Dataset Information

Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells.

ABSTRACT:

Background

An intergenic region at chromosome 4q31 is one of the most significant regions associated with COPD susceptibility and lung function in GWAS. In this region, the implicated causal gene HHIP has a unique epithelial expression pattern in adult human lungs, in contrast to dominant expression in fibroblasts in murine lungs. However, the mechanism underlying the species-dependent cell type-specific regulation of HHIP remains largely unknown.

Methods

We employed snATAC-seq analysis to identify open chromatin regions within the COPD GWAS region in various human lung cell types. ChIP-quantitative PCR, reporter assays, chromatin conformation capture assays and Hi-C assays were conducted to characterize the regulatory element in this region. CRISPR/Cas9-editing was performed in BEAS-2B cells to generate single colonies with stable knockout of the regulatory element. RT-PCR and Western blot assays were used to evaluate expression of HHIP and epithelial-mesenchymal transition (EMT)-related marker genes.

Findings

We identified a distal enhancer within the COPD 4q31 GWAS locus that regulates HHIP transcription at baseline and after TGFβ treatment in a SMAD3-dependent, but Hedgehog-independent manner in human bronchial epithelial cells. The distal enhancer also maintains chromatin topological domains near 4q31 locus and HHIP gene. Reduced HHIP expression led to increased EMT induced by TGFβ in human bronchial epithelial cells.

Interpretation

A distal enhancer regulates HHIP expression both under homeostatic condition and upon TGFβ treatment in human bronchial epithelial cells. The interaction between HHIP and TGFβ signalling possibly contributes to COPD pathogenesis.

Funding

Supported by NIH grants R01HL127200, R01HL148667 and R01HL162783 (to X. Z).

SUBMITTER: Guo F

PROVIDER: S-EPMC10944180 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Publications

Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells.

Guo Feng F Zhang Li L Yu Yuzhen Y Gong Lu L Tao Shiyue S Werder Rhiannon B RB Mishra Shreya S Zhou Yihan Y Anamika Wardatul Jannat WJ Lao Taotao T Inuzuka Hiroyuki H Zhang Yihan Y Pham Betty B Liu Tao T Tufenkjian Tiffany S TS Richmond Bradley W BW Wei Wenyi W Mou Hongmei H Wilson Andrew A AA Hu Ming M Chen Wei W Zhou Xiaobo X

EBioMedicine 20240227

<h4>Background</h4>An intergenic region at chromosome 4q31 is one of the most significant regions associated with COPD susceptibility and lung function in GWAS. In this region, the implicated causal gene HHIP has a unique epithelial expression pattern in adult human lungs, in contrast to dominant expression in fibroblasts in murine lungs. However, the mechanism underlying the species-dependent cell type-specific regulation of HHIP remains largely unknown.<h4>Methods</h4>We employed snATAC-seq an ...[more]

PMID: 38417378

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Project description:Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxin's antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts. We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs. In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.

Dataset Information

Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells.

Background

Methods

Findings

Interpretation

Funding

Publications

Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells.

Similar Datasets

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