Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNAi profiling of Drosophila after Nipped-B and Rad21 sister chromatid cohesin proteins in Drosophila ML-DmBG3 cells

ABSTRACT: Effects of Nipped-B and Rad21 sister chromatid cohesin proteins on gene expression data in ML-DmBG3 cells derived from Drosophila melanogaster larval central nervous system; We examined the effects of Nipped-B and Rad21 knockdown on gene expression in BG3 cells using microarrays that measure over 18,700 transcripts to (a) determine if the effects of cohesion on E(spl)-C and invected-engrailed expression are unique, (b) look for effects of cohesin on regulators of E(spl)-C and engrailed, and (c) obtain a comprehensive view of the effects of cohesin on gene expression. Experiment Overall Design: Effects of cohesin knockdown on E(spl)-C and invected-engrailed transcription vary over time, so we used two independent samples for three days after RNAi treatment, one four day and one six day sample for both Nipped-B and Rad21 knockdown, and mock RNAi controls for each time point. Experiment Overall Design: For RNAi treatment, cells were plated at 5x106 cells per 3 cm well. Media was replaced with 1 ml of Express Five SFM (Invitrogen) with 1% FCS, and 10 micrograms per ml insulin. The indicated amount of dsRNA was added per well. Media was adjusted to 3 ml and 10% FCS with Schneiderâs media after 2 hrs. Cells were replated as needed. Templates for dsRNA synthesis were made by PCR from cDNA templates using primers with T7 promoters (see supplementary file linked below). Equal amounts of two dsRNAs against each target were used.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Akbar Siddiqui

PROVIDER: E-GEOD-16152 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Regulation of the Drosophila Enhancer of split and invected-engrailed gene complexes by sister chromatid cohesion proteins.

Schaaf Cheri A CA Misulovin Ziva Z Sahota Gurmukh G Siddiqui Akbar M AM Schwartz Yuri B YB Kahn Tatyana G TG Pirrotta Vincenzo V Gause Maria M Dorsett Dale D

PloS one 20090709 7

The cohesin protein complex was first recognized for holding sister chromatids together and ensuring proper chromosome segregation. Cohesin also regulates gene expression, but the mechanisms are unknown. Cohesin associates preferentially with active genes, and is generally absent from regions in which histone H3 is methylated by the Enhancer of zeste [E(z)] Polycomb group silencing protein. Here we show that transcription is hypersensitive to cohesin levels in two exceptional cases where cohesin ...[more]

PMID: 19587787

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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.

Dataset Information

RNAi profiling of Drosophila after Nipped-B and Rad21 sister chromatid cohesin proteins in Drosophila ML-DmBG3 cells

Publications

Regulation of the Drosophila Enhancer of split and invected-engrailed gene complexes by sister chromatid cohesion proteins.

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