Dataset Information

The RNA Polymerase II factor RPAP1 is critical for Mediator-driven transcription and cell identity

ABSTRACT: The RNA Polymerase II Associated Protein 1 (RPAP1) is conserved across metazoa and critical for stem cell differentiation in plants, however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of lineage specifying factors and for viability. Accordingly, inhibition of RPAP1 triggers somatic cell de-differentiation and facilitates reprogramming into pluripotent stem cells. Conversely, interfering with RPAP1 in ES cells severely impairs their differentiation capacity. Mechanistically, we show that RPAP1 is essential for the interaction between Pol II and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific Pol II factor POLR2M/Gdown1 and the CTD phosphatase RPAP2. In agreement, depletion of RPAP1 disturbs the loading of Pol II and Pol II Ser5 phosphorylation levels and impairs expression of super-enhancer-driven genes. We conclude that Mediator-RPAP1-Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity.

OTHER RELATED OMICS DATASETS IN: PRJNA313941

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Nuria Ibarz

LAB HEAD: Javier Munoz

PROVIDER: PXD007114 | Pride | 2018-01-10

REPOSITORIES: Pride

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			Action	DRS
	20160823_NIF_Cian_IP_SCR_IgGA_Hph_F1.raw	Raw
	20160823_NIF_Cian_IP_SCR_IgGA_Hph_F2.raw	Raw
	20160823_NIF_Cian_IP_SCR_IgGA_Hph_F3.raw	Raw
	20160823_NIF_Cian_IP_SCR_IgGA_Hph_F4.raw	Raw
	20160823_NIF_Cian_IP_SCR_IgGA_Hph_FT.raw	Raw

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The RNA Polymerase II Factor RPAP1 Is Critical for Mediator-Driven Transcription and Cell Identity.

Lynch Cian J CJ Bernad Raquel R Calvo Isabel I Nóbrega-Pereira Sandrina S Ruiz Sergio S Ibarz Nuria N Martinez-Val Ana A Graña-Castro Osvaldo O Gómez-López Gonzalo G Andrés-León Eduardo E Espinosa Angarica Vladimir V Del Sol Antonio A Ortega Sagrario S Fernandez-Capetillo Oscar O Rojo Enrique E Munoz Javier J Serrano Manuel M

Cell reports 20180101 2

The RNA polymerase II-associated protein 1 (RPAP1) is conserved across metazoa and required for stem cell differentiation in plants; however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of cell identity genes and for cell viability. Depletion of RPAP1 triggers cell de-differentiation, facilitates reprogramming toward pluripotency, and impairs differentiation. Mechanistically, we show that RPAP1 is e ...[more]

PMID: 29320736

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Project description:Cell-type specific transcriptional profiling is key to understanding cell fate specification and function. In order to achieve this it has been necessary, to date, to isolate specific cell types from complex tissues. We have developed 'TaDa', a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation or affinity purification. The method is simple, sensitive, highly reproducible and is in principle transferable to any model system. Here we show that TaDa can be used to identify transcribed genes in a cell-type specific manner. We profile the genome-wide binding of RNA polymerase II (Pol II) in adjacent, clonally related neural stem cells in intact Drosophila brains. Our data reveal the activity of non-canonical metabolic pathways in proliferating neuroepithelial cells, and highlight a possible role for the retinal determination gene regulatory network in patterning neural stem cell fates. We also identify temporal differences in the activity of signalling pathways that control neuroepithelial cell fate by profiling Pol II occupancy at two different stages of brain development. Using RNA Pol II TaDa to profile, in a cell-type specific manner, the transcriptional state of neuroepithelial cells at two stages of larval brain development. Closely related asymmetrically dividing neural stem cells (neuroblasts) were also profiled, in order to compare the transcriptomes of two different types of neural stem cells. 3 biological relicates were performed for 3rd instar neuroepithelial cells (with one dye-swap). 2 biological relicates were performed for 3rd instar neuroblasts (with dye-swap). 2 biological relicates were performed for 1st instar neuroepithelial cells (with dye-swap). As additional supporting evidence for the Pol II TaDa technique, 2 biological relicates were performed for 3rd instar salivary glands (with dye-swap) in order to compare with previous Pol II-ChIP data for this tissue [PMID 22821985].