Rnf20 levels modulate the VEGF-Notch signaling circuits during sprouting angiogenesis [ChIP-seq]

Tight control of cell fate choices is crucial for proper vessel development. Here we show that Rnf20, the major E3 ubiquitin ligase of histone H2B, plays a key role in the tight control of VEGF-Notch signaling, which is crucial for proper vessel growth and patterning, as well as for the dynamic tip-stalk cell fate at the vascular front. We demonstrate that Rnf20 is dynamically expressed in the developing retina and controls vessel growth through two distinct modes of action. On the one hand, it restricts gene activation by the endothelial transcription factor ERG and on the other, it orchestrates large-scale mRNA processing events, which change the bioavailability and function of critical pro-angiogenic factors. We show that Rnf20 plays a key role for the upregulation of both VEGFR2 through alternative polyadenylation and VEGFA through intron inclusion, as well as for suppressing the ability of Notch1 to bind to chromatin. Interestingly, perturbations of RNF20 have been associated with complex cardiovascular malformations in human patients, suggesting that delineating the mechanisms that control Rnf20 expression and activity may provide new insights into diseases caused by vascular dysfunction. Overall design: HUVEC cells were transfected with SiCTR or siRNF20 to follow ChIP-seq with Pol 2 antibody following the protocol for ChiP-seq described in (PMID: 32065070)

精准调控细胞命运抉择，对于正常血管发育至关重要。本研究证实，作为组蛋白H2B（histone H2B）的主要E3泛素连接酶（E3 ubiquitin ligase），Rnf20在精准调控VEGF-Notch信号通路（VEGF-Notch signaling）中发挥关键作用；该信号通路对于正常血管生长与模式构建，以及血管前沿的动态尖端-茎部细胞命运维持均至关重要。
研究表明，Rnf20在发育中的视网膜中呈动态表达，并通过两种截然不同的作用模式调控血管生长：一方面，它可抑制内皮转录因子ERG介导的基因激活；另一方面，它可调控大规模mRNA加工过程，从而改变关键促血管生成因子的生物利用度与功能。
本研究证实，Rnf20可通过可变聚腺苷酸化（alternative polyadenylation）上调VEGFR2的表达，通过内含子保留（intron inclusion）上调VEGFA的表达，同时还可抑制Notch1与染色质结合的能力。
值得注意的是，人类患者中RNF20的功能异常与复杂心血管畸形相关，这提示阐明调控Rnf20表达与活性的机制，或可为血管功能障碍引发的疾病提供全新研究视角。
实验设计：将人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells, HUVEC）分别转染阴性对照小干扰RNA（SiCTR）或靶向RNF20的小干扰RNA（siRNF20），随后按照文献（PMID: 32065070）中描述的染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）实验方案，使用RNA聚合酶II（RNA polymerase II, Pol 2）抗体开展相关实验。