Genome binding/occupancy profiling by high throughput sequencing
Summary
Condensins are evolutionarily conserved protein complexes that are required for chromosome segregation during cell division and genome organization during interphase. In C. elegans, a specialized condensin, which forms the core of the dosage compensation complex (DCC), binds to and represses X chromosome transcription. Here, we analyzed DCC localization and the effect of DCC depletion on histone modifications, transcription factor binding, and gene expression using ChIP-seq and mRNA-seq. Across the X, DCC accumulates at accessible gene regulatory sites in active chromatin and not heterochromatin. DCC is required for reducing the levels of activating histone modifications, including H3K4me3 and H3K27ac, but not repressive modification H3K9me3. In X-to-autosome fusion chromosomes, DCC spreading into the autosomal sequences locally reduces gene expression, thus establishing a direct link between DCC binding and repression. Together, our results indicate that DCC-mediated transcription repression is associated with a reduction in the activity of X chromosomal gene regulatory elements.
Overall design
Included are ChIP-seq profiles data from C. elegans mixed stage embryos and L3 larva. Data was collected from two to three replicates. ChIP-seq data sets in N2, H3K9me3 null (GW638), X;A fusion (15eh1, YPT41), DCC mutant (CB428), and DCC RNAi knockdown (DPY-27 RNAi) strains were generated using antibodies against three dosage compensation complex proteins (SDC-3, CAPG-1, and DPY-27), histone modifications H3K4me3, H3K27ac, H4K16ac, H4panAc, H3ac, H3K4me1, H3K27me1, H3K27me2, H3K9me3, and H4K20me1, the histone protein H3, IgG, and chromatin associated proteins MDT-15, CBP-1, PQN-85, AMA-1, RNA Pol II, and PHA-4::GFP. PHA-4 was pulled down using anti-GFP in OP37[PHA-4::GFP] strain. We additionally include four replicates of RNA-seq data in L3 larva N2 and X;II and X;V fusion strains.
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