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Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

ABSTRACT: Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? Here we investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell lineage. We find that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global down-regulation of PRC2, recruitment of hypophosphorylated RNA Polymerase II (Pol II) to promoters, as well as expression and action of cell-type specific homologs of subunits of TFIID (tTAFs). In addition, action of tMAC, a tissue specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell-type specific localization of PRC1 components and tTAFs to the spermatocyte nucleolus. Together, action of the tMAC and tTAF cell-type specific chromatin and transcription machinery leads to loss of Polycomb and release of Pol II from the promoter of terminal differentiation genes to elongation, allowing robust transcription of the terminal differentiation genes.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE28728 | GEO | 2011/07/15

SECONDARY ACCESSION(S): PRJNA140535

REPOSITORIES: GEO

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Project description:Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? Here we investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell lineage. We find that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global down-regulation of PRC2, recruitment of hypophosphorylated RNA Polymerase II (Pol II) to promoters, as well as expression and action of cell-type specific homologs of subunits of TFIID (tTAFs). In addition, action of tMAC, a tissue specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell-type specific localization of PRC1 components and tTAFs to the spermatocyte nucleolus. Together, action of the tMAC and tTAF cell-type specific chromatin and transcription machinery leads to loss of Polycomb and release of Pol II from the promoter of terminal differentiation genes to elongation, allowing robust transcription of the terminal differentiation genes. Total RNA from approximately 200 pairs of fly testes (bam1/bam-delta-86; aly2/aly5P; sa1/sa2; y,w) was extracted using TRIzol (Invitrogen, #15596-018, Carlsbad, CA, USA) following the manufacturer's instructions. The genomic DNA was degraded using 2 Units of DNase I (Fermentas, #EN0521, Glen Burnie, MD, USA) at 37 degreeC for 20 minutes. RNA integrity was checked by gel electrophoresis (1% agarose). Approximately 4 ≈í¬∫g of total RNA from each biological replicate were used to generate labeling probes to hybridize with the Affymetix GeneChip Drosophila Genome 2.0 Array according to the Affymetrix protocol. Three biological replicates were performed for each genotype. Microarray hybridization was processed at the Core Facility at the Stanford University School of Medicine and the raw data were exported from the Affymetrix Microarray Suite (MAS). The CEL files were used for signal normalization with RMA as part of the limma package from the Bioconductor R packages (http://www.bioconductor.org).

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.

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Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

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