RESUMEN
During early development, gene expression is tightly regulated. However, how genome organization controls gene expression during the transition from naïve embryonic stem cells to epiblast stem cells is still poorly understood. Using single-molecule microscopy approaches to reach nanoscale resolution, we show that genome remodeling affects gene transcription during pluripotency transition. Specifically, after exit from the naïve pluripotency state, chromatin becomes less compacted, and the OCT4 transcription factor has lower mobility and is more bound to its cognate sites. In epiblast cells, the active transcription hallmark, H3K9ac, decreases within the Oct4 locus, correlating with reduced accessibility of OCT4 and, in turn, with reduced expression of Oct4 nascent RNAs. Despite the high variability in the distances between active pluripotency genes, distances between Nodal and Oct4 decrease during epiblast specification. In particular, highly expressed Oct4 alleles are closer to nuclear speckles during all stages of the pluripotency transition, while only a distinct group of highly expressed Nodal alleles are in close proximity to Oct4 when associated with a nuclear speckle in epiblast cells. Overall, our results provide new insights into the role of the spatiotemporal genome remodeling during mouse pluripotency transition and its correlation with the expression of key pluripotency genes.
Asunto(s)
Genoma , Estratos Germinativos , Células Madre Embrionarias de Ratones , Factor 3 de Transcripción de Unión a Octámeros , Animales , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Genoma/genética , Regulación del Desarrollo de la Expresión Génica , Cromatina/metabolismo , Cromatina/genética , Diferenciación Celular/genética , Imagen Individual de Molécula/métodos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Histonas/metabolismo , Histonas/genética , Ensamble y Desensamble de CromatinaRESUMEN
Transcription and genome architecture are interdependent, but it is still unclear how nucleosomes in the chromatin fiber interact with nascent RNA, and which is the relative nuclear distribution of these RNAs and elongating RNA polymerase II (RNAP II). Using super-resolution (SR) microscopy, we visualized the nascent transcriptome, in both nucleoplasm and nucleolus, with nanoscale resolution. We found that nascent RNAs organize in structures we termed RNA nanodomains, whose characteristics are independent of the number of transcripts produced over time. Dual-color SR imaging of nascent RNAs, together with elongating RNAP II and H2B, shows the physical relation between nucleosome clutches, RNAP II, and RNA nanodomains. The distance between nucleosome clutches and RNA nanodomains is larger than the distance measured between elongating RNAP II and RNA nanodomains. Elongating RNAP II stands between nascent RNAs and the small, transcriptionally active, nucleosome clutches. Moreover, RNA factories are small and largely formed by few RNAP II. Finally, we describe a novel approach to quantify the transcriptional activity at an individual gene locus. By measuring local nascent RNA accumulation upon transcriptional activation at single alleles, we confirm the measurements made at the global nuclear level.