RESUMO
Estrogen receptor α (ER) is the major driver of â¼75% of breast cancers, and multiple ER targeting drugs are routinely used clinically to treat patients with ER+ breast cancer. However, many patients relapse on these targeted therapies and ultimately develop metastatic and incurable disease, and understanding the mechanisms leading to drug resistance is consequently of utmost importance. It is now clear that, in addition to estrogens, ER function is modulated by other steroid receptors and multiple signaling pathways (e.g., growth factor and cytokine signaling), and many of these pathways affect drug resistance and patient outcome. Here, we review the mechanisms through which these pathways impact ER function and drug resistance as well as discuss the clinical implications.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptores de Esteroides/metabolismo , Transdução de Sinais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Citocinas/fisiologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Interactions between transcriptional promoters and their distal regulatory elements play an important role in transcriptional regulation; however, the extent to which these interactions are subject to rapid modulations in response to signals is unknown. Here, we use promoter capture Hi-C to demonstrate a rapid reorganization of promoter-anchored chromatin loops within 4 hr after inducing differentiation of 3T3-L1 preadipocytes. The establishment of new promoter-enhancer loops is tightly coupled to activation of poised (histone H3 lysine 4 mono- and dimethylated) enhancers, as evidenced by the acquisition of histone H3 lysine 27 acetylation and the binding of MED1, SMC1, and P300 proteins to these regions, as well as to activation of target genes. Intriguingly, formation of loops connecting activated enhancers and promoters is also associated with extensive recruitment of corepressors such as NCoR and HDACs, indicating that this class of coregulators may play a previously unrecognized role during enhancer activation.
Assuntos
Adipócitos/metabolismo , Adipogenia , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Regiões Promotoras Genéticas , Células 3T3-L1 , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Elementos Facilitadores Genéticos , Subunidade 1 do Complexo Mediador/genética , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Fatores de Tempo , Transcrição Gênica , Ativação TranscricionalRESUMO
Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-ß and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPß and -δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis.
Assuntos
Adipócitos/imunologia , Adipócitos/metabolismo , Apresentação de Antígeno , Antígenos CD1d/metabolismo , Proteínas de Transporte/metabolismo , Lipídeos/imunologia , Células 3T3-L1 , Adipogenia/genética , Adipogenia/imunologia , Animais , Antígenos CD1d/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteínas de Transporte/genética , Comunicação Celular/imunologia , Metabolismo Energético , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Células T Matadoras Naturais/imunologia , Transcrição GênicaRESUMO
Adipogenesis is tightly controlled by a complex network of transcription factors acting at different stages of differentiation. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBP) family members are key regulators of this process. We have employed DNase I hypersensitive site analysis to investigate the genome-wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides with cooperative binding of multiple early transcription factors (including glucocorticoid receptor, retinoid X receptor, Stat5a, C/EBPß and -δ) to transcription factor 'hotspots'. Our results demonstrate that C/EBPß marks a large number of these transcription factor 'hotspots' before induction of differentiation and chromatin remodelling and is required for their establishment. Furthermore, a subset of early remodelled C/EBP-binding sites persists throughout differentiation and is later occupied by PPARγ, indicating that early C/EBP family members, in addition to their well-established role in activation of PPARγ transcription, may act as pioneering factors for PPARγ binding.
Assuntos
Adipogenia/fisiologia , Montagem e Desmontagem da Cromatina , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Algoritmos , Animais , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Imunoprecipitação da Cromatina , Camundongos , RNA Mensageiro/genética , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC directly regulates enhancer activity to promote cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNA polymerase II (RNAPII), rather than regulating RNAPII pause-release, as is the case at promoters. This process is mediated by MYC-induced H3K9 demethylation and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. We propose that MYC drives prognostic cancer type-specific gene programs through induction of an enhancer-specific epigenetic switch, which can be targeted by BET and GCN5 inhibitors.
Assuntos
Neoplasias , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Proteínas Nucleares/genética , Prognóstico , Elementos Facilitadores Genéticos/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Epigênese Genética , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo Celular/genéticaRESUMO
The epithelial-to-mesenchymal transition (EMT) plays a central role in the development of cancer metastasis and resistance to chemotherapy. However, its pharmacological treatment remains challenging. Here, we used an EMT-focused integrative functional genomic approach and identified an inverse association between short-chain fatty acids (propionate and butanoate) and EMT in non-small cell lung cancer (NSCLC) patients. Remarkably, treatment with propionate in vitro reinforced the epithelial transcriptional program promoting cell-to-cell contact and cell adhesion, while reducing the aggressive and chemo-resistant EMT phenotype in lung cancer cell lines. Propionate treatment also decreased the metastatic potential and limited lymph node spread in both nude mice and a genetic NSCLC mouse model. Further analysis revealed that chromatin remodeling through H3K27 acetylation (mediated by p300) is the mechanism underlying the shift toward an epithelial state upon propionate treatment. The results suggest that propionate administration has therapeutic potential in reducing NSCLC aggressiveness and warrants further clinical testing.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Propionatos/farmacologia , Propionatos/uso terapêutico , Camundongos Nus , Linhagem Celular Tumoral , Pulmão/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Movimento CelularRESUMO
Nuclear receptors (NRs) are key transcriptional regulators of metazoan physiology and metabolism. Different NRs bind to similar or even identical core response elements; however, they regulate transcription in a highly receptor- and gene-specific manner. These differences in gene activation can most likely be accounted for by mechanisms involving receptor-specific interactions with DNA as well as receptor-specific interactions with protein complexes binding to adjacent and distant DNA sequences. Here, we review key molecular aspects of transactivation by NRs with special emphasis on the recent advances in the molecular mechanisms responsible for receptor- and gene-specific transcriptional activation. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.
Assuntos
Receptores Citoplasmáticos e Nucleares/fisiologia , Ativação Transcricional/genética , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptor Cross-Talk , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Estrogen receptor-α (ER) drives tumor development in ER-positive (ER+) breast cancer. The transcription factor GATA3 has been closely linked to ER function, but its precise role in this setting remains unclear. Quantitative proteomics was used to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, few proteins were lost from the ER complex in the absence of GATA3, with the only major change being depletion of the dioxygenase TET2. TET2 binding constituted a near-total subset of ER binding in multiple breast cancer models, with loss of TET2 associated with reduced activation of proliferative pathways. TET2 knockdown did not appear to change global methylated cytosine (5mC) levels; however, oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) was significantly reduced, and these events occurred at ER enhancers. These findings implicate TET2 in the maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.
Assuntos
5-Metilcitosina/análogos & derivados , Neoplasias da Mama/enzimologia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/metabolismo , 5-Metilcitosina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Bases de Dados Genéticas , Dioxigenases/genética , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/genética , Feminino , Fulvestranto/farmacologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens to understand endocrine drug resistance, we discovered ARID1A and other SWI/SNF complex components as the factors most critically required for response to two classes of estrogen receptor-alpha (ER) antagonists. In this context, SWI/SNF-specific gene deletion resulted in drug resistance. Unexpectedly, ARID1A was also the top candidate in regard to response to the bromodomain and extraterminal domain inhibitor JQ1, but in the opposite direction, with loss of ARID1A sensitizing breast cancer cells to bromodomain and extraterminal domain inhibition. We show that ARID1A is a repressor that binds chromatin at ER cis-regulatory elements. However, ARID1A elicits repressive activity in an enhancer-specific, but forkhead box A1-dependent and active, ER-independent manner. Deletion of ARID1A resulted in loss of histone deacetylase 1 binding, increased histone 4 lysine acetylation and subsequent BRD4-driven transcription and growth. ARID1A mutations are more frequent in treatment-resistant disease, and our findings provide mechanistic insight into this process while revealing rational treatment strategies for these patients.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilase 1/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Histona Desacetilase 1/genética , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cytokine interleukin-6 (IL6) and its downstream effector STAT3 constitute a key oncogenic pathway, which has been thought to be functionally connected to estrogen receptor α (ER) in breast cancer. We demonstrate that IL6/STAT3 signaling drives metastasis in ER+ breast cancer independent of ER. STAT3 hijacks a subset of ER enhancers to drive a distinct transcriptional program. Although these enhancers are shared by both STAT3 and ER, IL6/STAT3 activity is refractory to standard ER-targeted therapies. Instead, inhibition of STAT3 activity using the JAK inhibitor ruxolitinib decreases breast cancer invasion in vivo. Therefore, IL6/STAT3 and ER oncogenic pathways are functionally decoupled, highlighting the potential of IL6/STAT3-targeted therapies in ER+ breast cancer.
Assuntos
Neoplasias da Mama/genética , Elementos Facilitadores Genéticos/genética , Receptor alfa de Estrogênio/genética , Interleucina-6/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fulvestranto/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Metástase Neoplásica , Fator de Transcrição STAT3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Estrogen Receptor alpha (ERα) plays a major role in most breast cancers, and it is the target of endocrine therapies used in the clinic as standard of care for women with breast cancer expressing this receptor. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used against the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Assuntos
Anticorpos , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Receptor alfa de Estrogênio/imunologia , Imunoprecipitação/métodos , Especificidade de Anticorpos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7RESUMO
The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Fatores de Transcrição E2F/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Fatores de Transcrição E2F/metabolismo , Feminino , Humanos , Células MCF-7 , Camundongos SCID , Morfolinas/administração & dosagem , Piperazinas/administração & dosagem , Piridinas/administração & dosagem , Pirimidinas , Receptores de Estrogênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Understanding the dynamics of endogenous protein-protein interactions in complex networks is pivotal in deciphering disease mechanisms. To enable the in-depth analysis of protein interactions in chromatin-associated protein complexes, we have previously developed a method termed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins). Here, we present a quantitative multiplexed method (qPLEX-RIME), which integrates RIME with isobaric labelling and tribrid mass spectrometry for the study of protein interactome dynamics in a quantitative fashion with increased sensitivity. Using the qPLEX-RIME method, we delineate the temporal changes of the Estrogen Receptor alpha (ERα) interactome in breast cancer cells treated with 4-hydroxytamoxifen. Furthermore, we identify endogenous ERα-associated proteins in human Patient-Derived Xenograft tumours and in primary human breast cancer clinical tissue. Our results demonstrate that the combination of RIME with isobaric labelling offers a powerful tool for the in-depth and quantitative characterisation of protein interactome dynamics, which is applicable to clinical samples.
Assuntos
Cromatina/metabolismo , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Animais , Neoplasias da Mama/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Feminino , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
The three dimensional folding of mammalian genomes is cell type specific and difficult to alter suggesting that it is an important component of gene regulation. However, given the multitude of chromatin-associating factors, the mechanisms driving the colocalization of active chromosomal domains and the role of this organization in regulating the transcription program in adipocytes are not clear. Analysis of genome-wide chromosomal associations revealed cell type-specific spatial clustering of adipogenic genes in 3T3-L1 cells. Time course analysis demonstrated that the adipogenic 'hub', sampled by PPARγ and Lpin1, undergoes orchestrated reorganization during adipogenesis. Coupling the dynamics of genome architecture with multiple chromatin datasets indicated that among all the transcription factors (TFs) tested, RXR is central to genome reorganization at the beginning of adipogenesis. Interestingly, at the end of differentiation, the adipogenic hub was shifted to an H3K27me3-repressive environment in conjunction with attenuation of gene transcription. We propose a stage-specific hierarchy for the activity of TFs contributing to the establishment of an adipogenic genome architecture that brings together the adipogenic genetic program. In addition, the repositioning of this network in a H3K27me3-rich environment at the end of differentiation may contribute to the stabilization of gene transcription levels and reduce the developmental plasticity of these specialized cells. DATABASE: All sequence data reported in this paper have been deposited at GEO (http://www.ncbi.nlm.nih.gov/geo/) (GSE92475).
Assuntos
Adipócitos/metabolismo , Adipogenia/genética , Cromatina/química , Proteínas Nucleares/genética , PPAR gama/genética , Fosfatidato Fosfatase/genética , Receptores X de Retinoides/genética , Células 3T3-L1 , Adipócitos/citologia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , PPAR gama/metabolismo , Fosfatidato Fosfatase/metabolismo , Cultura Primária de Células , Receptores X de Retinoides/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key activators of adipogenesis. They mutually induce the expression of each other and have been reported to cooperate in activation of a few adipocyte genes. Recently, genome-wide profiling revealed a high degree of overlap between PPARγ and C/EBPα binding in adipocytes, suggesting that cooperativeness could be mediated through common binding sites. To directly investigate the interplay between PPARγ and C/EBPα at shared binding sites, we established a fibroblastic model system in which PPARγ and C/EBPα can be independently expressed. Using RNA sequencing, we demonstrate that coexpression of PPARγ and C/EBPα leads to synergistic activation of many key metabolic adipocyte genes. This is associated with extensive C/EBPα-mediated reprogramming of PPARγ binding and vice versa in the vicinity of these genes, as determined by chromatin immunoprecipitation combined with deep sequencing. Our results indicate that this is at least partly mediated by assisted loading involving chromatin remodeling directed by the leading factor. In conclusion, we report a novel mechanism by which the key adipogenic transcription factors, PPARγ and C/EBPα, cooperate in activation of the adipocyte gene program.
Assuntos
Adipócitos/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , PPAR gama/metabolismo , Células 3T3 , Animais , Sítios de Ligação/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Camundongos , PPAR gama/genética , Ligação Proteica/genéticaRESUMO
It is becoming increasingly clear that transcription factors operate in complex networks through thousands of genomic binding sites, many of which bind several transcription factors. However, the extent and mechanisms of crosstalk between transcription factors at these hotspots remain unclear. Using a combination of advanced proteomics and genomics approaches, we identify â¼12,000 transcription factor hotspots (â¼400 bp) in the early phase of adipogenesis, and we find evidence of both simultaneous and sequential binding of transcription factors at these regions. We demonstrate that hotspots are highly enriched in large super-enhancer regions (several kilobases), which drive the early adipogenic reprogramming of gene expression. Our results indicate that cooperativity between transcription factors at the level of hotspots as well as super-enhancers is very important for enhancer activity and transcriptional reprogramming. Thus, hotspots and super-enhancers constitute important regulatory hubs that serve to integrate external stimuli on chromatin.
Assuntos
Adipogenia , Elementos Facilitadores Genéticos , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Camundongos , Ligação Proteica , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Transcription factors have recently been shown to colocalize in hotspot regions of the genome, which are further clustered into super-enhancers. However, the detailed molecular organization of transcription factors at hotspot regions is poorly defined. Here, we have used digital genomic footprinting to precisely define factor localization at a genome-wide level during the early phase of 3T3-L1 adipocyte differentiation, which allows us to obtain detailed molecular insight into how transcription factors target hotspots. We demonstrate the formation of ATF-C/EBP heterodimers at a composite motif on chromatin, and we suggest that this may be a general mechanism for integrating external signals on chromatin. Furthermore, we find evidence of extensive recruitment of transcription factors to hotspots through alternative mechanisms not involving their known motifs and demonstrate that these alternative binding events are functionally important for hotspot formation and activity. Taken together, these findings provide a framework for understanding transcription factor cooperativity in hotspots.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Adipogenia , Células 3T3 , Fatores Ativadores da Transcrição/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/genética , Pegada de DNA , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Camundongos , Motivos de Nucleotídeos , Ligação Proteica , Ativação TranscricionalRESUMO
Adipocyte differentiation is tightly controlled by a transcriptional cascade, which directs the extensive reprogramming of gene expression required to convert fibroblast-like precursor cells into mature lipid-laden adipocytes. Recent global analyses of transcription factor binding and chromatin remodeling have revealed 'snapshots' of this cascade and the chromatin landscape at specific time-points of differentiation. These studies demonstrate that multiple adipogenic transcription factors co-occupy hotspots characterized by an open chromatin structure and specific epigenetic modifications. Such transcription factor hotspots are likely to represent key signaling nodes which integrate multiple adipogenic signals at specific chromatin sites, thereby facilitating coordinated action on gene expression.