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

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Supplementary Figures
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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


The eGFP—TRFl fusion protein tracks telomeres in viv0.


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The eGFP—TRFl fusion protein tracks telomeres in viv0.

CK15-negative cells, a bone fide marker of hair bulge stem cells

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TRF1 expression is downregulated
associated with tissue differentiation.


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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


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findings suggesting that
Lgr5-positive cells are dispensable for intestine homeostasiszs.

?What is the significance of Lgr5 again?

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

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TRF1 protein was
highly increased in wild-type iPS cells compared with the parental
MEFs, as indicated by westem blotting analysis

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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


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telomere elongation during reprogramming

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Intriguingly, eGFP-TRF1 fluorescence
greatly varied in different cells of the same iPS cell colony

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high TRF1 levels
can be re-established

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suggesting that elevated eGFP—TRF1
mRNA levels correlate with increased pluripotency

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These findings indicate that
high eGFP—TRFl expression in iPS cells is associated with
pluripotency rather than to high proliferation rates

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we repeatedly failed to obtain any iPS cell colonies
in the TRFI5/A p53‘ _-Cre cultures

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These results indicate that TRF1
does not influence reprogramming through a direct role in
transcriptional regulation.

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Together, these findings indicate that TRF1 is essential both for
the induction and maintenance of pluripotency in iPS cells.

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arguing that TRF1 is not a target of Nanog.

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we detected a signi?cant increase in TRF1
levels in Oct3/4-transduced MEFs only, even in the absence of
reprogramming


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In mESc, we detected signi?cant binding of
Oct3/-=1 protein to Oct3/4-Pr, Nanog-Pr and TRF1-Pr.

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failed to detect Nanog binding to the TRF1-Pr

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TRF1 is an essential protein during the acquisition of
pluripotency.

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the essential role of TRF1 in
reprogramming is unrelated to telomere maintenance.

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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.

Efficiency of reprogramming was determined by alkaline phosphatase activity


//still need to look at supplementary materials