Project description:Thousands of transcripts accumulate in an oscillatory manner during C. elegans larval development. Here, we confirmed these oscillations to result from rhythmic RNA polymerase II (RNAPII) binding to these oscillating genes by performing RNAPII ChIP-seq and total RNA-seq on the same samples (see related submission)
Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:Despite highly conserved chromatin states and cis-regulatory elements, studies of metazoan genomes reveal that gene organization and the strategies to control mRNA expression can vary widely among animal species. C. elegans gene regulation is often assumed to be similar to that of other model organisms, yet evidence suggests the existence of distinct molecular mechanisms to pattern the developmental transcriptome, including extensive post-transcriptional RNA control pathways, widespread splice leader (SL) trans-splicing of pre-mRNAs, and the organization of genes into operons. Here, we performed ChIP-seq for histone modifications in highly synchronized embryos cohorts representing three major developmental stages, with the goal of better characterizing whether the dynamic changes in embryonic mRNA expression are accompanied by changes to the chromatin state. We were surprised to find that thousands of promoters are persistently marked by active histone modifications, despite a fundamental restructuring of the transcriptome. We employed global run-on sequencing using a long-read nanopore format to map nascent RNA transcription across embryogenesis, finding that the invariant open chromatin regions are persistently transcribed by Pol II at all stages of embryo development, even though the mature mRNA is not produced. By annotating our nascent RNA sequencing reads into directional transcription units, we find extensive evidence of polycistronic RNA transcription genome-wide, suggesting that nearby genes in C. elegans are linked by shared transcriptional regulatory mechanisms. We present data indicating that the sharing of cis-regulatory sequences has constrained C. elegans gene positioning and likely explains the remarkable retention of syntenic gene pairs over long evolutionary timescales.
