Senescent cells cluster CTCF on nuclear speckles to sustain their splicing program [APEX-seq]

Senescence —the endpoint of replicative lifespan for normal cells— is established via a complex sequence of molecular events. One such event is the dramatic reorganization of CTCF into senescence-induced clusters (SICCs). However, the molecular determinants, genomic consequences, and functional purpose of SICCs remained unknown. Here, we combine functional assays, super-resolution imaging, and 3D genomics with computational modelling to dissect SICC emergence. We establish that the competition between CTCF-bound and non-bound loci dictates clustering propensity. Upon senescence entry, cells repurpose SRRM2 —a key component of nuclear speckles— and BANF1 —a 'molecular glue' for chromosomes— to cluster CTCF and rewire genome architecture. This CTCF-centric reorganization in reference to nuclear speckles functionally sustains the senescence splicing program, as SICC disruption fully reverts alternative splicing patterns. We therefore uncover a new paradigm, whereby cells translate changes in nuclear biochemistry into architectural changes directing splicing choices so as to commit to the fate of senescence. Overall design: Human lung fibroblasts (IMR90S) were cultured under two distinct conditions. One group was maintained in confluency (2*106 cells per 15 cm plate) using standard medium (MEM supplemented with 5% FBS, 1% NEAA, and 1% Pen/Strep). The other group was also grown in confluency (2*106 cells per 15 cm plate), in the same medium further supplemented with ICM to induce senescent phenotype. After mild fixation with 1% paraformaldehyde (PFA), the cells were scraped, centrifuged, and counted. Aliquots containing pellet of 1×106 cells per sample were then prepared and froze at -80°C. Pellets were then processed for RNA And DNA Interacting Complexes Ligation and Sequencing (RADICL-seq) to investigate possible changes between proliferating and ICM-treated IMR90S in terms of the interactions between RNA molecules and the genomic regions with which they associate.

细胞衰老（Senescence）——正常细胞复制寿命的终点——通过一系列复杂的分子事件得以确立。此类事件之一为CCCTC结合因子（CTCF）发生显著重排，聚集为衰老诱导簇（SICCs）。然而，此前对于SICCs的分子决定因素、基因组层面的影响以及其功能意义尚不清楚。本研究结合功能实验、超分辨率成像技术、三维基因组学与计算建模，对SICCs的形成机制进行解析。本研究证实，CTCF结合位点与非结合位点之间的竞争决定了聚集倾向。当细胞进入衰老状态时，会重新利用SRRM2——核斑的关键组分——与BANF1——染色体的“分子胶水”——来聚集CTCF并重塑基因组三维结构。这种以CTCF为中心、围绕核斑的重排在功能上维持了衰老相关的剪接程序，而破坏SICCs可完全逆转可变剪接模式的改变。因此本研究揭示了一种全新的范式：细胞可将核生物化学的变化转化为三维结构改变，从而调控剪接选择，最终使细胞走向衰老命运。实验设计概况：人类肺成纤维细胞（IMR90S）在两种不同条件下培养。一组在汇合状态下培养（每15cm培养皿接种2×10⁶个细胞），使用标准培养基：含5%胎牛血清（FBS）、1%非必需氨基酸（NEAA）以及1%青霉素-链霉素双抗（Pen/Strep）的最低必需培养基（MEM）。另一组同样在汇合状态下培养（每15cm培养皿接种2×10⁶个细胞），使用添加了诱导衰老表型的ICM的同款培养基。经1%多聚甲醛（PFA）轻度固定后，收集细胞、离心并计数。随后将每份样本分装为含1×10⁶个细胞的沉淀，置于-80℃冻存。随后对细胞沉淀进行RNA与DNA互作复合物连接测序（RADICL-seq），以探究增殖组与ICM处理组IMR90S之间，RNA分子与其结合的基因组区域之间互作模式的潜在变化。