RESUMO
PPARγ2 is a critical lineage-determining transcription factor that is essential for adipogenic differentiation. Here we report characterization of the three-dimensional structure of the PPARγ2 locus after the onset of adipogenic differentiation and the mechanisms by which it forms. We identified a differentiation-dependent loop between the PPARγ2 promoter and an enhancer sequence 10 kb upstream that forms at the onset of PPARγ2 expression. The arginine methyltransferase Prmt5 was required for loop formation, and overexpression of Prmt5 resulted in premature loop formation and earlier onset of PPARγ2 expression. Kinetic studies of regulatory factor interactions at the PPARγ2 promoter and enhancer revealed enhanced interaction of Prmt5 with the promoter that preceded stable association of Prmt5 with enhancer sequences. Prmt5 knockdown prevented binding of both MED1, a subunit of Mediator complex that facilitates enhancer-promoter interactions, and Brg1, the ATPase of the mammalian SWI/SNF chromatin remodeling enzyme required for PPARγ2 activation and adipogenic differentiation. The data indicate a dynamic association of Prmt5 with the regulatory sequences of the PPARγ2 gene that facilitates differentiation-dependent, three-dimensional organization of the locus. In addition, other differentiation-specific, long-range chromatin interactions showed Prmt5-dependence, indicating a more general role for Prmt5 in mediating higher-order chromatin connections in differentiating adipocytes.
Assuntos
Adipogenia/genética , Diferenciação Celular , Elementos Facilitadores Genéticos , Loci Gênicos , PPAR gama/genética , Regiões Promotoras Genéticas , Proteína-Arginina N-Metiltransferases/metabolismo , Células 3T3-L1 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Ligação Proteica , Transporte Proteico , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , DNA Helicases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Inibidores Enzimáticos/uso terapêutico , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Interferência de RNA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Differentiation signaling results in reprogramming of cellular gene expression that leads to morphological changes and functional specialization of a precursor cell. This global change in gene expression involves temporal regulation of differentiation-specific genes that are located throughout the genome, raising the idea that genome structure may also be re-organized during cell differentiation to facilitate regulated gene expression. Using in vitro adipocyte differentiation as a model, we explored whether gene organization within the nucleus is altered upon exposure of precursor cells to signaling molecules that induce adipogenesis. The peroxisome proliferator-activated receptor gamma (PPARγ) nuclear hormone receptor is a master determinant of adipogenesis and is required for adipose differentiation. We utilized the chromosome conformation capture (3C) assay to determine whether the position of the PPARγ locus relative to other adipogenic genes is changed during differentiation. We report that the PPARγ2 promoter is transiently positioned in proximity to the promoters of genes encoding adipokines and lipid droplet associated proteins at 6 hours post-differentiation, a time that precedes expression of any of these genes. In contrast, the PPARγ2 promoter was not in proximity to the EF1α promoter, which drives expression of a constitutively active, housekeeping gene that encodes a translation elongation factor, nor was the PPARγ2 promoter in proximity to the promoter driving the expression of the C/EBPα regulatory protein. The formation of the long-range, intergenic interactions involving the PPARγ2 promoter required the regulatory factor C/EBPß, elevated cyclic AMP (cAMP) levels, and protein kinase A (PKA) signaling. We conclude that genome organization is dynamically remodeled in response to adipogenic signaling, and we speculate that these transient inter-genic interactions may be formed for the purposes of selecting some of the transcriptionally silent tissue-specific loci for subsequent transcriptional activation.
Assuntos
Adipogenia , Cromatina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , PPAR gama/genética , Células 3T3-L1 , Adipocinas/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Transporte/genética , Loci Gênicos , Camundongos , Perilipina-1 , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Regulation of adipose tissue formation by adipogenic-regulatory proteins has long been a topic of interest given the ever-increasing health concerns of obesity and type 2 diabetes in the general population. Differentiation of precursor cells into adipocytes involves a complex network of cofactors that facilitate the functions of transcriptional regulators from the CCATT/enhancer binding protein, and the peroxisome proliferator-activated receptor (PPAR) families. Many of these cofactors are enzymes that modulate the structure of chromatin by altering histone-DNA contacts in an ATP-dependent manner or by posttranslationally modifying the histone proteins. Here we report that inhibition of protein arginine methyltransferase 5 (Prmt5) expression in multiple cell culture models for adipogenesis prevented the activation of adipogenic genes. In contrast, overexpression of Prmt5 enhanced adipogenic gene expression and differentiation. Chromatin immunoprecipitation experiments indicated that Prmt5 binds to and dimethylates histones at adipogenic promoters. Furthermore, the presence of Prmt5 promoted the binding of ATP-dependent chromatin-remodeling enzymes and was required for the binding of PPARγ2 at PPARγ2-regulated promoters. The data indicate that Prmt5 acts as a coactivator for the activation of adipogenic gene expression and promotes adipogenic differentiation.
Assuntos
Adipogenia/genética , Expressão Gênica , PPAR gama/genética , Proteínas Metiltransferases/fisiologia , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Montagem e Desmontagem da Cromatina , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Histonas/metabolismo , Gordura Intra-Abdominal/citologia , Gordura Intra-Abdominal/metabolismo , Metilação , Camundongos , PPAR gama/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases , Técnicas de Cultura de TecidosRESUMO
The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that specifies formation of the adipocyte lineage. PPARγ also serves as a primary target for the treatment of type 2 diabetes, illustrating both its medical relevance as well as the need to understand fundamental aspects of PPARγ expression and function. Here, we characterize molecular changes that occur at the PPARγ2 promoter within the first several hours of adipocyte differentiation in culture. Our results demonstrate that changes in chromatin accessibility at the PPARγ2 promoter and occupancy of the promoter by the c-Fos transcription factor occur within an hour of the onset of differentiation, followed closely by the binding of the CCAAT/enhancer binding protein beta (C/EBPß) transcription factor. All three events show a remarkable dependency on protein kinase A (PKA) activity. These results reflect novel requirements for the PKA signaling pathway and reinforce the importance of PKA function during the onset of adipocyte differentiation.
Assuntos
Adipogenia/fisiologia , Cromatina/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , PPAR gama/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , PPAR gama/genética , Ligação Proteica/fisiologiaRESUMO
BACKGROUND AND AIMS: Glomerular diseases are the third leading cause of kidney failure worldwide, behind only diabetes and hypertension. The molecular mechanisms underlying the cause of glomerular diseases are still largely unknown. The identification and characterization of new molecules associated with glomerular function should provide new insights into understanding the diverse group of glomerular diseases. The Chd2 protein belongs to a family of enzymes involved in ATP-dependent chromatin remodeling, suggesting that it likely functions as an epigenetic regulator of gene expression via the modification of chromatin structure. METHODS: In this study, we present a detailed histomorphologic characterization of mice containing a mutation in the chromodomain helicase DNA-binding protein 2 (Chd2). RESULTS: We show that Chd2-mutant mice present with glomerulopathy, proteinuria, and significantly impaired kidney function. Additionally, serum analysis revealed decreased hemoglobin and hematocrit levels in Chd2-mutant mice, suggesting that the glomerulopathy observed in these mice is associated with anemia. CONCLUSION: Collectively, the data suggest a role for the Chd2 protein in the maintenance of kidney function.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Nefropatias/genética , Mutação , Animais , Proteínas de Ligação a DNA/fisiologia , Epigênese Genética , Glomerulonefrite Membranosa , Rim/fisiologia , Rim/fisiopatologia , Camundongos , Fenótipo , ProteinúriaRESUMO
The E3 ubiquitin ligase Cbl-b is a negative regulator of TCR signaling that: 1) sets the activation threshold for T cells; 2) is induced in anergic T cells; and 3) protects against autoimmunity. However, the role of Cbl-b in regulating CD8 T cell activation and functions during physiological T cell responses has not been systematically examined. Using the lymphocytic choriomeningitis virus infection model, we show that Cbl-b deficiency did not significantly affect the clonal expansion of virus-specific CD8 T cells. However, Cbl-b deficiency not only increased the steady-state cell surface expression levels of TCR and CD8 but also reduced Ag-induced down-modulation of cell surface TCR expression by effector CD8 T cells. Diminished Ag-stimulated TCR down-modulation and sustained Ag receptor signaling induced by Cbl-b deficiency markedly augmented IFN-gamma production, which is known to require substantial TCR occupancy. By contrast, Cbl-b deficiency minimally affected cell-mediated cytotoxicity, which requires limited engagement of TCRs. Surprisingly, despite elevated expression of CD8 and reduced Ag-induced TCR down-modulation, the functional avidity of Cbl-b-deficient effector CD8 T cells was comparable to that of wild-type effectors. Collectively, these data not only show that Cbl-b-imposed constraint on TCR signaling has differential effects on various facets of CD8 T cell response but also suggest that Cbl-b might mitigate tissue injury induced by the overproduction of IFN-gamma by CD8 T cells. These findings have implications in the development of therapies to bolster CD8 T cell function during viral infections or suppress T cell-mediated immunopathology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Interferon gama/biossíntese , Vírus da Coriomeningite Linfocítica/imunologia , Proteínas Proto-Oncogênicas c-cbl/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Apresentação de Antígeno/imunologia , Antígenos CD8/biossíntese , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/virologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Epitopos de Linfócito T/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-cbl/biossíntese , Proteínas Proto-Oncogênicas c-cbl/deficiência , Receptores de Antígenos de Linfócitos T/biossíntese , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
Dominant mutations in the early growth response 2 (Egr2/Krox20) transactivator, a critical regulator of peripheral myelin development, have been associated with peripheral myelinopathies. These dominant mutants interfere with the expression of genes required for myelination by Schwann cells, including that for the most abundant peripheral myelin protein, Myelin protein zero (Mpz). In this study, we show that Egr2 mutants specifically affect an Egr2-responsive element within the Mpz first intron that also contains binding sites for the transcription factor Sox10. Furthermore, Egr2 activation through this element is impaired by mutation of the Sox10 binding sites. Using chromatin immunoprecipitation assays, we found that Egr2 and Sox10 bind to this element in myelinating sciatic nerve and that a dominant Egr2 mutant does not perturb Egr2 binding but rather attenuates binding of Sox10 to the Mpz intron element. Sox10 binding at other sites of Egr2/Sox10 synergy, including a novel site in the Myelin-associated glycoprotein (Mag) gene, is also reduced by the dominant Egr2 mutant. These results provide the first demonstration of binding of Egr2/Sox10 to adjacent sites in vivo and also demonstrate that neuropathy-associated Egr2 mutants antagonize binding of Sox10 at specific sites, thereby disrupting genetic control of the myelination program.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Mutação/genética , Proteína P0 da Mielina/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteínas de Grupo de Alta Mobilidade/genética , Íntrons/genética , Camundongos , Doenças do Sistema Nervoso Periférico/metabolismo , Ligação Proteica , Ratos , Fatores de Transcrição SOXE , Sensibilidade e Especificidade , Fatores de Transcrição/genéticaRESUMO
Egr2/Krox20 is a zinc finger transactivator that regulates a diverse array of genes required for peripheral nerve myelination. Although several studies have elucidated the Egr2-regulated gene network, it is not clear if Egr2 regulates its target genes directly or indirectly through induction of other transactivators. Moreover, very few Egr2 binding sites have been identified in regulatory elements of myelin genes. To address this issue, we have successfully adapted chromatin immunoprecipitation assays to test if Egr2 binds directly to target genes in myelinating rat sciatic nerve. These experiments demonstrate direct binding of Egr2 to previously described binding sites within the Schwann cell enhancer of the myelin basic protein gene. Furthermore, we show Egr2 binding to a conserved site within the myelin-associated glycoprotein gene. Finally, our experiments provide the first evidence that Egr2 directly regulates expression of desert hedgehog, which is critically involved in development, maintenance and regeneration of multiple nerve elements including myelinated fibers. Surprisingly, this analysis has identified an apparent preponderance of Egr2 binding sites within conserved intron sequences of several myelin genes. Application of chromatin immunoprecipitation analysis to myelination in vivo will prove to be a valuable asset in assaying transcription factor binding and chromatin modifications during activation of myelin genes.
Assuntos
Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Proteínas da Mielina/metabolismo , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação/fisiologia , Linhagem Celular , Imunoprecipitação da Cromatina/métodos , Proteína 2 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica/fisiologia , Proteínas Hedgehog , Humanos , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina/metabolismo , Proteínas da Mielina/genética , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/metabolismo , Ligação Proteica/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células de Schwann/metabolismo , Transativadores/metabolismoRESUMO
During myelination of the peripheral nervous system, the myelin protein zero (Mpz) gene is induced to produce the most abundant protein component (P(0)) of mature myelin. Although the basal embryonic expression of Mpz in Schwann cells has been attributed to regulation by Sox10, the molecular mechanism for the profound up-regulation of this gene during myelination has not been established. In this study, we have identified a highly conserved element within the first intron of the Mpz gene, which contains binding sites for the early growth response 2 (Egr2/Krox20) transcription factor, a critical regulator of peripheral nerve myelination. Egr2 can transactivate the intron element, and the induction is blocked by two known repressors of Egr2 activity. Using chromatin immunoprecipitation assays, we find that Egr2 binds in vivo to the intron element, but not to the Mpz promoter. Known inducers of Mpz expression such as forskolin and insulin-like growth factor-1 also activate the element in an Egr2-dependent manner. In addition, we found that Egr2 can act synergistically with Sox10 to activate this intron element, suggesting a model in which cooperative interactions between Egr2 and Sox10 mediate a large increase in Mpz expression to the high levels found in myelinating Schwann cells.
Assuntos
Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Regulação da Expressão Gênica , Proteína P0 da Mielina/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 20 , RNA Helicases DEAD-box , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Íntrons , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína P0 da Mielina/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Helicases/genética , RNA Helicases/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXE , Células de Schwann/citologia , Células de Schwann/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Myelination of peripheral nerves by Schwann cells requires a large amount of lipid and cholesterol biosynthesis. To understand the transcriptional coordination of the myelination process, we have investigated the developmental relationship between early growth response 2 (Egr2)/Krox20--a pivotal regulator of peripheral nerve myelination--and the sterol regulatory element binding protein (SREBP) pathway, which controls expression of cholesterol/lipid biosynthetic genes. During myelination of sciatic nerve, there is a very significant induction of SREBP1 and SREBP2, as well as their target genes, suggesting that the SREBP transactivators are important regulators in the myelination process. Egr2/Krox20 does not appear to directly regulate the levels of SREBP pathway components, but rather, we found that Egr2/Krox20 and SREBP transactivators can synergistically activate promoters of several SREBP target genes, indicating that direct induction of cholesterol/lipid biosynthetic genes by Egr2/Krox20 is a part of the myelination program regulated by this transactivator.