Phosphorylation of HORMA-domain protein HTP-3 at Serine 285 is dispensable for crossover formation

Abstract

Generation of functional gametes is accomplished through a multilayered and finely orchestrated succession of events during meiotic progression. In the Caenorhabditis elegans germline, the HORMA-domain-containing protein HTP-3 plays pivotal roles for the establishment of chromosome axes and the efficient induction of programmed DNA double-strand breaks, both of which are crucial for crossover formation. Double-strand breaks allow for accurate chromosome segregation during the first meiotic division and therefore are an essential requirement for the production of healthy gametes. Phosphorylation-dependent regulation of HORMAD protein plays important roles in controlling meiotic chromosome behavior. Here, we document a phospho-site in HTP-3 at Serine 285 that is constitutively phosphorylated during meiotic prophase I. pHTP-3S285 localization overlaps with panHTP-3 except in nuclei undergoing physiological apoptosis, in which pHTP-3 is absent. Surprisingly, we observed that phosphorylation of HTP-3 at S285 is independent of the canonical kinases that control meiotic progression in nematodes. During meiosis, the htp-3(S285A) mutant displays accelerated RAD-51 turnover, but no other meiotic abnormalities. Altogether, these data indicate that the Ser285 phosphorylation is independent of canonical meiotic protein kinases and does not regulate HTP-3-dependent meiotic processes. We propose a model wherein phosphorylation of HTP-3 occurs through noncanonical or redundant meiotic kinases and/or is likely redundant with additional phospho-sites for function in vivo.

Keywords: Caenorhabditis elegans meiosis; HORMA-domain proteins; HTP-3.

Fig. 1.

Phospho-HTP-3^S285 accumulation follows total HTP-3 staining pattern. a) Table showing several of the putative interactors identified by mass spectrometry analysis on the PARG-1::GFP pull downs. b) Annotated fragmentation mass spectrum of the HTP-3 peptide carrying pS285. Red and blue lines indicate b- and y-ions, respectively, y ions with phosphate losses are marked in light red. The illustration was generated using PeptideShaker ver. 2.2.0 (Vaudel et al. 2015). c) Whole-mount gonad stained with panHTP-3 and phosphoHTP-3^S285 antibodies. Scale bar 20 µm. d) htp-3(S285A) mutants stained with panHTP-3 and phosphoHTP-3^S285 antibodies showing specificity of the phospho-antibody. Scale bar 20 µm. e) Insets showing nuclei at different stages of meiotic prophase I. Note that pHTP-3^S285 antibody staining is absent in apoptotic cells (dotted circles). Scale bar 5 µm.

Fig. 2.

Phosphorylation of HTP-3^S285 is independent of synapsis and recombination. Mid-pachytene nuclei stained with pHTP-3^S285 and panHTP-3 antibodies in the indicated mutant backgrounds. Scale bar 5 µm.

Fig. 3.

HTP-3^S285 phosphorylation is independent of ERK, plk-2, chk-1/chk-2, and the DNA damage kinases atm-1 and atl-1. a) In vitro kinase assay of HTP-3 wild-type and HTP-3^S285A mutants with active recombinant ERK2. b) Mid-pachytene nuclei stained for pHTP-3^S285 and panHTP-3 in plk-2(ok1936). Scale bar 10 µm. c) HTP-3^S285 phosphorylation in ATM and ATR mutants. Dotted circles indicate nuclei with impaired axes morphogenesis. Scale bar 10 µm. d) HTP-3^S285 phosphorylation in chk-1^(RNAi). Scale bar 10 µm. e) HTP-3^S285 phosphorylation upon auxin-induced degradation of CHK-2. Scale bar 10 µm.

Fig. 4.

Phosphorylation of HTP-3^S285 does not require cdk-1 and cdk-2. HTP-3^S285 phosphorylation in mutant germlines upon loss of cdk-1 (a), cdk-2 (b), or both (c). Scale bar 10 µm.

Fig. 5.

htp-3(S285A) mutants display normal levels of fertility. a) Analysis of viability, brood size, and male progeny in the indicated mutants. n indicates the number of worms analyzed. b) Top: quantification of synapsis in the htp-3(S285A) mutants. Bottom: representative examples of mid-pachytene nuclei from the indicated genotypes labeled with HTP-3 and SYP-1. Scale bar 10 µm. c) Time-course analysis of RAD-51 foci formation and removal in htp-3(S285A) mutants and wild-type controls. Gonads were divided into 7 equal regions from the mitotic tip to diplotene entry and the number of RAD-51 foci in each nucleus was counted. Bars in the charts indicate mean and standard deviation (ns, nonsignificant, ****P < 0.0001, **P = 0.0072, *P = 0.047 as calculated by T-test). Insets show representative images of early-pachytene nuclei stained with anti-RAD-51 antibodies in the indicated genotypes. Scale bar 10 µm.