Single-Cell Analysis of the Multicellular Ecosystem in Viral Carcinogenesis by HTLV-1

成人T細胞白血病リンパ腫の多段階発がん分子メカニズムを解明 --難治性疾患の新規治療標的候補を複数同定--. 京都大学プレスリリース. 2021-09-07.Premalignant clonal expansion of human T-cell leukemia virus type-1 (HTLV-1)–infected cells occurs before viral carcinogenesis. Here we characterize premalignant cells and the multicellular ecosystem in HTLV-1 infection with and without adult T-cell leukemia/lymphoma (ATL) by genome sequencing and single-cell simultaneous transcriptome and T/B-cell receptor sequencing with surface protein analysis. We distinguish malignant phenotypes caused by HTLV-1 infection and leukemogenesis and dissect clonal evolution of malignant cells with different clinical behavior. Within HTLV-1–infected cells, a regulatory T-cell phenotype associates with premalignant clonal expansion. We also delineate differences between virus- and tumor-related changes in the nonmalignant hematopoietic pool, including tumor-specific myeloid propagation. In a newly generated conditional knockout mouse model recapitulating T-cell–restricted CD274 (encoding PD-L1) gene lesions found in ATL, we demonstrate that PD-L1 overexpressed by T cells is transferred to surrounding cells, leading to their PD-L1 upregulation. Our findings provide insights into clonal evolution and immune landscape of multistep virus carcinogenesis

Coincidence analysis to search for inspiraling compact binaries using TAMA300 and LISM data

Japanese laser interferometric gravitational wave detectors, TAMA300 and LISM, performed a coincident observation during 2001. We perform a coincidence analysis to search for inspiraling compact binaries. The length of data used for the coincidence analysis is 275 hours when both TAMA300 and LISM detectors are operated simultaneously. TAMA300 and LISM data are analyzed by matched filtering, and candidates for gravitational wave events are obtained. If there is a true gravitational wave signal, it should appear in both data of detectors with consistent waveforms characterized by masses of stars, amplitude of the signal, the coalescence time and so on. We introduce a set of coincidence conditions of the parameters, and search for coincident events. This procedure reduces the number of fake events considerably, by a factor $\sim 10^{-4}$ compared with the number of fake events in single detector analysis. We find that the number of events after imposing the coincidence conditions is consistent with the number of accidental coincidences produced purely by noise. We thus find no evidence of gravitational wave signals. We obtain an upper limit of 0.046 /hours (CL $= 90$) to the Galactic event rate within 1kpc from the Earth. The method used in this paper can be applied straightforwardly to the case of coincidence observations with more than two detectors with arbitrary arm directions.Comment: 28 pages, 17 figures, Replaced with the version to be published in Physical Review

Results of the search for inspiraling compact star binaries from TAMA300's observation in 2000-2004

We analyze the data of TAMA300 detector to search for gravitational waves from inspiraling compact star binaries with masses of the component stars in the range 1-3Msolar. In this analysis, 2705 hours of data, taken during the years 2000-2004, are used for the event search. We combine the results of different observation runs, and obtained a single upper limit on the rate of the coalescence of compact binaries in our Galaxy of 20 per year at a 90% confidence level. In this upper limit, the effect of various systematic errors such like the uncertainty of the background estimation and the calibration of the detector's sensitivity are included.Comment: 8 pages, 4 Postscript figures, uses revtex4.sty The author list was correcte

Integrated genetic and clinical prognostic factors for aggressive adult T-cell leukemia/lymphoma

成人T細胞白血病リンパ腫（ATL）におけるゲノム情報と臨床情報を統合したリスクモデルを確立 --ATLの個別化医療を推進--. 京都大学プレスリリース. 2023-04-10.The prognosis of aggressive adult T-cell leukemia/lymphoma (ATL) is poor, and allogeneic hematopoietic stem-cell transplantation (allo-HSCT) is a curative treatment. To identify favorable prognostic patients after intensive chemotherapy, and who therefore might not require upfront allo-HSCT, we aimed to improve risk stratification of aggressive ATL patients aged <70 years. The clinical risk factors and genetic mutations were incorporated into risk modeling for overall survival (OS). We generated the m7-ATLPI, a clinicogenetic risk model for OS, that included the ATL prognostic index (PI) (ATL-PI) risk category, and non-silent mutations in seven genes, namely TP53, IRF4, RHOA, PRKCB, CARD11, CCR7, and GATA3. In the training cohort of 99 patients, the m7-ATLPI identified a low-, intermediate-, and high-risk group with 2-year OS of 100%, 43%, and 19%, respectively (hazard ratio [HR] 5.46, p < 0.0001). The m7-ATLPI achieved superior risk stratification compared to the current ATL-PI (C-index 0.92 vs. 0.85, respectively). In the validation cohort of 84 patients, the m7-ATLPI defined low-, intermediate-, and high-risk groups with a 2-year OS of 81%, 30%, and 0%, respectively (HR 2.33, p = 0.0094), and the model again outperformed the ATL-PI (C-index 0.72 vs. 0.70, respectively). The simplified m7-ATLPI, which is easier to use in clinical practice, achieved superior risk stratification compared to the ATL-PI, as did the original m7-ATLPI; the simplified version was calculated by summing the following: high-risk ATL-PI category (+10), low-risk ATL-PI category (−4), and non-silent mutations in TP53 (+4), IRF4 (+3), RHOA (+1), PRKCB (+1), CARD11 (+0.5), CCR7 (−2), and GATA3 (−3)

Nap1 regulates proper CENP-B binding to nucleosomes

CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1

Observation results by the TAMA300 detector on gravitational wave bursts from stellar-core collapses

We present data-analysis schemes and results of observations with the TAMA300 gravitational-wave detector, targeting burst signals from stellar-core collapse events. In analyses for burst gravitational waves, the detection and fake-reduction schemes are different from well-investigated ones for a chirp-wave analysis, because precise waveform templates are not available. We used an excess-power filter for the extraction of gravitational-wave candidates, and developed two methods for the reduction of fake events caused by non-stationary noises of the detector. These analysis schemes were applied to real data from the TAMA300 interferometric gravitational wave detector. As a result, fake events were reduced by a factor of about 1000 in the best cases. The resultant event candidates were interpreted from an astronomical viewpoint. We set an upper limit of 2.2x10^3 events/sec on the burst gravitational-wave event rate in our Galaxy with a confidence level of 90%. This work sets a milestone and prospects on the search for burst gravitational waves, by establishing an analysis scheme for the observation data from an interferometric gravitational wave detector

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

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

Epigenetic engineering shows that a human centromere resists silencing mediated by H3K27me3/K9me3

Centromeres are characterized by the centromere-specific H3 variant CENP-A, which is embedded in chromatin with a pattern characteristic of active transcription that is required for centromere identity. It is unclear how centromeres remain transcriptionally active despite being flanked by repressive pericentric heterochromatin. To further understand centrochromatin’s response to repressive signals, we nucleated a Polycomb-like chromatin state within the centromere of a human artificial chromosome (HAC) by tethering the methyltransferase EZH2. This led to deposition of the H3K27me3 mark and PRC1 repressor binding. Surprisingly, this state did not abolish HAC centromere function or transcription, and this apparent resistance was not observed on a noncentromeric locus, where transcription was silenced. Directly tethering the reader/repressor PRC1 bypassed this resistance, inactivating the centromere. We observed analogous responses when tethering the heterochromatin Editor Suv39h1-methyltransferase domain (centromere resistance) or reader HP1α (centromere inactivation), respectively. Our results reveal that the HAC centromere can resist repressive pathways driven by H3K9me3/H3K27me3 and may help to explain how centromeres are able to resist inactivation by flanking heterochromatin