RESUMO
Lysine Specific Demethylase 1 (LSD1) removes mono- and dimethyl groups from lysine 4 of histone H3 (H3K4) or H3K9, resulting in repressive or activating (respectively) transcriptional histone marks. The mechanisms that control the balance between these two antagonist activities are not understood. We here show that LSD1 and the orphan nuclear receptor estrogen-related receptor α (ERRα) display commonly activated genes. Transcriptional activation by LSD1 and ERRα involves H3K9 demethylation at the transcriptional start site (TSS). Strikingly, ERRα is sufficient to induce LSD1 to demethylate H3K9 in vitro. The relevance of this mechanism is highlighted by functional data. LSD1 and ERRα coregulate several target genes involved in cell migration, including the MMP1 matrix metallo-protease, also activated through H3K9 demethylation at the TSS. Depletion of LSD1 or ERRα reduces the cellular capacity to invade the extracellular matrix, a phenomenon that is rescued by MMP1 reexpression. Altogether our results identify a regulatory network involving a direct switch in the biochemical activities of a histone demethylase, leading to increased cell invasion.
Assuntos
Histona Desmetilases/metabolismo , Histonas/metabolismo , Receptores de Estrogênio/metabolismo , Movimento Celular , Regulação da Expressão Gênica , Células HEK293 , Histona Desmetilases/genética , Humanos , Lisina/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metilação , Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Sítio de Iniciação de Transcrição , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Several physiopathological processes require orientated cellular migration. This phenomenon highly depends on members of the RHO family of GTPases. Both excessive and deficient RHO activity impair directional migration. A tight control is thus exerted on these proteins through the regulation of their activation and of their stability. Here we show that the estrogen-related receptor α (ERRα) directly activates the expression of TNFAIP1, the product of which [BTB/POZ domain-containing adapter for Cullin3-mediated RhoA degradation 2 (BACURD2)] regulates RHOA protein turnover. Inactivation of the receptor leads to enhanced RHOA stability and activation. This results in cell disorientation, increased actin network, and inability to form a lamellipodium at the migration edge. As a consequence, directional migration, but not cell motility per se, is impaired in the absence of the receptor, under pathological as well as physiological conditions. Altogether, our results show that the control exerted by ERRα on RHOA stability is required for directional migration.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Receptores de Estrogênio/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Culina/metabolismo , Matriz Extracelular/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Cicatrização , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
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-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.
Assuntos
Mapas de Interação de Proteínas , Humanos , Microscopia de Fluorescência/métodosRESUMO
BACKGROUND: 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 in vitro and in vivo the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells. RESULTS: We show that Ens-1 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 Ens-1 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 Ens-1. CONCLUSION: Our results show that Ens-1 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, Ens-1 LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.
Assuntos
DNA Viral/genética , Células-Tronco Embrionárias/virologia , Retrovirus Endógenos/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Embrião de Galinha , Ligação Proteica , Sequências Repetidas TerminaisRESUMO
Estrogen 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.
Assuntos
Neoplasias da Mama , Receptores de Estrogênio , Neoplasias da Mama/genética , RNA Helicases DEAD-box/genética , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Cell 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.
Assuntos
Actinas , Adesões Focais , Fatores de Despolimerização de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Movimento Celular/fisiologia , Adesões Focais/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Endocrine-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.
Assuntos
Disruptores Endócrinos/química , Disruptores Endócrinos/farmacologia , Fibroblastos/citologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Células Cultivadas , Disruptores Endócrinos/classificação , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , HumanosRESUMO
Lysine-specific demethylase 1 (LSD1) exerts dual effects on histone H3, promoting transcriptional repression via Lys4 (H3K4) demethylation or transcriptional activation through Lys9 (H3K9) demethylation. These activities are often exerted at transcriptional start sites (TSSs) and depend on the type of enhancer-bound transcription factor (TFs) with which LSD1 interacts. In particular, the Estrogen-Receptor Related α (ERRα) TF interacts with LSD1 and switches its activities toward H3K9 demethylation, resulting in transcriptional activation of a set of common target genes. However, how are the LSD1-TF and, in particular LSD1-ERRα, complexes determined to act at TSSs is not understood. Here we show that promoter-bound nuclear respiratory factor 1 (NRF1), but not ERRα, is essential to LSD1 recruitment at the TSSs of positive LSD1-ERRα targets. In contrast to ERRα, NRF1 does not impact on the nature of LSD1 enzymatic activity. We propose a three factor model, in which the LSD1 histone modifier requires a TSS tethering factor (NRF1) as well as an activity inducer (ERRα) to transcriptionally activate common targets. The relevance of this common network is illustrated by functional data, showing that all three factors are required for cell invasion in an MMP1 (Matrix MetalloProtease 1)-dependent manner, the expression of which is regulated by NRF1/LSD1/ERRα-mediated H3K9me2 demethylation.
Assuntos
Histona Desmetilases/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Receptores de Estrogênio/metabolismo , Linhagem Celular , Cromatina/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica , Ativação Transcricional , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
The TP63 gene, a member of the TP53 gene family, encodes several isoforms with (TAp63) or without (DeltaNp63) transactivating properties. Whereas the role of p63 in the normal development of squamous epithelia is well established, its function in other cell types remains to be elucidated. Here, we have analysed the expression of TA and DeltaNp63 isoforms in liver cells, by using both primary hepatocytes from wild type and p53-null mice and three human hepatocellular carcinoma (HCC) cell lines, according to the transformation state and the TP53 status of the cells. We observed the expression of DeltaNp63 isoforms only in a p53-null context. On the other hand, the expression of TAp63 isoforms was restricted to the HCC cell lines, whatever the TP53 status. We then studied the expression of TP63 upon genotoxic treatment. When treated with UVB or H(2)O(2), hepatocytes did not exhibit any change in p63 mRNA level. At the opposite, upon treatment with topoisomerase II inhibitors (doxorubicin or etoposide), the expression of TAp63 isoforms was clearly induced, independently of the TP53 status of cells. The same treatment did not induce any variation in the expression of DeltaNp63 isoforms, both at mRNA and protein levels. In HCC cell lines, doxorubicin or etoposide treatment also resulted in an increase of TAp63 transcripts only. This increase was accompanied by an increase in the intracellular level of TAp63 alpha protein. In parallel, we observed an upregulation of some p53-target genes related to cell cycle regulation, such as WAF1/CIP1, PIG3, 14-3-3sigma or GADD45, independently of the TP53 status of cells. In conclusion, we report for the first time that TA and DeltaNp63 alpha proteins are present in liver cells. Furthermore, our results suggest that p63 may partially substitute for wild-type p53, in counteracting uncontrolled liver cell proliferation in response to certain forms of DNA-damage.
Assuntos
Genes p53 , Fosfoproteínas/genética , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Células 3T3 , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/genética , Camundongos , Família Multigênica , Isoformas de Proteínas , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/metabolismo , RNA Neoplásico/metabolismo , Receptores de Estrogênio/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Neoplásico/genética , Receptores de Estrogênio/genética , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
ERRα is an orphan member of the nuclear receptor family, the complete inactivation of which confers resistance to bone loss induced by ageing and estrogen withdrawal to female mice in correlation with increased bone formation in vivo. Furthermore ERRα negatively regulates the commitment of mesenchymal cells to the osteoblast lineage ex vivo as well as later steps of osteoblast maturation. We searched to determine whether the activities of ERRα on osteoblast maturation are responsible for one or both types of in vivo induced bone loss. To this end we have generated conditional knock out mice in which the receptor is normally present during early osteoblast differentiation but inactivated upon osteoblast maturation. Bone ageing in these animals was similar to that observed for control animals. In contrast conditional ERRαKO mice were completely resistant to bone loss induced by ovariectomy. We conclude that the late (maturation), but not early (commitment), negative effects of ERRα on the osteoblast lineage contribute to the reduced bone mineral density observed upon estrogen deficiency.
Assuntos
Receptor alfa de Estrogênio/fisiologia , Estrogênios/deficiência , Osteoblastos/citologia , Osteoporose/fisiopatologia , Animais , Linhagem da Célula , Receptor alfa de Estrogênio/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Osteoporose/patologia , CoelhosRESUMO
PGC-1α 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-1α is repressed by estrogens in the mouse specifically in the uterus. In the absence of estrogens, expression of PGC-1α target genes involved in mitochondrial activity is activated, but not mitochondrial biogenesis. Regulation of PGC-1α expression by estrogens also occurs in Ishikawa human uterine cells at the promoter level and involve modulation of c-jun expression.
Assuntos
Estrogênios/farmacologia , Proteínas de Choque Térmico/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismo , Animais , Regulação para Baixo/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Proteínas de Choque Térmico/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Diferenciação Celular/genética , Proliferação de Células , Proteínas Cromossômicas não Histona/genética , Células-Tronco Embrionárias/metabolismo , Animais , Western Blotting , Ciclo Celular/genética , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Epigenômica , Perfilação da Expressão Gênica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
High expression of the estrogen receptor-related receptor (ERR)-alpha in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRalpha reduces the proliferation of various cell lines and blocks the G(1)/S transition of the cell cycle in an ERRalpha-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21(waf/cip)(1) at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice.