Mouse CCDC79 (TERB1) is a meiosis-specific telomere associated protein

Background: Telomeres have crucial meiosis-specific roles in the orderly reduction of chromosome numbers and in ensuring the integrity of the genome during meiosis. One such role is the attachment of telomeres to trans-nuclear envelope protein complexes that connect telomeres to motor proteins in the cytoplasm. These trans-nuclear envelope connections between telomeres and cytoplasmic motor proteins permit the active movement of telomeres and chromosomes during the first meiotic prophase. Movements of chromosomes/telomeres facilitate the meiotic recombination process, and allow high fidelity pairing of homologous chromosomes. Pairing of homologous chromosomes is a prerequisite for their correct segregation during the first meiotic division. Although inner-nuclear envelope proteins, such as SUN1 and potentially SUN2, are known to bind and recruit meiotic telomeres, these proteins are not meiosis-specific, therefore cannot solely account for telomere-nuclear envelope attachment and/or for other meiosis-specific characteristics of telomeres in mammals. Results: We identify CCDC79, alternatively named TERB1, as a meiosis-specific protein that localizes to telomeres from leptotene to diplotene stages of the first meiotic prophase. CCDC79 and SUN1 associate with telomeres almost concurrently at the onset of prophase, indicating a possible role for CCDC79 in telomere-nuclear envelope interactions and/or telomere movements. Consistent with this scenario, CCDC79 is missing from most telomeres that fail to connect to SUN1 protein in spermatocytes lacking the meiosis-specific cohesin SMC1B. SMC1B-deficient spermatocytes display both reduced efficiency in telomere-nuclear envelope attachment and reduced stability of telomeres specifically during meiotic prophase. Importantly, CCDC79 associates with telomeres in SUN1-deficient spermatocytes, which strongly indicates that localization of CCDC79 to telomeres does not require telomere-nuclear envelope attachment. Conclusion: CCDC79 is a meiosis-specific telomere associated protein. Based on our findings we propose that CCDC79 plays a role in meiosis-specific telomere functions. In particular, we favour the possibility that CCDC79 is involved in telomere-nuclear envelope attachment and/or the stabilization of meiotic telomeres. These conclusions are consistent with the findings of an independently initiated study that analysed CCDC79/TERB1 functions

Alignment of Homologous Chromosomes and Effective Repair of Programmed DNA Double-Strand Breaks during Mouse Meiosis Require the Minichromosome Maintenance Domain Containing 2 (MCMDC2) Protein.

Orderly chromosome segregation during the first meiotic division requires meiotic recombination to form crossovers between homologous chromosomes (homologues). Members of the minichromosome maintenance (MCM) helicase family have been implicated in meiotic recombination. In addition, they have roles in initiation of DNA replication, DNA mismatch repair and mitotic DNA double-strand break repair. Here, we addressed the function of MCMDC2, an atypical yet conserved MCM protein, whose function in vertebrates has not been reported. While we did not find an important role for MCMDC2 in mitotically dividing cells, our work revealed that MCMDC2 is essential for fertility in both sexes due to a crucial function in meiotic recombination. Meiotic recombination begins with the introduction of DNA double-strand breaks into the genome. DNA ends at break sites are resected. The resultant 3-prime single-stranded DNA overhangs recruit RAD51 and DMC1 recombinases that promote the invasion of homologous duplex DNAs by the resected DNA ends. Multiple strand invasions on each chromosome promote the alignment of homologous chromosomes, which is a prerequisite for inter-homologue crossover formation during meiosis. We found that although DNA ends at break sites were evidently resected, and they recruited RAD51 and DMC1 recombinases, these recombinases were ineffective in promoting alignment of homologous chromosomes in the absence of MCMDC2. Consequently, RAD51 and DMC1 foci, which are thought to mark early recombination intermediates, were abnormally persistent in Mcmdc2-/- meiocytes. Importantly, the strand invasion stabilizing MSH4 protein, which marks more advanced recombination intermediates, did not efficiently form foci in Mcmdc2-/- meiocytes. Thus, our work suggests that MCMDC2 plays an important role in either the formation, or the stabilization, of DNA strand invasion events that promote homologue alignment and provide the basis for inter-homologue crossover formation during meiotic recombination

MCMDC2 is not required for extensive non-homologous synaptonemal complex formation in the <i>Spo11</i>^<i>-/-</i> background.

<p>(<b>a</b>) SYCP3 (axis marker) and SYCP1 (synaptonemal complex marker) were detected by immunofluorescence on nuclear surface spreads of zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i>, <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. Whereas comparatively few synaptonemal complex stretches are detected in the <i>Mcmdc2</i>^<i>-/-</i>spermatocyte, extensive non-homologous synaptonemal complex formation is seen in the <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. Scale bars; 10μm (<b>b</b>) Quantification of SYCP1 stretch numbers in zygotene-pachytene spermatocytes with fully condensed chromosome axes of the indicated genotypes. The numbers of synaptonemal complex stretches is significantly higher in <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes than in <i>Mcmdc2</i>^<i>-/-</i> (Mann Whitney test). The numbers of synaptonemal complex stretches are not significantly different in <i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes (p = 0.8639, Mann Whitney test). Median numbers of foci are marked, and n corresponds to the number of analyzed spermatocytes in two (<i>Spo11</i>^<i>-/-</i> or <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/-</i>) or three (<i>Mcmdc2</i>^<i>-/-</i>) pooled experiments.</p

Preferential expression of <i>Mcmdc2</i> in the gonads, and <i>Mcmdc2</i> targeting in mice.

<p>(<b>a</b>) Expression of <i>Mcmdc2</i> and a “house-keeping” gene (<i>S9</i>) in testis and a somatic tissue mix measured by RT-PCR. cDNAs were prepared from four RNA mixtures: (1) Equal amounts of RNAs from 17 somatic tissues (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006393#sec012" target="_blank">Materials and Methods</a> for the tissue list) were mixed and 1μg of the resulting mixture was used for RT (17 somatic tissues). (2) Mixture “1” supplemented with testis RNA at a concentration equal to that of the individual somatic RNAs (17 somatic tissues + 1x testis). (3) Mixture “1” supplemented with testis RNA at a concentration equal to five times that of the individual somatic RNAs (17 somatic tissues + 5 x testis) (4) Mixture “3” with no RT (17 somatic tissues + 5xtestis noRT). <i>Mcmdc2</i>-specific PCR-products were amplified preferentially from templates that contained testis cDNA. (<b>b</b>) <i>Mcmdc2</i> targeting strategy. Schematics of the targeting construct, the wild-type (WT) and the modified <i>Mcmdc2</i> genomic locus. Black boxes represent exons (not to scale). Recombination at the homology arms (HA) of the targeting construct modifies intron 4 by introducing: 1) an additional exon (SA-IRES-LacZ) that contains a strong splice acceptor site (SA) and poly-adenylation site (left grey box), 2) a transcriptional unit that contains the strong housekeeping human ß-Actin promotor <i>(hBactP)</i> driving the neomycin (Neo) resistance gene as a selection marker. This modification of intron 4 also disrupts the <i>Mcmdc2</i> open reading frame after the 95th codon <i>(Mcmdc2</i>^{<i>insertion</i>} allele). Recombination catalyzed by FLPe at FRT sites removes the SA-IRES-LacZ exon and the hBactP-Neo gene, and restores the MCMDC2 ORF (<i>Mcmdc2</i>^{<i>restored</i>}). <i>Mcmdc2</i>^{<i>restored</i>} is a functional allele that can be disrupted by Cre-mediated recombination between loxP sites (<i>Mcmdc2</i>^{<i>deletion</i>}). Excision of exon 5–7 causes a frameshift after the 80th codon. Cre-mediated recombination between loxP sites of a <i>Mcmdc2</i>^{<i>insertion</i>} allele results in <i>Mcmdc2</i>^{<i>insertion-deletion</i>} allele. The positions of PCR-genotyping primers are indicated. Red bars mark the 3`and the internal Southern blot probes; the predicted length of restriction fragments is indicated. (<b>c</b>) Southern blot of DNA from wild-type (+/+) and targeted <i>Mcmdc2</i>^{<i>+/insertion</i>} (<i>+/i</i>) embryonic stem cell clones (C6 and F7) that were used to derive two independent mouse lines. DNA was digested with Eco31I and hybridized with an internal probe for LacZ (left panel), or DNA was digested with BclI and hybridized with a 3’ probe (right panel). The blots indicate a single integration of the targeting cassette in the <i>Mcmdc2</i> locus. (<b>d</b>) RT-PCR was used to detect <i>Mcmdc2</i> and "house-keeping" <i>Rps9 (S9)</i> transcripts in testes of wild-type and <i>Mcmdc2</i>^<i>-/-</i> (insertion-deletion) mice. Oligo-pairs specific to <i>Mcmdc2</i> exon 3 and 4, 5 and 6, 6 and 7, 8 and 9, or 10 and 11 were used.</p

MutSγ and MutLγ foci formation are defective in <i>Mcmdc2</i>^<i>-/-</i> meiocytes.

<p>(<b>a, b, e, f</b>) Immunostaining of SYCP3 together with MSH4 (<b>a, b</b>) or MLH1 (<b>e, f</b>) on nuclear surface spreads of pachytene <i>Mcmdc2</i>^<i>+/+</i> or zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i> meiocytes. (<b>a, b</b>) MSH4 foci are readily detected along synapsed axes of pachytene spermatocytes and oocytes (16dpc). MSH4 foci numbers are much lower in <i>Mcmdc2</i>^<i>-/-</i> meiocytes. (<b>e, f</b>) Typically, a single MLH1 focus is detected along each synapsed axis pair of <i>Mcmdc2</i>^<i>+/+</i> pachytene spermatocytes and oocytes (from ovaries of newborn mice). MLH1 foci are not present along the unsynapsed axes of <i>Mcmdc2</i>^<i>-/-</i> meiocytes. Scale bars; 10μm. (<b>c, d</b>) Numbers of MSH4 foci in <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i> spermatocytes and oocytes. (<b>c</b>) Spermatocytes were examined at leptotene (lepto), early zygotene (e zygo) in <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i>, late zygotene (l zygo) and early-mid pachytene (e-m pa) in <i>Mcmdc2</i>^<i>+/+</i> and zygotene-pachytene (zyg-pa) in <i>Mcmdc2</i>^<i>-/-</i> mice. MSH4 foci numbers are significantly lower in <i>Mcmdc2</i>^<i>-/-</i> than in <i>Mcmdc2</i>^<i>+/+</i> spermatocytes from early-zygotene stage onwards (Mann Whitney test). (<b>d</b>) Oocytes with fully formed axes (late zygotene and early pachytene) were examined from fetal ovaries at the 16dpc developmental time point. MSH4 foci numbers are significantly lower in <i>Mcmdc2</i>^<i>-/-</i> than in <i>Mcmdc2</i>^<i>+/+</i> oocytes (Mann Whitney test). (<b>c, d</b>) Median numbers of foci are marked, and n corresponds to the number of analyzed meiocytes in two pooled experiments.</p

RAD51 and DMC1 foci persist in <i>Mcmdc2</i>^<i>-/-</i> oocytes.

<p>(<b>a, c, e)</b> Immunostaining of SYCP3 along with RAD51 (<b>a</b>), DMC1 (<b>c</b>) or γH2AX (<b>e</b>) on nuclear surface spreads of pachytene <i>Mcmdc2</i>^<i>+/+</i>, or zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i> oocytes. Oocytes were collected from the ovaries of littermate fetuses at 18dpc, which is a time point when most wild-type oocytes are in the late pachytene stage. RAD51 and DMC1 foci are largely absent from synapsed chromosomes in <i>Mcmdc2</i>^<i>+/+</i> oocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of <i>Mcmdc2</i>^<i>-/-</i> oocytes. (<b>e</b>) γH2AX is largely absent from the synapsed chromosomes of the <i>Mcmdc2</i>^<i>+/+</i> oocyte. γH2AX associates with chromatin throughout the nucleus in the <i>Mcmdc2</i>^<i>-/-</i> oocyte. Scale bars; 10μm. (<b>b, d</b>) Numbers of RAD51 (<b>b</b>) or DMC1 (<b>d</b>) foci are shown in <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i> oocytes at 18dpc. Median numbers of foci are marked, and n corresponds to the number of analyzed oocytes in two pooled experiments. DMC1 and RAD51 foci numbers are significantly higher in <i>Mcmdc2</i>^<i>-/-</i> than in <i>Mcmdc2</i>^<i>+/+</i> oocytes (Mann Whitney test).</p

<i>Mcmdc2</i>^<i>-/-</i> mice are deficient in germ cells from late meiotic prophase onwards in both sexes.

<p>(<b>a, b</b>) Growth curves of five (<b>a</b>) or three (<b>b</b>) independent lines of <i>Mcmdc2</i>^<i>+/</i>+ (+/+) and <i>Mcmdc2</i>^<i>-/-</i> mouse embryonic fibroblasts. Cells were grown either without aphidicolin treatment (<b>a</b>) or with aphidicolin treatment for the first 24 hours (<b>b</b>), where 1μM aphidicolin was added at day 0. (<b>a, b</b>) Cell numbers were determined at the indicated time points in three technical replicates of each fibroblast line. Means and standard deviations of the medians of technical triplicates are shown. Growth curves of <i>Mcmdc2</i>^<i>+/</i>+ and <i>Mcmdc2</i>^<i>-/-</i> mouse embryonic fibroblasts are not significantly different (<b>a</b>: p = 0.8201, <b>b</b>: p = 0.9932, two-way ANOVA test). (<b>c</b>) Images of <i>Mcmdc2</i>^<i>+/</i>+ (+/+) and <i>Mcmdc2</i>^<i>-/-</i> (-/-) testes (upper panel) and ovaries (lower panel). Scale bars; 500μm. (<b>d</b>) Cryosections of testes from adult <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i> mice. DNA was detected by DAPI, histone H1T (marker of spermatocytes after mid-pachytene) and nuclear cleaved PARP1 (marker of apoptotic cells) were detected by immunostaining. Outlines of testis tubules are marked by dashed lines. The upper panels of <b>d</b> show stage V-VI and VII-VIII wild-type testis tubules, which contain several layers of germ cells at distinct spermatogenic stages: Sertoli cells (Se), spermatogonia B (SgB, stage V-VI), preleptotene (pl, stage VII-VIII), mid-pachytene (pa, stage V-VI), late-pachytene (pa, stage VII-VIII) spermatocytes, post-meiotic spermatids (sd) and spermatozoa (sp). Lower panels of <b>d</b> show that <i>Mcmdc2</i>^<i>-/-</i> meiocytes underwent apoptosis at a stage corresponding to wild-type mid-pachytene in stage IV tubules. Consequently, spermatocytes were not found in the inner layers of testis tubules beyond stage IV, and post-meiotic spermatids and spermatozoa were also missing from <i>Mcmdc2</i>^<i>-/-</i> testes. To illustrate this, stage IV, V-VI and VII-VIII tubules of <i>Mcmdc2</i>^<i>-/-</i> mice are shown. Apoptotic (ap) and non-apoptotic early-mid pachytene (pa) spermatocytes are shown in the stage IV tubule, which was identified by the presence of mitotic intermediate spermatogonia (m) and intermediate spermatogonia (Int). Stage V-VI and VII-VIII tubules contain somatic Sertoli cells (Se) and spermatogonia B (SgB) or preleptotene (pl) spermatocytes, respectively, but more advanced spermatogenic cells are missing. Due to elimination at mid-pachytene, histone H1T positive cells are missing from <i>Mcmdc2</i>^<i>-/-</i> testis tubules. (<b>e</b>) NOBOX (oocyte marker) was detected by immunofluorescence on cryosections of ovaries from 6-week-old mice. DNA was stained by DAPI. Oocytes in primordial (pd) and secondary (s) follicles are shown in the section of a wild-type ovary. In contrast, oocytes are not detected in the shown <i>Mcmdc2</i>^<i>-/-</i> ovary section. (<b>d, e</b>) Scale bars; 50μm.</p

RAD51 and DMC1 foci persist in <i>Mcmdc2</i>^<i>-/-</i> <i>s</i>permatocytes.

<p>(<b>a, b, e</b>) Immunostaining showing SYCP3 together with RAD51 (<b>a</b>), DMC1 (<b>b</b>) or γH2AX (<b>e</b>) on nuclear surface spreads of pachytene <i>Mcmdc2</i>^<i>+/+</i>, late zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i>, <i>Spo11</i>^<i>-/-</i>, and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. RAD51 and DMC1 foci are present at comparatively high density along the axes of unsynapsed sex chromosomes (<b>a, b</b>, asterisk), and are largely absent from synapsed autosomes of <i>Mcmdc2</i>^<i>+/+</i> spermatocytes. Both RAD51 and DMC1 foci are present in high numbers along the unpaired axes of <i>Mcmdc2</i>^<i>-/-</i> spermatocytes. Absence of RAD51 and DMC1 foci is shown in <i>Spo11</i>^<i>-/-</i> and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. (<b>e</b>) γH2AX preferentially accumulates on the partially synapsed sex chromosomes of the <i>Mcmdc2</i>^<i>+/+</i> spermatocyte. γH2AX associates with chromatin throughout the nucleus in the <i>Mcmdc2</i>^<i>-/-</i> spermatocytes. γH2AX is largely restricted to the sex chromatin in wild-type pachytene spermatocytes, and to pseudo-sex bodies in <i>Spo11</i>^<i>-/-</i> and <i>Spo11</i>^<i>-/-</i> <i>Mcmdc2</i>^<i>-/—</i>spermatocytes. Scale bars; 10μm. (<b>c, d</b>) Numbers of RAD51 (<b>c</b>) or DMC1 (<b>d</b>) foci are shown in leptotene (lepto), early zygotene (e zygo) in <i>Mcmdc2</i>^<i>+/+</i> and <i>Mcmdc2</i>^<i>-/-</i>, late zygotene (l zygo) and early-mid pachytene (e-m pa) in <i>Mcmdc2</i>^<i>+/+</i> and zygotene-pachytene (zyg-pa) in <i>Mcmdc2</i>^<i>-/-</i> spermatocytes. Median numbers of foci are marked, and n corresponds to the number of analyzed spermatocytes in three pooled experiments. DMC1 and RAD51 foci numbers are significantly higher in zygotene-pachytene <i>Mcmdc2</i>^<i>-/-</i> spermatocytes than in late-zygotene or early-mid-pachytene <i>Mcmdc2</i>^<i>+/+</i> spermatocytes (Mann Whitney test).</p

Proline-rich protein PRR19 functions with cyclin-like CNTD1 to promote meiotic crossing over in mouse.

Orderly chromosome segregation is enabled by crossovers between homologous chromosomes in the first meiotic division. Crossovers arise from recombination-mediated repair of programmed DNA double-strand breaks (DSBs). Multiple DSBs initiate recombination, and most are repaired without crossover formation, although one or more generate crossovers on each chromosome. Although the underlying mechanisms are ill-defined, the differentiation and maturation of crossover-specific recombination intermediates requires the cyclin-like CNTD1. Here, we identify PRR19 as a partner of CNTD1. We find that, like CNTD1, PRR19 is required for timely DSB repair and the formation of crossover-specific recombination complexes. PRR19 and CNTD1 co-localise at crossover sites, physically interact, and are interdependent for accumulation, indicating a PRR19-CNTD1 partnership in crossing over. Further, we show that CNTD1 interacts with a cyclin-dependent kinase, CDK2, which also accumulates in crossover-specific recombination complexes. Thus, the PRR19-CNTD1 complex may enable crossover differentiation by regulating CDK2

Proline-rich protein PRR19 functions with cyclin-like CNTD1 to promote meiotic crossing over in mouse.

Orderly chromosome segregation is enabled by crossovers between homologous chromosomes in the first meiotic division. Crossovers arise from recombination-mediated repair of programmed DNA double-strand breaks (DSBs). Multiple DSBs initiate recombination, and most are repaired without crossover formation, although one or more generate crossovers on each chromosome. Although the underlying mechanisms are ill-defined, the differentiation and maturation of crossover-specific recombination intermediates requires the cyclin-like CNTD1. Here, we identify PRR19 as a partner of CNTD1. We find that, like CNTD1, PRR19 is required for timely DSB repair and the formation of crossover-specific recombination complexes. PRR19 and CNTD1 co-localise at crossover sites, physically interact, and are interdependent for accumulation, indicating a PRR19-CNTD1 partnership in crossing over. Further, we show that CNTD1 interacts with a cyclin-dependent kinase, CDK2, which also accumulates in crossover-specific recombination complexes. Thus, the PRR19-CNTD1 complex may enable crossover differentiation by regulating CDK2