Conditional TRF1 Knockout in Haematopoietic Progenitor Cells Causes Telomere Shortening and Bone Marrow Failure by Induction of Cellular Senescence

Mutations in the telomerase complex may cause bone marrow failure syndromes due to loss-of-function and consecutive telomere shortening. In addition to the telomerase complex, the six “shelterin” proteins (TRF1, TRF2, TIN2, RAP1, POT1 and TPP1) are required for telomere maintenance. TRF1 has a prominent role in chromosome capping function and prevents the recognition of telomeres by DNA repair mechanisms. At the moment, only TIN2 mutations have been linked to bone marrow failure. Here we aimed to identify other shelterin proteins might cause bone marrow failures. A previous study reported an clinical association between TRF1 mutations and acquired aplastic anemia, however the proof-of-principle that TRF1 can cause bone marrow failure is still missing (Savage SA Exp Hematol 2006).

To address this issue, we used the Mx1-Cre system in combination with the recently generated TRF1 allele in which the exon 1 of TRF1 is flanked by floxP (Martinez P Gen Dev 2009). The bone marrow of the bitransgenic mice was transplanted into B6 wildtype mice and poly (P:I) injections allowed the conditional knockout of TRF1.

Initiation of poly (P:I) injections 4 weeks after transplantation resulted in a failure of all three haematopoietic lineages after 17 days and histopathology revealed massive hypocellular bone marrow consistent with a bone marrow failure. Transplanted control animals showed normal histopathology and even increased neutrophil and thrombocyte counts. Further detailed FACS analysis 7 days after initiation of poly (P:I) injections showed a significant depletion of common myeloid, megakaryocte-erythocyte and common lymphoid progenitor cells, but only a slight decrease of lin-, c-kit+,Sca-1+ haematopoietic stem cells. Interesting, we found no increased rate of apoptosis for the decrease of the progenitor cells, but ß-galactosidase staining showed significant higher amounts of senescent cells in the bone marrow. Further detailed analysis of FACS sorted bone marrow cells showed that especially the c-kit positive progenitor fraction underwent senescence and cell cycle analysis showed an increased G2-M phase indicating a G2-M arrest. In line with these findings RT-PCR of FACS sorted BM revealed increased levels of p21 in the c-kit positive fraction. In addition BrdU injections into the mice on day 7 after poly (P:I) initiation showed increased incorporation and telomere length analysis of transplanted animals with and without poly (P:I) injections revealed massive telomere shortening on day 17.

Our data indicates that TRF1 knockout especially affects haematopoietic progenitor cells by inducing G2-M arrest, induction of p21, and subsequent senescence. Further, compensation of the progenitor cell depletion leads to higher cell turnover and consecutively massive telomere shortening. Taken together this is the first report proving that TRF1 can cause a bone marrow failure and is accompanied with significant telomere shortening.

No relevant conflicts of interest to declare.