HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence [RNA-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. Two biological replicates (IMR90 I10 and I79) were treated with NTC or HMGB1 siRNA.of proliferating or senescent IMR90 were used.

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得以维持与调控。当细胞进入衰老状态后，这一过程会发生紊乱：高丰度表达的HMGB1蛋白会从细胞核中耗竭，并转而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，目前我们对其在染色质上的定位以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结合位点定位实验。我们将染色质免疫共沉淀测序（ChIP-seq）、染色体构象捕获测序（Hi-C）与图论分析相结合，揭示了被HMGB1标记的拓扑结构域的聚集现象，这类结构域携带了介导旁分泌衰老所需的相关基因。通过紫外交联免疫沉淀测序（sCLIP）与功能实验，我们证实HMGB1同时也是一种真正的RNA结合蛋白（RNA-binding protein, RBP），能够结合数百种信使RNA（messenger RNA, mRNA）。HMGB1的互作组中富含与衰老调控相关的RNA结合蛋白，这类RNA结合蛋白中许多的信使RNA可直接被HMGB1结合，并调控与衰老相关分泌表型（senescence-associated secretory phenotype, SASP）相关转录本的可用性。我们的研究结果表明，HMGB1的功能范围远超此前的认知：它能够协同调控染色质折叠与RNA稳态，以此作为调控细胞自主衰老与旁分泌衰老的调控环路的一部分发挥作用。本研究使用了处于增殖或衰老状态的IMR90细胞的两份生物学重复样本（IMR90 I10与IMR90 I79），这些样本分别经非靶向对照（non-targeting control, NTC）或HMGB1小干扰RNA（small interfering RNA, siRNA）处理。