Role of the Epigenetic Regulator HP1γ in the Control of Embryonic Stem Cell Properties

The unique properties of embryonic stem cells (ESC) rely on long-lasting self-renewal and their ability to switch in all adult cell type programs. Recent advances have shown that regulations at the chromatin level sustain both ESC properties along with transcription factors. We have focused our interest on the epigenetic modulator HP1γ (Heterochromatin Protein 1, isoform γ) that binds histones H3 methylated at lysine 9 (meH3K9) and is highly plastic in its distribution and association with the transcriptional regulation of specific genes during cell fate transitions. These characteristics of HP1γ make it a good candidate to sustain the ESC flexibility required for rapid program changes during differentiation. Using RNA interference, we describe the functional role of HP1γ in mouse ESC. The analysis of HP1γ deprived cells in proliferative and in various differentiating conditions was performed combining functional assays with molecular approaches (RT-qPCR, microarray). We show that HP1γ deprivation slows down the cell cycle of ESC and decreases their resistance to differentiating conditions, rendering the cells poised to differentiate. In addition, HP1γ depletion hampers the differentiation to the endoderm as compared with the differentiation to the neurectoderm or the mesoderm. Altogether, our results reveal the role of HP1γ in ESC self-renewal and in the balance between the pluripotent and the differentiation programs

The endogenous retrovirus ENS-1 provides active binding sites for transcription factors in embryonic stem cells that specify extra embryonic tissue

<p>Abstract</p> <p>Background</p> <p>Long terminal repeats (LTR) from endogenous retroviruses (ERV) are source of binding sites for transcription factors which affect the host regulatory networks in different cell types, including pluripotent cells. The embryonic epiblast is made of pluripotent cells that are subjected to opposite transcriptional regulatory networks to give rise to distinct embryonic and extraembryonic lineages. To assess the transcriptional contribution of ERV to early developmental processes, we have characterized <it>in vitro </it>and <it>in vivo </it>the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells.</p> <p>Results</p> <p>We show that <it>Ens-1 </it>LTR activity is controlled by two transcriptional pathways that drive pluripotent cells to alternative developmental fates. Indeed, both Nanog that maintains pluripotency and Gata4 that induces differentiation toward extraembryonic endoderm independently activate the LTR. Ets coactivators are required to support Gata factors' activity thus preventing inappropriate activation before epigenetic silencing occurs during differentiation. Consistent with their expression patterns during chick embryonic development, Gata4, Nanog and Ets1 are recruited on the LTR in embryonic stem cells; in the epiblast the complementary expression of Nanog and Gata/Ets correlates with the <it>Ens-1 </it>gene expression pattern; and Ens-1 transcripts are also detected in the hypoblast, an extraembryonic tissue expressing Gata4 and Ets2, but not Nanog. Accordingly, over expression of Gata4 in embryos induces an ectopic expression of <it>Ens-1</it>.</p> <p>Conclusion</p> <p>Our results show that <it>Ens-1 </it>LTR have co-opted conditions required for the emergence of extraembryonic tissues from pluripotent epiblasts cells. By providing pluripotent cells with intact binding sites for Gata, Nanog, or both, <it>Ens-1 </it>LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.</p

A Cell-Based Method to Detect Agonist and Antagonist Activities of Endocrine-Disrupting Chemicals on GPER

International audienceEndocrine-disrupting chemicals (EDCs) are exogenous compounds that impact endogenous hormonal systems, resulting in adverse health effects. These chemicals can exert their actions by interfering with several pathways. Simple biological systems to determine whether EDCs act positively or negatively on a given receptor are often lacking. Here we describe a low-to-middle throughput method to screen the agonist/antagonist potential of EDCs specifically on the GPER membrane estrogen receptor. Application of this assay to 23 candidate EDCs from different chemical families reveals the existence of six agonists and six antagonists

LSD1-ERRα complex requires NRF1 to positively regulate transcription and cell invasion

International audienc

Estrogens repress PGC1-alpha expression in the uterus

International audiencePGC-1 alpha is a transcriptional coactivator that is highly involved in several aspects of regulation of metabolism, including mitochondrial biogenesis and activity. Using several in vivo models, we here report that the expression of PGC-1 alpha is repressed by estrogens in the mouse specifically in the uterus. In the absence of estrogens, expression of PGC-1 alpha target genes involved in mitochondrial activity is activated, but not mitochondrial biogenesis. Regulation of PGC-1 alpha expression by estrogens also occurs in Ishikawa human uterine cells at the promoter level and involve modulation of c-jun expression. (C) 2010 Elsevier Ireland Ltd. All rights reserved

ERRα coordinates actin and focal adhesion dynamics

International audienceCell migration depends on the dynamic organisation of the actin cytoskeleton and assembly and disassembly of focal adhesions (FAs). However, the precise mechanisms coordinating these processes remain poorly understood. We previously identified the oestrogen-related receptor α (ERRα) as a major regulator of cell migration. Here, we show that loss of ERRα leads to abnormal accumulation of actin filaments that is associated with an increased level of inactive form of the actin-depolymerising factor cofilin. We further show that ERRα depletion decreases cell adhesion and results in defective FA formation and turnover. Interestingly, specific inhibition of the RhoA-ROCK-LIMK-cofilin pathway rescues the actin polymerisation defects resulting from ERRα silencing, but not cell adhesion. Instead, we found that MAP4K4 is a direct target of ERRα and down-regulation of its activity rescues cell adhesion and FA formation in the ERRα-depleted cells. Altogether, our results highlight a crucial role of ERRα in coordinating the dynamic of actin network and FAs through the independent regulation of the RhoA and MAP4K4 pathways

miR-135a Inhibits the Invasion of Cancer Cells via Suppression of ERRα.

MicroRNA-135a (miR-135a) down-modulates parameters of cancer progression and its expression is decreased in metastatic breast cancers (as compared to non-metastatic tumors) as well as in prostate tumors relative to normal tissue. These expression and activity patterns are opposite to those of the Estrogen-Related Receptor α (ERRα), an orphan member of the nuclear receptor family. Indeed high expression of ERRα correlates with poor prognosis in breast and prostate cancers, and the receptor promotes various traits of cancer aggressiveness including cell invasion. Here we show that miR-135a down-regulates the expression of ERRα through specific sequences of its 3'UTR. As a consequence miR-135a also reduces the expression of downstream targets of ERRα. miR-135a also decreases cell invasive potential in an ERRα-dependent manner. Our results suggest that the decreased expression of miR-135a in metastatic tumors leads to elevated ERRα expression, resulting in increased cell invasion capacities

Computational identification of new potential transcriptional partners of ERRα in breast cancer cells: specific partners for specific targets

International audienceEstrogen related receptors are orphan members of the nuclear receptor superfamily acting as transcription factors (TFs). In contrast to classical nuclear receptors, the activities of the ERRs are not controlled by a natural ligand. Regulation of their activities thus relies on availability of transcriptional co-regulators. In this paper, we focus on ERRα, whose involvement in cancer progression has been broadly demonstrated. We propose a new approach to identify potential co-activators, starting from previously identified ERRα-activated genes in a breast cancer (BC) cell line. Considering mRNA gene expression from two sets of human BC cells as major endpoint, we used sparse partial least squares modeling to uncover new transcriptional regulators associated with ERRα. Among them, DDX21, MYBBP1A, NFKB1, and SETD7 are functionally relevant in MDA-MB-231 cells, specifically activating the expression of subsets of ERRα-activated genes. We studied SET7 in more details and showed its co-localization with ERRα and its ERRα-dependent transcriptional and phenotypic effects. Our results thus demonstrate the ability of a modeling approach to identify new transcriptional partners from gene expression. Finally, experimental results show that ERRα cooperates with distinct co-regulators to control the expression of distinct sets of target genes, thus reinforcing the combinatorial specificity of transcription

A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein&ndash;protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context