Histones can receive many different modifications, and the presence/absence of certain modifications is implicated in transcriptional control and chromosomal events. One modification, histone H3 lysine 9 dimethylation (H3K9me2), is associated with unsynapsed chromatin during meiosis in many organisms, including C. elegans [1-3]. For example, in the male C. elegans germ line, the single X chromosome does not synapse and, during pachytene, is detected as a strong focus of H3K9me2 by immuno-labeling. In contrast, a much lower level of H3K9me2 staining is detected on synapsed autosomes. This interesting phenomenon led to the question of how H3K9me2 marks are specifically targeted to their genomic sites.
In order to address this question, we are identifying H3K9me2 methyltransferase (MET-2) interactors. To do so, we generated epitope-tagged met-2 transgenes and antibody against MET-2. Using these reagents, we performed immunoprecipitation and tandem mass spec analysis to recover potential interactors. One candidate protein that we recovered is the smrc-1 (C16A3.1) gene product. SMRC-1 is the C. elegans ortholog of mammalian SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein). Mammalian and Drosophila SMARCAL1 family members are annealing helicases that provide protection from DNA replication stress. Little is known about SMARCAL1 function in the germ line.
Using CRISPR-Cas9 methods, we generated smrc-1 nonsense mutations and flag-tagged the endogenous smrc-1 gene. We immunoprecipitated 3xFLAG::SMRC-1 and confirmed the interaction with MET-2. We evaluated 3xFLAG::SMRC-1 expression and observed that it localizes to mitotic and meiotic nuclei in both male and hermaphrodite germ lines. smrc-1 nonsense mutations cause germline defects, including meiotic defects consistent with impaired recombination. Meiotic H3K9me2 is elevated and abnormally distributed in smrc-1 males. smrc-1 hermaphrodites exhibit maternal-effect embryonic lethality and, under conditions of temperature stress, exhibit a progressive reduction in brood size leading to complete infertility within several generations. We are investigating the relationships among SMRC-1 function, MET-2 function, and DNA repair.
References: (1) Shiu et al. (2001) Cell 107:905-916. (2) Kelly et al. (2002) Development 129:479-492. (3) Turner et al. (2005) Nat Genet 37:41-47.