. Wnt5a expression was not responsive to DNA methyltransferase inhibitor 5-aza-cytidine treatment. However, histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate (NaBt) significantly increased Wnt5a mRNA expression in SW620. Importantly, lower transcription of Wnt5a in SW620 than SW480 corresponded to multiple histone modifications, including lower levels of acetylated histone H3, H4 and H3K4me2 and higher levels of H3K27me3 in the promoter region. The increase of H3Ac, H4Ac and H3K4me2 after NaBt treatment in SW620 confirmed the involvement of histone modifications in the transcriptional regulation of Wnt5a. Additionally, NaBt treatment increased β-catenin signaling and diminished the difference in cell adhesion ability between non-metastatic SW480 and metastatic SW620, suggesting that the HDAC inhibitor plays critical roles in the WNT signaling pathway and cell physiology that relate to metastasis. In conclusion, our study suggests the importance of Wnt5a in colon cancer metastasis and also indicates that Wnt5a silencing in the highly invasive human colon cancer cell line might result from transcriptional regulation of the gene by histone modifications.
|Genome-Wide Approaches Reveal Functional Interleukin-4-Inducible STAT6 Binding to the Vascular Cell Adhesion Molecule 1 Promoter. |
Tozawa H, Kanki Y, Suehiro J, Tsutsumi S, Kohro T, Wada Y, Aburatani H, Aird WC, Kodama T, Minami T.
Molecular and cellular biology 31 2196-209 2011
Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis and inflammation. Here, we show that interleukin-4 (IL-4) markedly induced vascular cell adhesion molecule 1 (VCAM-1), both in cultured endothelial cells and in the intact endothelium in mice. Combined treatment with IL-4 and tumor necrosis factor alpha (TNF-α) resulted in further, sustained induction of VCAM-1 expression. IL-4-mediated induction of VCAM-1 and secondary monocyte adhesion was predominantly regulated by the transcription factor STAT6. Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin-immunoprecipitation with deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) in endothelial cells revealed regions of transient and sustained transcription factor binding. Through the combination of DNA microarrays and ChIP-seq at the same time points, the majority of IL-4-responsive genes were shown to be STAT6 dependent and associated with direct STAT6 binding to their promoter. IL-4-mediated stable binding of STAT6 led to sustained target gene expression. Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within 16 kb upstream of the VCAM-1 gene. Taken together, these findings support a critical role for STAT6 in mediating IL-4 signal transduction in endothelial cells. Identification of a novel IL-4-mediated VCAM-1 enhancer may provide a foundation for targeted therapy in vascular disease.
|The mouse RANKL gene locus is defined by a broad pattern of histone H4 acetylation and regulated through distinct distal enhancers. |
Martowicz ML, Meyer MB, Pike JW.
Journal of cellular biochemistry 112 2030-45 2011
RANKL is a stromal cell-derived tumor necrosis factor (TNF)-like factor that plays a primary role in osteoclast formation and function. Recent studies suggest that 1,25(OH)(2) D(3) induces Rankl expression via vitamin D receptor (VDR) interaction at several enhancers located up to 76 kb upstream of the gene's transcriptional start site (TSS). In the current studies, we explored these interactions further using ChIP-chip and RNA analysis. We confirm VDR and RXR binding to the five enhancers described previously and identify two additional sites, one located within the Rankl coding region. We also show that RNA polymerase II is recruited to these enhancers, most likely through transcription factors TBP, TFIIB, and TAF(II) 250. Interestingly, the recruitment of these factors leads to the production of RNA transcripts, although their role at present is unknown. We also discovered that histone H4 acetylation (H4ac) marks many upstream Rankl enhancers under basal conditions and that H4ac is increased upon 1,25(OH)(2) D(3) treatment. Surprisingly, the hormone also induces C/EBPβ binding across the Rankl locus. C/EBPβ binding correlates directly with increased H4ac activity following 1,25(OH)(2) D(3) treatment. Finally, elevated H4ac is restricted to an extended region located between two potential insulator sites occupied by CTCF and Rad21. These data suggest a mechanism whereby 1,25(OH)(2) D(3) functions via the VDR and C/EBPβ to upregulate Rankl expression. J. Cell. Biochem. 112: 2030-2045, 2011. © 2011 Wiley-Liss, Inc.
|Perturbation of BRD4 protein function by BRD4-NUT protein abrogates cellular differentiation in NUT midline carcinoma. |
Yan J, Diaz J, Jiao J, Wang R, You J
The Journal of biological chemistry 286 27663-75. Epub 2011 Jun 7. 2011
NUT midline carcinoma (NMC) belongs to a class of highly lethal and poorly differentiated epithelial cancers arising mainly in human midline organs. NMC is caused by the chromosome translocation-mediated fusion of the NUT (nuclear protein in testis) gene on chromosome 15 to a few other genes, most frequently the BRD4 gene on chromosome 19. The mechanism by which the BRD4-NUT fusion product blocks NMC cellular differentiation and contributes to oncogenesis remains elusive. In this study, we show that BRD4-NUT and BRD4 colocalize in discrete nuclear foci that are hyperacetylated but transcriptionally inactive. BRD4-NUT recruits histone acetyltransferases to induce histone hyperacetylation in these chromatin foci, which provide docking sites for accumulation of additional BRD4 and associated P-TEFB (positive transcription elongation factor b) complexes in the transcriptionally inactive BRD4-NUT foci. These molecular events lead to repression of a BRD4·P-TEFB downstream target gene c-fos, a component of activator protein 1 (AP-1), that directly regulates epithelial differentiation. Knockdown of BRD4-NUT in NMC cells disperses the transcriptionally inactive chromatin foci and releases the transcriptional activators to stimulate c-fos expression, leading to restoration of cellular differentiation. Our study provides a novel mechanism by which the BRD4-NUT oncogene perturbs BRD4 functions to block cellular differentiation and to contribute to the oncogenic progression in the highly aggressive NMC.
|p53-paralog DNp73 oncogene is repressed by IFNαSTAT2 through the recruitment of the Ezh2 polycomb group transcriptional repressor. |
Testoni B, Schinzari V, Guerrieri F, Gerbal-Chaloin S, Blandino G, Levrero M
The DNp73 proteins act as trans-repressors of p53 and p73-dependent transcription and exert both anti-apoptotic activity and pro-proliferative activity. DNp73s are frequently up-regulated in a variety of human cancers, including human hepatocellular carcinomas (HCCs). Increased levels of DNp73 proteins confer to HCC cells resistance to apoptosis and, irrespective to p53 status, a chemoresistant phenotype. Here, we show that interferon (IFN)α down-regulates DNp73 expression in primary human hepatocytes (PHHs) and HCC cell lines. IFNα has been used as pro-apoptotic agent in the treatment of malignancies and there is increasing evidence of IFNα effectiveness in HCC treatment and prevention of recurrence. The precise mechanisms by which class I IFNs exert their anti-proliferative and anti-tumor activity remain unclear. IFNα binding to its receptor activates multiple intracellular signaling cascades regulating the transcription of numerous direct target genes through the recruitment of a complex comprising of STAT1, STAT2 and IFN regulatory factor (IRF)9 to their promoters. We found that, in response to IFNα, the P2p73 promoter undergoes substantial chromatin remodeling. Histone deacetylases (HDACs) replace histone acetyl transferases. STAT2 is recruited onto the endogenous P2p73 promoter together with the polycomb group protein Ezh2, leading to increased H3K27 methylation and transcriptional repression. The reduction of DNp73 levels by IFNα is paralleled by an increased susceptibility to IFNα-triggered apoptosis of Huh7 hepatoma cells. Our results show, for the first time, that IFN-stimulated gene factor 3 recruitment may serve both in activating and repressing gene expression and identify the down-regulation of DNp73 as an additional mechanism to counteract the chemoresistance of liver cancer cells.Oncogene advance online publication, 14 March 2011; doi:10.1038/onc.2010.635.
|H3K4 trimethylation by set1 promotes efficient termination by the nrd1-Nab3-Sen1 pathway. |
Terzi N, Churchman LS, Vasiljeva L, Weissman J, Buratowski S
Molecular and cellular biology 2011
In Saccharomyces cerevisiae, the Nrd1-Nab3-Sen1 pathway mediates termination of snoRNAs and cryptic unstable transcripts (CUTs). Both Nrd1 and the Set1 histone H3K4 methyltransferase complex interact with the RNA polymerase II (Pol II) during early elongation, leading us to test whether these two processes are functionally linked. Deletion of SET1 exacerbates the growth rate and termination defects of nrd1 mutants. Set1 is important for appropriate recruitment of Nrd1. Additionally, Set1 modulates histone acetylation levels in the promoter proximal region via the Rpd3L deacetylase and NuA3 acetyltransferase complexes, both of which contain PHD finger proteins that bind methylated H3K4. Increased levels of histone acetylation reduce the efficiency of Nrd1-dependent termination. We speculate that Set1 promotes proper early termination by the Nrd1-Nab3-Sen1 complex by affecting the kinetics of Pol II transcription in early elongation.
|Secreted frizzled-related protein-5 is epigenetically downregulated and functions as a tumor suppressor in kidney cancer. |
Kazumori Kawakami,Soichiro Yamamura,Hiroshi Hirata,Koji Ueno,Sharanjot Saini,Shahana Majid,Yuichiro Tanaka,Ken Kawamoto,Hideki Enokida,Masayuki Nakagawa,Rajvir Dahiya
International journal of cancer. Journal international du cancer 128 2011
Secreted frizzled-related protein-5 (sFRP-5) has been identified as 1 of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated, whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.
|Mouse Rankl expression is regulated in T cells by c-Fos through a cluster of distal regulatory enhancers designated the T cell control region. |
Bishop KA, Coy HM, Nerenz RD, Meyer MB, Pike JW
The Journal of biological chemistry 2011
Rankl is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. While T cell activation upregulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this upregulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II (RNA pol II) density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between -123 to -156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-κB and NFAT. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-κB was observed at the TSS and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, 3C analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.
|Specific histone lysine 4 methylation patterns define TR-binding capacity and differentiate direct T3 responses. |
Bilesimo P, Jolivet P, Alfama G, Buisine N, Le Mevel S, Havis E, Demeneix BA, Sachs LM
Mol Endocrinol 25 225-37. Epub 2011 Jan 14. 2011
The diversity of thyroid hormone T(3) effects in vivo makes their molecular analysis particularly challenging. Indeed, the current model of the action of T(3) and its receptors on transcription does not reflect this diversity. Here, T(3)-dependent amphibian metamorphosis was exploited to investigate, in an in vivo developmental context, how T(3) directly regulates gene expression. Two, direct positively regulated T(3)-response genes encoding transcription factors were analyzed: thyroid hormone receptor β (TRβ) and TH/bZIP. Reverse transcription-real-time quantitative PCR analysis on Xenopus tropicalis tadpole brain and tail fin showed differences in expression levels in premetamorphic tadpoles (lower for TH/bZIP than for TRβ) and differences in induction after T(3) treatment (lower for TRβ than for TH/bZIP). To dissect the mechanisms underlying these differences, chromatin immunoprecipitation was used. T(3) differentially induced RNA polymerase II and histone tail acetylation as a function of transcriptional level. Gene-specific patterns of TR binding were found on the different T(3) -responsive elements (higher for TRβ than for TH/bZIP), correlated with gene-specific modifications of H3K4 methylation (higher for TRβ than for TH/bZIP). Moreover, tissue-specific modifications of H3K27 were found (lower in brain than in tail fin). This first in vivo analysis of the association of histone modifications and TR binding/gene activation during vertebrate development for any nuclear receptor indicate that chromatin context of thyroid-responsive elements loci controls the capacity to bind TR through variations in histone H3K4 methylation, and that the histone code, notably H3, contributes to the fine tuning of gene expression that underlies complex physiological T(3) responses.
|Chromatin Immunoprecipitation (ChIP)||Xenopus||21239616|
|A small molecule differentiation inducer increases insulin production by pancreatic β cells. |
Elhadji M Dioum,Jihan K Osborne,Sean Goetsch,Jamie Russell,Jay W Schneider,Melanie H Cobb
Proceedings of the National Academy of Sciences of the United States of America 108 2011
New drugs for preserving and restoring pancreatic β-cell function are critically needed for the worldwide epidemic of type 2 diabetes and the cure for type 1 diabetes. We previously identified a family of neurogenic 3,5-disubstituted isoxazoles (Isx) that increased expression of neurogenic differentiation 1 (NeuroD1, also known as BETA2); this transcription factor functions in neuronal and pancreatic β-cell differentiation and is essential for insulin gene transcription. Here, we probed effects of Isx on human cadaveric islets and MIN6 pancreatic β cells. Isx increased the expression and secretion of insulin in islets that made little insulin after prolonged ex vivo culture and increased expression of neurogenic differentiation 1 and other regulators of islet differentiation and insulin gene transcription. Within the first few hours of exposure, Isx caused biphasic activation of ERK1/2 and increased bulk histone acetylation. Although there was little effect on histone deacetylase activity, Isx increased histone acetyl transferase activity in nuclear extracts. Reconstitution assays indicated that Isx increased the activity of the histone acetyl transferase p300 through an ERK1/2-dependent mechanism. In summary, we have identified a small molecule with antidiabetic activity, providing a tool for exploring islet function and a possible lead for therapeutic intervention in diabetes.
|Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii. |
Daniela Strenkert,Stefan Schmollinger,Michael Schroda
Plant methods 7 2011
ABSTRACT: We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.Full Text Article
|Direct Inhibition of TNF-α Promoter Activity by Fanconi Anemia Protein FANCD2. |
Matsushita N, Endo Y, Sato K, Kurumizaka H, Yamashita T, Takata M, Yanagi S.
PloS one 6 e23324 2011
Fanconi anemia (FA), an inherited disease, is associated with progressive bone marrow failure, predisposition to cancer, and genomic instability. Genes corresponding to 15 identified FA complementation groups have been cloned, and each gene product functions in the response to DNA damage induced by cross-linking agents and/or in protection against genome instability. Interestingly, overproduction of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and aberrant activation of NF-κB-dependent transcriptional activity have been observed in FA cells. Here we demonstrated that FANCD2 protein inhibits NF-κB activity in its monoubiquitination-dependent manner. Furthermore, we detected a specific association between FANCD2 and an NF-κB consensus element in the TNF-α promoter by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assay. Therefore, we propose FANCD2 deficiency promotes transcriptional activity of the TNF-α promoter and induces overproduction of TNF-which then sustains prolonged inflammatory responses. These results also suggest that artificial modulation of TNFα production could be a promising therapeutic approach to FA.
|Dynamics of DNA methylation and Histone H4 acetylation during floral bud differentiation in azalea. |
Meijón M, Feito I, Valledor L, Rodríguez R, Cañal MJ
BMC Plant Biol 10 10. 2010
BACKGROUND: The ability to control the timing of flowering is a key strategy for planning production in ornamental species such as azalea, however it requires a thorough understanding of floral transition. Floral transition is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Dynamic changes between chromatin states facilitating or inhibiting DNA transcription regulate the expression of floral induction pathways in response to environmental and developmental signals. DNA methylation and histone modifications are involved in controlling the functional state of chromatin and gene expression.Full Text Article
|CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer. |
Wang J, Liu X, Wu H, Ni P, Gu Z, Qiao Y, Chen N, Sun F, Fan Q
Nucleic Acids Res 2010
Long non-coding RNA (lncRNA), highly up-regulated in liver cancer (HULC) plays an important role in tumorigenesis. Depletion of HULC resulted in a significant deregulation of several genes involved in liver cancer. Although up-regulation of HULC expression in hepatocellular carcinoma has been reported, the molecular mechanisms remain unknown. In this study, we used in vivo and in vitro approaches to characterize cancer-dependent alterations in the chromatin organization and find a CREB binding site (encompassing from -67 to -53 nt) in the core promoter. Besides, we also provided evidence that PKA pathway may involved in up-regulation of HULC. Furthermore, we demonstrated HULC may act as an endogenous \'sponge\', which down-regulates a series of microRNAs (miRNAs) activities, including miR-372. Inhibition of miR-372 leads to reducing translational repression of its target gene, PRKACB, which in turn induces phosphorylation of CREB. Over-expression of miR-372 decreases the association of CREB with the proximal promoter, followed by the dissociation of P300, resulting in a change of the histone \'code\', such as in deacetylation and methylation. The study elucidates that fine tuning of HULC expression is part of an auto-regulatory loop in which it\'s inhibitory to expression and activity of miR-372 allows lncRNA up-regulated expression in liver cancer.
|Animals lacking link protein have attenuated perineuronal nets and persistent plasticity. |
Carulli D, Pizzorusso T, Kwok JC, Putignano E, Poli A, Forostyak S, Andrews MR, Deepa SS, Glant T, Fawcett JW
Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage link protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.
|15-hydroxyeicosatetraenoic acid is a preferential peroxisome proliferator-activated receptor beta/delta agonist. |
S Naruhn, W Meissner, T Adhikary, K Kaddatz, T Klein, B Watzer, S Muller-Brusselbach, R Muller
Molecular pharmacology 77 171-84 2010
Peroxisome proliferator-activated receptor (PPARs) modulate target gene expression in response to unsaturated fatty acid ligands, such as arachidonic acid (AA). Here, we report that the AA metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) activates the ligand-dependent activation domain (AF2) of PPARbeta/delta in vivo, competes with synthetic agonists in a PPARbeta/delta ligand binding assay in vitro, and triggers the interaction of PPARbeta/delta with coactivator peptides. These agonistic effects were also seen with PPARalpha and PPARgamma, but to a significantly weaker extent. We further show that 15-HETE strongly induces the expression of the bona fide PPAR target gene Angptl4 in a PPARbeta/delta-dependent manner and, conversely, that inhibition of 15-HETE synthesis reduces PPARbeta/delta transcriptional activity. Consistent with its function as an agonistic ligand, 15-HETE triggers profound changes in chromatin-associated PPARbeta/delta complexes in vivo, including the recruitment of the coactivator cAMP response element-binding protein binding protein. Both 15R-HETE and 15S-HETE are similarly potent at inducing PPARbeta/delta coactivator binding and transcriptional activation, indicating that 15-HETE enantiomers generated by different pathways function as PPARbeta/delta agonists.
|Jmjd1a Demethylase-regulated Histone Modification Is Essential for cAMP-response Element Modulator-regulated Gene Expression and Spermatogenesis. |
Liu Z, Zhou S, Liao L, Chen X, Meistrich M, Xu J
The Journal of biological chemistry 285 2758-70 2010
Spermatogenesis, a fundamental process in the male reproductive system, requires a series of tightly controlled epigenetic and genetic events in germ cells ranging from spermatogonia to spermatozoa. Jmjd1a is a key epigenetic regulator expressed in the testis. It specifically demethylates mono- and di-methylated histone H3 lysine 9 (H3K9me1 and H3K9me2) but not tri-methylated H3K9 (H3K9me3). In this study, we generated a Jmjd1a antibody for immunohistochemistry and found Jmjd1a was specifically produced in pachytene and secondary spermatocytes. Disruption of the Jmjd1a gene in mice significantly increased H3K9me1 and H3K9me2 levels in pachytene spermatocytes and early elongating spermatids without affecting H3K9me3 levels. Concurrently, the levels of histone acetylation were decreased in Jmjd1a knock-out germ cells. This suggests Jmjd1a promotes transcriptional activation by lowering histone methylation and increasing histone acetylation. Interestingly, the altered histone modifications in Jmjd1a-deficient germ cells caused diminished cAMP-response element modulator (Crem) recruitment to chromatin and decreased expression of the Crem coactivator Act and their target genes Tnp1 (transition protein 1), Tnp2, Prm1 (protamine 1), and Prm2, all of which are essential for chromatin condensation in spermatids. In agreement with these findings, Jmjd1a deficiency caused extensive germ cell apoptosis and blocked spermatid elongation, resulting in severe oligozoospermia, small testes, and infertility in male mice. These results indicate that the Jmjd1a-controlled epigenetic histone modifications are crucial for Crem-regulated gene expression and spermatogenesis.Full Text Article
|Valproate treatment of human cord blood CD4-positive effector T cells confers on them the molecular profile (microRNA signature and FOXP3 expression) of natural regulatory CD4-positive cells through inhibition of histone deacetylase. |
Hussein Fayyad-Kazan,Redouane Rouas,Makram Merimi,Nabil El Zein,Philippe Lewalle,Fadi Jebbawi,Mohamad Mourtada,Hussein Badran,Mohamad Ezzeddine,Bruno Salaun,Pedro Romero,Arsène Burny,Philippe Martiat,Bassam Badran
The Journal of biological chemistry 285 2010
Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.Full Text Article
|Oxidized Low-Density Lipoprotein-Induced Matrix Metalloproteinase-9 Expression via PKC-delta/p42/p44 MAPK/Elk-1 Cascade in Brain Astrocytes. |
HH Wang, HL Hsieh, CY Wu, CM Yang
Neurotoxicity research 17 50-65 2010
After ischemic injury to brain, disruption of the blood-brain barrier (BBB) raises the possibility of exposing the central nervous system (CNS) to oxidized low-density lipoprotein (oxLDL), a risk factor implicated in neurodegenerative diseases. Matrix metalloproteinases (MMPs), especially MMP-9, contribute to extracellular matrix (ECM) remodeling during the CNS diseases. However, the molecular mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes remained unclear. Here, we reported that oxLDL induced MMP-9 expression via a PKC-delta/p42/p44 MAPK-dependent Elk-1 activation in rat brain astrocyte (RBA)-1 cells, revealed by gelatin zymography, RT-PCR, and Western blotting analyses. These responses were attenuated by pretreatment with pharmacological inhibitors and transfection with dominant negative mutants. Moreover, Elk-1-mediated MMP-9 gene transcription was confirmed by transfection with an Elk-1 binding site-mutated MMP-9 promoter construct (mt-Ets-MMP9), which blocked oxLDL-stimulated MMP-9 luciferase activity. Understanding the regulatory mechanisms by which oxLDL induced MMP-9 expression in astrocytes might provide a new therapeutic strategy of brain diseases.
|Distal regions of the human IFNG locus direct cell type-specific expression. |
Collins PL, Chang S, Henderson M, Soutto M, Davis GM, McLoed AG, Townsend MJ, Glimcher LH, Mortlock DP, Aune TM
J Immunol 185 1492-501. Epub 2010 Jun 23. 2010
Genes, such as IFNG, which are expressed in multiple cell lineages of the immune system, may employ a common set of regulatory elements to direct transcription in multiple cell types or individual regulatory elements to direct expression in individual cell lineages. By employing a bacterial artificial chromosome transgenic system, we demonstrate that IFNG employs unique regulatory elements to achieve lineage-specific transcriptional control. Specifically, a one 1-kb element 30 kb upstream of IFNG activates transcription in T cells and NKT cells but not in NK cells. This distal regulatory element is a Runx3 binding site in Th1 cells and is needed for RNA polymerase II recruitment to IFNG, but it is not absolutely required for histone acetylation of the IFNG locus. These results support a model whereby IFNG uses cis-regulatory elements with cell type-restricted function.Full Text Article
|Variations in DNA methylation, acetylated histone H4, and methylated histone H3 during Pinus radiata needle maturation in relation to the loss of in vitro organogenic capability. |
Luis Valledor,Mónica Meijón,Rodrigo Hasbún,Maria Jesús Cañal,Roberto Rodríguez
Journal of plant physiology 167 2010
Needle differentiation is a very complex process associated with the formation of a mature photosynthetic organ. From meristem differentiation to leaf maturation, gene control must play an important role switching required genes on and off to define tissue functions, with the epigenetic code being one of the main regulation mechanisms. In this work, we examined the connections between the variation in the levels of some epigenetic players (DNA methylation, acetylated histone H4 and histone H3 methylation at Lys 4 and Lys 9) at work during needle maturation. Our results indicate that needle maturation, which is associated with a decrease in organogenic capability, is related to an increase in heterochromatin-related epigenetic markers (high DNA methylation and low acetylated histone H4 levels, and the presence of histone H3 methylated at lys 9). Immunohistochemical analyses also showed that the DNA methylation of palisade parenchyma cell layers during the transition from immature to mature scions is associated with the loss of the capacity to induce adventitious organs.
|Genetic and maternal effects on valproic acid teratogenesis in C57BL/6J and DBA/2J mice. |
Downing C, Biers J, Larson C, Kimball A, Wright H, Ishii T, Gilliam D, Johnson T
Toxicol Sci 116 632-9. Epub 2010 May 10. 2010
Valproic acid (VPA) is used worldwide to treat epilepsy, migraine headaches, and bipolar disorder. However, VPA is teratogenic and in utero exposure can lead to congenital malformations. Using inbred C57BL/6J (B6) and DBA/2J (D2) mice, we asked whether genetic variation could play a role in susceptibility to VPA teratogenesis. Whereas B6 fetuses were more susceptible than D2 fetuses to digit and vertebral malformations, D2 fetuses were more susceptible to rib malformations. In a reciprocal cross between B6 and D2, genetically identical F1 mice carried in a B6 mother had a greater percentage of vertebral malformations following prenatal VPA exposure than F1 mice carried in a D2 mother. This reciprocal F1 difference is known as a maternal effect and shows that maternal genotype/uterine environment is an important mediator of VPA teratogenecity. VPA is a histone deacetylase inhibitor, and it is possible that the differential teratogenesis in B6 and D2 is because of strain differences in histone acetylation. We observed strain differences in acetylation of histones H3 and H4 in both embryo and placenta following in utero VPA exposure, but additional studies are needed to determine the significance of these changes in mediating teratogenesis. Our results provide additional support that genetic factors, both maternal and fetal, play a role in VPA teratogenesis. Lines of mice derived from B6 and D2 will be a useful model for elucidating the genetic architecture underlying susceptibility to VPA teratogenesis.Full Text Article
|Epigenetic Regulation of Thy-1 by Histone Deacetylase Inhibitor in Rat Lung Fibroblasts. |
Sanders YY, Tollefsbol TO, Varisco BM, Hagood JS
Am J Respir Cell Mol Biol 2010
Thy-1 is a cell surface glycoprotein present on normal lung fibroblasts but absent from the fibroblastic foci of idiopathic pulmonary fibrosis (IPF). Thy-1 correlates inversely with fibrogenic phenotypic characteristics and function as a âfibrosis suppressorâ. Promoter region hypermethylation can silence Thy-1 expression in fibroblastic foci, suggesting that epigenetic regulation is important in programming the fibrotic phenotype. We examined whether histone modifications are important in regulating Thy-1 expression in lung fibroblasts. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) restored Thy-1 expression in Thy-1- cells in a time- and dosage- dependent fashion, and was associated with enrichment of histone acetylation. Chromatin immunoprecipitation (ChIP) demonstrated Thy-1 depletion of trimethylated H3K27 after 24 hours of TSA treatment, concurrent with enrichment of trimethylated H3K4 and acetylated H4. Bisulfite sequencing of the Thy-1 promoter region revealed demethylation of the previously hypermethylated CpG sites after treatment with TSA. Although Thy-1 was hypermethylated in Thy-1- lung fibroblasts, we observed that Thy-1- cells have lower global DNA methylation when compared to Thy-1+ lung fibroblasts, which was partially reversed by TSA treatment. TSA treatment upregulates total methyltransferases activity in these cells. Our data indicate that Thy-1 silencing is regulated by histone modifications in addition to promoter hypermethylation in lung fibroblasts. Additionally, our findings indicate that alteration of histone modifications alters DNA methylation. Understanding the molecular hierarchy of events with respect to reactivation of transcription and reversal of histone modification will be critical to understanding and modifying the regulated expression of Thy-1, a tumor- and fibrosis-suppressor gene.
|Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation. |
Hydbring P, Bahram F, Su Y, TronnersjÃ¶ S, HÃ¶gstrand K, von der Lehr N, Sharifi HR, Lilischkis R, Hein N, Wu S, Vervoorts J, Henriksson M, Grandien A, LÃ¼scher B, Larsson LG
Proceedings of the National Academy of Sciences of the United States of America 107 58-63. Epub 2009 Dec 4. 2010
The MYC and RAS oncogenes are frequently activated in cancer and, together, are sufficient to transform rodent cells. The basis for this cooperativity remains unclear. We found that although Ras interfered with Myc-induced apoptosis, Myc repressed Ras-induced senescence, together abrogating two main barriers of tumorigenesis. Inhibition of cellular senescence required phosphorylation of Myc at Ser-62 by cyclin E/cyclin-dependent kinase (Cdk) 2. Cdk2 interacted with Myc at promoters, where it affected Myc-dependent regulation of genes, including Bmi-1, p16, p21, and hTERT, which encode proteins known to control senescence. Repression of senescence by Myc was abrogated by the Cdk inhibitor p27Kip1, which is induced by antiproliferative signals like IFN-gamma or by pharmacological inhibitors of Cdk2 but not by inhibitors of other Cdks. In contrast, a phospho-mimicking Myc-S62D mutant was resistant to these manipulations. Inhibition of cyclin E/Cdk2 reversed the senescence-associated gene expression pattern imposed by Myc/cyclin E/Cdk2. This indicates a role of Cdk2 as a transcriptional cofactor and activator of the antisenescence function of Myc and provides mechanistic insight into the Myc-p27Kip1 antagonism. Finally, our findings highlight that pharmacological inhibition of Cdk2 activity is a potential therapeutical principle for cancer therapy, in particular for tumors with activated Myc or Ras.
|Maintenance of a constitutive heterochromatin domain in vertebrates by a Dicer-dependent mechanism. |
KE Giles, R Ghirlando, G Felsenfeld
Nature cell biology 12 94-9; sup pp 1-6 2010
The 16 kilobase (kb) heterochromatin domain between the chicken beta-globin locus and the folate receptor gene is used here to study the roles of RNA-dependent mechanisms and histone modifications in the maintenance of a constitutive heterochromatic structure. Inhibition of histone deacetylase (HDAC) activity is shown to both increase intergenic transcription and render the heterochromatin more accessible to MspI digestion. We show that short interfering RNA (siRNA)-mediated downregulation of the enzyme Dicer has similar effects: histone acetylation is increased, transcript levels rise and the compact chromatin structure becomes more accessible to restriction endonucleases. We also show that the chicken Argonaute 2 homologue binds the 16 kb region in a Dicer-dependent manner and is necessary for a condensed chromatin structure. Heterochromatic domains of this kind, which are widely distributed in vertebrate genomes, thus seem to be maintained in their condensed form by highly conserved mechanisms.
|A novel mammalian complex containing Sin3B mitigates histone acetylation and RNAPII progression within transcribed loci. |
Jelinic P, Pellegrino J, David G
Mol Cell Biol 2010
Transcription requires the progression of RNA-polymerase II (RNAPII) through a permissive chromatin structure. Recent studies in S. cerevisiae have demonstrated that the yeast Sin3 protein contributes to the restoration of the repressed chromatin structure at actively transcribed loci. Yet, the mechanisms underlying the restoration of the repressive chromatin structure at transcribed loci and its significance in gene expression has not been investigated in mammals. We report here the identification of a mammalian complex containing the co-repressor Sin3B, the histone deacetylase HDAC1, Mrg15 and the PHD finger-containing Pf1 and show that this complex plays important roles in regulation of transcription. We demonstrate that this complex localizes at discrete loci approximately one kilobase downstream of the transcription start site of transcribed genes, and this localization requires both Pf1's and Mrg15's interaction with chromatin. Inactivation of this mammalian complex promotes increased RNAPII progression within transcribed regions and subsequent increased transcription. Our results define a novel mammalian complex that contributes to the regulation of transcription, and point to divergent uses of the Sin3 proteins homologues throughout evolution in the modulation of transcription.
|SP1 is required for basal activation and chromatin accessibility of CD151 promoter in liver cancer cells. |
Wang J, Liu X, Ni P, Gu Z, Fan Q
Biochemical and biophysical research communications 393 291-6 Epub 2010 Feb 10 2010
CD151 plays an important role in liver cancer metastasis. The mechanism on how CD151 is expressed remains unclear. Here we have identified SP1 is a protein functioning in constitutive activation of CD151. Applying a PCR-based chromatin accessibility assay, an open chromatin conformation was discovered localized around the transcription start site of the CD151 gene. Deletion constructs of the 5' flanking region were fused to a luciferase reporter gene. After transient transfection in HepG2 and Hep3B cells, a minimal region -171/-53 bearing three SP1-binding sites was identified as the core promoter. Results obtained from electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that SP1 is bound to the core promoter. Deletion of SP1 consensus sequence resulted in the total loss of the promoter activity. Moreover, knockdown of SP1 reduced both CD151 promoter activity and chromatin accessibility. Conclusively, SP1 is pivotal to CD151 transcription partly via the construction of a local open chromatin configuration across the promoter. 2010 Elsevier Inc. All rights reserved.
|Essential role of Tip60-dependent recruitment of ribonucleotide reductase at DNA damage sites in DNA repair during G1 phase. |
Niida H, Katsuno Y, Sengoku M, Shimada M, Yukawa M, Ikura M, Ikura T, Kohno K, Shima H, Suzuki H, Tashiro S, Nakanishi M
Genes & development 24 333-8 2010
A balanced deoxyribonucleotide (dNTP) supply is essential for DNA repair. Here, we found that ribonucleotide reductase (RNR) subunits RRM1 and RRM2 accumulated very rapidly at damage sites. RRM1 bound physically to Tip60. Chromatin immunoprecipitation analyses of cells with an I-SceI cassette revealed that RRM1 bound to a damage site in a Tip60-dependent manner. Active RRM1 mutants lacking Tip60 binding failed to rescue an impaired DNA repair in RRM1-depleted G1-phase cells. Inhibition of RNR recruitment by an RRM1 C-terminal fragment sensitized cells to DNA damage. We propose that Tip60-dependent recruitment of RNR plays an essential role in dNTP supply for DNA repair.Full Text Article
|Genome-wide analysis of the VDRRXR cistrome in osteoblast cells provides new mechanistic insight into the actions of the vitamin D hormone. |
Meyer MB, Goetsch PD, Pike JW
The Journal of steroid biochemistry and molecular biology 2010
The vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) in target cells and tissues by orchestrating the expression of gene networks responsible for vitamin D-induced phenotypes. The molecular mechanisms of these regulatory systems have been studied for decades under the principle that transcriptional regulation occurs near the transcriptional start site of the gene. However, this now appears to be an outdated view of transcriptional control. In this study, we examined the genome-wide chromatin immunoprecipitation on microarray (ChIP-chip) across pre-osteoblastic cells for VDR, retinoid X receptor (RXR), RNA polymerase II, and histone H4 acetylation (H4ac). We uncovered potential regulatory mechanisms for genes important to osteoblast biology as well as skeletal formation under the control of 1,25(OH)(2)D(3). We found that VDR, along with RXR and H4ac, binds to distal regions 43% of the time; and within gene introns and exons 44%, leaving only 13% of activation at traditional promoter regions. Here, we briefly summarize our findings for all the VDR/RXR cis-acting transcriptional elements (VDR/RXR cistrome) in pre-osteoblastic cells, MC3T3-E1, provide a few examples of this dynamic control by VDR and 1,25(OH)(2)D(3), and demonstrate that distal transcriptional control contributes to the majority of vitamin D(3)-mediated transcription. Copyright Â© 2010 Elsevier Ltd. All rights reserved.
|Asf1 can promote trimethylation of h3 k36 by set2. |
Lin LJ, Minard LV, Johnston GC, Singer RA, Schultz MC
Molecular and cellular biology 30 1116-29 2010
Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.Full Text Article
|Histone deacetylase 1 (HDAC1), but not HDAC2, controls embryonic stem cell differentiation. |
Dovey, Oliver M, et al.
Proc. Natl. Acad. Sci. U.S.A., 107: 8242-7 (2010) 2010
Histone deacetylases (HDAC) 1 and 2 are highly similar enzymes that help regulate chromatin structure as the core catalytic components of corepressor complexes. Although tissue-specific deletion of HDAC1 and HDAC2 has demonstrated functional redundancy, germ-line deletion of HDAC1 in the mouse causes early embryonic lethality, whereas HDAC2 does not. To address the unique requirement for HDAC1 in early embryogenesis we have generated conditional knockout embryonic stem (ES) cells in which HDAC1 or HDAC2 genes can be inactivated. Deletion of HDAC1, but not HDAC2, causes a significant reduction in the HDAC activity of Sin3A, NuRD, and CoREST corepressor complexes. This reduced corepressor activity results in a specific 1.6-fold increase in histone H3 K56 acetylation (H3K56Ac), thus providing genetic evidence that H3K56Ac is a substrate of HDAC1. In culture, ES cell proliferation was unaffected by loss of either HDAC1 or HDAC2. Rather, we find that loss of HDAC1 affects ES cell differentiation. ES cells lacking either HDAC1 or HDAC2 were capable of forming embryoid bodies (EBs), which stimulates differentiation into the three primary germ layers. However, HDAC1-deficient EBs were significantly smaller, showed spontaneous rhythmic contraction, and increased expression of both cardiomyocyte and neuronal markers. In summary, our genetic study of HDAC1 and HDAC2 in ES cells, which mimic the embryonic epiblast, has identified a unique requirement for HDAC1 in the optimal activity of HDAC1/2 corepressor complexes and cell fate determination during differentiation.
|Developmental changes in DNA methylation and covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin gene promoters in Papio anubis. |
Donald Lavelle, Kestis Vaitkus, Maria Hankewych, Mahipal Singh, Joseph DeSimone
Blood cells, molecules diseases 36 269-78 2006
The baboon is a suitable and relevant animal model to study the mechanism of human globin gene switching. This investigation addresses the role of DNA methylation and histone coding in globin gene switching in the baboon, Papio anubis. Bisulfite sequencing and chromatin immunoprecipitation studies were performed in erythroid cells purified from fetuses of varying gestational ages and from adult bone marrow to analyze the manner that changes in DNA methylation of the epsilon-, gamma-, and beta-globin promoters and association of ac-H3, ac-H4, H3-dimeK4, H3-dimeK36, and H3-dimeK79 with the epsilon-, gamma-, and beta-globin promoters occur during development. Changes in DNA methylation of the epsilon- and gamma-globin gene promoters during transitional stages of globin gene switching were consistent with the stochastic model of methylation and a role of DNA methylation in gene silencing. Enrichment of ac-H3, ac-H4, and pol II at the promoters of developmentally active genes was observed, while the pattern of distribution of H3-dimeK4 and H3-dimeK79 suggests that these modifications are found near both currently and formerly active promoters. Enrichment of H3-dimeK36 at the silenced epsilon-globin gene promoter was observed. These studies demonstrate that coordinated epigenetic modifications in the chromatin structure of the beta-like globin gene promoters accompany the highly regulated changes in expression patterns of these genes during development.
|How was this antibody made? What modifications will it recognize?||The Anti-acetyl-Histone H4 was raised against the tetra acetylated H4. This antibody will recognize intermediately acetylated H4, but we are unsure what the reactivity for the antibody is against tetra-acetylated H4. We know it will not recognize acetylation on lysine 16, however it does recognize acetylation on the other three sites. A protein does not require acetylation on all four sites for this antibody to recognize it.|