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Heidelberg, Germany

Junion G.,French Institute of Health and Medical Research | Junion G.,Gene Expression Unit | Jagla K.,French Institute of Health and Medical Research
Methods in Molecular Biology | Year: 2012

Mapping the cis-regulatory modules (CRMs) to which bind myogenic transcription factors is an -obligatory step towards understanding gene regulatory networks governing muscle development and function. This can be achieved in silico or by chromatin immunoprecipitation (ChIP) approaches. We have developed a ChIP-enriched in silico targets (ChEST) strategy designed for mapping the CRMs by combining in silico and ChIP methods. ChEST involves a software-assisted prediction of transcription factor (TF) - specific CRMs, which are spotted to produce a computed genomic CRM microarray. In parallel, the in vivo pool of targets of a given TF is isolated by ChIP and used as a probe for hybridization with the array generated. Here we describe ChEST strategy applied to identify direct targets of Myogenic Enhancer Factor, Dmef2 in Drosophila embryos.

Wolden-Kirk H.,University Hospital Gasthuisberg | Wolden-Kirk H.,University of Southern Denmark | Rondas D.,University Hospital Gasthuisberg | Bugliani M.,University of Pisa | And 11 more authors.
Endocrinology | Year: 2014

Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D 3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH) 2D3. Effects on insulin secretion and β-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in β-cell apoptosis, which was almost completely prevented by 1,25(OH) 2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic β-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D 3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes. Copyright © 2014 by the Endocrine Society.

Forst A.H.,RWTH Aachen | Karlberg T.,Karolinska Institutet | Herzog N.,RWTH Aachen | Thorsell A.-G.,Karolinska Institutet | And 14 more authors.
Structure | Year: 2013

ADP-ribosyltransferases (ARTs) catalyze the transfer of ADP-ribose from NAD+ onto substrates. Some ARTs generate in an iterative process ADP-ribose polymers that serve as adaptors for distinct protein domains. Other ARTs, exemplified by ARTD10, function as mono-ADP-ribosyltransferases, but it has been unclear whether this modification occurs in cells and how it is read. We observed that ARTD10 colocalized with ARTD8 and defined its macrodomains 2 and 3 as readers of mono-ADP-ribosylation both in vitro and in cells. The crystal structures of these two ARTD8 macrodomains and isothermal titration calorimetry confirmed their interaction with ADP-ribose. These macrodomains recognized mono-ADP-ribosylated ARTD10, but not poly-ADP-ribosylated ARTD1. This distinguished them from the macrodomain of macroH2A1.1, which interacted with poly- but not mono-ADP-ribosylated substrates. Moreover, Ran, an ARTD10 substrate, was also read by ARTD8 macrodomains. This identifies readers of mono-ADP-ribosylated proteins, defines their structures, and demonstrates the presence of this modification in cells. © 2013 Elsevier Ltd. All rights reserved.

Andersen D.S.,Cancer Research UK Research Institute | Andersen D.S.,Institute for Signaling | Raja S.J.,Gene Expression Unit | Raja S.J.,Max Planck Institute For Immunbiologie | And 8 more authors.
Molecular and Cellular Biology | Year: 2010

Drosophila MCRS2 (dMCRS2; MCRS2/MSP58 and its splice variant MCRS1/p78 in humans) belongs to a family of forkhead-associated (FHA) domain proteins. Whereas human MCRS2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 has been largely uncharacterized. Recent data show that MCRS2 is purified as part of a complex containing the histone acetyltransferase MOF (males absent on first) in both humans and flies. MOF mediates H4K16 acetylation and regulates the expression of a large number of genes, suggesting that MCRS2 could also have a function in transcription regulation. Here, we show that dMCRS2 copurifies with RNA polymerase II (RNAP II) complexes and localizes to the 5′ ends of genes. Moreover, dMCRS2 is required for optimal recruitment of RNAP II to the promoter regions of cyclin genes. In agreement with this, dMCRS2 is required for normal levels of cyclin gene expression. We propose a model whereby dMCRS2 promotes gene transcription by facilitating the recruitment of RNAP II preinitiation complexes (PICs) to the promoter regions of target genes. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

De Bruyn M.,University of Hospital Gasthuisberg | De Bruyn M.,Rega Institute for Medical Research | Machiels K.,University of Hospital Gasthuisberg | Vandooren J.,Rega Institute for Medical Research | And 16 more authors.
Inflammatory Bowel Diseases | Year: 2014

Background: Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), a disintegrin and metalloprotease with thrombospondin motifs [ADAM(TS)s] and growth factors are involved in inflammation and tissue damage and repair, all occurring in inflammatory bowel disease (IBD). We studied the impact of anti-inflammatory therapy with infliximab on mucosal expression of these tissue remodeling genes in patients with IBD. Methods: Mucosal gene expression of 23 MMPs, 4 TIMPs, 50 ADAM(TS)s, and 158 growth factors was investigated in 61 patients with IBD before and after the first infliximab therapy and in 12 controls, with microarrays and quantitative RT-PCR. Protein localization, mucosal gelatinase levels, and net gelatinolytic activity were investigated by immunohistochemistry, zymography analysis, and gelatin degradation assay, respectively. Results: In patients with active IBD before infliximab versus controls, gene expression of many MMPs, TIMPs, ADAM(TS)s, and growth factors was upregulated, whereas colonic expression of MMP28 and TGFA and ileal expression of ADAMDEC1 and AGT were downregulated. After controlling inflammation with infliximab, most gene dysregulations observed at baseline were restored in responders. Increased ratio of MMP1/TIMP1 expression at baseline in active IBD was restored in responders with colonic mucosal healing. With immunohistochemistry, protein localization differences of MMP1, MMP3, REG1A, and TIMP1 were shown between active IBD and control mucosa. With zymography analysis and gelatin degradation assay, higher gelatinase levels and net gelatinolytic activity were measured before infliximab and levels normalized after infliximab. Conclusions: Our data suggest that suppression of inflammation results in the arrest of epithelial damage and subsequent mucosal healing. Therefore, the therapeutic potential of agents targeting MMPs or growth factors as primary therapy seems rather complex. Copyright © 2013 Crohn's & Colitis Foundation of America, Inc.

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