TFR2-mediated telomere protection is dispensable in pluripotent stem cells

The shelterin complex stabilizes the 3’ overhangs in telomeres (D- and t-loops) so that degradation by exonucleases as well as the undesirable end-joining of chromosomes is inhibited.

TRF2 – Telomeric Repeat-binding Factor 2 • essential for proliferation in >700 cell lines • gene deletion in mice is lethal

• tissue-specific gene deletions lead to chromosomal fusions (breakage-fusion-bridge cycles)

Studies of Trf2 function to date have been in somatic cells.

Somatic Cell: Any cell in a multicellular organism except for germ line cells. The ones used in this paper have a fibroblast-like morphology and are called FLICs. (Germ Cells: Sex cells or gametes; e.g., sperm/egg) Stem Cells: Unspecialized cells with self-renewal abilities, but which can also differentiate into other cell types. Embryonic Stem Cells: Stem cells from the inner mass of an embryo. They are pluripotent. They are called mESCs in this paper.

What is the effect of TRF2 deletion on the growth of embryonic stem cells versus the more differentiated fibroblasts they created: The fibroblasts went into a growth arrest with OHT treatment (as expected) while the stem cells, albeit slowed down, kept growing.

Why do the embryonic stem cells stay alive despite TRF2 ablation: They don’t form those chromosomal fusions that are so apparent in the fibroblast population.

Do the embryonic stem cells form telomeric dysfunction-induced foci in response to TRF2 deletion: When there are breaks in dsDNA, the histone protein variant called H2AX gets phosphorylated and this can be detected with an antibody.

Do the embryonic stem cells form telomeric dysfunction-induced foci in response to TRF2 deletion: When there are breaks in dsDNA, there is also an accumulation of 53BP1 at those sites. It also accumulates at sites of chromosomal fusions when telomeres are defective.

Do the embryonic stem cells form telomeric dysfunction-induced foci in response to TRF2 deletion: Not often, and not in a very significant way.

What happens to embryonic stem cells when dsDNA breaks are induced by irradiation? This time markers of dsDNA breaks light up, and they do so just as often as they do in the fibroblasts.

The lack of telomere fusions in stem cells deleted in TRF2 is not caused by some intrinsic defect in how these cells respond to DNA damage. They can do so, but just not at telomeres.

So if it isn’t Trf2, what is helping protect telomeres in the stem cell populations: Candidates were identified by performing a ‘genome-wide synthetic lethal CRISPR-Cas9-knockout screen’.

Neutral: Gene does not impact growth of cells under any condition. Essential: Gene impacts growth of cells under all conditions. Synthetic Lethal: Gene impacts growth, but only under conditions when TRF2 is knocked out.

Pot1b is a telomere-binding protein, but it is known to be ‘dispensable’ for telomere protection.

Brd2 is a bromodomain and BET domain-containing protein that regulates gene expression. It may also have a role in suppressing DNA damage responses downstream of ATM kinase signaling.

So if it isn’t Trf2, what is helping protect telomeres in the stem cell populations? Neither Pot1b nor Brd2 impact stem cell growth on their own, but when TRF2 is knocked out, the synthetic lethal effect was observed.

What about Pot1B helps it promote survival of the stem cells harboring a TRF2 knockout: Pot1B appears to protect the telomeres from forming chromosomal fusions in the stem cells depleted of Trf2. Similar results are seen with the related protein Pot1A. Similar results are seen with a drug inhibitor of the DNA damage sensor ATR. Pot1B appears to protect the telomeres from forming chromosomal fusions in the stem cells depleted of Trf2.

Brd2 deletion increase basal levels of DNA damage response signaling as indicated by the increase in KAP1 phosphorylation. This DNA damage response signaling is accentuated when both Brd2 and Trf2 are no longer expressed. This DNA damage response signaling also appears to be regulated by the ATM kinase.

In stem cells, there are conditions, like the loss of Pot1A/B or Brd2, that would lead to DNA damage response signaling downstream of ATR or ATM that cause chromosomal fusions at telomeres.

So, maybe that’s it, telomeres are protected in stem cells because they do express Pot1A/B and Brb2.

So if it isn’t Pot1A/B or Brd2, what is helping protect telomeres in the stem cell populations: Most of the up-regulated genes in the stem cells (up to 18-fold) were genes common to the totipotent 2-cell-stage of embryogenesis; i.e., genes of the zinc finger and SCAN domain-containing gene cluster (Zscan4).

Is there a functional role for Zscan4 in stem cells depleted of Trf2? Yes, the silencing of Zscan4 led to a loss in telomere protection resulting in chromosomal fusions. The induction of Zscan4 led to an increase in telomere protection resulting in fewer chromosomal fusions.

Telomere protection in stem cells is mediated by an attenuation of the ATM/ATR signaling pathways and by the expression of genes that help maintain totipotency.

Connections between Zscan4 and Cancer: It has a role in telomere maintenance in a telomerase-independent manner in cancer cells. It works with shelterin complex proteins in telomere lengthening in cancer cells. It is up-regulated in response to the DNA damage induced by chemotherapy drugs. It can alter the epigenetic state in such a way as to promote a cancer stem cell phenotype.