Novel insights from a multiomics dissection of the Hayflick limit
- Calico Life Sciences, LLC, United States
- Calico Life Sciences LLC, United States
Abstract
The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick’s original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by the transcription factors YAP1/TEAD1 and TGF-𝛽2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.
Data availability
Sequencing data have been deposited in GEO under accession code GSE175533.Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository under accession code EBI PRIDECode for processing and analyzing data modalities have been deposited at https://github.com/dghendrickson/hayflickSource data for figures and analysis have been deposited at https://github.com/dghendrickson/hayflick and/or uploaded as source data files.
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Transcriptome analysis of oxdative-stress induced senescence in human astrocytesNCBI Gene Expression Omnibus, GSE58910.
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Mapping of nucleolus-associated chromosomal domains during replicative senescence.NCBI Gene Expression Omnibus, GSE78043.
Article and author information
Author details
Jonathon J O'Brien
David G Hendrickson
Funding
The authors are employed by Calico Sciences, and no external funding was received.
Copyright
© 2022, Chan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Novel insights from a multiomics dissection of the Hayflick limit
eLife 11:e70283.
https://doi.org/10.7554/eLife.70283
Further reading
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- Cell Biology
- Chromosomes and Gene Expression
During oncogene-induced senescence there are striking changes in the organisation of heterochromatin in the nucleus. This is accompanied by activation of a pro-inflammatory gene expression programme – the senescence-associated secretory phenotype (SASP) – driven by transcription factors such as NF-κB. The relationship between heterochromatin re-organisation and the SASP has been unclear. Here, we show that TPR, a protein of the nuclear pore complex basket required for heterochromatin re-organisation during senescence, is also required for the very early activation of NF-κB signalling during the stress-response phase of oncogene-induced senescence. This is prior to activation of the SASP and occurs without affecting NF-κB nuclear import. We show that TPR is required for the activation of innate immune signalling at these early stages of senescence and we link this to the formation of heterochromatin-enriched cytoplasmic chromatin fragments thought to bleb off from the nuclear periphery. We show that HMGA1 is also required for cytoplasmic chromatin fragment formation. Together these data suggest that re-organisation of heterochromatin is involved in altered structural integrity of the nuclear periphery during senescence, and that this can lead to activation of cytoplasmic nucleic acid sensing, NF-κB signalling, and activation of the SASP.
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- Chromosomes and Gene Expression
- Evolutionary Biology
Gene regulation is essential for life and controlled by regulatory DNA. Mutations can modify the activity of regulatory DNA, and also create new regulatory DNA, a process called regulatory emergence. Non-regulatory and regulatory DNA contain motifs to which transcription factors may bind. In prokaryotes, gene expression requires a stretch of DNA called a promoter, which contains two motifs called –10 and –35 boxes. However, these motifs may occur in both promoters and non-promoter DNA in multiple copies. They have been implicated in some studies to improve promoter activity, and in others to repress it. Here, we ask whether the presence of such motifs in different genetic sequences influences promoter evolution and emergence. To understand whether and how promoter motifs influence promoter emergence and evolution, we start from 50 ‘promoter islands’, DNA sequences enriched with –10 and –35 boxes. We mutagenize these starting ‘parent’ sequences, and measure gene expression driven by 240,000 of the resulting mutants. We find that the probability that mutations create an active promoter varies more than 200-fold, and is not correlated with the number of promoter motifs. For parent sequences without promoter activity, mutations created over 1500 new –10 and –35 boxes at unique positions in the library, but only ~0.3% of these resulted in de-novo promoter activity. Only ~13% of all –10 and –35 boxes contribute to de-novo promoter activity. For parent sequences with promoter activity, mutations created new –10 and –35 boxes in 11 specific positions that partially overlap with preexisting ones to modulate expression. We also find that –10 and –35 boxes do not repress promoter activity. Overall, our work demonstrates how promoter motifs influence promoter emergence and evolution. It has implications for predicting and understanding regulatory evolution, de novo genes, and phenotypic evolution.
