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  • Cellular and viral chromatin proteins are positive factors in the regulation of adenovirus gene expression. 20926393

    The adenovirus genome forms chromatin-like structure with viral core proteins. This complex supports only a low level of transcription in a cell-free system, and thus core proteins have been thought to be negative factors for transcription. The mechanism how the transcription from the viral DNA complexed with core proteins is activated in infected cells remains unclear. Here, we found that both core proteins and histones are bound with the viral DNA in early phases of infection. We also found that acetylation of histone H3 occurs at the promoter regions of viral active genes in a transcription-independent manner. In addition, when a plasmid DNA complexed with core proteins was introduced into cells, core proteins enhanced transcription. Knockdown of TAF-I, a remodeling factor for viral core protein-DNA complexes, reduces the enhancement effect by core proteins, indicating that core proteins positively regulate viral transcription through the interaction with TAF-I. We would propose a possible mechanism that core proteins ensure transcription by regulating viral chromatin structure through the interaction with TAF-I.
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  • Analysis of model replication origins in Drosophila reveals new aspects of the chromatin landscape and its relationship to origin activity and the prereplicative complex. 22049023

    Epigenetic regulation exerts a major influence on origins of DNA replication during development. The mechanisms for this regulation, however, are poorly defined. We showed previously that acetylation of nucleosomes regulates the origins that mediate developmental gene amplification during Drosophila oogenesis. Here we show that developmental activation of these origins is associated with acetylation of multiple histone lysines. Although these modifications are not unique to origin loci, we find that the level of acetylation is higher at the active origins and quantitatively correlated with the number of times these origins initiate replication. All of these acetylation marks were developmentally dynamic, rapidly increasing with origin activation and rapidly declining when the origins shut off and neighboring promoters turn on. Fine-scale analysis of the origins revealed that both hyperacetylation of nucleosomes and binding of the origin recognition complex (ORC) occur in a broad domain and that acetylation is highest on nucleosomes adjacent to one side of the major site of replication initiation. It was surprising to find that acetylation of some lysines depends on binding of ORC to the origin, suggesting that multiple histone acetyltransferases may be recruited during origin licensing. Our results reveal new insights into the origin epigenetic landscape and lead us to propose a chromatin switch model to explain the coordination of origin and promoter activity during development.
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  • Transcription of the transforming growth factor beta activating integrin beta8 subunit is regulated by SP3, AP-1, and the p38 pathway. 20519498

    Integrin alphavbeta8 is a critical regulator of transforming growth factor beta activation in vasculogenesis during development, immune regulation, and endothelial/epithelial-mesenchymal homeostasis. Recent studies have suggested roles for integrin beta8 in the pathogenesis of chronic obstructive pulmonary disease, brain arteriovenous malformations, and select cancers (Araya, J., Cambier, S., Markovics, J. A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V. C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D. J., and Nishimura, S. L. (2007) J. Clin. Invest. 117, 3551-3562; Su, H., Kim, H., Pawlikowska, L., Kitamura, H., Shen, F., Cambier, S., Markovics, J., Lawton, M. T., Sidney, S., Bollen, A. W., Kwok, P. Y., Reichardt, L., Young, W. L., Yang, G. Y., and Nishimura, S. L. (2010) Am. J. Pathol. 176, 1018-1027; Culhane, A. C., and Quackenbush, J. (2009) Cancer Res. 69, 7480-7485; Cambier, S., Mu, D. Z., O'Connell, D., Boylen, K., Travis, W., Liu, W. H., Broaddus, V. C., and Nishimura, S. L. (2000) Cancer Res. 60, 7084-7093). Here we report the first identification and characterization of the promoter for ITGB8. We show that a SP binding site and a cyclic AMP response element (CRE) in the ITGB8 core promoter are required for its expression and that Sp1, Sp3, and several AP-1 transcription factors form a complex that binds to these sites in a p38-dependent manner. Furthermore, we demonstrate the requirement for Sp3, ATF-2, and p38 for the transcription and protein expression of integrin beta8. Additionally, reduction of SP3 or inhibition of p38 blocks alphavbeta8-mediated transforming growth factor beta activation. These results place integrin beta8 expression and activity under the control of ubiquitous transcription factors in a stress-activated and pro-inflammatory pathway.
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  • Epigenetic profile of the euchromatic region of human Y chromosome. 21252296

    The genome of a multi-cellular organism acquires various functional capabilities in different cell types by means of distinct chromatin modifications and packaging states. Acquired during early development, the cell type-specific epigenotype is maintained by cellular memory mechanisms that involve epigenetic modifications. Here we present the epigenetic status of the euchromatic region of the human Y chromosome that has mostly been ignored in earlier whole genome epigenetic mapping studies. Using ChIP-on-chip approach, we mapped H3K9ac, H3K9me3, H3K27me3 modifications and CTCF binding sites while DNA methylation analysis of selected CpG islands was done using bisulfite sequencing. The global pattern of histone modifications observed on the Y chromosome reflects the functional state and evolutionary history of the sequences that constitute it. The combination of histone and DNA modifications, along with CTCF association in some cases, reveals the transcriptional potential of all protein coding genes including the sex-determining gene SRY and the oncogene TSPY. We also observe preferential association of histone marks with different tandem repeats, suggesting their importance in genome organization and gene regulation. Our results present the first large scale epigenetic analysis of the human Y chromosome and link a number of cis-elements to epigenetic regulatory mechanisms, enabling an understanding of such mechanisms in Y chromosome linked disorders.
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  • Epigenetic regulation of the estrogen receptor alpha promoter in the cerebral cortex following ischemia in male and female rats. 18353557

    Permanent middle cerebral artery occlusion (MCAO) causes neuronal cell death in the striatum and cortex. In rodents, estradiol treatment protects the cortex from cell death in an estrogen receptor alpha (ERalpha) dependent manner. ERalpha is only transiently expressed in the cortex during neonatal development and is very low in uninjured adult cortex. Following MCAO, ERalpha mRNA expression is upregulated in the cortex of female rats, but the mechanism of this increase is still unknown. It is also unknown whether a similar increase in ERalpha expression in seen in males. In the following studies, male and vehicle or estradiol-treated ovariectomized (OVX) female rats underwent MCAO to investigate the regulation of ERalpha expression after ischemia. Twenty-four hours after surgery, mRNA or genomic DNA was collected from 1 mm micropunches taken from 300 mum brain sections for quantitative reverse transcription-polymerase chain reaction (RT-PCR) or methylation-specific (MSP) PCR, respectively. Additionally, adjacent 20 mum sections were processed for ERalpha immunohistochemistry. In OVX females, ERalpha mRNA and protein were increased in the ischemic cortex, but unchanged in males. We hypothesized that this increase in ERalpha in females is due to a reversal of gene silencing by DNA methylation. Using MSP targeting of CpG islands within the 5' untranslated region (UTR) of the rat ERalpha gene, we found that ischemia decreased methylation in the ischemic cortex of both groups of females, but there was no change in methylation in males. Using chromatin immunoprecipitation, we found that MeCP2 associates with ERalpha 5'UTR corresponding with the methylation status of the promoter. These data are the first to demonstrate a difference in the regulation of ERalpha expression in response to MCAO between males and females and that methylation of the ERalpha gene corresponds with mRNA levels in the brain.
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  • Ikaros is a regulator of Il10 expression in CD4+ T cells. 19828627

    IL-10 is a regulatory cytokine critical for controlling inflammatory responses. Here we show that Ikaros, a zinc finger DNA-binding protein, plays an important role in the regulation of Il10 in murine CD4(+) T cells. Upon initial stimulation of the TCR, T cells deficient in Ikaros express significantly lower levels of IL-10 compared with wild-type T cells. In addition, under Th2 skewing conditions, which induce IL-10 production by wild-type T cells, Ikaros null T cells are unable to properly differentiate, producing only low levels of IL-10. Expression of a dominant-negative isoform of Ikaros in wild-type Th2 cells represses IL-10 production but does not significantly alter expression levels of the genes encoding the transcription factors GATA-3 and T-bet. Furthermore, expression of Ikaros in Ikaros null T cells restores expression of the Th2 cytokines IL-10 and IL-4 while reducing production of the Th1 cytokine, IFN-gamma. Coexpression of Ikaros and GATA-3 further increases IL-10 production, showing that these two factors have an additive effect on activating Il10 expression. Finally, we show that Ikaros binds to conserved regulatory regions of the Il10 gene locus in Th2 cells, supporting a direct role for Ikaros in Il10 expression. Thus, we provide evidence for Ikaros as a regulator of Il10 and Ifng gene expression and suggest a role for Ikaros in directing lineage-specific cytokine gene activation and repression.
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  • Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis. 21986577

    The promoter of human interleukin-10 (IL10), a cytokine crucial for suppressing inflammation and regulating immune responses, contains an interspecies-conserved sequence with CpG motifs. The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA).Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human, macaque and mouse IL10 genes. Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected. The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 μmol/L). The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA, respectively. The methylation of CpGs in the IL10 promoter was determined by pyrosequencing. Chromatin immunoprecipitation (ChIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions.One interspecies-conserved sequence was found within the IL10 promoter. The upstream CpGs at -408, -387, -385, and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls. In contrast, the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016), which was correlated with higher IL10 mRNA and serum levels. In the 5-azacytidine-treated PBMCs, the CpG motifs were demethylated, and the expression levels of IL10 mRNA and protein was significantly increased. CHIP assays revealed increased phospho-CREB binding to the IL10 promoter.The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.
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