Abstract
RAP1 is a well-known telomere-binding protein, but its functions in human stem cells have remained unclear. Here we generated RAP1-deficient human embryonic stem cells (hESCs) by using CRISPR/Cas9 technique and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via directed differentiation. In both hMSCs and hNSCs, RAP1 not only negatively regulated telomere length but also acted as a transcriptional regulator of RELN by tuning the methylation status of its gene promoter. RAP1 deficiency enhanced self-renewal and delayed senescence in hMSCs, but not in hNSCs, suggesting complicated lineage-specific effects of RAP1 in adult stem cells. Altogether, these results demonstrate for the first time that RAP1 plays both telomeric and nontelomeric roles in regulating human stem cell homeostasis.
Highlights
Telomeres, the short repeat sequences at the chromosome ends in eukaryotic organisms (Greider, 1991; Blackburn, 2001), are indispensable for the maintenance of genome stability (Londono-Vallejo, 2004; Zhang et al, 2018)
With the aid of the CRISPR/Cas9-mediated gene-editing technique, our study revealed for the first time that repressor/activator protein 1 (RAP1) negatively regulated telomere length as a telomere-binding protein and positively regulated the expression of RELN as a potential epigenetic regulator in both human mesenchymal stem cells (hMSCs) and human neural stem cells (hNSCs)
RAP1 functioned as a proliferation/senescence regulator only in hMSCs (Fig. 6), but not in hNSCs
Summary
The short repeat sequences at the chromosome ends in eukaryotic organisms (Greider, 1991; Blackburn, 2001), are indispensable for the maintenance of genome stability (Londono-Vallejo, 2004; Zhang et al, 2018). Shelterin is involved in the maintenance of advanced telomere structures and regulates the telomere niche via interaction with numerous protein components (Palm and de Lange, 2008; Xin et al, 2008; Bandaria et al, 2016). RAP1 (repressor/activator protein 1), known as TERF2IP (TERF2 interaction protein), was first reported as a transcriptional regulator in Saccharomyces cerevisiae (Shore and Nasmyth, 1987). In addition to the role in regulating telomere length, RAP1 has been reported to suppress the expression of telomeric repeat-containing RNA (TERRA) and subtelomeric genes (Nanavaty et al, 2017). Emerging evidences have suggested that mammalian RAP1 may play a nontelomeric role by occupying specific extratelomeric DNA regions as a transcriptional factor and regulating gene expression (Martinez et al, 2010, 2013, 2016; Yang et al, 2011).
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