The DNA damage response (DDR) is essential for preserving genomic integrity in eukaryotes. At the heart of the DDR network are the ATM and ATR kinases, which impose their regulation on multiple repair pathways via damage-induced phosphorylation. The Fanconi anemia (FA) pathway is one such arm of the DDR, and is exquisitely regulated by ATM and ATR. The FA network functions as an ubiquitylation cascade which consists of at least fifteen proteins, and is essential for the repair of interstrand crosslinks (ICL) that arise as a result of numerous cellular processes and/or chemotherapeutic agents. Additionally, the FA pathway has been implicated in the maintenance of hematopoeitic stem cells via mechanisms that have yet to be elucidated. The newest FA member is Slx4 (or Fancp), which has been shown by the PI’s laboratory to be essential for gametogenesis in the mouse. Newborn male pups lacking Slx4 show a drastic reduction of SSC numbers as a result of gonocyte apoptosis in late gestation. This phenotype is apparent in a number of other FA gene mutations although, surprisingly, no studies to date have elucidated the precise role of the FA machinery in germ cell proliferation. We hypothesize that FA members are crucial for driving DDR events in proliferating germ cells to ensure genome stability in SSC precursors, and that this role is regulated via phosphorylation by ATM/ATR kinases.
Our interest in SLX4 was buoyed by the observations that, in addition to its role as a substrate of ATM/ATR, it also interacts with a wide range of key structure-specific nucleases, as well as with components of the DNA mismatch repair (MMR) pathway, including MLH1, and with the RecQ helicase, BLM. All of these interactors are known to play key roles in meiotic recombination, implying that SLX4 may serve as a key integrator between recombination pathways in mouse meiosis.
In addition to its meiotic role, we have shown that loss of SLX4 results in a dramatic loss of SSC precursors late in gestation, suggesting that SLX4 plays an essential role in facilitating the proliferation and/or viability of these cells. This phenotype is remarkably similar to a number of other Fanc gene mutations,and yet no groups have yet attempted to understand how this Fanconi network functions in the establishment of the mature SSC pool within the developing testis, nor is it clear how Slx4 integrates in to this network. The Cohen lab is investigating the role of the FA pathway in these events during gonadal development and beyond.
FA is a genomic instability syndrome characterized by developmental abnormalities and increased cancer predisposition. FA deficient cells exhibit increased chromosome aberrations and hypersensitivity to interstrand crosslinks (ICL), which can occur as a result of chemotherapeutic agents, or naturally as a result of lipid peroxidation. ICLs can cause cellular damage in at least three ways: (1) by inhibiting replisome progression at the replication fork leading to “mitotic catastrophe”, (2) by stalling transcription, or (3) by impairing DNA maintenance by preventing access to DNA by transcription factors, helicases etc.
Fifteen FA genes have been identified, eight of which (a, b, c, e, f, g, l, m) form the core complex, while two (d2, i) form the ID complex. DNA damage induces monoubiquitylation of the ID complex by the core complex, which leads to downstream interactions with a number of proteins, including FANCD1 (BRCA2), FANCN, and FANCJ. While the function of the FA complexes have not been fully elucidated, clearly they act in concert to repair ICLs via homologous recombination repair. A number of mutant mouse lines for FA genes exhibit germ cell proliferation defects, all of which show a drastically reduced number of germ cells in newborn mice, the loss always occurring after entry into the gonad, leading us to suggest a role for the FA pathway in genome stabilization in the germline.
Holloway J.K., Mohan S., Balmus G., Sun X., Modzelewski A., Borst P.L., Freire R. Weiss R.S., Cohen P.E. Mammalian BTBD12 (SLX4) protects against genomic instability during mammalian spermatogenesis. PLoS Genetics 7(6): e1002094 (2011) DOWNLOAD