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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.
    Document Type:
    Reference
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    Multiple
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    Multiple

  • The homeobox gene MSX2 determines chemosensitivity of pancreatic cancer cells via the regulation of transporter gene ABCG2. 21465479

    Pancreatic cancer is one of the life-threatening cancers due to the difficulty in the curative surgery and resistance against conventional therapeutic strategies. Recent studies indicated that cancer stem cells, which exist as a small number of cells within the entire cancer tissue, contribute to the disease progression. Cancer stem cells reveal resistance against conventional chemotherapy, which is derived from the high-expression of multiple transporter genes. Our previous study demonstrated the aggravating role of the homeobox gene MSX2 as an inducer of epithelial-mesenchymal transition, and MSX2 turned out to correlate with the chemoresistance in the current study. Comprehensive analysis of the MSX2-target gene has identified ABCG2 as the responsible gene. Since previous studies reported the pivotal role of ABCG2 as a determining factor of cancer stem cells, the detailed regulatory mechanism of ABCG2 expression by MSX2 was investigated. As a result, the MSX2 expression level in each cell line well correlated with the ABCG2 expression level, and alteration of the MSX2 expression level by over-expression or siRNA-based knockdown affected the ABCG2 expression accordingly. Finally, we identified the functional cooperation of MSX2 and SP1 in the transcriptional regulation of ABCG2 via the SP1 binding elements within the ABCG2 promoter. These findings clarified the intriguing regulatory mechanism of the cancer stem cell-related gene, and will delineate a novel therapeutic target in pancreatic cancer.
    Document Type:
    Reference
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    Multiple
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    Multiple

  • 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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    Reference
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    Multiple
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    Multiple

  • 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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    Reference
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    Multiple
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    Multiple

  • Chromatin immunoprecipitation for identifying transcription factor targets in keratinocytes. 19908003

    Protein-DNA interactions, such as those that are necessary for transcription, are critical in regulating cellular function and behavior. The identification of DNA sequences that interact with transcriptional regulatory proteins is an important step necessary to better understand the molecular mechanisms regulating gene expression. Chromatin immunoprecipitation (ChIP) is one such procedure that provides a snapshot of which transcription factors are occupying specific DNA sequences. This method allows one not only to determine whether a particular genomic region is occupied by transcription factors but also to identify specific regulatory sequences that potentially control expression of their target genes. Recently, ChIP has been combined with both microarray analysis and a new generation of sequencing allowing a true genome-wide examination of transcription factor binding. Identifying the exact DNA sequence that a transcriptional regulatory protein binds, the precise timing of this association, and what other factors are involved in these interactions are important steps that will shed light on the transcriptional control mechanisms that dictate the biology of all cells, including keratinocytes.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • IFN-γ activated JAK1 shifts CD40-induced cytokine profiles in human antigen-presenting cells toward high IL-12p70 and low IL-10 production. 20709027

    CD40Ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. CD40L induces activation of antigen-presenting cells (APCs) such as DCs, monocytes, B-cells and endothelial cells. However, CD40 activation alone, whilst inducing IL-10 production, is insufficient to induce interleukin (IL)-12p70 release in human APCs suggesting that additional cytokine signals (e.g. GM-CSF, IL-4 or IFN-γ) are required for the induction of a pro-inflammatory cytokine profile. We demonstrate that IFN-γ-induced Janus kinase 1 (JAK1) enhances CD40-induced IL-12p70 release whilst simultaneously inhibiting IL-10 synthesis, resulting in a pro-inflammatory phenotype of CD40L-activated dendritic cells (DCs). JAK2 mediated enhancing effects on IL-12p70 but did not inhibit IL-10 release, whereas Tyk2 mediated inhibitory effects on IL-12p70 release in this system. The mechanism by which complementary IFN-γ/JAK activities affect IL-12p70 production involves STAT1 activation and de novo induction of interferon-responsive factors (IRF)-1 and IRF-8. Simultaneously, JAK1 was unique in inhibiting IL-10 synthesis via STAT1 and IRF-8 with both transcription factors binding to the IL-10 promoter. We demonstrate that CD40- and JAK/STAT/IRF-signalling pathways are strictly complementary for the induction of a pro-inflammatory cytokine profile in human APCs. This suggests that a number of CD40 effects in chronic inflammatory diseases might be weakened by targeting JAK/STAT.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Acid ceramidase-mediated production of sphingosine 1-phosphate promotes prostate cancer invasion through upregulation of cathepsin B. 22322590

    Invasiveness is one of the key features of aggressive prostate cancer; however, our understanding of the precise mechanisms effecting invasion remains limited. The ceramide hydrolyzing enzyme acid ceramidase (AC), overexpressed in most prostate tumors, causes an aggressive and invasive phenotype through downstream effectors that have not yet been well characterized. Here, we demonstrate that AC, through generation of sphingosine-1-phosphate (S1P), promotes Ets1 nuclear expression and binding to the promoter region of matrix-degrading protease cathepsin B. Through confocal microscopy and flow cytometry, we found that AC overexpression promotes pericellular localization of cathepsin B and its translocation to the outer leaflet of the cell membrane. AC overexpressing cells have an increased abundance of cathepsin B-enriched invasive structures and enhanced ability to invade through a collagen matrix, but not in the presence of an inhibitor of cathepsin B. In human prostate tissues, AC and cathepsin B overexpression were strongly associated and may relate to poor outcome. These results demonstrate a novel pathway by which AC, through S1P, promotes an invasive phenotype in prostate cancer by causing overexpression and secretion of cathepsin B through activation and nuclear expression of Ets1. As prostate cancer prognosis is dramatically worse when invasion has occurred, this study provides critical insight into the progression toward lethal prostate cancer.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Direct regulation of complex I by mitochondrial MEF2D is disrupted in a mouse model of Parkinson disease and in human patients. 21393861

    The transcription factors in the myocyte enhancer factor 2 (MEF2) family play important roles in cell survival by regulating nuclear gene expression. Here, we report that MEF2D is present in rodent neuronal mitochondria, where it can regulate the expression of a gene encoded within mitochondrial DNA (mtDNA). Immunocytochemical, immunoelectron microscopic, and biochemical analyses of rodent neuronal cells showed that a portion of MEF2D was targeted to mitochondria via an N-terminal motif and the chaperone protein mitochondrial heat shock protein 70 (mtHsp70). MEF2D bound to a MEF2 consensus site in the region of the mtDNA that contained the gene NADH dehydrogenase 6 (ND6), which encodes an essential component of the complex I enzyme of the oxidative phosphorylation system; MEF2D binding induced ND6 transcription. Blocking MEF2D function specifically in mitochondria decreased complex I activity, increased cellular H(2)O(2) level, reduced ATP production, and sensitized neurons to stress-induced death. Toxins known to affect complex I preferentially disrupted MEF2D function in a mouse model of Parkinson disease (PD). In addition, mitochondrial MEF2D and ND6 levels were decreased in postmortem brain samples of patients with PD compared with age-matched controls. Thus, direct regulation of complex I by mitochondrial MEF2D underlies its neuroprotective effects, and dysregulation of this pathway may contribute to PD.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Myocardin-like protein 2 regulates TGFβ signaling in embryonic stem cells and the developing vasculature. 22899851

    The molecular mechanisms that regulate and coordinate signaling between the extracellular matrix (ECM) and cells contributing to the developing vasculature are complex and poorly understood. Myocardin-like protein 2 (MKL2) is a transcriptional co-activator that in response to RhoA and cytoskeletal actin signals physically associates with serum response factor (SRF), activating a subset of SRF-regulated genes. We now report the discovery of a previously undescribed MKL2/TGFβ signaling pathway in embryonic stem (ES) cells that is required for maturation and stabilization of the embryonic vasculature. Mkl2(-/-) null embryos exhibit profound derangements in the tunica media of select arteries and arterial beds, which leads to aneurysmal dilation, dissection and hemorrhage. Remarkably, TGFβ expression, TGFβ signaling and TGFβ-regulated genes encoding ECM are downregulated in Mkl2(-/-) ES cells and the vasculature of Mkl2(-/-) embryos. The gene encoding TGFβ2, the predominant TGFβ isoform expressed in vascular smooth muscle cells and embryonic vasculature, is activated directly via binding of an MKL2/SRF protein complex to a conserved CArG box in the TGFβ2 promoter. Moreover, Mkl2(-/-) ES cells exhibit derangements in cytoskeletal organization, cell adhesion and expression of ECM that are rescued by forced expression of TGFβ2. Taken together, these data demonstrate that MKL2 regulates a conserved TGF-β signaling pathway that is required for angiogenesis and ultimately embryonic survival.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Nickel-induced Epithelial-Mesenchymal Transition by Reactive Oxygen Species Generation and E-cadherin Promoter Hypermethylation. 22648416

    Epithelial-mesenchymal transition (EMT) is considered a critical event in the pathogenesis of lung fibrosis and tumor metastasis. During EMT, the expression of differentiation markers switches from cell-cell junction proteins such as E-cadherin to mesenchymal markers such as fibronectin. Although nickel-containing compounds have been shown to be associated with lung carcinogenesis, the role of nickel in the EMT process in bronchial epithelial cells is not clear. The aim of this study was to examine whether nickel contributes to EMT in human bronchial epithelial cells. We also attempted to clarify the mechanisms involved in NiCl(2)-induced EMT. Our results showed that NiCl(2) induced EMT phenotype marker alterations such as up-regulation of fibronectin and down-regulation of E-cadherin. In addition, the potent antioxidant N-acetylcysteine blocked EMT and expression of HIF-1α induced by NiCl(2), whereas the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored the down-regulation of E-cadherin induced by NiCl(2). Promoter hypermethylation of E-cadherin, determined by quantitative real time methyl-specific PCR and bisulfate sequencing, was also induced by NiCl(2). These results shed new light on the contribution of NiCl(2) to carcinogenesis. Specifically, NiCl(2) induces down-regulation of E-cadherin by reactive oxygen species generation and promoter hypermethylation. This study demonstrates for the first time that nickel induces EMT in bronchial epithelial cells.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit