ABSTRACT
Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. In this study, using budding yeast and normal human fibroblasts, we found that cellular senescence-stable cell cycle arrest contributing to organismal aging-is the long-term outcome of PMD. Our genetic screening using budding yeast unexpectedly identified a close genetic association between PMD response and replicative lifespan regulations. Furthermore, PMD limits replicative lifespan in budding yeast; upregulation of membrane repair factors ESCRT-III (SNF7) and AAA-ATPase (VPS4) extends it. In normal human fibroblasts, PMD induces premature senescence via the Ca2+-p53 axis but not the major senescence pathway, DNA damage response pathway. Transient upregulation of ESCRT-III (CHMP4B) suppressed PMD-dependent senescence. Together with mRNA sequencing results, our study highlights an underappreciated but ubiquitous senescent cell subtype: PMD-dependent senescent cells.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Humans , Saccharomyces cerevisiae/genetics , Longevity , Tumor Suppressor Protein p53/genetics , Fibroblasts , Cell Membrane/metabolism , Cellular Senescence/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Adenosine Triphosphatases/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The pluripotent state of embryonic stem cells (ESCs) is regulated by a sophisticated network of transcription factors. High expression of KLF17 has recently been identified as a hallmark of naive state of human ESCs (hESCs). However, the functional role of KLF17 in naive state is not clear. Here, by employing various gain and loss-of-function approaches, we demonstrate that KLF17 is essential for the maintenance of naive state and promotes the primed to naive state transition in hESCs. Mechanistically, we identify MAPK3 and ZIC2 as two direct targets repressed by KLF17. Overexpression of MAPK3 or ZIC2 partially blocks KLF17 from promoting the naive pluripotency. Furthermore, we find that human and mouse homologs of KLF17 retain conserved functions in promoting naive pluripotency of both species. Finally, we show that Klf17 may be essential for early embryo development in mouse. These findings demonstrate the important and conserved function of KLF17 in promoting naive pluripotency and reveal two essential transcriptional targets of KLF17 that underlie its function.
Subject(s)
Human Embryonic Stem Cells , Animals , Cell Differentiation/genetics , Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/metabolism , Humans , Mice , Mitogen-Activated Protein Kinase 3 , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
microRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in plants, animals and viruses. Measuring miRNA activity in vivo remains a big challenge. Here, using an miRNA-mediated single guide RNA (sgRNA)-releasing strategy and dCas9-VPR to drive a transgene red fluorescent protein, we create an miRNA sensor that can faithfully measure miRNA activity at cellular levels and use it to monitor differentiation status of stem cells. Furthermore, by designing sgRNAs to target endogenous loci, we adapted this system to control the expression of endogenous genes or mutate specific DNA bases upon induction by cell-type-specific miRNAs. Finally, by miRNA sensor library screening, we discover a previously undefined layer of heterogeneity associated with miR-21a activity in mouse embryonic stem cells. Together, these results highlight the utility of an miRNA-induced CRISPR-Cas9 system as miRNA sensors and cell-type-specific genome regulation tools.
