Genome binding/occupancy profiling by high throughput sequencing
Summary
The Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The interrelatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population, and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control.
Overall design
Mouse embryonic stem cells in which PRC1 can be depleted via the auxin-inducible degron (AID) system were profiled for genomic distribution of Polycomb factors (RING1B, SUZ12), histone modifications (H2AK119ub1, H3K27me3, H3K4me3) and RNA polymerase II (total Pol II, Ser5P-Pol II, Ser2P-Pol II) using spike-in calibrated ChIP-seq. Cells were treated with auxin for 2, 4, 8 or 24 hours in three independent biological replicates. Another mouse ESC line in which PRC2 can be depleted via the dTAG-inducible degron system was profiled in three independent biological replicates for genomic distribution of H3K27me3 before and after 2 hours treatment with dTAG-13 compound. Additionally, RING1B binding was compared between wild type (TIR1) and PRC1deg cells before auxin treatment, and H3K27me3 enrichment was compared between wild type (E14) and PRC2deg cell before dTAG-13 treatment.
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