Post-GWAS functional analysis identifies CUX1 as a regulator of p16INK4a and cellular senescence

Accumulation of senescent cells with age is believed to be an important driver of aging and age-related diseases. However, the underlying mechanisms and the signaling pathways that regulate senescence remain elusive. In this report, we couple high throughput (HTP) mapping of disease-associated functional SNPs (fSNPs) with proteomics analysis of fSNP-binding regulatory proteins to dissect the CDKN2A/B locus, a locus known to be associated with multiple age-related diseases, as well as overall human longevity. We demonstrate that the homeodomain transcription factor CUX1 specifically binds to a fSNP (rs1537371) associated with atherosclerosis within the CDKN2A/B locus. Although it binds at a considerable distance (> 100 kb), CUX1 is shown to regulate the CDKN2A/B encoded genes p14ARF, p15INK4b, p16INK4a, and ANRIL in endothelial cells (ECs). In-depth analysis demonstrates that 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 premature senescence via activating p16INK4A expression. Consistent with these findings, we also detect elevated expression of CUX1 and p16INK4A in the plaques of patients with carotid artery disease. Our HTP strategy therefore identifies CUX1 as a regulator of p16INK4A expression and endothelial senescence and a potential therapeutic target for atherosclerosis and other age-related diseases. Overall design: mRNA profiles from human endothelial cells treated with a scrambled siRNA control or a siRNA targeting human CUX1

随着年龄增长，衰老细胞的积累被认为是衰老及年龄相关疾病的重要驱动因素。然而，调控细胞衰老的潜在机制与信号通路仍不明确。本研究将疾病相关功能单核苷酸多态性（functional SNPs, fSNPs）的高通量（high throughput, HTP）定位，与fSNP结合调控蛋白的蛋白质组学分析相结合，以解析CDKN2A/B基因座——这一已知与多种年龄相关疾病及人类整体长寿相关的基因座。我们证实，同源域转录因子CUX1可特异性结合CDKN2A/B基因座内与动脉粥样硬化相关的fSNP（rs1537371）。尽管二者结合位点相距超过100 kb，研究显示CUX1可在内皮细胞（endothelial cells, ECs）中调控CDKN2A/B编码的p14ARF、p15INK4b、p16INK4a及ANRIL基因。深入分析表明，内皮细胞中CUX1的表达与端粒长度相关，且可被DNA损伤剂与氧化应激诱导。此外，CUX1表达的诱导可通过激活p16INK4A的表达，同时触发复制性衰老与应激诱导的早衰。与上述发现一致，我们在颈动脉疾病患者的斑块中也检测到CUX1与p16INK4A的表达升高。因此，本研究的高通量策略将CUX1鉴定为p16INK4A表达、内皮细胞衰老的调控因子，同时也是动脉粥样硬化及其他年龄相关疾病的潜在治疗靶点。整体实验设计：对经阴性对照siRNA或靶向人CUX1的siRNA处理的人内皮细胞进行mRNA表达谱检测。