Optimization of Light-Inducible Gal4/UAS Gene Expression System in Mammalian Cells

遺伝子発現を光で制御できる新技術を開発 --発生・幹細胞研究への貢献に期待--. 京都大学プレスリリース. 2020-08-28.Light-inducible gene expression systems represent powerful methods for studying the functional roles of dynamic gene expression. Here, we developed an optimized light-inducible Gal4/UAS gene expression system for mammalian cells. We designed photoactivatable (PA)-Gal4 transcriptional activators based on the concept of split transcription factors, in which light-dependent interactions between Cry2-CIB1 PA-protein interaction modules can reconstitute a split Gal4 DNA-binding domain and p65 transcription activation domain. We developed a set of PA-Gal4 transcriptional activators (PA-Gal4cc), which differ in terms of induced gene expression levels following pulsed or prolonged light exposure, and which have different activation/deactivation kinetics. These systems offer optogenetic tools for the precise manipulation of gene expression at fine spatiotemporal resolution in mammalian cells

Glucose-Dependent Insulinotropic Polypeptide Prevents the Progression of Macrophage-Driven Atherosclerosis in Diabetic Apolipoprotein E-Null Mice

Aim: We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe 2/2) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes. Methods: Nondiabetic Apoe 2/2 mice, streptozotocin-induced diabetic Apoe 2/2 mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages. Results: Diabetic Apoe 2/2 mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe 2/2 mice of the same age. GIP infusion in diabetic Apoe 2/2 mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro 3]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe 2/2 mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15 % in macrophages from diabetic Apoe 2/2 mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by onl

Rpd3/CoRest-mediated activity-dependent transcription regulates the flexibility in memory updating in Drosophila

Consolidated memory can be preserved or updated depending on the environmental change. Although such conflicting regulation may happen during memory updating, the flexibility of memory updating may have already been determined in the initial memory consolidation process. Here, we explored the gating mechanism for activity-dependent transcription in memory consolidation, which is unexpectedly linked to the later memory updating in Drosophila. Through proteomic analysis, we discovered that the compositional change in the transcriptional repressor, which contains the histone deacetylase Rpd3 and CoRest, acts as the gating mechanism that opens and closes the time window for activity-dependent transcription. Opening the gate through the compositional change in Rpd3/CoRest is required for memory consolidation, but closing the gate through Rpd3/CoRest is significant to limit future memory updating. Our data indicate that the flexibility of memory updating is determined through the initial activity-dependent transcription, providing a mechanism involved in defining memory state

Glucagon-like Peptide-1 Suppresses the Proliferation and Migration of Vascular Smooth Muscle Cells: Implications for Preventive Effects on Atherosclerosis

Our group previously demonstrated the suppressive effect of glucagon-like peptide-1 (GLP-1) on macrophage-driven atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice. In the present study we investigated the suppressive effect of GLP-1 on the atherogenic phenotype of vascular smooth muscle cells (VSMCs) in vivo using apoE-/- mice, and the proliferation and migration of human VSMCs in vitro. A 4-week infusion of GLP-1 in 17-week-old apoE-/- mice significantly reduced the proliferative VSMC phenotype stained with SMemb. Platelet-derived growth factor (PDGF) -BB significantly stimulated the proliferation of human aortic VSMCs by three fold. Both 0.1 and 1nmol/l GLP-1 significantly suppressed the PDGF-induced VSMC proliferation, and this suppressive effect was significantly abolished by the GLP-1 receptor antagonist exendin (9-39) (50nmol/l). The GLP-1 receptor agonists liraglutide (100nmol/l) and exendin-4 (100nmol/l) mimicked GLP-1, significantly suppressing PDGF-induced VSMC proliferation. PDGF-BB significantly stimulated the migration of human aortic VSMCs by 1.7 -fold, and this effect was significantly suppressed by 1nmol/l GLP-1. These findings suggest that GLP-1-related treatments may prevent the progression of atherosclerotic lesions by suppressing the proliferation and migration of VSMCs, which are characteristic features of atherosclerosis

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

Requirement of the Mre11 Complex and Exonuclease 1 for Activation of the Mec1 Signaling Pathway

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block