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TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells

2015-04-29

TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-11-2014d0913\TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells.pdf" Supplementary Figures "C:\Users\Kurt Whittemore\Box Sync\DocDR\2014\02-11-2014d0913\TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells Supplementary.pdf" Abstract TRF1 is a component of the shelterin complex that protects chromosome ends. TRF1 deficiency leads to early embryonic lethality and to severe organ atrophy when deleted in adult tissues. Here we generate a reporter mouse carrying a knock-in eGFP-TRF1 fusion allele to study the role of TRF1 in stem cell biology and tissue homeostasis. We find that eGFP-TRF1 expression in mice is maximal in known adult stem cell compartments and show that TRF1 ensures their functionality. eGFP-TRF1 is highly expressed in induced pluripotent stem cells, uncoupled from the telomere elongation associated with reprogramming. Selection of eGFP-TRF1-high induced pluripotent stem cells correlates with higher pluripotency as indicated by their ability to form teratomas and chimeras. We further show that TRF1 is necessary for both induction and maintenance of pluripotency, and that TRF1 is a direct transcriptional target of Oct3/4. [TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells Supplementary Data notes] Some notes -ASU doesn't have access to this article --got it from ill -Nature communications journal -CNIO paper -corresponding author: Maria Blasco The eGFP—TRFl fusion protein tracks telomeres in viv0. q The eGFP—TRFl fusion protein tracks telomeres in viv0. CK15-negative cells, a bone fide marker of hair bulge stem cells q TRF1 expression is downregulated associated with tissue differentiation. q eGFP—TRFl levels do not correlate with proliferation indexe eGFP—TRF1 expression marks adult stem cell compartments. Paneth +4 are less differentiated cells and +7 cells are more differentiated cells q findings suggesting that Lgr5-positive cells are dispensable for intestine homeostasiszs. ?What is the significance of Lgr5 again? -It is a biomarker for stem cells Intestinal TRF1 ablation leads to severe GI atrophy the less differentiated compartments show higher sensitivity to the DDR induced by TRF1 ablation These results are in line with recent findings showing telomere shortening associated with TRF1 deletion in the hematopoetic system as the result of increased compensatory proliferation of the remaining stem cell pools q TRF1 protein was highly increased in wild-type iPS cells compared with the parental MEFs, as indicated by westem blotting analysis q Of note, the increased TRF1 levels associated with pluripotency were not paralleled to the same extent by other shelterin proteins, such as TRF2 or RAPI q telomere elongation during reprogramming q Intriguingly, eGFP-TRF1 fluorescence greatly varied in different cells of the same iPS cell colony q high TRF1 levels can be re-established q suggesting that elevated eGFP—TRF1 mRNA levels correlate with increased pluripotency q These findings indicate that high eGFP—TRFl expression in iPS cells is associated with pluripotency rather than to high proliferation rates q we repeatedly failed to obtain any iPS cell colonies in the TRFI5/A p53‘ _-Cre cultures q These results indicate that TRF1 does not influence reprogramming through a direct role in transcriptional regulation. q Together, these findings indicate that TRF1 is essential both for the induction and maintenance of pluripotency in iPS cells. q arguing that TRF1 is not a target of Nanog. q we detected a signi?cant increase in TRF1 levels in Oct3/4-transduced MEFs only, even in the absence of reprogramming q In mESc, we detected signi?cant binding of Oct3/-=1 protein to Oct3/4-Pr, Nanog-Pr and TRF1-Pr. q failed to detect Nanog binding to the TRF1-Pr q TRF1 is an essential protein during the acquisition of pluripotency. q the essential role of TRF1 in reprogramming is unrelated to telomere maintenance. q Finally supporting the notion that TRF1 is a key factor for pluripotency, we make the Lmprecedented ?nding that TRF1 is a direct target of Oct3/4, which binds TRF1 promoter and is suf?cient to upregulate TRF1, thus providing a mechanistic link between TRF1 and pluripotency. -q Efficiency of reprogramming was determined by alkaline phosphatase activity //still need to look at supplementary materials
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