(Genome-wide Association Studies) GWAS have identified ∼42 late-onset Alzheimer’s disease (LOAD)-associated loci, each of which contains multiple single nucleotide polymorphisms (SNPs) in linkage disequilibrium (LD) and most of these SNPs are in the non-coding region of human genome. However, how these SNPs regulate risk gene expression remains unknown. In this work, by using a set of novel techniques, we identified 6 functional SNPs (fSNPs) rs9271198, rs9271200, rs9281945, rs9271243, and rs9271247 on the LOAD-associated HLA-DRB1/DQA1 locus and 42 proteins specifically binding to five of these 6 fSNPs. As a proof of evidence, we verified the allele-specific binding of GATA2 and GATA3, ELAVL1 and HNRNPA0, ILF2 and ILF3, NFIB and NFIC, as well as CUX1 to these five fSNPs, respectively. Moreover, we demonstrate that all these nine proteins regulate the expression of both HLA-DQA1 and HLA-DRB1 in human microglial cells. The contribution of HLA class II to the susceptibility of LOAD is discussed.

Accumulation of senescent cells with age is an important driver of aging and age-related diseases. However, the mechanisms and signaling pathways that regulate senescence remain elusive. In this report, we performed post-genome-wide association studies (GWAS) functional studies on the CDKN2A/B locus, a locus known to be associated with multiple age-related diseases and overall human lifespan. We demonstrate that transcription factor CUX1 (Cut-Like Homeobox 1) specifically binds to an atherosclerosis-associated functional single-nucleotide polymorphism (fSNP) (rs1537371) within the locus and regulates the CDKN2A/B-encoded proteins p14^ARF, p15^INK4b and p16^INK4a and the antisense noncoding RNA in the CDK4 (INK4) locus (ANRIL) in endothelial cells (ECs). Endothelial CUX1 expression correlates with telomeric length and is induced by both DNA-damaging agents and oxidative stress. Moreover, induction of CUX1 expression triggers both replicative and stress-induced senescence via activation of p16^INK4a expression. Thus, our studies identify CUX1 as a regulator of p16^INK4a-dependent endothelial senescence and a potential therapeutic target for atherosclerosis and other age-related diseases.

Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)–dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N -methyltransferase 2E-antisense 1 ( KMT2E-AS1 ) and histone lysine N-methyltransferase 2E ( KMT2E ). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α–dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)–specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1 /KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.

GWAS cannot identify functional SNPs (fSNP) from disease-associated SNPs in linkage disequilibrium (LD). Here, we report developing three sequential methodologies including Reel-seq (Regulatory element-sequencing) to identify fSNPs in a high-throughput fashion, SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify fSNP-bound proteins and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to detect allele-specific protein:fSNP binding. We first apply Reel-seq to screen a library containing 4316 breast cancer-associated SNPs and identify 521 candidate fSNPs. As proof of principle, we verify candidate fSNPs on three well-characterized loci: FGFR2, MAP3K1 and BABAM1. Next, using SDCP-MS and AIDP-Wb, we rapidly identify multiple regulatory factors that specifically bind in an allele-imbalanced manner to the fSNPs on the FGFR2 locus. We finally demonstrate that the factors identified by SDCP-MS can regulate risk gene expression. These data suggest that the sequential application of Reel-seq, SDCP-MS, and AIDP-Wb can greatly help to translate large sets of GWAS data into biologically relevant information.

Genome‐wide association studies (GWAS) have validated a strong association of atherosclerosis with the CDKN2A/B locus, a locus harboring three tumor suppressor genes: p14^ARF, p15^INK4b, and p16^INK4a. Post‐GWAS functional analysis reveals that CUX is a transcriptional activator of p16^INK4a via its specific binding to a functional SNP (fSNP) rs1537371 on the atherosclerosis‐associated CDKN2A/B locus, regulating endothelial senescence. In this work, we characterize SATB2, another transcription factor that specifically binds to rs1537371. We demonstrate that even though both CUX1 and SATB2 are the homeodomain transcription factors, unlike CUX1, SATB2 is a transcriptional suppressor of p16^INK4a and overexpression of SATB2 competes with CUX1 for its binding to rs1537371, which inhibits p16^INK4a and p16^INK4a‐dependent cellular senescence in human endothelial cells (ECs). Surprisingly, we discovered that SATB2 expression is transcriptionally repressed by CUX1. Therefore, upregulation of CUX1 inhibits SATB2 expression, which enhances the binding of CUX1 to rs1537371 and subsequently fine‐tunes p16^INK4a expression. Remarkably, we also demonstrate that IL‐1β, a senescence‐associated secretory phenotype (SASP) gene itself and a biomarker for atherosclerosis, induces cellular senescence also by upregulating CUX1 and/or downregulating SATB2 in human ECs. A model is proposed to reconcile our findings showing how both primary and secondary senescence are activated via the atherosclerosis‐associated p16^INK4a expression.