Differential Impact of a Telomeropathy-causing Mutation in Shelterin Protein TPP1 on Mouse Hematopoiesis and Germline

Abstract

Chromosome ends face two problems: the end-protection (end-to-end fusions) and the end-replication (progressive telomere shortening) problems. The protein complex shelterin binds to the telomeric DNA repeats at chromosome ends to protect them from illicit end joining and other unwanted recombination or resection events. The ribonucleoprotein complex telomerase extends chromosome ends in somatic and germline stem cells to overcome the end-replication problem and ensure continued proliferation. Mutations in genes important for telomerase and/or shelterin function often result in diseases termed telomeropathies, the most prominent example of which being dyskeratosis congenita (DC). Severe shortening of telomeres in patients with DC results in depletion of stem cells and bone marrow (BM) failure, the primary cause of death.

TPP1 is the only shelterin component known to both protect chromosome ends and recruit telomerase to telomeres. We have previously defined regions of TPP1 that are critical for recruiting telomerase and characterized the consequences of a patient-derived DC mutation in TPP1 (K170Δ) resulting in decreased telomerase activity, impaired recruitment, and short telomeres in cultured human cells. While these studies provide a direct cause-effect relationship, they do not provide insights into stem cell dysfunction in vivo.

A DC mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. In this dissertation, I first present work on how we used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic (Chapter 2) and germline (Chapter 3) compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous BM failure but rather succumb to infertility at steady-state. Our work suggests a species-specific sensitivity in the germline rather than the soma in mice, which is reversed in human patients with this disease. We speculate that the species-specific differences in the response to severe telomere shortening arises from the distinct proliferation burdens on the mouse and human, soma and germline. Small and short-lived species like mice may boost germline proliferation to increase gamete production and maximize the number of offspring produced in a lifetime, while large and long-lived species like humans prioritize somatic development to survive to the age of reproduction (and nurturing) to produce fewer, but healthy, offspring.