HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence [ChIP-seq]

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome-wide in two primary cell lines. We integrated ChIP-seq and Hi-C with graph theory to uncover clustering of HMGB1-marked topological domains that harbour genes required for paracrine senescence. Using sCLIP and functional tests, we show that HMGB1 is also a bona fide RNA-binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate the availability of SASP-relevant transcripts. Our findings highlight a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell-autonomous and paracrine senescence. Overall design: Two replicates of proliferating/senescent IMR90 and one replicate of proliferating HUVEC cells were used for ChIP-seq experiment for immunoprecipitation with HMGB1 and inputs.

哺乳动物染色体的空间构象与基因表达会伴随细胞周期进程进行维持与调控。当细胞进入衰老状态后，这一稳态便会被打破：丰度极高的高迁移率族蛋白B1（HMGB1）会从细胞核中耗竭，并作为细胞外促炎刺激因子发挥作用。尽管HMGB1具有重要的生理功能，但目前我们对其在染色质上的定位及核内功能仍知之甚少。为解决这一问题，我们在两种原代细胞系中完成了全基因组范围内的HMGB1结合位点图谱绘制。我们将染色质免疫共沉淀测序（ChIP-seq）、高通量染色体构象捕获测序（Hi-C）与图论相结合，揭示了带有HMGB1标记的拓扑结构域的聚集现象，这类结构域携带了旁分泌衰老所需的相关基因。通过链特异性交联免疫沉淀测序（sCLIP）与功能实验，我们证实HMGB1亦是一类可结合数百种mRNA的名副其实的RNA结合蛋白（RBP），其相互作用组富含与衰老调控相关的RBP。其中多数RBP的mRNA会被HMGB1直接结合，并调控与衰老相关分泌表型（SASP）相关转录本的可获得性。我们的研究结果表明，HMGB1的功能范围远超此前认知，其可协同调控染色质折叠与RNA稳态，作为调控细胞自主衰老与旁分泌衰老的反馈环路的一部分发挥作用。实验设计：针对增殖态/衰老态IMR90细胞设置两组生物学重复，增殖态人脐静脉内皮细胞（HUVEC）设置一组生物学重复，上述样本均开展针对HMGB1的染色质免疫共沉淀测序（ChIP-seq）实验，并设置Input对照样本。