Contact Information: Email: firstname.lastname@example.org; Phone: 607-253-3636
Sponsor: New York State Department of Health-NYSTEM
Grant Number: C026442
Title: Genome Maintenance in Germline Stem Cells
Annual Direct Cost: $146,702
Project Period: 09/01/10-08/31/13
DESCRIPTION (provided by applicant): For any sexually reproducing species, the germline is the most crucial lineage because it contains the genetic information that is transmitted to subsequent generations. Moreso than somatic cells, it is important that germline stem cells have effective mechanisms for minimizing mutations that would be deleterious to offspring. Primordial germ cells (PGCs) proliferate during embryonic development, colonize the primitive gonads, and ultimately form cells that can become oocytes or sperm. We and others have noted that mutations in certain genes that are important for DNA repair and DNA replication can deplete the reservoir of germline stem cells, often leading to infertility. This project uses two mouse models of germ cell depletion to study the DNA repair and cell cycle checkpoint control systems in germline stem cells, and to determine why these cells are exquisitely sensitive to replication stress and defects in DNA repair.
One model contains a gene trap disruption of Mcm9, an unique member of the minichromosome maintenance (MCM) family of DNA replication licensing factors. Its evolutionarily conserved relatives MCM2-7 comprise the replicative helicase that is essential for cell viability. The other model is a mutant allele of Fancm, a component of the Fanconi anemia DNA repair complementation group, that we recovered in a forward genetic screen for genomic instability mutants. The FANC complex is particularly important in repair of damage at DNA replication forks. Both of these recessive, viable mutations cause loss of germ cells in male and female mice. We hypothesize that germ cells are hypersensitive to the types of DNA damage caused (Mcm9) or left unrepaired by (Fancm) these mutations (presumably double strand breaks caused during DNA replication), leading to enhanced (relative to somatic cells) cell death by checkpoint activation. This proposed project has two major Aims. The first is to identify the exact stage of development that germ cell depletion occurs in these mutants, and whether it is attributable to decreased proliferation, survival, or impaired ability of stem cell self-renewal. In the second Aim, genetic and molecular approaches will be used to better characterize the types of damage occuring in mutant cells, and the genome surveillence mechanisms that are operating in these cells. In the genetic approach, we will determine if mice containing mutations in checkpoint genes that respond to either double strand breaks or replication fork damage can rescue the germ cell depletion phenotypes of Mcm9 or Fancm. These data will implicate the pathway responsible for eliminating defective cells. In the molecular approach, markers of DNA damage and checkpoint response pathways will be examined directly in PGCs and spermatogonia. These studies will give insight into how the cells holding the keys to our species - the germline - are affected by genetic and environmental factors can impact fertility and the well-being of offspring.