BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation

Summary: BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR. : Following induction of DNA double-strand break, a pro-end-joining environment is created in G2 by transient 53BP1 phosphorylation and RIF1 recruitment. Here, Isono et al. show that, if timely repair does not ensue, BRCA1 promotes 53BP1 dephosphorylation and RIF1 release, favoring repair by homologous recombination. Keywords: ATM, DNA-end resection, BRCA1, 53BP1, RIF1, PP4C, NHEJ, H

Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe

Background and Purpose: To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.Copyright:Materials and Methods: DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53+/ + and p53-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA doublestrand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.Results: The p53-/- cells were more resistant than the p53+/+ cells to X-ray irradiation, while the sensitivities of the p53+/+ and p53-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53+/+ cells but not the p53-/- cells. In the p53-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring longretained DSBs at 24 h post-irradiation.Conclusions: Efficient induction of mitotic catastrophe in apoptosis-resistant p53- deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Effects of X-irradiation on embryonic stem cells-derived neural stem cells

The neural stem cells increase in number and differentiate intoneurons, astrocytes and oligodendrocytes in developmental brainduring fetal period. Radiation exposure during the fetal period leadsto disturbances in development of the brain and can inducemicrocephaly or anencephalia. Here we investigated biologicaleffects of the X-irradiation on the neural stem cells, prepared frommouse embryonic stem (ES) cells by the Neural Stem Sphere (NSS)method. Cultured with mitogen fibroblast growth factor-2, theneural stem cells proliferated exponentially. When the neural stemcells were exposed to various doses of X-ray, the proliferation of theneural stem cells was dose-dependently inhibited by X-irradiationand almost completely stopped by 5 Gy-radiation. Morphology ofthe multipolar neural stem cells was affected by the radiation, andthe surviving cells after radiation were bipolar and larger than theneural stem cells. However, immunofluorescent study showed thatthe surviving bipolar cells were positive for nestin, and geneexpression of nestin and musashi-1 was also demonstrated in thecells by RT-PCR study. These results suggested that radiationinhibits proliferation of the neural stem cells but the surviving cellsafter radiation maintain basic properties of the neural stem cells

Radiosensitivity of neural stem cells derived from mouse embryonic stem cells: Proliferation, cell cycle regulation and DNA double-strand break repair

Radiation exposure to developing fetal period is known to cause neuronal disorders, such as neuronal dysfunction, microcephaly and mental retardation, which are considered mainly due to the radiation damage produced in neural stem cells (NSCs). NSCs, which are the dominant number in the fetal brain, have an ability to proliferate itself, and differentiate into other cell types that construct the central nervous system (CNS), such as neurons, astrocytes and oligodendrocytes. Recently, it has been reported that NSCs exist in an adult brain. Thus, investigation on radiation biological effects against NSCs would be one of the important studies for radiation risk assessment, such as for the field of radiation therapy, and health risks against space environmental radiation exposure. The purpose of this study was to examine the cellular responses of proliferating NSCs against X-ray irradiation. Following biological endpoints were measured to demonstrate the radio-sensitivity of NSCs: maintenance of its ability to proliferate as NSCs, growth curve, cell cycle regulation and DNA repair. The NSCs were prepared from embryonic stem cells (HK cell line) of C57BL/6 mice using Neural Stem Sphere method that has established originally. The cells were irradiated up to 10 Gy by 200 kVp X-ray. Our findings demonstrated that NSCs, which survived after X-ray irradiation, maintained its ability as stem cells. Cellular responses, such as cell cycle arrest due to the DNA damage recognized at G1 and G2/M check points, and the linear-quadratic repair kinetics of DNA double-strand break repair, were similar to other mammalian cell lines. Further investigation on radiation sensitivities of NSCs in proliferation condition is necessary, and clarifying the correlation between the radiation-induced DNA damage in proliferative NSCs and its effect in the differentiation pathway will give the important information on radiation risks against in CNS.14th International Congress of Radiation Researc

Proliferation and differentiation of neural stem cells irradiated by X-ray in logarithmic growth phase

Ionizing radiation to a fetal brain can cause microcephaly and mental retardation, which is thought to result from defects in neurogenesis during the developing stage. In the central nervous system of a brain, neural stem cells (NSCs) have important roles, which are considered to be the major characteristic, such as enabling itself to proliferate so as to increase it numbers, and to differentiate into neurons, astrocytes, and oligodendrocytes. To understand the cellular responses of NSCs to radiation exposure, purified NSCs derived from embryonic stem cells by the Neural Stem Sphere (NSS) method were irradiated with a 6 MV X-ray source and analyzed with growth curve measurements, real-time RT-PCR and immunofluorescence. The 1 Gy-irradiated cells recovered an ability to proliferate and maintained expression of the NSCs marker Nestin. Cells irradiated with more than 5 Gy also showed the expression of Nestin, although they lost their ability to proliferate. The sub-cultured cells after 1 Gy-irradiation proliferated logarithmically and differentiated into neurons and astrocytes. These results provided the basic information for defects in neurogenesis in a brai