Revisiting the Hayflick Limit: Insights from an Integrated Analysis of Changing Transcripts, Proteins, Metabolites, and Chromatin

Replicative senescence (RS) as a model has become the central focus of research into cellular aging in vitro. Despite decades of study, this process through which cells cease dividing is not fully understood in culture, and even much less so in vivo during development and with aging. Here, we revisit Hayflick’s original observation of RS in WI-38 human fetal lung fibroblasts equipped with a battery of high dimensional modern techniques and analytical methods to deeply profile the process of RS across each aspect of the central dogma and beyond. We applied and integrated RNA-seq, proteomics, metabolomics, and ATAC-seq to a high resolution RS time course. We found that the transcriptional changes that underlie RS manifest early, gradually increase, and correspond to a concomitant global increase in accessibility in nucleolar and lamin associated domains. During RS WI-38 fibroblast gene expression patterns acquire a striking resemblance to those of myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT). This observation is supported at the transcriptional, proteomic, and metabolomic levels of cellular biology. In addition, we provide evidence suggesting that t s conversion is regulated by the transcription factors YAP1/TEAD1 and the signaling molecule TGF-β