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.

Highlights d yylncRNAs are divergently expressed with transcripts from cell fate regulatory gene loci d yylncT is a yylncRNA encoded by and physically associated with the active T locus d yylncT binds DNMT3B and affects DNA methylation at the active T locus d yylncT is essential for mesoderm commitment of human PSCs

How cytokine‐driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)‐1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF‐κB at regions that are highly enriched for inflammatory disease‐relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL‐1α‐inducible IL8 and CXCL1‐3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL‐1α/TAK1‐inducible manner. Microdeletions of p65‐binding sites in either of the two enhancers impair NF‐κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher‐order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 “master” enhancer in the regulation of sustained IL‐1α signaling, as well as for IL‐8 and IL‐6 secretion. CRISPR‐guided transactivation of the IL8 locus or cross‐TAD regulation by TNFα‐responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF‐κB.

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure‐to‐function relationship. However, all 3C‐based methods rely on chemical cross‐linking to stabilize spatial interactions. This step remains a “black box” as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed “i3C”, a novel approach for capturing spatial interactions without a need for cross‐linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation‐based orthogonal validation method, “TALE‐iD”, we show that native interactions resemble cross‐linked ones, but display improved signal‐to‐noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions.