Project description:Here, we identify that a human DNA helicase RTEL1, which is known to play a role in the maintenance of telomeres by interacting with proteins in the shelterin complex by resolving G4 quadruplexes, is essential for prevention of R-loop accumulation during replication and for MiDAS in mitosis at both CFSs and telomeres. Our findings indicate that RTEL1 plays a genome-wide role in preventing the collapse of replication forks at sites of G-quadruplexes and R-loop formation. Considering cancer cells rely on RTEL1 function to resolve the elevated transcription-replication conflicts caused by oncogene activation, RTEL1 is a potential drug target for cancer therapy.
Project description:Strand asymmetry in the distribution of guanines and cytosines, measured by GC skew, predisposes DNA sequences towards R-loop formation upon transcription. Previous work revealed that GC skew and R-loop formation associate with a core set of unmethylated CpG island (CGI) promoters in the human genome. Here, we show that GC skew can distinguish four classes of promoters, including three types of CGI promoters, each associated with unique epigenetic and gene ontology signatures. In particular, we identify a strong and a weak class of CGI promoters and show that these loci are enriched in distinct chromosomal territories reflecting the intrinsic strength of their protection against DNA methylation. Interestingly, we show that strong CGI promoters are depleted from the X chromosome while weak CGIs are enriched, a property consistent with the acquisition of DNA methylation during dosage compensation. Furthermore, we identify a third class of CGI promoters based on its unique GC skew profile and show that this gene set is enriched for Polycomb group targets. Lastly, we show that nearly 2,000 genes harbor GC skew at their 3’ ends and that these genes are preferentially located in gene-dense regions and tend to be closely arranged. Genomic profiling of R-loops accordingly showed that a large proportion of genes with terminal GC skew form R-loops at their 3’-ends, consistent with a role for these structures in permitting efficient transcription termination. Altogether, we show that GC skew and R-loop formation offer significant insights into the epigenetic regulation, genomic organization, and function of human genes. DRIP-seq was performed on genomic DNA extracted from human pluripotent Ntera2 cells. The DNA was either fragmented using HindIII, EcoRI, BsrGI, XbaI and SspI (DRIP-seq 1) or BamHI, NcoI, ApaLI, NheI and PvuII (DRIP-seq 2, two technical replicates). Input DNA was also fragmented with each restriction enzyme cocktail and sequenced alongside.
Project description:We used eXcision Repair-sequencing (XR-seq) to map UV induced damage in U2OS cells and in U2OS cells in which CSA or UVSSA genes were knocked out. Complementation of UVSSA knockout with WT or mutant proteins shows UVSSA is a core component of human transcription coupled repair.
Project description:ORCA is an ORC associated protein that plays important roles in replication initiation as well as heterochromatin organization. We carried out ORCA ChIP-seq in U2OS cells synchronized at different stage of G1 phase to determine its genome wide localization. To understand the genomic features of ORCA binding regions, we also carried out Methylated DNA IP (MeDIP) followed by deep sequencing in U2OS cells to determine the genome wide localizatoin of methyl-CpG sites in U2OS cells and how ORCA bidning regions co-localize with this important repressive mark.
Project description:CRISPR screen: U2OS or U2OS p53KO cells expressing Cas9 were transduced with a whole-genome library of CRISPR sgRNAs, then treated with either DMSO or etoposide. Differential sgRNA abundances were calculated for each condition and used to determine the effect of each single gene knockout on fitness and the drug-induced death rate. RNA-Seq: U2OS or U2OS p53KO cells were cultured with either DMSO or etoposide for 48 hours, and then U2OS cells were incubated in this conditioned media for 8 hours. RNA was collected and use to observe differential expression changes between conditions.
Project description:ChIP followed by deep sequencing was performed with antibodies to ERalpha in U2OS-ERalpha cells treated with 17beta-estradiol. Examination of Eralpha binding sites in U2OS-Eralpha cells. Sequenced input was used as a control
Project description:We performed paired-end poly A+ RNA sequencing using total RNA isolated from U2OS cells that are synchronised into G1, G1/S, S, G2 and M phase of cell cycle.
