Project description:Chromosomal rearrangements are essential events in the pathogenesis of both malignant and nonmalignant disorders, yet the factors affecting their formation are incompletely understood. We developed a zinc finger nuclease translocation reporter (ZITR) and screened for factors that modulate rearrangements in human cells. We identified UBC9 and RAD50 as suppressors and 53BP1, DDB1, and PARP3 as promoters of chromosomal rearrangements across human cell types. We focused on poly(ADP)ribose polymerase 3 (PARP3) as it is dispensable for murine viability and has druggable catalytic activity. We found that PARP3 regulated G quadruplex (G4) DNA in response to DNA damage, which suppressed repair by nonhomologous end-joining and homologous recombination. Chemical stabilization of G4 DNA in PARP3-/- cells led to widespread DNA double-strand breaks and synthetic lethality. We propose a model in which PARP3 suppresses G4 DNA and facilitates DNA repair by multiple pathways.
Project description:G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and they are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. We found that drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation, and oppositely, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. To study the underlying mechanisms by which native G4 affects transcriptional regulation, we annealed the biotin-labeled core promoter DNA to form G4s and performed pull-down assays with nuclear extraction proteins in the presence or absence of TMPyP4. Mass spectrometry analysis was performed to identify the interacting proteins with G4-forming core promoter DNA.
Project description:SPO11-promoted DNA double-strand breaks (DSBs) formation is a crucial step for meiotic recombination, and it is indispensable to detect the broken DNA ends accurately for dissecting the molecular mechanisms behind. Here, we report a novel technique, named DEtail-seq (DNA End tailing followed by sequencing), that can directly and quantitatively capture the meiotic DSB 3’ overhang hotspots at single-nucleotide resolution.
Project description:DNA sequences of high guanine (G) content have the potential to fold into G quadruplex (G4) structures. DNA G4 is known to play important roles in various cellular processes including DNA replication, transcriptional regulation, and maintenance of genomic integrity. A more complete understanding about the biological functions of G4 DNA requires the investigation about how these structures are recognized by cellular proteins. Here, we conducted exhaustive quantitative proteomic experiments to profile the interaction proteomes of three well-defined G4 structures derived from the human telomere and the promoters of cMYC and cKIT genes. Our results led to the identification of a number of candidate G4-interacting proteins; some were previously reported, e.g., FUS, TOP1, and PARP1, and many others were discovered here for the first time. These included three proteins that can bind to all three DNA G4 structures and many proteins that can bind specifically to selected DNA G4 structure(s). We also validated that GRSF1 can bind directly and selectively toward G4 DNA structure derived from the cMYC promoter. Taken together, we uncovered a number of cellular proteins that exhibit general and selective recognitions of G4 folding patterns, which underscore the complexity of G4 DNA in biology and the importance in understanding fully the G4-interaction proteome.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
