xnd-1 regulates the global recombination landscape in Caenorhabditis elegans

Abstract

Meiotic crossover (CO) recombination establishes physical linkages between homologous chromosomes that are required for their proper segregation into developing gametes, and promotes genetic diversity by shuffling genetic material between parental chromosomes. COs require the formation of double strand breaks (DSBs) to create the substrate for strand exchange. DSBs occur in small intervals called hotspots and significant variation in hotspot usage exists between and among individuals. This variation is thought to reflect differences in sequence identity and chromatin structure, DNA topology and/ or chromosome domain organization. Chromosomes show different frequencies of nondisjunction (NDJ), reflecting inherent differences in meiotic crossover control, yet the underlying basis of these differences remains elusive. Here we show that a novel chromatin factor, X non-disjunction factor 1 (xnd-1), is responsible for the global distribution of COs in C. elegans. xnd-1 is also required for formation of double-strand breaks (DSBs) on the X, but surprisingly XND-1 protein is autosomally enriched. We show that xnd-1 functions independently of genes required for X chromosome-specific gene silencing, revealing a novel pathway that distinguishes the X from autosomes in the germ line, and further show that xnd-1 exerts its effects on COs, at least in part, by modulating levels of H2A lysine 5 acetylation.

Figure 1

xnd-1 is needed for the normal recombination landscape in C. elegans. (a) C05D2.5 was identified by its increase in the number of recombinant progeny with the phenotype of the middle of three genetic markers, either unc-45 dpy-18 unc-64 (wt, n=8750, std. dev= 0.06; xnd-1, n=3980, std. dev= 0.15) (i) or dpy-1 lon-1 dpy-18 (wt, n=2229, std. dev= 0; xnd-1, n=1863, std dev= 0.21) (ii) Error bars represent the standard deviation from three or two independent experiments, respectively. (b) Chromosome I recombination maps for wt and xnd-1 oocytes (top) and sperm (bottom). (c) X chromosome recombination maps for wt and xnd-1 oocytes (data for b,c are found in Supplementary Tables 1-3). Genetic and physical markers are shown above and below the graphic representation of each chromosome, respectively. Boxes represent the relative map size for each interval as determined by SNP analysis (see Methods). Significant differences between wt and xnd-1 are marked (*p<0.05; **p<0.001).

Figure 2

xnd-1 is required for efficient DSB formation on the X chromosome. (a-c) Achiasmate chromosomes are observed at diakinesis in xnd-1. FISH probes mark Chromosomes V (yellow) and X (magenta); DNA is stained with DAPI, (green). (a) Six DAPI-staining bodies indicate that all chromosomes have recombined, (b) seven reveal the achiasmate X’s in xnd-1 oocytes. (c) Quantification of achiasmate X frequency. Chromosome V was never achiasmate in wt or xnd-1. (d) Ionizing radiation (IR) rescues the CO defects of xnd-1. Quantification of IR rescue as assessed by the number of DAPI staining bodies at diakinesis 24 hrs post-irradiation. (wt −/+ IR, N=91, 82; spo-11 −/+ IR, N=73, 100; xnd-1 −/+ IR, N= 190, 157; him-8 −/+ IR, N=83, 65). (e) Fewer RAD-51 foci are observed on the X chromosome (white circles) in xnd-1. rad-54(RNAi) treated animals were dissected and germlines co-stained for DNA (green), HTZ-1 (which marks autosomes, yellow), and the DNA repair protein, RAD-51 (magenta), which acts as a marker of DSBs9). The percentage of nuclei in which RAD-51 foci were observed on the X was quantified (wt, N=369; xnd-1, N=391).

Figure 3

XND-1 is an autosomal protein that regulates X chromosome crossing over. (a) Anti-XND-1 antibody staining of a wt hermaphrodite germline. (b) Close-up of wt nuclei reveals the absence of staining on one chromosome (yellow arrowheads). (c) Co-staining of wt pachytene nuclei with anti-XND-1 and anti-H4K12Ac reveals that these proteins are coincident, indicating that XND-1 is enriched on autosomes. A yellow arrowhead indicates the unstained X. (d) Localization of XND-1 is independent of the X chromosome silencing gene mes-2. XND-1 antibody staining in mes-2 (M-Z-) mutants with rare pachytene nuclei reveals normal XND-1 localization (X marked by white arrowhead). (e) Activating HPTMs remain excluded from the X in xnd-1mutants (yellow arrowheads). Histone H4K12Ac (magenta) is enriched on autosomes in wt (top) and xnd-1 (bottom). (f) Suppression of xnd-1 HIM phenotype by mes-2(bn11) and mes-3(RNAi ) (xnd-1, n = >8000; mes-2= >2000; mes-2; xnd-1, n=733; GFP(RNAi), n= 192; mes-3RNAi), n= 283). Error bars represent standard deviation from at least three experiments.

Figure 4

Germline chromatin is altered in xnd-1 mutants. (a) Histone H2A lysine 5 acetylation is increased in the xnd-1 mutant. wt (upper) and xnd-1 (lower) mutant gonads were stained on one slide and imaged identically revealing the more intense anti-histone H2A K5Ac staining in the xnd-1 gonad. (b) Mid-pachytene nuclei from wt and xnd-1 gonads (DNA, green; anti-H2A K5Ac, magenta) showing the more intense and uniformly distributed staining in the mutant. The gain in the wt image has been increased to reveal the modification in these nuclei. (c) mys-1(RNAi) suppresses the X chromosome CO defect of xnd-1 mutants. Quantification of DAPI staining bodies at diakinesis: 6 foci, gray; 7 foci, red (untreated, n= 94 nuclei; GFP(RNAi), n=124; mys-1(RNAi), n=288). (d) Proposed model for XND-1 function. The presence of XND-1 on autosomes allows for SPO-11 to gain access to the transcriptionally silent, heterochromatin-like X chromosomes. In the absence of XND-1, SPO-11 targets the gene-rich clusters (more transcriptionally active regions) on autosomes and titrates DSBs away from the silent X chromosomes and the less transcriptionally active autosomal arms.