Publications by authors named "Gilles Salbert"

28 Publications

  • Page 1 of 1

The hydroxymethylome of multiple myeloma identifies FAM72D as a 1q21 marker linked to proliferation.

Haematologica 2020 03 20;105(3):774-783. Epub 2019 Jun 20.

SPARTE, IGDR, CNRS UMR6290, University Rennes 1, Rennes, France

Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2019.222133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049362PMC
March 2020

Interplay between transcription regulators RUNX1 and FUBP1 activates an enhancer of the oncogene c-KIT and amplifies cell proliferation.

Nucleic Acids Res 2018 11;46(21):11214-11228

Univ Rennes, CNRS, IGDR (Institut de Génétique et Développement de Rennes) - UMR 6290, F-35000 Rennes, France.

Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265458PMC
November 2018

Reading cytosine modifications within chromatin.

Transcription 2018 6;9(4):240-247. Epub 2018 Feb 6.

a University of Rennes 1, UMR6290 CNRS , Institute of Genetics and Development of Rennes , Campus de Beaulieu, Rennes Cedex , France.

Zinc-finger and homeodomain transcription factors have been shown in vitro to bind to recognition motifs containing a methylated CpG. However, accessing these motifs in vivo might be seriously impeded by the inclusion of DNA in nucleosomes and by the condensed structure adopted by chromatin formed on methylated DNA. Here, we discuss how oxidation of 5-methylcytosine into 5-hydroxymethylcytosine could provide the initial destabilizing clue for such transcription factors to get access to nucleosomal DNA and read epigenetic information.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/21541264.2017.1406435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104689PMC
October 2018

Cytosine modifications modulate the chromatin architecture of transcriptional enhancers.

Genome Res 2017 06 10;27(6):947-958. Epub 2017 Apr 10.

CNRS UMR6290, Equipe SP@RTE, Institut de Génétique et Développement de Rennes, Campus de Beaulieu, 35042 Rennes Cedex, France.

Epigenetic mechanisms are believed to play key roles in the establishment of cell-specific transcription programs. Accordingly, the modified bases 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been observed in DNA of genomic regulatory regions such as enhancers, and oxidation of 5mC into 5hmC by Ten-eleven translocation (TET) proteins correlates with enhancer activation. However, the functional relationship between cytosine modifications and the chromatin architecture of enhancers remains elusive. To gain insights into their function, 5mC and 5hmC levels were perturbed by inhibiting DNA methyltransferases and TETs during differentiation of mouse embryonal carcinoma cells into neural progenitors, and chromatin characteristics of enhancers bound by the pioneer transcription factors FOXA1, MEIS1, and PBX1 were interrogated. In a large fraction of the tested enhancers, inhibition of DNA methylation was associated with a significant increase in monomethylation of H3K4, a characteristic mark of enhancer priming. In addition, at some specific enhancers, 5mC oxidation by TETs facilitated chromatin opening, a process that may stabilize MEIS1 binding to these genomic regions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.211466.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453328PMC
June 2017

Inactivation of the Nuclear Orphan Receptor COUP-TFII by Small Chemicals.

ACS Chem Biol 2017 03 13;12(3):654-663. Epub 2017 Jan 13.

Université Rennes 1 , F-35042 Rennes, France.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan member of the nuclear receptor family of transcription factors whose activities are modulated upon binding of small molecules into an hydrophobic ligand-binding pocket (LBP). Although the LBP of COUP-TFII is filled with aromatic amino-acid side chains, alternative modes of ligand binding could potentially lead to regulation of the orphan receptor. Here, we screened a synthetic and natural compound library in a yeast one-hybrid assay and identified 4-methoxynaphthol as an inhibitor of COUP-TFII. This synthetic inhibitor was able to counteract processes either positively or negatively regulated by COUP-TFII in different mammalian cell systems. Hence, we demonstrate that the true orphan receptor COUP-TFII can be targeted by small chemicals which could be used to study the physiological functions of COUP-TFII or to counteract detrimental COUP-TFII activities in various pathological conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.6b00593DOI Listing
March 2017

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies.

PLoS Genet 2016 Dec 29;12(12):e1006482. Epub 2016 Dec 29.

CNRS, UMR 6290, Institut de Génétique et Développement de Rennes, Rennes, France.

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1006482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198995PMC
December 2016

5-hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system.

J Comp Neurol 2017 02 29;525(3):478-497. Epub 2016 Jul 29.

Inserm, UMR 1085, Research Institute in Health, Environment and Occupation, Institut National de la Santé et de la Recherche Médicale U1085, Université de Rennes 1, SFR Biosite, 9 avenue du Prof. Léon Bernard - Rennes, France.

The epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, its precise functions still remain poorly understood and information about its distribution in non-mammalian species is still lacking. Here, the distribution of 5hmC was investigated in the brain of adult zebrafish, African claw frog, and mouse in a comparative manner. We show that zebrafish neurons are endowed with high levels of 5hmC, whereas quiescent or proliferative neural progenitors show low to undetectable levels of the modified cytosine. In the brain of larval and juvenile Xenopus, 5hmC is also detected in neurons, while ventricular proliferative cells do not display this epigenetic mark. Similarly, 5hmC is enriched in neurons compared to neural progenitors of the ventricular zone in the mouse developing cortex. Interestingly, 5hmC colocalized with the methylated DNA binding protein MeCP2 and with the active chromatin histone modification H3K4me2 in mouse neurons. Taken together, our results show an evolutionarily conserved cerebral distribution of 5hmC between fish and tetrapods and reinforce the idea that 5hmC fulfills major functions in the control of chromatin activity in vertebrate neurons. J. Comp. Neurol. 525:478-497, 2017. © 2016 Wiley Periodicals, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cne.24077DOI Listing
February 2017

Changes in Gene Expression and Estrogen Receptor Cistrome in Mouse Liver Upon Acute E2 Treatment.

Mol Endocrinol 2016 Jul 10;30(7):709-32. Epub 2016 May 10.

Equipe Spatio-Temporal Regulation of Transcription in Eukaryotes (SP@RTE) (G.P., C.L.P., S.A., G.S., R.M.), Unité Mixte de Recherche 6290 Centre National de la Recherche Scientifique (Institut de Genétique et Développement de Rennes), Université de Rennes 1, Campus de Beaulieu, and Equipe Transcription, Environment and Cancer (TREC) (G.F.), Inserm U1085-Institut de Recherche en Santé, Environnement et Travail, Rennes 35042 Cedex, France; and Equipe 9 "Estrogen Receptor: In Vivo Dissection and Modulation" (A.F., R.S., F.L., C.F., J.-F.A.), Inserm Unité 1048 (Institut des Maladies Métaboliques et Cardiovasculaires), Toulouse 31432 Cedex 4, France.

Transcriptional regulation by the estrogen receptor-α (ER) has been investigated mainly in breast cancer cell lines, but estrogens such as 17β-estradiol (E2) exert numerous extrareproductive effects, particularly in the liver, where E2 exhibits both protective metabolic and deleterious thrombotic actions. To analyze the direct and early transcriptional effects of estrogens in the liver, we determined the E2-sensitive transcriptome and ER cistrome in mice after acute administration of E2 or placebo. These analyses revealed the early induction of genes involved in lipid metabolism, which fits with the crucial role of ER in the prevention of liver steatosis. Characterization of the chromatin state of ER binding sites (BSs) in mice expressing or not ER demonstrated that ER is not required per se for the establishment and/or maintenance of chromatin modifications at the majority of its BSs. This is presumably a consequence of a strong overlap between ER and hepatocyte nuclear factor 4α BSs. In contrast, 40% of the BSs of the pioneer factor forkhead box protein a (Foxa2) were dependent upon ER expression, and ER expression also affected the distribution of nucleosomes harboring dimethylated lysine 4 of Histone H3 around Foxa2 BSs. We finally show that, in addition to a network of liver-specific transcription factors including CCAAT/enhancer-binding protein and hepatocyte nuclear factor 4α, ER might be required for proper Foxa2 function in this tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2015-1311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426578PMC
July 2016

Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease digestion identifies evolutionarily unconserved CpGs as TET targets.

Genome Biol 2016 Mar 29;17:56. Epub 2016 Mar 29.

CNRS UMR6290, Equipe SP@RTE, Institut de Génétique et Développement de Rennes, Campus de Beaulieu, Rennes cedex, 35042, France.

Conventional techniques for single-base resolution mapping of epigenetic modifications of DNA such as 5-hydroxymethylcytosine (5hmC) rely on the sequencing of bisulfite-modified DNA. Here we present an alternative approach called SCL-exo which combines selective chemical labeling (SCL) of 5hmC in genomic DNA with exonuclease (exo) digestion of the bead-trapped modified DNA molecules. Associated with a straightforward bioinformatic analysis, this new procedure provides an unbiased and fast method for mapping this epigenetic mark at high resolution. Implemented on mouse genomic DNA from in vitro-differentiated neural precursor cells, SCL-exo sheds light on an intrinsic lack of conservation of hydroxymethylated CpGs across vertebrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-016-0919-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810514PMC
March 2016

Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells.

Cell Rep 2015 Nov 22;13(5):1059-71. Epub 2015 Oct 22.

INSERM, UMR917, Equipe labellisée Ligue contre le Cancer, Rennes 35043, France; Pôle de Biologie, Centre Hospitalier Universitaire (CHU), Rennes 35033, France; Université de Rennes 1, Rennes 35065, France; Etablissement Français du Sang de Bretagne, Rennes 35016, France. Electronic address:

Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.09.051DOI Listing
November 2015

Dynamic estrogen receptor interactomes control estrogen-responsive trefoil Factor (TFF) locus cell-specific activities.

Mol Cell Biol 2014 Jul 21;34(13):2418-36. Epub 2014 Apr 21.

Equipe SP@RTE, UMR CNRS 6290, Equipe Labellisée Ligue contre le Cancer, Université de Rennes I, Rennes, France

Estradiol signaling is ideally suited for analyzing the molecular and functional linkages between the different layers of information directing transcriptional regulations: the DNA sequence, chromatin modifications, and the spatial organization of the genome. Hence, the estrogen receptor (ER) can bind at a distance from its target genes and engages timely and spatially coordinated processes to regulate their expression. In the context of the coordinated regulation of colinear genes, identifying which ER binding sites (ERBSs) regulate a given gene still remains a challenge. Here, we investigated the coordination of such regulatory events at a 2-Mb genomic locus containing the estrogen-sensitive trefoil factor (TFF) cluster of genes in breast cancer cells. We demonstrate that this locus exhibits a hormone- and cohesin-dependent reduction in the plasticity of its three-dimensional organization that allows multiple ERBSs to be dynamically brought to the vicinity of estrogen-sensitive genes. Additionally, by using triplex-forming oligonucleotides, we could precisely document the functional links between ER engagement at given ERBSs and the regulation of particular genes. Hence, our data provide evidence of a formerly suggested cooperation of enhancers toward gene regulation and also show that redundancy between ERBSs can occur.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00918-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054307PMC
July 2014

Peroxisome proliferator-activated receptor γ regulates genes involved in insulin/insulin-like growth factor signaling and lipid metabolism during adipogenesis through functionally distinct enhancer classes.

J Biol Chem 2014 Jan 27;289(2):708-22. Epub 2013 Nov 27.

From INSERM UMR U1011, F-59000 Lille, France.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) is a transcription factor whose expression is induced during adipogenesis and that is required for the acquisition and control of mature adipocyte functions. Indeed, PPAR induces the expression of genes involved in lipid synthesis and storage through enhancers activated during adipocyte differentiation. Here, we show that PPAR also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is more restricted to adipocytes. Thus, PPAR utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of genes involved in the adipogenic process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.526996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887199PMC
January 2014

The elongation complex components BRD4 and MLLT3/AF9 are transcriptional coactivators of nuclear retinoid receptors.

PLoS One 2013 10;8(6):e64880. Epub 2013 Jun 10.

European Genomic Institute for Diabetes-EGID, FR 3508, Lille, France.

Nuclear all-trans retinoic acid receptors (RARs) initiate early transcriptional events which engage pluripotent cells to differentiate into specific lineages. RAR-controlled transactivation depends mostly on agonist-induced structural transitions in RAR C-terminus (AF-2), thus bridging coactivators or corepressors to chromatin, hence controlling preinitiation complex assembly. However, the contribution of other domains of RAR to its overall transcriptional activity remains poorly defined. A proteomic characterization of nuclear proteins interacting with RAR regions distinct from the AF-2 revealed unsuspected functional properties of the RAR N-terminus. Indeed, mass spectrometry fingerprinting identified the Bromodomain-containing protein 4 (BRD4) and ALL1-fused gene from chromosome 9 (AF9/MLLT3), known to associate with and regulates the activity of Positive Transcription Elongation Factor b (P-TEFb), as novel RAR coactivators. In addition to promoter sequences, RAR binds to genomic, transcribed regions of retinoid-regulated genes, in association with RNA polymerase II and as a function of P-TEFb activity. Knockdown of either AF9 or BRD4 expression affected differentially the neural differentiation of stem cell-like P19 cells. Clusters of retinoid-regulated genes were selectively dependent on BRD4 and/or AF9 expression, which correlated with RAR association to transcribed regions. Thus RAR establishes physical and functional links with components of the elongation complex, enabling the rapid retinoid-induced induction of genes required for neuronal differentiation. Our data thereby extends the previously known RAR interactome from classical transcriptional modulators to components of the elongation machinery, and unravel a functional role of RAR in transcriptional elongation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064880PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677938PMC
February 2014

Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers.

Nucleic Acids Res 2012 Sep 22;40(17):8255-65. Epub 2012 Jun 22.

Université de Rennes 1, CNRS UMR6290, Team SP@RTE, Campus de Beaulieu, Rennes F-35042, France.

Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458548PMC
http://dx.doi.org/10.1093/nar/gks595DOI Listing
September 2012

The novel antibacterial compound walrycin A induces human PXR transcriptional activity.

Toxicol Sci 2012 May 7;127(1):225-35. Epub 2012 Feb 7.

Univ Lille Nord de France, F-59000 Lille, France.

The human pregnane X receptor (PXR) is a ligand-regulated transcription factor belonging to the nuclear receptor superfamily. PXR is activated by a large, structurally diverse, set of endogenous and xenobiotic compounds and coordinates the expression of genes central to metabolism and excretion of potentially harmful chemicals and therapeutic drugs in humans. Walrycin A is a novel antibacterial compound targeting the WalK/WalR two-component signal transduction system of Gram (+) bacteria. Here, we report that, in hepatoma cells, walrycin A potently activates a gene set known to be regulated by the xenobiotic sensor PXR. Walrycin A was as efficient as the reference PXR agonist rifampicin to activate PXR in a transactivation assay at noncytotoxic concentrations. Using a limited proteolysis assay, we show that walrycin A induces conformational changes at a concentration which correlates with walrycin A ability to enhance the expression of prototypic target genes, suggesting that walrycin A interacts with PXR. The activation of the canonical human PXR target gene CYP3A4 by walrycin A is dose and PXR dependent. Finally, in silico docking experiments suggest that the walrycin A oxidation product Russig's blue is the actual ligand for PXR. Taken together, these results identify walrycin A as a novel human PXR activator.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/toxsci/kfs073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435511PMC
May 2012

Tracking genomic hydroxymethylation by the base.

Nat Methods 2011 Dec 28;9(1):45-6. Epub 2011 Dec 28.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.1813DOI Listing
December 2011

SORGOdb: Superoxide Reductase Gene Ontology curated DataBase.

BMC Microbiol 2011 May 16;11:105. Epub 2011 May 16.

CNRS UMR 6026, ICM, Equipe Sp@rte, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes, France.

Background: Superoxide reductases (SOR) catalyse the reduction of superoxide anions to hydrogen peroxide and are involved in the oxidative stress defences of anaerobic and facultative anaerobic organisms. Genes encoding SOR were discovered recently and suffer from annotation problems. These genes, named sor, are short and the transfer of annotations from previously characterized neelaredoxin, desulfoferrodoxin, superoxide reductase and rubredoxin oxidase has been heterogeneous. Consequently, many sor remain anonymous or mis-annotated.

Description: SORGOdb is an exhaustive database of SOR that proposes a new classification based on domain architecture. SORGOdb supplies a simple user-friendly web-based database for retrieving and exploring relevant information about the proposed SOR families. The database can be queried using an organism name, a locus tag or phylogenetic criteria, and also offers sequence similarity searches using BlastP. Genes encoding SOR have been re-annotated in all available genome sequences (prokaryotic and eukaryotic (complete and in draft) genomes, updated in May 2010).

Conclusions: SORGOdb contains 325 non-redundant and curated SOR, from 274 organisms. It proposes a new classification of SOR into seven different classes and allows biologists to explore and analyze sor in order to establish correlations between the class of SOR and organism phenotypes. SORGOdb is freely available at http://sorgo.genouest.org/index.php.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2180-11-105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116461PMC
May 2011

Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers.

Genome Res 2011 Apr 13;21(4):555-65. Epub 2011 Jan 13.

Université de Rennes 1, Rennes, France.

Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.111534.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065703PMC
April 2011

Biological and biophysical properties of the histone deacetylase inhibitor suberoylanilide hydroxamic acid are affected by the presence of short alkyl groups on the phenyl ring.

J Med Chem 2010 Mar;53(5):1937-50

Equipe SPARTE, UMR CNRS 6026-Université Rennes 1, France, Cedex.

Inhibition of histone deacetylases (HDACs) leads to growth arrest, differentiation, or apoptosis of tumor cell lines, suggesting HDACs as promising targets for cancer therapy. At present, only one HDAC inhibitor (HDACi) is used in therapy: suberoylanilide hydroxamic acid (SAHA). Here, we describe the synthesis and biological evaluation of a new series of compounds derived from SAHA by substituting short alkyl chains at various positions of the phenyl ring. Such modifications induced variable effects ranging from partial loss of activity to increased potency. Through molecular modeling, we describe a possible interaction between HDAC7 proline 809, a residue that is strictly conserved within class 2 enzymes only, and the amide group of HDACi, while nuclear magnetic resonance experiments indicated that dimethyl m-substitution may stabilize the inhibitor in the active site. Our data provide novel information on the structure-activity relationship of HDACi and suggest new ways for developing second generation SAHA-like molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm901561uDOI Listing
March 2010

Defining specificity of transcription factor regulatory activities.

J Cell Sci 2009 Nov;122(Pt 22):4027-34

Université de Rennes I, CNRS, UMR 6026, Equipe SPARTE, 35042 Rennes Cedex, France.

Mammalian transcription factors (TFs) are often involved in differential cell-type- and context-specific transcriptional responses. Recent large-scale comparative studies of TF recruitment to the genome, and of chromatin structure and gene expression, have allowed a better understanding of the general rules that underlie the differential activities of a given TF. It has emerged that chromatin structure dictates the differential binding of a given TF to cell-type-specific cis-regulatory elements. The subsequent regulation of TF activity then ensures the functional activation of only the precise subset of all regulatory sites bound by the TF that are required to mediate appropriate gene expression. Ultimately, the organization of the genome within the nucleus, and crosstalk between different cis-regulatory regions involved in gene regulation, also participate in establishing a specific transcriptional program. In this Commentary, we discuss how the integration of these different and probably intimately linked regulatory mechanisms allow for TF cell-type- and context-specific modulation of gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.054916DOI Listing
November 2009

Identification of small molecule regulators of the nuclear receptor HNF4alpha based on naphthofuran scaffolds.

Bioorg Med Chem 2009 Oct 5;17(19):7021-30. Epub 2009 Aug 5.

Equipe SPARTE, Université de Rennes 1, UMR6026 CNRS, Campus de Beaulieu, Bat 13, 35042 Rennes Cedex, France.

Nuclear receptors are ligand-activated transcription factors involved in all major physiological functions of complex organisms. In this respect, they are often described as drugable targets for a number of pathological states including hypercholesterolemia and atherosclerosis. HNF4alpha (NR2A1) is a recently 'deorphanized' nuclear receptor which is bound in vivo by linoleic acid, although this natural ligand does not seem to promote transcriptional activation. In mouse, HNF4alpha is a major regulator of liver development and hepatic lipid metabolism and mutations in human have been linked to diabetes. Here, we have used a yeast one-hybrid system to identify small molecule activators of HNF4alpha in a library of synthetic compounds and found one hit bearing a methoxy group branched on a nitronaphthofuran backbone. A collection of molecules deriving from the discovered hit was generated and tested for activity toward HNF4alpha in yeast one-hybrid system. It was found that both the nitro group and a complete naphthofuran backbone were required for full activity of the compounds. Furthermore, adding a hydroxy group at position 7 of the minimal backbone led to the most active compound of the collection. Accordingly, a direct interaction of the hydroxylated compound with the ligand binding domain of HNF4alpha was detected by NMR and thermal denaturation assays. When used in mammalian cell culture systems, these compounds proved to be highly toxic, except when methylated on the furan ring. One such compound was able to modulate HNF4alpha-driven transcription in transfected HepG2C3A cells. These data indicate that HNF4alpha activity can be modulated by small molecules and suggest new routes for targeting the receptor in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2009.07.079DOI Listing
October 2009

Cyclical DNA methylation of a transcriptionally active promoter.

Nature 2008 Mar;452(7183):45-50

Université de Rennes I, CNRS, UMR 6026 Equipe SPARTE, IFR 140 GFAS, Campus de Beaulieu, 35042 Rennes cedex, France.

Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature06544DOI Listing
March 2008

Deoxyribonucleic acid methyl transferases 3a and 3b associate with the nuclear orphan receptor COUP-TFI during gene activation.

Mol Endocrinol 2007 Sep 19;21(9):2085-98. Epub 2007 Jun 19.

Unité Mixte de Recherche 6026, Centre National de la Recherche Scientifique, Université Rennes I, Institut Fédératif de Recherche 140 Génomique Fonctionnelle Agronomie Santé, Equipe Information et Programmation Cellulaire, France.

Transcriptional activation of silent genes can require the erasure of epigenetic marks such as DNA methylation at CpGs (cytosine-guanine dinucleotide). Active demethylation events have been observed, and associated processes are repeatedly suspected to involve DNA glycosylases such as mCpG binding domain protein 4, thymine DNA glycosylase (TDG), Demeter, and repressor of silencing 1. A complete characterization of the molecular mechanisms occurring in metazoan is nonetheless awaited. Here, we report that activation of the endogenous vitronectin gene in P19 cells by the nuclear receptor chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is observed in parallel with the recruitment of TDG and p68 RNA helicase, two components of a putative demethylation complex. Interestingly, when activated, the vitronectin gene was loaded with DNA methyltransferases 3a and 3b (Dnmt3a/b), and a strand-biased decrease in CpG methylation was detected. Dnmt3a was further found to associate with COUP-TFI and TDG in vivo, and cotransfection experiments demonstrated that Dnmt3a/b can enhance COUP-TFI-mediated activation of a methylated reporter gene. These results suggest that Dnmt3a/b could cooperate with the orphan receptor COUP-TFI to regulate transcription of the vitronectin gene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2006-0490DOI Listing
September 2007

A neural-specific splicing event generates an active form of the Wiskott-Aldrich syndrome protein.

EMBO Rep 2004 Sep;5(9):895-900

Equipe 'Information et Programmation Cellulaire', UMR 6026 CNRS, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France.

Actin polymerization is required for cellular events such as podosome, lamellipode or filopode formation in migrating cells, and members of the Wiskott-Aldrich syndrome protein (WASP) family have essential roles in regulating actin dynamics at the cell leading edge. However, WASP proteins need first to be activated in order to be able to target actin polymerization. Here, we show the occurrence of a neural-specific splicing event, which is favoured by the nuclear orphan receptor chicken ovalbumin upstream promoter-transcription factor I, and generates a truncated WASP protein deleted of exon 2-encoded amino acids. This deletion relocates the protein to the plasma membrane and induces the formation of actin-rich podosome-like structures that also contain paxillin and vinculin. Furthermore, expression of the truncated protein in PC12 cells, as well as in primary neurons, stimulates neuritogenesis. These data underscore the importance of the neural-specific splicing of WASP RNA during development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.embor.7400239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299140PMC
September 2004

Formation of an hER alpha-COUP-TFI complex enhances hER alpha AF-1 through Ser118 phosphorylation by MAPK.

EMBO J 2002 Jul;21(13):3443-53

Equipe d'Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026, Université de Rennes I, 35042 Rennes Cedex, France.

The enhancement of the human estrogen receptor alpha (hER alpha, NR3A1) activity by the orphan nuclear receptor COUP-TFI is found to depend on the establishment of a tight hER alpha-COUP-TFI complex. Formation of this complex seems to involve dynamic mechanisms different from those allowing hER alpha homodimerization. Although the hER alpha-COUP-TFI complex is present in all cells tested, the transcriptional cooperation between the two nuclear receptors is restricted to cell lines permissive to hER alpha activation function 1 (AF-1). In these cells, the physical interaction between COUP-TFI and hER alpha increases the affinity of hER alpha for ERK2/p42(MAPK), resulting in an enhanced phosphorylation state of the hER alpha Ser118. hER alpha thus acquires a strengthened AF-1 activity due to its hyperphosphorylation. These data indicate an alternative interaction process between nuclear receptors and demonstrate a novel protein intercommunication pathway that modulates hER alpha AF-1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/emboj/cdf344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC126093PMC
July 2002

Multiple phosphorylation events control chicken ovalbumin upstream promoter transcription factor I orphan nuclear receptor activity.

Mol Endocrinol 2002 Jun;16(6):1332-51

Equipe Information et Programmation Cellulaire, Unité Mixte de Recherche 6026 Centre National de la Recherche Scientifique, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France.

Chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) is an orphan member of the nuclear hormone receptor superfamily that comprises key regulators of many biological functions, such as embryonic development, metabolism, homeostasis, and reproduction. Although COUP-TFI can both actively silence gene transcription and antagonize the functions of various other nuclear receptors, the COUP-TFI orphan receptor also acts as a transcriptional activator in certain contexts. Moreover, COUP-TFI has recently been shown to serve as an accessory factor for some ligand-bound nuclear receptors, suggesting that it may modulate, both negatively and positively, a wide range of hormonal responses. In the absence of any identified cognate ligand, the mechanisms involved in the regulation of COUP-TFI activity remain unclear. The elucidation of several putative phosphorylation sites for MAPKs, PKC, and casein kinase II within the sequence of this orphan receptor led us to investigate phosphorylation events regulating the various COUP-TFI functions. After showing that COUP-TFI is phosphorylated in vivo, we provide evidence that in vivo inhibition of either MAPK or PKC signaling pathway leads to a specific and pronounced decrease in COUP-TFI-dependent transcriptional activation of the vitronectin gene promoter. Focusing on the molecular mechanisms underlying the MAPK- and PKC-mediated regulation of COUP-TFI activity, we show that COUP-TFI can be directly targeted by PKC and MAPK. These phosphorylation events differentially modulate COUP-TFI functions: PKC-mediated phosphorylation enhances COUP-TFI affinity for DNA and MAPK-mediated phosphorylation positively regulates the transactivation function of COUP-TFI, possibly through enhancing specific coactivator recruitment. These data provide evidence that COUP-TFI is likely to integrate distinct signaling pathways and raise the possibility that multiple extracellular signals influence biological processes controlled by COUP-TFI.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/mend.16.6.0840DOI Listing
June 2002

Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins.

J Chromatogr B Analyt Technol Biomed Life Sci 2002 Mar;769(1):133-44

Department of Biochemistry and Biophysics, Stockholm University, Sweden.

In this report, we describe a flexible, efficient and rapid protein purification strategy for the isolation and cleavage of glutathione-S-transferase (GST) fusion proteins. The purification and on-column cleavage strategy was developed to work for the purification of difficult proteins and for target proteins where efficient fusion-tag cleavage is essential for downstream processes, such as structural and functional studies. To test and demonstrate the flexibility of this method, seven diverse unrelated target proteins were assayed. A purification technique is described that can be applied to a wide range of both soluble and membrane inserted recombinant target proteins of differing function, structure and chemical nature. This strategy is performed in a single chromatographic step applying an on-column cleavage method, yielding "native" proteins in the 200 microg to 40 mg/l scale of 95-98% purity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s1570-0232(01)00637-7DOI Listing
March 2002