RESUMEN
The transcription factor Zic3 is required for maintenance of ESC pluripotency. By genome-wide chromatin immunoprecipitation (ChIP-chip) in ESCs, we have identified 379 direct Zic3 targets, many of which are functionally associated with pluripotency, cell cycle, proliferation, oncogenesis, and early embryogenesis. Through a computational analysis of Zic3 target sequences, we have identified a novel Zic3 consensus binding motif (5'-CC(C)/(T) GCTGGG-3'). ChIP results and in vitro DNA binding assays revealed that Zic3 binds with high affinity and specificity on the Nanog promoter. Here, we demonstrate that Zic3 functions as a transcriptional activator of the Nanog promoter in three ways: (a) Nanog transcript levels are sustained with Zic3 overexpression in differentiating ESCs, (b) Zic3 depletion in ESCs downregulates Nanog promoter activity, and (c) Zic3 overexpression leads to increased Nanog promoter activity. Furthermore, the activity of a mutant Nanog promoter with ablated Oct4/Sox2 binding is rescued by Zic3 overexpression to nearly wild-type levels. This indicates that Nanog is a positive transcriptional target of Zic3 in a mechanism that is independent of Oct4/Sox2 binding. Hence, we demonstrate an important pathway for regulation of Nanog expression in pluripotent ESCs through direct activation by Zic3.
Asunto(s)
Células Madre Embrionarias/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Línea Celular , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Ratones , Proteína Homeótica Nanog , Reacción en Cadena de la Polimerasa , Unión Proteica , Interferencia de ARN , Factores de Transcripción/genéticaRESUMEN
Zfp206 (recently renamed Zscan10) encodes a zinc finger transcription factor specifically expressed in human and mouse embryonic stem cells (ESC). It has been shown that Zfp206 is required to maintain ESC in an undifferentiated, pluripotent state. Presented here are data showing that Zfp206 works together with two other transcription factors, Oct4 and Sox2, which are also essential regulators of ESC pluripotency. We show that Zfp206 binds to the Oct4 promoter and directly regulates Oct4 expression. Genome-wide mapping of Zfp206-binding sites in ESC identifies more than 3000 target genes, many of which encode transcription factors that are also targeted for regulation by Oct4 and Sox2. In addition, we show that Zfp206 physically interacts with both Oct4 and Sox2. These data demonstrate that Zfp206 is a key component of the core transcriptional regulatory network and together with Oct4 and Sox2 regulates differentiation of ESC.