Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis
Tomohito Doke, Shizheng Huang, Chengxiang Qiu, Hongbo Liu, Yuting Guan, Hailong Hu, Ziyuan Ma, Junnan Wu, Zhen Miao, Xin Sheng, Jianfu Zhou, Aili Cao, Jianhua Li, Lewis Kaufman, Adriana Hung, Christopher D. Brown, Richard Pestell, Katalin Susztak
Tomohito Doke, Shizheng Huang, Chengxiang Qiu, Hongbo Liu, Yuting Guan, Hailong Hu, Ziyuan Ma, Junnan Wu, Zhen Miao, Xin Sheng, Jianfu Zhou, Aili Cao, Jianhua Li, Lewis Kaufman, Adriana Hung, Christopher D. Brown, Richard Pestell, Katalin Susztak
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Research Article Genetics Nephrology

Transcriptome-wide association analysis identifies DACH1 as a kidney disease risk gene that contributes to fibrosis

Abstract

Genome-wide association studies (GWAS) for kidney function identified hundreds of risk regions; however, the causal variants, target genes, cell types, and disease mechanisms remain poorly understood. Here, we performed transcriptome-wide association studies (TWAS), summary Mendelian randomization, and MetaXcan to identify genes whose expression mediates the genotype effect on the phenotype. Our analyses identified Dachshund homolog 1 (DACH1), a cell-fate determination factor. GWAS risk variant was associated with lower DACH1 expression in human kidney tubules. Human and mouse kidney single-cell open chromatin data (snATAC-Seq) prioritized estimated glomerular filtration rate (eGFR) GWAS variants located on an intronic regulatory region in distal convoluted tubule cells. CRISPR-Cas9–mediated gene editing confirmed the role of risk variants in regulating DACH1 expression. Mice with tubule-specific Dach1 deletion developed more severe renal fibrosis both in folic acid and diabetic kidney injury models. Mice with tubule-specific Dach1 overexpression were protected from folic acid nephropathy. Single-cell RNA sequencing, chromatin immunoprecipitation, and functional analysis indicated that DACH1 controls the expression of cell cycle and myeloid chemotactic factors, contributing to macrophage infiltration and fibrosis development. In summary, integration of GWAS, TWAS, single-cell epigenome, expression analyses, gene editing, and functional validation in different mouse kidney disease models identified DACH1 as a kidney disease risk gene.

Authors

Tomohito Doke, Shizheng Huang, Chengxiang Qiu, Hongbo Liu, Yuting Guan, Hailong Hu, Ziyuan Ma, Junnan Wu, Zhen Miao, Xin Sheng, Jianfu Zhou, Aili Cao, Jianhua Li, Lewis Kaufman, Adriana Hung, Christopher D. Brown, Richard Pestell, Katalin Susztak

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Figure 8

DACH1 transcriptionally controls cell cycle genes in kidney tubule cells.

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DACH1 transcriptionally controls cell cycle genes in kidney tubule cells...
(A) DACH1-ChIP 19,460-binding sites, corresponding to 5111 genes in the K562 cell (human immortalized myelogenous leukemia). Lower panel shows the distribution of peaks compared with TSS distance. (B) Top 10 most significant pathways from the Molecular Signatures Database (MsigDB) using the Genomic Region of Enrichment of Annotations (GREAT) package (see Supplemental Methods). (C) Primary mouse kidney tubule cells were isolated from Dach1fl/fl mice and infected with adenovirus-GFP (Ade-GFP; control [CTRL]) or adenovirus-Cre-GFP (Ade-Cre-GFP; Dach1 KO). Representative Western blots of DACH1 protein expression in control and Dach1 KO tubule cells. GAPDH was used as loading control. (D) DACH1-ChIP-PCR for Ccnd1 and Ccnd2 in control kidney tubule cells. Three independent experiments were performed. (E) Integrated Genome Browser view of the human CCND2 locus. From top to bottom, the labels indicate the CCND2 locus, DACH1–ChIP-Seq, followed by human kidney snATAC-Seq in DCT, proximal tubule, loop of Henle, principal cell, intercalated cell. The box highlights the overlapping peaks between DACH1–ChIP-Seq and snATAC-Seq. (F) Relative gene expression of Ccna1, Ccnd1, Ccnd2, and Ccne1 in Ade-GFP or Ade-Cre-GFP transfected cells (n = 3). Gapdh was used as internal control. (G) Results of cell proliferation analysis using the MTT assay at 24 and 48 hours after Ade-GFP or Ade-Cre-GFP infection. (n = 3 in triplicate). *P < 0.05, **P < 0.01, 1-way ANOVA and Tukey’s post hoc test.