Chromatin landscape, DSB levels, and cKU-70/80 contribute to patterning of meiotic DSB processing along chromosomes in C. elegans.

Programmed DNA double-strand break (DSB) formation is essential for achieving accurate chromosome segregation during meiosis. DSB repair timing and template choice are tightly regulated. However, little is known about how DSB distribution and the choice of repair pathway are regulated along the length of chromosomes, which has direct effects on the recombination landscape and chromosome remodeling at late prophase I. Here, we use the spatiotemporal resolution of meiosis in the Caenorhabditis elegans germline along with genetic approaches to study distribution of DSB processing and its regulation. High-resolution imaging of computationally straightened chromosomes immunostained for the RAD-51 recombinase marking DSB repair sites reveals that the pattern of RAD-51 foci throughout pachytene resembles crossover distribution in wild type. Specifically, RAD-51 foci occur primarily along the gene-poor distal thirds of the chromosomes in both early and late pachytene, and on both the X and the autosomes. However, this biased off-center distribution can be abrogated by the formation of excess DSBs. Reduced condensin function, but not an increase in total physical axial length, results in a homogeneous distribution of RAD-51 foci, whereas regulation of H3K9 methylation is required for the enrichment of RAD-51 at off-center positions. Finally, the DSB recognition heterodimer cKU-70/80, but not the non-homologous end-joining canonical ligase LIG-4, contributes to the enriched off-center distribution of RAD-51 foci. Taken together, our data supports a model by which regulation of the chromatin landscape, DSB levels, and DSB detection by cKU-70/80 collaborate to promote DSB processing by homologous recombination at off-center regions of the chromosomes in C. elegans.

GRAS-1 is a novel regulator of early meiotic chromosome dynamics in C. elegans.

Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 coordinates the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I.

ATM/ATR kinases link the synaptonemal complex and DNA double-strand break repair pathway choice.

DNA double-strand breaks (DSBs) are deleterious lesions, which must be repaired precisely to maintain genomic stability. During meiosis, programmed DSBs are repaired via homologous recombination (HR) while repair using the nonhomologous end joining (NHEJ) pathway is inhibited, thereby ensuring crossover formation and accurate chromosome segregation.1,2 How DSB repair pathway choice is implemented during meiosis is unknown. In C. elegans, meiotic DSB repair takes place in the context of the fully formed, highly dynamic zipper-like structure present between homologous chromosomes called the synaptonemal complex (SC).3,4,5,6,7,8,9 The SC consists of a pair of lateral elements bridged by a central region composed of the SYP proteins in C. elegans. How the structural components of the SC are regulated to maintain the architectural integrity of the assembled SC around DSB repair sites remained unclear. Here, we show that SYP-4, a central region component of the SC, is phosphorylated at Serine 447 in a manner dependent on DSBs and the ATM/ATR DNA damage response kinases. We show that this SYP-4 phosphorylation is critical for preserving the SC structure following exogenous (γ-IR-induced) DSB formation and for promoting normal DSB repair progression and crossover patterning following SPO-11-dependent and exogenous DSBs. We propose a model in which ATM/ATR-dependent phosphorylation of SYP-4 at the S447 site plays important roles both in maintaining the architectural integrity of the SC following DSB formation and in warding off repair via the NHEJ repair pathway, thereby preventing aneuploidy.