RATIONALE Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap. OBJECTIVE To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated. METHODS AND RESULTS Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation. CONCLUSIONS By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

中文翻译：

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内皮增强子的基因组表征揭示了 NR2F2 在动静脉基因表达调节中的多功能作用。


基本原理 重大进展揭示了动脉和静脉内皮细胞命运调节背后的转录输入。然而，人们对这一过程的全基因组调控知之甚少。因此，有必要进行此类研究来解决这一差距。目的 鉴定和表征人类基因组中动脉和静脉特异性内皮增强子，从而深入了解血管特性的调节机制。方法和结果 利用染色质免疫沉淀和深度测序对人动脉和静脉内皮细胞中活性染色质标记进行测序，我们鉴定了数千个动脉和静脉特异性调节元件。计算分析显示，NR2F2（核受体亚家族 2，F 组，成员 2）位点在静脉特异性增强子中过多，表明在促进静脉识别方面具有直接作用。随后染色质免疫沉淀和深度测序数据集与 RNA 测序的整合揭示了 NR2F2 调节与动静脉身份相关的 3 个不同方面。首先，与之前的遗传观察一致，NR2F2直接激活细胞周期基因侧翼的增强子元件以驱动其表达。其次，NR2F2 对于直接激活静脉特异性增强子及其相关基因至关重要。我们的基因组方法进一步揭示，NR2F2 在许多位点与 ERG（ETS 相关基因）共同作用，驱动静脉特异性基因表达。最后，NR2F2 仅直接抑制静脉细胞中的少量动脉增强子以防止其激活，包括动脉特异性转录因子 HEY2（具有 YRPW 基序 2 的相关家族 bHLH 转录因子）上游的远端元件。 在动脉内皮细胞中，该增强子通常与 ERG 结合，这也是动脉 HEY2 表达所必需的。相比之下，在静脉内皮细胞中，NR2F2 与 ERG 一起结合到该位点，并阻止其激活。结论 通过利用全基因组方法，我们揭示了 NR2F2 如何发挥多种作用以维持静脉特性的机制见解。重要的是，对其在 HEY2 上游关键动脉增强子中的作用的表征建立了一种新的机制，通过该机制可以实现动脉特异性表达。