RLSuite: An Integrative R-Loop Bioinformatics Framework.

We recently described the development of a database of 810 R-loop mapping datasets and used this data to conduct a meta-analysis of R-loops. R-loops are three-stranded nucleic acid structures containing RNA:DNA hybrids and we were able to verify that 30% of expressed genes have an associated R-loop in a location conserved manner.. Moreover, intergenic R-loops map to enhancers, super enhancers and with TAD domain boundaries. This work demonstrated that R-loop mapping via high-throughput sequencing can reveal novel insight into R-loop biology, however the analysis and quality control of these data is a non-trivial task for which few bioinformatic tools exist. Herein we describe RLSuite, an integrative R-loop bioinformatics framework for pre-processing, quality control, and downstream analysis of R-loop mapping data. RLSuite enables users to compare their data to hundreds of public datasets and generate a user-friendly analysis report for sharing with non-bioinformatician colleagues. Taken together, RLSuite is a novel analysis framework that should greatly benefit the emerging R-loop bioinformatics community in a rapidly expanding aspect of epigenetic control that is still poorly understood.

p21 controls patterning but not homologous recombination in RPE development.

p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53.

Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay.

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.