the 2 huvec cell lines used in the study were derived from male caucasian donors genotyped as heterozygotes for the 9p21 cad associated snps . the 4 ebv transformed lymphoblastoid cell lines ( lcl ) were selected for their genotypes ( 2 homozygous cad risk or 2 homozygous cad non - risk ) using the hapmap data27 . experiments were performed within 24 passages by treating cells with 100 ng / ml of ifn ( r&d ) for 24 hrs . treated and untreated cells were subjected to rt - pcr ( huvec ) , chip ( huvec and lcl ) , 3c analysis ( huvec ) or fish ( huvec ) . probe design : we designed 12 acceptor probes in the interval chr9:22100523 - 22126469 ( hg18 ; spanning from ecad7 to the et2d2 enhancer ) , on both strands immediately 3 of the 6 bamhi sites in the region . we designed 290 donor probes on both strands immediately 5 of the 145 bamhi sites in the interval chr9:21035934 - 22494089 ( hg18 ; except where acceptor probes were designed ) . a universal sequence added to the probes is compatible with illumina ga adapters for direct sequencing . the 12 acceptor probes and 290 donor probes ( supplemental table 8) were pooled in equimolar amounts , separately . 3d - dsl sequencing : the dsl ligation products were prepared as described in kwon et al.23 . 3c was performed as per lieberman - aiden et al 28 and the products were sheared by sonication . donor and acceptor probes pools were annealed to the biotinylated 3c samples and the biotinated dna was bound on to streptavidin magnetic beads . the 5 phosphate of acceptor probes and 3 oh of donor probes were ligated using taq dna ligase . the ligated products were washed and eluted from the streptavidin magnetic beads , followed by pcr amplification and deep sequencing on the illumina ga2 ( supplemental information ) . the 2 huvec cell lines used in the study were derived from male caucasian donors genotyped as heterozygotes for the 9p21 cad associated snps . the 4 ebv transformed lymphoblastoid cell lines ( lcl ) were selected for their genotypes ( 2 homozygous cad risk or 2 homozygous cad non - risk ) using the hapmap data27 . experiments were performed within 24 passages by treating cells with 100 ng / ml of ifn ( r&d ) for 24 hrs . treated and untreated cells were subjected to rt - pcr ( huvec ) , chip ( huvec and lcl ) , 3c analysis ( huvec ) or fish ( huvec ) . probe design : we designed 12 acceptor probes in the interval chr9:22100523 - 22126469 ( hg18 ; spanning from ecad7 to the et2d2 enhancer ) , on both strands immediately 3 of the 6 bamhi sites in the region . we designed 290 donor probes on both strands immediately 5 of the 145 bamhi sites in the interval chr9:21035934 - 22494089 ( hg18 ; except where acceptor probes were designed ) . a universal sequence added to the probes is compatible with illumina ga adapters for direct sequencing . the 12 acceptor probes and 290 donor probes ( supplemental table 8) were pooled in equimolar amounts , separately . 3d - dsl sequencing : the dsl ligation products were prepared as described in kwon et al.23 . 3c was performed as per lieberman - aiden et al 28 and the products were sheared by sonication . donor and acceptor probes pools were annealed to the biotinylated 3c samples and the biotinated dna was bound on to streptavidin magnetic beads . the ligated products were washed and eluted from the streptavidin magnetic beads , followed by pcr amplification and deep sequencing on the illumina ga2 ( supplemental information ) .
genome wide association studies ( gwas ) have identified snps in the 9p21 gene desert associated with coronary artery disease ( cad)14 and type 2 diabetes ( t2d)57 . despite evidence for a role of the associated interval in neighboring gene regulation810 , the biological underpinnings of these genetic associations to cad or t2d have not yet been explained . 
 here we identify 33 enhancers in 9p21 ; the interval is the second densest gene - desert for predicted enhancers and 6 times denser than the whole genome ( p<6.55 1033 ) . 
 the cad risk alleles of snps rs10811656/rs10757278 are located in one of these enhancers and disrupt a binding site for stat1 . 
 lymphoblastoid cell lines ( lcl ) homozygous for the cad risk haplotype exhibit no binding of stat1 , and in lcl homozygous for the cad non - risk haplotype binding of stat1 inhibits cdkn2bas expression , which is reversed by sirna knock - down of stat1 . using a new , open - ended approach to detect long - distance interactions ( 3d - dsl ) , we find that in human vascular endothelium cells ( huvec ) the enhancer interval containing the cad locus physically interacts with the cdkn2a / b locus , the mtap gene and an interval downstream of infa21 . in huvec 
 , ifn activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus , including stat1 binding , long - range enhancer interactions and altered expression of neighboring genes . 
 our findings establish a link between cad genetic susceptibility and the response to inflammatory signaling in a vascular cell type and thus demonstrate the utility of gwas findings to direct studies to novel genomic loci and biological processes important for disease etiology .