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

Antagonistic Actions of HLH/bHLH Proteins Are Involved in Grain Length and Weight in Rice

Grain size is a major yield component in rice, and partly controlled by the sizes of the lemma and palea. Molecular mechanisms controlling the sizes of these organs largely remain unknown. In this study, we show that an antagonistic pair of basic helix-loop-helix (bHLH) proteins is involved in determining rice grain length by controlling cell length in the lemma/ palea. Overexpression of an atypical bHLH, named POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1), in lemma/palea increased grain length and weight in transgenic rice. PGL1 is an atypical non-DNA-binding bHLH and assumed to function as an inhibitor of a typical DNA-binding bHLH through heterodimerization. We identified the interaction partner of PGL1 and named it ANTAGONIST OF PGL1 (APG). PGL1 and APG interacted in vivo and localized in the nucleus. As expected, silencing of APG produced the same phenotype as overexpression of PGL1, suggesting antagonistic roles for the two genes. Transcription of two known grain-length-related genes, GS3 and SRS3, was largely unaffected in the PGL1-overexpressing and APG-silenced plants. Observation of the inner epidermal cells of lemma revealed that are caused by increased cell length. PGL1-APG represents a new grain length and weight-controlling pathway in which APG is a negative regulator whose function is inhibited by PGL1

Localization of PGL1 and APG protein in plant cells.

<p>Fluorescence signal detected using a light microscope from GFP-APG (upper) and GFP-PGL1 (midle) and GFP protein (lower) expression under the 35S promoter in <i>N. benthiamina</i> leaf epidermis cells; GFP, green fluorescent; MERGE, merged view of the GFP and DAPI images. (bar = 50 µm).</p

Inner epidermal cells observed by confocal microscopy.

<p>A) Lemma inner epidermal cells of NiWT and transgenic PGL1:OX (Ni9) and APG RNAi (Ri-12) (bar = 100 µm). B) Distribution of the number of cells at various cell lengths. C) Distribution of the number of cells at various cell widths; NiWT, Nippobare wild type cyan; T₀ transgenic PGL1:OX line Ni9, red; Ni23 green; RNAi T₀ line Ri-1, purple; and Ri-12, blue. Triangles represent average values of the respective lines.</p

Grain traits and lemma inner epidermis cell of PGL1:OX and APG RNAi lines.

a,b:<p> data are the average of 10 samples (±sd).</p>c,d:<p> data are the average of 250 samples (±sd).</p>ns<p>, none-significant;</p><p>*p<0.05;</p><p>**p<0.01;</p><p>***p<0.001.</p><p>NiWT, Nipponbare wild type.</p><p>Ri#, APG RNAi line.</p><p>Ni#, PGL:Ox line.</p

Interaction between PGL1 and APG.

<p>A) Interaction between PGL1 and APG <i>in vitro</i> detected by pull-down assay. Amylose resin–bound MBP-APG or MBP was incubated with an equal amount of GST-PGL1. Proteins co-precipitated with the amylose resin were detected by immunoblotting using anti-GST antibody. B) <i>In vitro</i> homodimerization of APG detected by pull-down assay. Amylose resin–bound MBP-APG or MBP was incubated with an equal amount of GFP-APG extracted from <i>N. benthiamiana</i> leaves. Proteins co-precipitated with the amylose resin were detected by immunoblotting using anti-GFP antibody. C) Confocal images of interaction <i>in vivo</i> between PGL1 and APG revealed by BiFC assay in <i>N. benthiamiana</i> leaf epidermis. BF, bright field image; YFP, yellow fluorescent protein; DAPI, 4′,6-diamidino-2 phenylindole for nuclear staining; MERGE, merged view of the YFP and DAPI images. YN-PGL1+YC-APG indicates <i>Agrobacterium</i> mediated co-infiltration of constructs encoding N-EYFP-PGL1 and C-EYFP-APG (upper); YN-PGL1+YC, co-infitration of N-EYFP-PGL1 and C-EYFP alone (middle); YN+YC-APG, co-infiltration of N-EYFP alone and C-EYFP-APG (lower). (bar = 75 µm) D) Light microscopic images of homodimerization <i>in vivo</i> of APG revealed by BiFC assay. YFP was detected in the nucleus when YN-APG was co-infiltrated with YC-APG, suggesting that the protein forms a homodimer to reconstruct the YFP signal (upper). In contrast, the YFP signal was not detected when YN-APG or YC-APG was used alone. (bar = 50 µm).</p

Overexpression of <i>PGL1</i> increased grain size in rice.

<p>A) Structure of the chitinase promoter and <i>PGL1</i> gene in the pPZP2H-lac binary vector. B) Grain phenotype of T₀ transgenic plants (Ni#) compared with the Nipponbare wild type (NiWT) (bar = 1 cm) C) Quantitative PCR expression analysis of <i>PGL1</i> in the lemma/palea of T₀ plants compared with the wild type (WT = 1) normalized by <i>OsActin</i>. Error bar indicates ±sd over three biological repeats. D) Comparison of grain length and width of transgenic T₀ and wild type plants (error bar, ±sd, n = 10). Asterisks denote a significant difference from the wild type as determined by Student's t tests (ns, not significant; **, p<0.01; ***,p<0.001).</p