BRCA1-BARD1 associate with the synaptonemal complex and pro-crossover factors and influence RAD-51 dynamics during Caenorhabditis elegans meiosis.

During meiosis, the maternal and paternal homologous chromosomes must align along their entire length and recombine to achieve faithful segregation in the gametes. Meiotic recombination is accomplished through the formation of DNA double-strand breaks, a subset of which can mature into crossovers to link the parental homologous chromosomes and promote their segregation. Breast and ovarian cancer susceptibility protein BRCA1 and its heterodimeric partner BARD1 play a pivotal role in DNA repair in mitotic cells; however, their functions in gametogenesis are less well understood. Here we show that localization of BRC-1 and BRD-1 (Caenorhabditis elegans orthologues of BRCA1 and BARD1) is dynamic during meiotic prophase I; they ultimately becoming concentrated at regions surrounding the presumptive crossover sites, co-localizing with the pro-crossover factors COSA-1, MSH-5 and ZHP-3. The synaptonemal complex and PLK-2 activity are essential for recruitment of BRC-1 to chromosomes and its subsequent redistribution towards the short arm of the bivalent. BRC-1 and BRD-1 form in vivo complexes with the synaptonemal complex component SYP-3 and the crossover-promoting factor MSH-5. Furthermore, BRC-1 is essential for efficient stage-specific recruitment/stabilization of the RAD-51 recombinase to DNA damage sites when synapsis is impaired and upon induction of exogenous damage. Taken together, our data provide new insights into the localization and meiotic function of the BRC-1-BRD-1 complex and highlight its essential role in DNA double-strand break repair during gametogenesis

Separable Roles for a Caenorhabditis elegans RMI1 Homolog in Promoting and Antagonizing Meiotic Crossovers Ensure Faithful Chromosome Inheritance.

During the first meiotic division, crossovers (COs) between homologous chromosomes ensure their correct segregation. COs are produced by homologous recombination (HR)-mediated repair of programmed DNA double strand breaks (DSBs). As more DSBs are induced than COs, mechanisms are required to establish a regulated number of COs and to repair remaining intermediates as non-crossovers (NCOs). We show that the Caenorhabditis elegans RMI1 homolog-1 (RMH-1) functions during meiosis to promote both CO and NCO HR at appropriate chromosomal sites. RMH-1 accumulates at CO sites, dependent on known pro-CO factors, and acts to promote CO designation and enforce the CO outcome of HR-intermediate resolution. RMH-1 also localizes at NCO sites and functions in parallel with SMC-5 to antagonize excess HR-based connections between chromosomes. Moreover, RMH-1 also has a major role in channeling DSBs into an NCO HR outcome near the centers of chromosomes, thereby ensuring that COs form predominantly at off-center positions

RMH-1 promotes the bias for CO formation on chromosome arms.

<p>(A) Schematics of crosses to obtain the progeny of singled F2 individuals subjected to Next Generation Sequencing (NGS) for SNP analysis. White insert indicates the WT (Bristol) background, and black insert indicates the Hawaiian background. (B) Quantification of the overall recombination frequencies for assayed chromosomes; stacked bar graph indicates the fraction of meiotic products with zero, one, or two COs. For WT (<i>n</i> = 36 chromatids), for <i>rmh-1(jf54)</i> (<i>n</i> = 40 chromatids), and for <i>rmh-1(tn309)</i> (<i>n</i> = 45 chromatids). The frequency of COs was not found to be different between WT and both mutants (Chi² test). (C) Scheme of the different chromosomes used during the recombination assay. The chromosome domains (left arm in blue, center in yellow, and right arm in purple) are correlated with the physical map of each chromosome. (D) Locations of the recombination events (assayed for chromosomes X, IV, and V) in WT (<i>n</i> = 17 COs: three events on X, four on II, four on IV, and six on V), for <i>rmh-1(jf54)</i> (<i>n</i> = 20 COs: 11 events on II and 9 on V), and <i>rmh-1(tn309)</i> (<i>n</i> = 21 COs: nine events on X, nine on IV, and three on V); also see the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002412#sec015" target="_blank">Experimental Procedures</a> section. The relative distribution of COs in the center versus arm domains differed from the WT for <i>tn309</i> (<i>p</i> = 0.046, Chi² test) and for <i>jf54</i>, (p = 0.062, Chi² test).</p

RMH-1 and SMC-5 cooperate to prevent accumulation of aberrant interhomolog connections.

<p>(A) In <i>smc-5(ok2421)</i>, RMH-1 foci increase in mid pachytene nuclei (MP) (green square), while in late pachytene (LP) (yellow square), a zone with fewer foci is still present, as in WT. (B) Quantification of RMH-1 foci in MP nuclei in WT (<i>n</i> = 205) and <i>smc-5(ok2421)</i> (<i>n</i> = 203). In the mutant, we frequently observe nuclei with more than 25 foci, never seen in the WT. Distribution of mid pachytene GFP::RMH-1 foci is significantly different between WT and <i>smc-5</i> mutant (Mann Whitney test, **** <i>p</i> < 0.0001). (C) Quantification of RMH-1 foci in LP nuclei; data are represented as mean +/- SD with ns (not significant). Quantification of hatch rate (D), larval arrest (E), and DAPI bodies in diakinesis oocytes (F) for WT, <i>rmh-1(jf54)</i>, <i>smc-5(ok2421)</i>, and <i>rmh-1(jf54); smc-5</i> (<i>n</i> = 35–45 hermaphrodites per genotype). Data are represented as mean +/- SD with ns (not significant) and * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001. (G–J) Images of individual diakinesis bivalents stained for long arm and short arm markers. Both <i>rmh-1(jf54)</i> and <i>smc-5</i> single mutants exhibit abnormally structured bivalents at low frequency (H,I). In <i>rmh-1; smc-5</i>, all diakinesis nuclei contain bivalents with abnormal structures; typical of these abnormalities is a side-by-side organization of the long arms of the bivalents (J′), presumably reflecting the presence of persistent interhomolog associations at NCO sites. (K) Quantification of the frequencies of diakinesis nuclei (-2 and -1) containing at least one abnormal bivalent (<i>n</i> = 13–25 nuclei per genotype). Data are represented as percentage with ns (not significant) and ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, and **** <i>p</i> < 0.0001 (Chi² test) (L–N) Images of chromosomes in diakinesis nuclei from <i>zhp-3; smc-5</i> and <i>rmh-1(jf54) zhp-3; smc-5</i> worms. Despite the absence of the canonical meiotic CO machinery component ZHP-3, fewer than 12 DAPI structures are observed in some <i>zhp-3; smc-5</i>–1 oocytes, indicating the presence of ectopic connections (L,M). Such ectopic connections occur at high frequency in the <i>rmh-1(jf54) zhp-3; smc-5</i> triple mutant (N–N′). The quantification is presented in (O) with <i>n</i> = 13–36 oocytes per genotype. Data are represented as mean +/- SD with ns (not significant), * <i>p</i> < 0.05 and **** <i>p</i> < 0.0001.</p

RMH-1 (but not RMH-2) contributes to reliable chiasma formation and chromosome segregation in meiosis.

<p>(A) Schematics of RMH-1 and RMH-2. (A′) Location of the three <i>rmh-1</i> mutations. In the <i>jf92</i> allele, the coding sequence was disrupted after the START codon by the insertion of the <i>unc-119</i> gene by the CRISPR technology. In the <i>jf54</i> allele, the G-to-A transition affects the first nucleotide of intron 1 and therefore, destroys the splice donor site of the preceding exon 1. qRT-PCR revealed the presence of different splicing variants (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002412#pbio.1002412.s001" target="_blank">S1 Fig</a>). In the <i>tn309</i> allele, the G-to-A transition introduces a premature STOP codon at position aa 640, leading to the deletion of the OB2 domain (for more details, see the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002412#sec015" target="_blank">Experimental Procedures</a> section). (B–E) Oocyte nuclei at the diakinesis stage of meiotic prophase. Each image shows the complete set of DAPI-stained chromosomes from a single nucleus. The wild-type (WT) and <i>rmh-2(jf94)</i> nuclei contain six bivalents (homolog pairs connected by chiasmata), whereas the <i>rmh-1</i> mutant nuclei contain a mixture of bivalents and univalents. (F) Quantification of the average number of DAPI-positive structures in diakinesis oocytes in the -1 position. WT <i>n</i> = 25, <i>rmh-1(jf92) n</i> = 21, <i>rmh-1(jf54) n</i> = 36, <i>rmh-1(tn309) n</i> = 30, and <i>rmh-2(jf94) n</i> = 18. (G–I) Quantification of embryonic hatch rates (G). Frequencies of male offspring (H) and larval arrest (I) among the progeny of WT and <i>rmh-1</i> and <i>rmh-2</i> mutant worms (<i>n</i> = 35–45 hermaphrodites per genotype). Data for F–I are represented as mean +/- SD; ns stands for not significant, differences are highlighted with stars (* <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001. and **** <i>p</i> < 0.0001). Scale bars, 2 μm.</p

RMH-1 contributes to successful bivalent formation at several levels during prophase I.

<p>Top: scheme of <i>C</i>. <i>elegans</i> gonad showing prophase I divided in zones: transition zone (leptotene/zygotene), pachytene (early, mid, and late), diplotene, and diakinesis. Bottom: summary of localization and functions of RMH-1. Bold: key words referring to localization or function; italics: used when mechanistic insight is proposed. First row: localization of RMH-1: numerous foci in mid pachytene, six bright foci in late pachytene: RMH-1 is absent in diakinesis. Second row: genetic requirements and characteristics of RMH-1 localization. Third row: anti CO function: RMH-1 prevents accumulation of joint molecules and discourages CO formation in chromosome centers. Last row: pro CO functions: role of RMH-1 in CO designation, in assurance of chiasma formation and, finally, its potential function in supporting the geometry of recombination intermediates. We propose timing for the different functions based on our data and previous publications. However, the <i>C</i>. <i>elegans</i> gonad is a continuous production line of meiocytes, and we do not intend to imply sharper transitions between meiotic stages than exist.</p

Separable and interdependent functions of RMH-1 and HIM-6 (BLM).

<p>(A-C) Comparison of RMH-1::GFP and HIM-6(BLM)::HA localization in wild-type germ cells. (B–B′′) In mid pachytene (MP) nuclei, a high number of GFP::RMH-1 and HIM-6::HA foci are present, and most colocalize. (C–C′′) In late pachytene (LP), HIM-6 and RMH-1 colocalize in bright foci, but additional HIM-6 signals are present at other sites. (D) In <i>rmh-1(tn309)</i>, HIM-6 foci are very small and faint (two gonads) or completely undetectable (three gonads). Inset: residual HIM-6 foci do not colocalize (white arrows) with ZHP-3. (E) In <i>rmh-1(jf54)</i>, HIM-6 is still present in foci. Insets for MP (1) and LP (2); in (2), HIM-6 foci can be found in proximity of ZHP-3 at some putative CO-designated sites; however, most do not colocalize. (F) Stacked bar graph showing the percentage of nuclei containing a defined number of COSA-1 foci either in WT (<i>n</i> = 428), <i>rmh-1(jf54)</i> (<i>n</i> = 291), <i>rmh-1(tn309)</i> (<i>n</i> = 329), <i>him-6(ok412)</i> (<i>n</i> = 204), or <i>rmh-1(jf54)</i>; <i>him-6</i> (<i>n</i> = 177). The data presented for WT, <i>rmh-1(jf54)</i>, and <i>rmh-1(tn309)</i> are the same as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002412#pbio.1002412.g004" target="_blank">Fig 4F</a>. Difference in COSA-1 foci distribution was assessed by a Mann Whitney test: ns stands for not significant and **** <i>p</i> < 0.0001. (G) Quantification of the number of DAPI positive structures in the -1 oocyte for WT; single mutants (<i>rmh-1(tn309)- him-6(ok412)- rmh-1(jf54)) and</i> the double <i>rmh-1(tn309)</i>; <i>him-6</i> and <i>rmh-1(jf54)</i>; <i>him-6</i> (<i>n</i> = 15–36 nuclei per genotype). Data are represented as mean +/- SEM with ns (not significant) and **** <i>p</i> < 0.001. (H) In <i>him-6(ok412)</i>, RMH-1 is not detectable in MP (I,I′), but bright RMH-1 foci are present in LP (J,J′), albeit quantification (K) indicates that their numbers are reduced relative to WT (<i>p</i> < 0.001). Data are represented as mean +/- SD. Scale bar 20 μm for gonads, 5 μm for pachytene nuclei insets, and 2.5 μm for single nuclei.</p

Dynamic localization of RMH-1 to distinct foci in pachytene.

<p>(A) In early pachytene (EP), GFP::RMH-1 is diffuse and few foci are detected on DNA. In mid pachytene (MP), high numbers of foci (up to 25 per nucleus) are observed (yellow square and B and B′). In late pachytene (LP), GFP::RMH-1 concentrates into bright foci, on average six per nucleus (red square and C and C′). (D) Quantification of the average number of RMH-1 foci per nucleus in EP, MP, and LP (<i>n</i> = 123 nuclei for EP, <i>n</i> = 205 for MP, and <i>n</i> = 180 for LP). Data are represented as mean +/- SEM. (E) Histogram showing the percentage of late pachytene nuclei containing a certain number of RMH-1 foci per nucleus (one to nine) (<i>n</i> = 180 nuclei). (F) Staining for RAD-51 and GFP::RMH-1. RMH-1 localization starts after and persists longer than the RAD-51 positive zone. (F′) GFP::RMH-1 and RAD-51 mark different recombination intermediates, as they do not colocalize. (G–I) Staining for MSH-5 and GFP::RMH-1. (H–H′′) RMH-1 and MSH-5 partially colocalize in MP (yellow square). (I–I′′) Both proteins become enriched at brighter foci as pachytene progresses (red square). (J) Staining for COSA-1::GFP and mCherry::RMH-1. (K) Staining for ZHP-3 and GFP::RMH-1. In LP, RMH-1 colocalizes at CO sites with MSH-5 (G′), COSA-1 (J′–J′′′) and ZHP-3 (K′–K′′′). Scale bar = 20 μm for gonads, 5 μm for pachytene nuclei insets, and 2.5 μm for single nucleus.</p

Foci of RMH-1 and BLM are resolved as doublets or elongated structures during the pachytene stage.

<p>(A–G) Single pachytene nuclei imaged by SIM. (A–B) In the WT, HIM-6 and RMH-1 colocalize in MP. Foci appear elongated or as a doublet (see insets). (C–E) In LP, RMH-1 and HIM-6 are concentrated at CO sites contained in a structure resolvable into a doublet (see insets). (F) Colocalization of HIM-6 and COSA-1 at CO sites in WT. (G) In <i>rmh-1(jf54)</i>, HIM-6 does not colocalize with COSA-1 at CO sites (white arrow) but can be found in close proximity (white arrows). Scale bar 2μm.</p

DNA double strand break repair is delayed in <i>rmh-1</i> mutant.

<p>(B–C) Immunostaining for DNA strand exchange protein RAD-51, with insets for mid pachytene (MP) and late pachytene (LP) nuclei. (B) Almost no RAD-51 foci are detected in LP nuclei in wild-type (WT). (C) Abundant RAD-51 foci are still detected in LP nuclei in the <i>rmh-1(jf54)</i> mutant, but disappear in diplotene. (B′,C′) Quantification of the numbers of RAD-51 foci per nucleus. For quantification, gonads were divided into six equal zones (A). In both WT and <i>rmh-1(jf54)</i>, zones 1 and 2 correspond to the mitotic zone; zone 3 to transition zone and early pachytene; zones 4 to 5 to pachytene. Analysis of <i>n</i> = 124–231 nuclei per zone for each genotype. Distribution of RAD-51 foci is significantly different between WT and mutant for the zones 3 to 6 (Mann Whitney test, **** <i>p</i> < 0.0001). Scale bar = 20 μm for gonads and 5 μm for insets.</p