Project description:Use Global Run-On assay (GRO)-seq, chromatin immunoprecipitation (ChIP)-seq, and DNA: RNA immunoprecipitation (DRIP)-seq to investigate whether and how hNABP2 (hSSB1) regualte transcription.
Project description:DNA Double Strands Breaks (DSBs) are highly detrimental since they can lead to mutations and chromosomes rearrangements (amplification, deletion, translocation and chromosome loss). Here, we set to assess the role of senataxin, a RNA:DNA helicase involved in the regulation of transcription and the maintenance of genome integrity, at sites of DNA Double Strand Breaks. We performed ChIP-Seq mapping of senataxin before and after damage, genome-wide DNA:RNA hybrids (DRIP) mapping before and after damage. We also performed RNA-Seq and RNA pol II mapping (total and phosphorylated on serine 2 of the CTD) by ChIP-Seq in undamaged cells to discriminate between damage in active versus inactive regions.
Project description:DNA topoisomerases assist DNA replication & transcription events by controlling supercoiling alterations. We investigated supercoil distribution across the yeast genome and compared with the accumulation of RNA pol2 and DNA topoisomerases particularly in S-phase. Our data indicate that Top2 along with Hmo1 maintain negative supercoil at gene boundaries by stabilizing alternative DNA structures. To understand how DNA superhelical tension accumulates across the genome we have adopted previously described method [Naughton C et al., 2013] to budding yeast where a biotin molecule was attached to TMP via a linker (bTMP). The Chip on chip analysis for proteins was carried out as described (Bermejo R et al., 2009). For RNA-DNA hybrids DRIP-chip is carried out as described previously (Chan YA et al., 2014). Supercoiled regions are then compared with RNA pol2 (RPB3-chip), DNA Topoisomerase (Top1-chip) & RNA-DNA hybrid (DRIP-chip).
Project description:R-loops are three-stranded nucleic acid structures consisting of a DNA-RNA hybrid and a displaced single-stranded DNA (ssDNA). R-loops are facilitators of gene expression and genome stability that play both regulatory and potentially deleterious roles in cells. To elucidate the protein-based mechanisms of R-loop regulation, we developed an APEX-based proximity proteomics using a catalytically inactive mutant of RNase H1 (APEX-RNH1D210N) to profile the proximal proteome of R-loops in cells. Our proteomics results identified a list of known and potential R-loop-binding proteins with diverse molecular functions, and confirmed YY1 as a novel R-loop-binding protein through a series of in-vitro binding assays. YY1 is a DNA-binding protein recognizing a consensus motif or G-quadruplex (G4) structure. Our binding results indicated YY1's notably stronger affinity for R-loops compared to RNA/DNA hybrid, DNA G4 and DNA without a consensus motif, implying R-loops' role in YY1 recruitment to genomic regions lacking a YY1-binding motif. To investigate YY1-R-loop interaction in cells, we introduced R-loop structures into specific genomic regions vis CRISPR-dCas9 targeting, followed by DRIP-qPCR, YY1-ChIP-qPCR and RT-qPCR experiments. Increased R-loop levels corresponded with heightened YY1 occupancy and differential gene expression, suggesting YY1-R-loop interactions contribute to transcriptional regulation. More importantly, our RNA-seq and YY1-ChIP-seq results revealed that the interactions between YY1 and promoter R-loops were involved in global transcriptional regulation, especially in the positive regulation of transcription. Together, we identified YY1 as a novel R-loop-binding protein and uncovered a mechanism wherein YY1-R-loop interactions regulate gene expression.
Project description:To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we constructed CCRF-CEM (T-ALL) stable cell lines expressing HA-AMPKα2 and performed ChIP-seq and RNA-seq assays in control or glucose deprivation conditions. ChIP-seq and RNA-seq analysis identified the histone genes as a subset of genes regulated by AMPK with direct AMPKα2 occupancy at their TSS region.
Project description:H3K27me3 signals on the chromatin were determined using ChIP-seq assay to assess if deletin of AMPKa1 and AMPKa2, which inactivates the AMPK kinase, affected global H3K27me3 abundance and distribution on the chromatin.