Transcription-dependent cohesin repositioning rewires chromatin loops in cellular senescence

Senescence is a state of stable proliferative arrest, generally accompanied by the senescence-associated secretory phenotype, which modulates tissue homeostasis. Enhancer-promoter interactions, facilitated by chromatin loops, play a key role in gene regulation but their relevance in senescence remains elusive. Here, we use Hi-C to show that oncogenic RAS-induced senescence in human diploid fibroblasts is accompanied by extensive enhancer-promoter rewiring, which is closely connected with dynamic cohesin binding to the genome. We find de novo cohesin peaks often at the 3′ end of a subset of active genes. RAS-induced de novo cohesin peaks are transcription-dependent and enriched for senescence-associated genes, exemplified by IL1B, where de novo cohesin binding is involved in new loop formation. Similar IL1B induction with de novo cohesin appearance and new loop formation are observed in terminally differentiated macrophages, but not TNFα-treated cells. These results suggest that RAS-induced senescence represents a cell fate determination-like process characterised by a unique gene expression profile and 3D genome folding signature, mediated in part through cohesin redistribution on chromatin.


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Sample size
We used the following R packages for data analysis and visualization (mentioned in the Methods Sample sizes for ChIP-seq and Hi-C experiments were determined by comparison with previous studies (see below) and by taking into consideration the fact that we are studying one condition (RAS-induced senescence) against control (growing, proliferating cells). The dynamic range of DNA binding of the proteins studied (CTCF, RAD21, SMC3) appeared saturated based on the number of common peaks between replicates and on the Pearson correlation coefficients between normalised signal tracks. The number of Hi-C and cHi-C replicates was assessed based on their good agreement (as determined with two independent methods) and the cis/trans interactions ratio, which was similar to other studies.
Experience of the authors performing Hi-C and ChIP-seq experiments in previous studies, as well as their experience in developing Capture HiC (cHi-C), allowed us to confidently select the number of replicates and library sizes for each of our experiments. Please refer to the following studies which use two Hi-C/cHi-C replicates per condition and similar library sizes per sample: One of the RAS-induced Senescence Hi-C samples was excluded due to lower number of available valid reads and low complexity, which rendered its comparison to control samples inadequate and thus we included two Hi-C RAS-induced Senescence replicates and three Growing replicates. No other data exclusions were performed as all other experiments (cHi-C, all ChIP-seq) were replicated with success. Our Hi-C dataset consists of three Growing control replicates and two RAS-induced Senescence replicates, as well as two capture Hi-C Growing replicates and two capture Hi-C RAS-induced Senescence replicates. We used HiC-Spector and HiCRep in order to check the similarity between Hi-C samples and good agreement was reported. For ChIP-seq experiments, we checked agreement between replicates in terms of number of common peaks detected and overall Pearson correlation between normalized signal (with subtracted input), calculated genome-wide using 10kb bins. The number of ChIP-seq replicates for each condition is described in detail below. qPCR experiments were performed to confirm the up-regulation of HMGA2, IL1A and IL1B with the following number of replicates: 8 replicates (8 growing, 8 senescence) for HMGA2, and 9 replicates for growing and 9 senescence replicates for IL1A and IL1B. FISH experiments for NRG1 and HMGA2 genes and nearby genomic regions were performed in two replicates of Growing and two replicates of senescence.
All attempts at replication were successful, except for one of the Hi-C samples which exhibited low complexity and was removed from further analysis.
Only two experimental groups are analysed mainly throughout our study: control and senescence. All the comparisons we performed were pairwise between control and senescent cells or between control and other treatment (TNFa/DRB). We prioritized handling samples in ways which ensure no accidental mislabeling or switching can occur and batch effects are minimized. The readout from all the experiments was sequencing.
Blinding is not applicable in our case because no grouping was present besides the two conditions studied, which have to be prepared in different ways and cannot be mislabeled. Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication.
Cells were regularly tested for mycoplasma contamination and always found to be negative.
No commonly misidentified cell lines were used.
The following secure token has been created to allow review of record GSE135093 while it remains in private status: szqfkgugzxeljmt.