Integrative high-throughput enhancer surveying and functional verifying divulges YY2 condensed regulatory axis conferring risk for osteoporosis

The precise roles of chromatin organizations at osteoporosis risk loci remains largely elusive. Here we combined chromatin interaction conformation (Hi-C) profiling and self-transcribing active regulatory region sequencing (STARR-seq) to qualify enhancer activities of prioritized osteoporosis-associated SNPs. We identified 319 SNPs with biased allelic enhancer activity effect (baaSNPs) which linked to hundreds of candidate target genes through chromatin interactions across 146 loci. Functional characterizations revealed active epigenetic enrichment for baaSNPs, and prevailing osteoporosis-relevant regulatory roles for their chromatin interacted genes. Further motif enrichment and network mapping prioritized several putative key transcription factors (TFs) controlling osteoporosis binding to baaSNPs. Specifically, we selected on top ranked TF and deciphered that an intronic baaSNP (rs11202530) could allele-preferentially bind to YY2 to augment PAPSS2 expression through chromatin interactions and promote osteoblast differentiation. Our results underlined roles of TF-mediated enhancer-promoter contacts for osteoporosis, which may help better understand the intricate molecular regulatory mechanisms underlying osteoporosis-risk loci. Overall design: We prioritized 5,642 candidate osteoporosis-associated SNPs interacted with nearby genes promoters through combing GWAS fine-mapping and Hi-C profiling for high-throughput biased enhancer qualification by STARR-seq. A 120-bp genomic sequence centered on each SNP was extracted, with identical adapter sequence added on the 5' and 3' region. Oligonucleotides for 150-bp length SNPs on both reference and alternative alleles were synthesized in a CustomArray. Firstly, STARR-seq plasmid library was constructed using hSTARR-seq_ORI vector and amplified oligonucleotides. Secondly, The plasmid library was transfected into human osteoblast-like cell line U2OS for 24h. Finally, the plasmid library and RNA extracted after transfection were used to construct input and output sequencing libraries respectively. The library preparations were sequenced on an Illumina Novaseq 6000 platform (PE150) and paired-end reads were generated. Three biological replicates were performed for input or output library.