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. 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.