Coupling High-Throughput Mapping With Proteomics Analysis Delineates CAD-Associated CDKN2A/B Locus

Objective:Both promoters and enhancers contain short DNA sequences that serve as cis-regulatory elements（cis-REs）regulating gene transcription by recruiting regulatory proteins.In addition,epigenetic modifications,alter DNA accessibility by opening or closing chromatin structure,thereby regulating gene transcription by making the underlying cis-REs accessible to regulatory proteins.Nevertheless,growing evidence also suggests that cis-REs exist in epigenetically unmarked sites and play unexpected roles in controlling gene transcription.Therefore,the ability to identify cis-REs and regulatory proteins in the human genome represents a yet unresolved challenge.Recently,the incidence and mortality of coronary artery disease（CAD）have increased.Genome-wide association studies（GWAS）have identified that cyclin-dependent kinase inhibitor 2A/B（CDKN2A/B）locus on chromosome 9p21.3 is associated with CAD.Therefore,we aim to design and apply new technology to continuously analyze human genome region in a single base pair resolution using specific cells.Coupling regulatory elements sequencing（Reel-seq）and flanking restriction DNA pulldown-mass spectrometry（FREP-MS）screens cis-REs and regulatory protein that may be related to the pathogenesis of CAD,which is great significance to further elucidate the CDKN2A/B locus.Methods:1、We generated DNA Library 1 and Library 2 covering the entire CAD related CDKN2A/B locus,and applied Reel-seq to screen.After a total of 10 rounds,polymerase chain reaction（PCR）product from rounds 1,4,7 and 10 was prepared for high-throughput sequencing and result analysis.2、To demonstrate the functionality of the cis-REs,we performed an electrophresis mobility shift assay（EMSA）on ten randomly picked candidate cis-REs and negative control.3、To confirm the functionality of the S1606 and S961,we performed luciferase reporter assay.CRISPR/cas9 gene editing was carried out using a sg RNA that targets either S1606 or S961.Two independent CRISPR edited cell lines were obtained with mutations in each of these two elements.To determine if these mutations change the expression of downstream genes,a quantitative real-time polymerase chain reaction（q RT-PCR）analysis was performed using total RNA isolated from these clones together with their controls.4、We applied FREP-MS to determine the regulatory proteins that specifically bind to the cis-REs on S1606 and S961.We performed a DNA-pulldown western blot to verify these specific binding.5、We performed luciferase reporter assay and chromatin immunoprecipitation assay to verify the specific binding of PABPC1 to S1606 and the highly enriched binding of FOXC2 to S961.6、We performed Western blot and q RT-PCR to determine if p14^ARF,p15^INK4b,p16^INK4a and antisense non-coding RNA（ANRIL）are regulated by regulatory proteins using scrambled and knockdown group.7、We performed SA-β-gal,γ-H₂AX and q RT-PCR detecting senescence-associated secretory phenotype expression to determine if cellular senescence are regulated by regulatory proteins.Results:1、We identified a total of 209 cis-REs in Library 1 and 199cis-REs in Library 2.2、8 of the 10 candidate cis-REs showed clear and unique EMSA bands.3、S1606 and S961 sequence regulated the expression of downstream gene.4、PABPC1 and FOXC2 could specifically bind to S1606 and S961,respectively.5、PABPC1 and FOXC2 regulated p14^ARF,p15^INK4b,p16^INK4a and ANRIL expression.6、PABPC1 and FOXC2 participated in cellular senescence by regulating the p16^INK4a expression.Conclusions:1、Reel seq,a new technology,maps cis-REs at high resolution over a large region of the human genome in a systematic and continuous manner.Coupling Reel-seq with FREP-MS well characterized regulatory proteins bound to cis-REs.2、CDKN2A/B locus may be involved in the pathological process of CAD by regulating cellular senescence.