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Super-Enhancer-Associated Aortic Valve Stenosis Risk Locus 1p21.2 Alters NFATC2 Binding Site and Promotes Fibrogenesis [H3K27ac HiChIP]

ABSTRACT: Genome-wide association studies identified a strong signal for non coding variants at the 1p21.2 locus associated with calcific aortic valve stenosis (CAVS). Regulation at the locus and impact on the biology of the aortic valve is presently unknown. Integrative mapping of genetic association data was performed. The locus was evaluated by 3D genome mapping, analysis of atomic resolution data, DNA-binding assay and CRISPR activation (CRISPRa). Weighted gene co-expression network analysis (WGCNA) was performed to determine regulatory network in CAVS. The functional impact of the locus was assessed in isolated VICs. Fine-grained mapping at 1p21.2 risk locus identified rs6702619 as being located in open chromatin and a distant-acting super-enhancer including chromatin interaction with the promoter of PALMD in VICs. Atomic-level data showed that the risk variant modified base readout and DNA shape, which prevented the recruitment of NFATC2, a transcription factor involved in heart valve morphogenesis, and lowered the expression of PALMD. CRISPRa confirmed that rs6702619 exerts a control on the expression of PALMD. WGCNA performed in 233 calcified aortic valves identified a co-expression network encompassing PALMD, which was enriched in actin-based process. In LC-MS/MS and co-immunoprecipitation assay, actin was identified as a protein interacting with PALMD. Lower expression of PALMD in VICs promoted the polymerization of actin, the activation of myocardin-related transcription factor and fibrogenesis. Risk allele at rs6702619 disrupts a NFATC2 binding site and decreases enhancer-mediated expression of PALMD, which results in actin polymerization, a myofibroblast-like phenotype in VICs and fibrogenesis, a key underpinning process in the pathogenesis of CAVS.

ORGANISM(S): Homo sapiens

PROVIDER: GSE154512 | GEO | 2020/07/16

REPOSITORIES: GEO

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Project description:Background: Palmdelphin (PALMD), a protein of unknown function, belongs to the family of Paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms (SNPs) in the PALMD locus, which reduce its expression, are strong risk factors for development of calcific aortic valve stenosis (CAVS) and for severity of the disease. Methods: Public database screening and immunodetection of PALMD showed dominant expression in endothelial cells (ECs). Mass spectrometry allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in siRNA silenced EC cultures, in knockout mice and in human valve samples and EC cultures from patients. RNA sequencing on siRNA silenced cells and transcript arrays on valve samples from a Swedish SNP rs7543130 patient cohort informed about gene regulatory changes. Results: ECs express the cytoplasmic PALMD-KKVI splice variant, which was shown to exist in complex with RAN GTPase activating protein1 (RANGAP1). RANGAP1 regulates the activity of the GTPase RAN and thereby, nucleocytoplasmic shuttling via Exportin1 (XPO1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, providing a mechanism for the gene regulatory changes established in PALMD-deficiency. Gene ontology analysis showed enrichment of actin and RHO GTPAse related terms in PALMD-silenced ECs. In accordance, PALMD-silenced ECs showed multiple changes in adhesive and migratory properties and cells were softer. In particular, PALMD-deficient ECs failed to form a perinuclear actin cap when exposed to flow. The perinuclear actin cap is known to provide protection against mechanical stress. Lack of the actin cap correlated with tilting of the nuclear long axis relative to the cell body, which was established in PALMD-deficient ECs, mice and patient valve samples. The loss of the actin cap may contribute to further dysregulated gene transcription eventually promoting calcific precipitations and inflammation in CAVS. Conclusions: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex changes the subcellular localization of essential nucleocytoplasmic shuttling components, thereby instigating the loss of actin-dependent nuclear resilience, which in turn promotes further deterioration of gene expression patterns predisposing to the establishment of CAVS.

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Super-Enhancer-Associated Aortic Valve Stenosis Risk Locus 1p21.2 Alters NFATC2 Binding Site and Promotes Fibrogenesis [H3K27ac HiChIP]

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