GSE115304
Chromatin conformation links enhancers in intracranial aneurysm associated regions to potential candidate genes
Background and Purpose: We previously showed that intracranial aneurysm (IA)-associated single nucleotide polymorphisms (SNPs) are enriched in promoters and putative enhancers of the human circle of Willis (CoW), on which IAs develop, suggesting a role for such regulatory regions in IA. In this study we further investigated the role of these putative enhancers in the pathogenesis of IA by identifying the target genes of these enhancers and validating their regulatory activity.   Methods: We selected putative enhancers in IA-associated regions from genome wide association studies, using our previously published CoW chromatin immunoprecipitation and sequencing data. Next, we used a chromatin conformation capture technique (4C) to identify physical interactions between these putative enhancers and the promoters of their target genes. Subsequently, we assessed the activity of the identified enhancers in vivo in zebrafish embryos and we analyzed gene expression of the target genes identified with 4C in CoW RNA-sequencing data and by in situ hybridization on CoW paraffin sections.  Results: We identified fifteen enhancers that interact with the promoters of six target genes: SOX17, CDKN2B, MTAP, CNNM2, RPEL1 and GATA6. For eight out of eleven enhancers that could be tested, we confirmed in vivo activity in the zebrafish embryos. These eight enhancers reside in four out of six IA-associated GWAS regions. All six target genes are expressed in the CoW and in situ hybrdization showed that these genes are expressed in multiple cell types in the CoW.   Conclusions: In four out of six IA-associated GWAS regions we identified eight enhancers that are active in vivo and interact with six nearby genes, suggesting that these genes are regulated by the identified enhancers. These genes, SOX17, CDKN2B, MTAP, CNNM2, RPEL1 and GATA6, are potential candidate genes involved in IA pathogenesis and should be studied using animal models in the future. Overall design: Two circles of Willis samples were used to investigate enhancer-promoter interactions through 40 different 4C experiments, 7 of these experiments were performend in both circles of Willis samples