Project description:Cohesin and Polycomb proteins functionally interact to control transcription at silenced, restrained, and active genes [expression array data]
Project description:Cohesin is crucial for proper chromosome segregation, but also regulates gene transcription and organism development by poorly understood mechanisms. We find that in Drosophila, cohesin functionally interacts with Polycomb group (PcG) silencing proteins at both silenced and active genes. Cohesin unexpectedly facilitates binding of Polycomb Repressive Complex 1 (PRC1) to many active genes. In contrast, cohesin and PRC1 binding are mutually antagonistic at silenced genes. PRC1 depletion decreases phosphorylated RNA polymerase and mRNA at many active genes, but increases them at silenced genes. Cohesin also facilitates long-range interactions between Polycomb Response Elements in the invected-engrailed gene complex where it represses transcription. These multiple distinct cohesin-PcG interactions reveal a previously unrecognized role for PRC1 in facilitating productive gene transcription, and provide new insights into how cohesin and PRC1 control development. We extracted RNA from control and Ph RNAi-treated BG3 cells and measured changes in gene expression following Ph dose depletion by hybridization to Affymetrix arrays. We also extracted RNA from wild-type wing imaginal disc and measured control wing disc expression levels by hybridization to Affymetrix arrays.
Project description:Cohesin is crucial for proper chromosome segregation, but also regulates gene transcription and organism development by poorly understood mechanisms. We find that in Drosophila, cohesin functionally interacts with Polycomb group (PcG) silencing proteins at both silenced and active genes. Cohesin unexpectedly facilitates binding of Polycomb Repressive Complex 1 (PRC1) to many active genes. In contrast, cohesin and PRC1 binding are mutually antagonistic at silenced genes. PRC1 depletion decreases phosphorylated RNA polymerase and mRNA at many active genes, but increases them at silenced genes. Cohesin also facilitates long-range interactions between Polycomb Response Elements in the invected-engrailed gene complex where it represses transcription. These multiple distinct cohesin-PcG interactions reveal a previously unrecognized role for PRC1 in facilitating productive gene transcription, and provide new insights into how cohesin and PRC1 control development. ChIP-chip of cohesin, Polycomb group proteins, and RNA Polymerase II was performed in whole wing imaginal discs in developing wing imaginal disc, revealing that cohesin and Polycomb Repressive Complex 1 (PRC1) components co-bind with cohesin proteins at active genes. We then measured cohesin, Pc, and H3K27me3 separately in anterior and posterior wing imaginal discs and compared their binding at the invected-engrailed complex, which is silenced in the anterior disc, and expressed in its posterior. This confirmed that cohesin and PRC1 (Pc) co-bind at inv-en in its active state, and H3K27me3 and PRC1 (Pc) co-target inv-en in its silenced state. Comparison of binding between Pc-RJ and Pc-VP was performed, and revealed that Pc-VP is subject to epitope masking specifically at active genes. Finally, we measured cohesin and Pc binding in Drosophila ML-DmBG3-c2 cells, and found that they co-bind active genes in this cell line in as well as in wing imaginal discs. ChIP-chip of cohesin subunit Rad21 after PRC1 component Ph depletion, and ChIP-chip of PRC1 subunit Pc after Rad21 RNAi depletion, revealed that these two complexes affect one another's binding. Finally, ChIP-chip of Rpb3 (representing total Pol II) and Ser2P-Pol II (representing elongating Pol II) after PRC1 component Ph depletion revealed that PRC1 restrains entry of non-phosphorylated Pol II into gene bodies.
Project description:Cohesin is crucial for proper chromosome segregation, but also regulates gene transcription and organism development by poorly understood mechanisms. We find that in Drosophila, cohesin functionally interacts with Polycomb group (PcG) silencing proteins at both silenced and active genes. Cohesin unexpectedly facilitates binding of Polycomb Repressive Complex 1 (PRC1) to many active genes. In contrast, cohesin and PRC1 binding are mutually antagonistic at silenced genes. PRC1 depletion decreases phosphorylated RNA polymerase and mRNA at many active genes, but increases them at silenced genes. Cohesin also facilitates long-range interactions between Polycomb Response Elements in the invected-engrailed gene complex where it represses transcription. These multiple distinct cohesin-PcG interactions reveal a previously unrecognized role for PRC1 in facilitating productive gene transcription, and provide new insights into how cohesin and PRC1 control development.
Project description:Cohesin is crucial for proper chromosome segregation, but also regulates gene transcription and organism development by poorly understood mechanisms. We find that in Drosophila, cohesin functionally interacts with Polycomb group (PcG) silencing proteins at both silenced and active genes. Cohesin unexpectedly facilitates binding of Polycomb Repressive Complex 1 (PRC1) to many active genes. In contrast, cohesin and PRC1 binding are mutually antagonistic at silenced genes. PRC1 depletion decreases phosphorylated RNA polymerase and mRNA at many active genes, but increases them at silenced genes. Cohesin also facilitates long-range interactions between Polycomb Response Elements in the invected-engrailed gene complex where it represses transcription. These multiple distinct cohesin-PcG interactions reveal a previously unrecognized role for PRC1 in facilitating productive gene transcription, and provide new insights into how cohesin and PRC1 control development.
Project description:The essential histone variant H2A.Z localises to both active and silent chromatin sites. In embryonic stem cells (ESCs), H2A.Z is also reported to co-localise with polycomb repressive complex 2 (PRC2) at developmentally silenced genes. The mechanism of H2A.Z targeting is not clear, but a role for the PRC2 component Suz12 has been suggested. Given this association, we wished to determine if polycomb functionally directs H2A.Z incorporation in ESCs. We demonstrate that the PRC1 component Ring1B interacts with multiple complexes in ESCs. Moreover, we show that although the genomic distribution of H2A.Z co-localises with PRC2, Ring1B and with the presence of CpG islands, H2A.Z still blankets polycomb target loci in the absence of Suz12, Eed (PRC2) or Ring1B (PRC1). Therefore we conclude that H2A.Z accumulates at developmentally silenced genes in ESCs in a polycomb independent manner. Array design includes 2 biological replicates for all samples and technical replication (dye swaps for H3K27me3_WT_ES, EZH2_WT_ES, EZH2_RING1b_KO_ES, H2AZ_WT_ES(2), H2AZ_Eed_KO_ES and H2AZ_Suz12_KO_ES). Ring1B_WT_ES is represented by a single replicate.
Project description:The key transcription factors that control the embryonic stem cell gene expression program have been identified, but how they function to implement this program is not well understood. While screening for genes essential for maintenance of ES cell state, we identified many components of the Mediator and Cohesin complexes. Mediator and Cohesin were found to physically and functionally connect the enhancers and core promoters of active genes. An ES cell Mediator complex was found to copurify with Cohesin and its loading factor Nipbl, and normal levels of these proteins were essential for expression of the genes they occupy and for maintenance of ES cell state. See associated publication.
Project description:The key transcription factors that control the embryonic stem cell gene expression program have been identified, but how they function to implement this program is not well understood. While screening for genes essential for maintenance of ES cell state, we identified many components of the Mediator and Cohesin complexes. Mediator and Cohesin were found to physically and functionally connect the enhancers and core promoters of active genes. An ES cell Mediator complex was found to copurify with Cohesin and its loading factor Nipbl, and normal levels of these proteins were essential for expression of the genes they occupy and for maintenance of ES cell state. See associated publication.
Project description:The essential histone variant H2A.Z localises to both active and silent chromatin sites. In embryonic stem cells (ESCs), H2A.Z is also reported to co-localise with polycomb repressive complex 2 (PRC2) at developmentally silenced genes. The mechanism of H2A.Z targeting is not clear, but a role for the PRC2 component Suz12 has been suggested. Given this association, we wished to determine if polycomb functionally directs H2A.Z incorporation in ESCs. We demonstrate that the PRC1 component Ring1B interacts with multiple complexes in ESCs. Moreover, we show that although the genomic distribution of H2A.Z co-localises with PRC2, Ring1B and with the presence of CpG islands, H2A.Z still blankets polycomb target loci in the absence of Suz12, Eed (PRC2) or Ring1B (PRC1). Therefore we conclude that H2A.Z accumulates at developmentally silenced genes in ESCs in a polycomb independent manner.