RESUMEN
Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pull-down assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17ß-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an "activated" state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a "natural" ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Receptor alfa de Estrógeno/metabolismo , Regulación Neoplásica de la Expresión Génica , Nucleosomas/metabolismo , Proteómica , Elementos de Respuesta/genética , Neoplasias de la Mama/genética , Inmunoprecipitación de Cromatina , ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Receptor alfa de Estrógeno/genética , Estrógenos/farmacología , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HeLa , Humanos , Células MCF-7 , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coactivador 3 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/metabolismo , Nucleosomas/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transactivadores , Transcripción Genética , Activación TranscripcionalRESUMEN
The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.