Project description:The gene nhr-6 encodes the sole C. elegans NR4A nuclear receptor homolog which has a critical role in organogenesis by regulating the development of the spermatheca organ. Here we utilized chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify NHR-6 binding sites during both the late L3/early L4 and mid L4 developmental stages. Our results revealed 30,745 enriched binding sites for NHR-6 with sites specific to each developmental stage and sites found in both stages. Additionally, ~70% of enriched sites were found within 3 kb upstream of a gene transcription start site. Binding sites for a cohort of candidate target genes with probable functions in spermatheca organogenesis were validated through qPCR. Reproductive and spermatheca phenotypes were also evaluated for these genes following a loss-of-function RNAi screen which revealed several genes with critical functions during spermatheca organogenesis. Our results uncovered a complex nuclear receptor regulatory network whereby NHR-6 regulates multiple cellular processes including proliferation, differentiation, and metabolism.
Project description:Dosage Compensation is required to correct for uneven gene dose between the sexes. We utilized global run-on sequencing (GRO-seq) to examine how Caenorhabditis elegans dosage compensation reduces transcription of X-linked genes. To facilitate these experiments, we required accurate 5M-bM-^@M-^Y-ends of genes that have been missing due to a co-transcriptional trans-splicing event common in nematodes. We developed a modified GRO-seq protocol to identify TSSs that are supported by transcription, and determined that TSSs lie more than 1 kb upstream of the previously annotated TSS for nearly one-quarter of all genes. We then investigated the changes that occur in transcriptionally engaged RNA Polymerase when dosage compensation is disrupted, and find that dosage compensation controls recruitment of RNA Polymerase to X-linked genes. GRO-cap reactions were performed with TAP, and without TAP as a control.
Project description:In this study we have used ChIP followed by high throughput sequencing to profile the genome-wide recruitment of wildtype and non-oligomerizing Groucho (Gro) at high resolution in single cell types (Kc167 and S2R+) using Drosophila cell culture. Our results reveal that Gro is typically recruited to discrete peaks in active chromatin and that blocking Gro oligomerization does not change the width of the peaks to which it is recruited. We have also investigated acetylated histone H3 and H4 and RNA polymerase II profiles around Gro binding sites, along with gene expression, in wildtype, Gro knockdown, Gro-GFP transfected, and non-oligomerizing Gro-GFP transfected Kc167 cells and found that Gro associates with chromatin containing hypoacetylated histones and frequently overlaps the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing.
