Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.

Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 39 end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 39 end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 39 end trimming, which might serve to enhance snoRNA stability.

SummaryReplicative senescence has a major impact on function and integrity of cell preparations. This process is reflected by continuous DNA methylation (DNAm) changes at specific CpG dinucleotides in the course of in vitro culture, and such modifications can be used to estimate the state of cellular senescence for quality control of cell preparations. Still, it is unclear how senescence‐associated DNAm changes are regulated and whether they occur simultaneously across a cell population. In this study, we analyzed global DNAm profiles of human mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) to demonstrate that senescence‐associated DNAm changes are overall similar in these different cell types. Subsequently, an Epigenetic‐Senescence‐Signature, based on six CpGs, was either analyzed by pyrosequencing or by bar‐coded bisulfite amplicon sequencing. There was a good correlation between predicted and real passage numbers in bulk populations of MSCs (R 2 = 0.67) and HUVECs (R 2 = 0.97). However, when we analyzed the Epigenetic‐Senescence‐Signature in subclones of MSCs, the predictions revealed high variation and they were not related to the adipogenic or osteogenic differentiation potential of the subclones. Notably, in clonally derived subpopulations, the DNAm levels of neighboring CpGs differed extensively, indicating that these genomic regions are not synchronously modified during senescence. Taken together, senescence‐associated DNAm changes occur in a highly reproducible manner, but they are not synchronously co‐regulated. They rather appear to be acquired stochastically—potentially evoked by other epigenetic modifications.

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 harbor genes involved in paracrine senescence. Using simplified Cross-Linking and Immuno-Precipitation 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 availability of SASPrelevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cellautonomous and paracrine senescence.

The oncofetal IGF2 mRNA-binding protein 1 (IGF2BP1) controls the migration and invasiveness of primary as well as tumor-derived cells in vitro. Whether the protein also modulates epithelial-mesenchymal-transition (EMT), a hallmark of tumor progression involved in tumor cell dissemination, remained elusive. In this study, we reveal that IGF2BP1 enhances mesenchymal-like cell properties in tumor-derived cells by promoting the expression of the transcriptional regulators LEF1 and SLUG (SNAI2). IGF2BP1 associates with LEF1 transcripts and prevents their degradation in a 3′-UTR-dependent manner resulting in an upregulation of LEF1 expression. LEF1 promotes transcription of the mesenchymal marker fibronectin by associating with the fibronectin 1 promoter. Moreover, LEF1 enforces the synthesis of the ‘EMT-driving’ transcriptional regulator SNAI2. Accordingly, IGF2BP1 knockdown causes MET-like (mesenchymal-epithelial-transition) morphological changes, enhances the formation of cell–cell contacts and reduces cell migration in various mesenchymal-like tumor-derived cells. However, in epithelial-like tumor-derived cells characterized by a lack or low abundance of IGF2BP1, the protein fails to induce EMT. These findings identify IGF2BP1 as a pro-mesenchymal post-transcriptional determinant, which sustains the synthesis of ‘EMT-driving’ transcriptional regulators, mesenchymal markers and enhances tumor cell motility. This supports previous reports, suggesting a role of IGF2BP1 in tumor cell dissemination.