A novel function for CDK2 activity at meiotic crossover sites

Abstract

Genetic diversity in offspring is induced by meiotic recombination, which is initiated between homologs at >200 sites originating from meiotic double-strand breaks (DSBs). Of this initial pool, only 1-2 DSBs per homolog pair will be designated to form meiotic crossovers (COs), where reciprocal genetic exchange occurs between parental chromosomes. Cyclin-dependent kinase 2 (CDK2) is known to localize to so-called "late recombination nodules" (LRNs) marking incipient CO sites. However, the role of CDK2 kinase activity in the process of CO formation remains uncertain. Here, we describe the phenotype of 2 Cdk2 point mutants with elevated or decreased activity, respectively. Elevated CDK2 activity was associated with increased numbers of LRN-associated proteins, including CDK2 itself and the MutL homolog 1 (MLH1) component of the MutLγ complex, but did not lead to increased numbers of COs. In contrast, reduced CDK2 activity leads to the complete absence of CO formation during meiotic prophase I. Our data suggest an important role for CDK2 in regulating MLH1 focus numbers and that the activity of this kinase is a key regulatory factor in the formation of meiotic COs.

Fig 1. SC formation, meiotic staging analysis, and synaptic defects analysis in Cdk2^T160A spermatocytes.

Chromosome spread preparations from adult (postnatal day 40) testes coimmunostained for SYCP1 (green) and SYCP3 (red) are shown for WT (A–G) or Cdk2^T160A spermatocytes (H–M) for selected stages of meiotic prophase I. For staging purposes, histone H1t (white) is shown to the right of each image. H1t positivity is indicative of mid-pachytene stage onwards. During early-pachytene, both WT (C) and Cdk2^T160A (J) spermatocytes show SYCP3 colocalization with SYCP1, indicating the normal formation of the SC scaffold structure. WT spermatocytes progress normally into mid-pachytene (D), late-pachytene (E), diplotene (F), and diakinesis stages (G). Mid-pachytene–stage Cdk2^T160A (K) spermatocytes show premature desynapsis. Yellow arrows in insets I and II highlight areas of desynapsis between homologs. Late-pachytene stage is not observed in the Cdk2^T160A mutant. Instead, meiosis progresses to a diplotene-like stage with desynaspsis observed between both autosomal homologs and the X–Y bivalent (L and M and insets III and IV). All main panels are representative of at least 20 images taken for specified stages. Similar staining patterns were confirmed in at least 3 biological replicates. In all main panels, scale bars are representative of 5 μm; in all inset pictures, scale bars are representative of 1.25 μm. Meiotic staging analysis (N) shows the percentages of spermatocytes observed in each stage of meiotic prophase. Asynapsis was quantified specifically for mid-pachytene stages by counting the percentages nuclei containing incompletely synapsed autosomal bivalents, classified as having incomplete colocalization of SYCP1 and SYCP3 along the entire chromosomal length (O). X–Y bivalent synapsis was quantified specifically for mid-pachytene stages by counting the percentage of nuclei containing fully synapsed X–Y chromosomes, classified as being physically associated with no clear separation (P). End-to-end associations were quantified specifically for mid-pachytene stages by counting the percentages nuclei containing bivalents that were joined end-to-end; the joining of 2 ends between distinct bivalents was quantified as a single event (Q). For N–Q, WT or Cdk2^T160A data are shown using orange and blue bars, respectively. Each data point is a mean percentage ± SD determined from 3 biological replicates. For each biological replicate, percentages were determined from at least 200 spermatocyte images. All data were assumed to be non-normally distributed. Statistical significance between genotypes was determined by unpaired t test. Significance and P-values are reported directly over each comparison. The underlying data for (N, O, P, Q) can be found in S1 Data. CDK2, cyclin-dependent kinase 2; SC, synaptonemal complex; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type.

Fig 2. Analysis of telomeric fusion defects in Cdk2^T160A spermatocytes.

Chromosome spread preparations from adult (postnatal day 40) testes immunostained with ACA (green) and the telomeric shelterin protein RAP1 (red) in conjunction with SYCP3 (blue) are shown for WT (A–B), Cdk2^T160A (C–D), and Cdk2^−/− (E) for selected stages of meiotic prophase I. For each of the 3 genotypes, RAP1 and ACA can be detected at the telomeres and centromeres as specific foci, respectively. The centromeric ACA signal is always detected proximal to a telomeric RAP1 signal because of the proximity of the centrosome to telomeric ends in mouse chromosomes. In pachytene-like Cdk2^−/− spermatocytes (E) and mid-pachytene–stage Cdk2^T160A spermatocytes (D), telomere fusions can be observed. Quantification of fusion events (F) specifically for mid-pachytene–stage nuclei was performed for WT (orange bars, N = 3 total events counted) and Cdk2^T160A spermatocytes (blue bars, N = 30 total events counted). Here, data are presented as the total number of NC–NC, NC–C, and C–C fusions, respectively. For each genotype, fusion events were counted from 90 images pooled from 3 biological replicates in which at least 20 images were taken from each replicate; no statistical test is applied because of the low numbers of countable events. Examples of C–C, NC–C, and NC–NC fusions are shown in panels D–E by pink arrows. Additional staining is shown for the pericentromeric chromatin marker, H3K9me3 (green) in conjunction with SYCP3 (blue) for WT (G–H), Cdk2^T160A (I–J), and Cdk2^−/− (K), for selected stages of meiotic prophase I. Histone H1t (red) positivity also shown and is indicative of mid-pachytene stage onwards. In all WT and Cdk2^T160A images, pericentric chromatin can be visualized as a flare-like staining emanating from the end of bivalents. In Cdk2^−/−, aggregates of H3K9me3-positive pericentromeric chromatin show the extensive interactions between centromeric ends. All images within Fig 2 are representative of at least 20 images taken for specified stages. Similar staining patterns were confirmed in at least 3 biological replicates. In all main panels, scale bars are representative of 5 μm; in all inset pictures, scale bars are representative of 1.25 μm. The underlying data for (F) can be found in S1 Data. ACA, autocentromere antibody; CDK2, cyclin-dependent kinase 2; C–C, centromeric end to centromeric end; H3K9me3, trimethylated lysine 9 of histone H3; NC–C, noncentromeric end to centromeric end; NC–NC, noncentromeric end to noncentromeric end; RAP1, TERF2 interacting protein; SYCP, synaptonemal complex protein; WT, wild-type.

Fig 3. CDK2/Speedy A localization in Cdk2^T160A spermatocytes.

Chromosome spread preparations from adult (postnatal day 40) testes immunostained with CDK2 (green) and Speedy A (red) in conjunction with SYCP3 (blue) are shown for WT (A–D) and Cdk2^T160A (G–J) spermatocytes for selected stages of meiotic prophase I. CDK2 and Speedy A can be detected as colocalized foci at all telomeric ends for all meiotic stages in WT (A–D and insets E–F) and Cdk2^T160A spermatocytes (G–J and insets K–L). Interstitial CDK2 foci—absent of Speedy A positivity—marking LRNs are transiently observed in mid-pachytene–stage WT (D) and insets (E–F), but not Cdk2^T160A spermatocytes (J and insets K, L). X–Y bivalents (blue XY labels) were also found to be positive for both CDK2 and Speedy A from early-pachytene onwards in both genotypes. All images are representative of at least 20 images taken for specified stages. Similar staining patterns were confirmed in at least 3 biological replicates. In all main panels, scale bars are representative of 5 μm; in all inset pictures, scale bars are representative of 1.25 μm. Interstitial CDK2 foci (M) were quantified specifically for mid-pachytene stages by counting the average numbers of nontelomeric CDK2 foci counted per nucleus. Data are presented as individual foci counts for WT (orange bars, N = 72) and Cdk2^T160A (blue bars, N = 50); error bars are indicative of the mean and SD. For mid-pachytene, Cdk2^T160A spermatocytes (J) with decreased CDK2/Speedy A signal in a subset of nuclei was observed (as marked by red arrows in insets K, L). The average telomeric CDK2 focus intensity/nuclei were quantified specifically for mid-pachytene stages (N). Data are presented as a mean intensity in AU ± SD determined from 3 biological replicates (N = 48 overall nuclei counted for WT (orange bars) and N = 48 overall nuclei counted (blue bars) for Cdk2^T160A. Telomeric signals were only counted from autosomes and were excluded if involved in telomeric fusion events. All intensity values calculated for a single nucleus were normalized to the background intensity of that nuclei. Percentages of mid-pachytene–stage nuclei with at least one telomere showing a decrease in telomeric CDK2 intensity of ≥50% (as compared with the average telomeric CDK2 intensity for that cell) are quantified in panel O. Individual data used to make panel O are shown in S3G Fig. All data were assumed to be non-normally distributed. Statistical significance between genotypes was determined by unpaired t test. Significance and P-values are reported directly over each comparison. The underlying data for (M, N, O) can be found in S1 Data. AU, arbitrary unit; CDK2, cyclin-dependent kinase 2; LRN, late recombination nodule; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type.

Fig 4. γH2AX analysis of meiotic DSB repair in WT, Cdk2^T160, and Cdk2^−/− spermatocytes.

Chromosome spread preparations from adult (postnatal day 40) testes immunostained with the DNA damage marker γH2AX (green) and SYCP3 (red) are shown for WT (A–D), Cdk2^T160A (E–H), and Cdk2^−/− (I–K) spermatocytes, for selected stages of meiotic prophase I. γH2AX can be detected as a panchromatin stain in leptotene through zygotene stage in all genotypes and all stages for the Cdk2^−/− mutant. At early-pachytene, γH2AX localizes to the chromatin surrounding the X–Y bivalent (sex body)—outlined with dashed lines—in WT (C) and Cdk2^T160A (G), indicating normal MSCI. This pattern is retained into mid-pachytene (D and H) but does not occur in the Cdk2^−/− mutant (K). Percentages of nuclei retaining γH2AX signal specifically on autosomes, are quantified for all genotypes from leptotene to early-pachytene (L). Data are presented as a mean percentage of cells ± SD determined from 3 biological replicates (N = 60 for the overall nuclei counted for WT leptotene, zygotene, and early-pachytene stages [orange bars]; N = 60 for the overall nuclei counted for Cdk2^T160A leptotene, zygotene, and early-pachytene stages [blue bars]; and N = 60 for the overall nuclei counted for Cdk2^−/− leptotene, zygotene, and early-pachytene stages [gray bars]). Additional staining is shown for P40 chromosome spread preparations immunostained for RAD51 (green) and SYCP3 (red). WT (M–P), Cdk2^T160A spermatocytes (Q–T), and Cdk2^−/− (U–W) images are shown for selected stages of meiotic prophase I. During leptotene and zygotene stages in WT (M–N), Cdk2^T160A (Q–R), and Cdk2^−/− (U–V), RAD51 focus formation precedes the synapsis of homologs visualized using SYCP3. From the early-pachytene stage, fully paired homologs from WT and Cdk2^T160A spermatocytes lose RAD51 foci (O–P and S–T, respectively). In pachytene-like Cdk2^−/− spermatocytes, only a pachytene-like arrest state is achieved (W). Here, RAD51 foci are observed to remain bound to stretches chromosomal axes despite extensive nonhomologous synapsis. These are sites of presumed failed strand invasion events. All images are representative of at least 20 images taken for specified stages. Similar staining patterns were confirmed in at least 3 biological replicates. In all main panels, scale bars are representative of 5 μm. RAD51 foci were quantified specifically for leptotene, zygotene, and early-pachytene stages (X) by counting the average numbers of RAD51 foci per nucleus. Data are presented as individual foci counts for WT (orange bars, N = 90 for leptotene, zygotene, and early-pachytene stages), Cdk2^T160A (blue bars, N = 90 for leptotene, zygotene, and early-pachytene stages) and Cdk2^−/− (gray bars, N = 90 for leptotene, zygotene, and early-pachytene stages). For panels L and X, error bars are indicative of the mean and SD. All data were assumed to be non-normally distributed. Statistical significance between genotypes was determined by unpaired t test. Significance and P-values are reported directly over each comparison. The underlying data for (L, X) can be found in S1 Data. CDK2, cyclin-dependent kinase 2; DSB, double-strand break; MSCI, meiotic sex chromosome inactivation; RAD51, RAD51 recombinase; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type; γH2AX, phosphoserine 139 H2AX.

Fig 5. Comparison of MSH4 and RNF212 dynamics in WT and Cdk2^T160A spermatocytes.

P40 chromosome spread preparations immunostained for MSH4 (green) and SYCP3 (red) are shown for WT (A–B) and Cdk2^T160A spermatocytes (C–D) for selected stages of meiotic prophase I. Inset images are shown in their corresponding inverted color for better visualization of MSH4 foci. In early-pachytene WT and Cdk2^T160A spermatocytes, MSH4 foci can be observed along the length of paired axes (A and C, insets I–II and V–VI). MSH4 foci decrease in number as WT spermatocytes progress to mid-pachytene (B, insets III–IV). MSH4 focus numbers remain high in mid-pachytene Cdk2^T160A spermatocytes (D, insets VII–VIII). An identical analysis is shown for P40 chromosome spread preparations immunostained for RNF212 (magenta) and SYCP3 (white). Like MSH4, RNF212 foci in early-pachytene WT and Cdk2^T160A spermatocytes can be observed along the length of paired axes (E and G, insets IX and XIII). RNF212 foci decrease in number as WT spermatocytes progress to mid-pachytene. At this stage, RNF212 foci mark LRNs (F, insets X–XII). RNF212 focus numbers remain high in mid-pachytene Cdk2^T160A spermatocytes (H, inset XIV). All images are representative of at least 20 images taken for equivalent stages. Similar staining patterns were confirmed in at least 3 biological replicates. In all main panels, scale bars are representative of 5 μm; in all inset pictures, scale bars are representative of 1.25 μm. Quantification of MSH4 foci and RNF212 foci counts are shown in panels I and J, respectively. Data are presented as individual foci counts, and WT and Cdk2^T160A data are represented using orange or blue bars, respectively. Error bars are indicative of the mean and SD. All data were assumed to be non-normally distributed. Statistical significance between genotypes was determined by unpaired t test. Significance and P-values are reported directly over each comparison. For WT data in I and J, N = 90 for both early-pachytene and mid-pachytene stages. For Cdk2^T160A data in I, N = 90 for early-pachytene and N = 42 for mid-pachytene stages. For Cdk2^T160A data in J, N = 90 for early-pachytene and N = 45 for mid-pachytene stages. The underlying data for (I, J) can be found in S1 Data. CDK2, cyclin-dependent kinase 2; LRN, late recombination nodule; MSH4/5, MutS protein homolog 4/5; RNF212, ring finger protein 212; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type.

Fig 6. Analysis of MLH1 dynamics in Cdk2^T160A spermatocytes.

Chromosome spread preparations from adult (postnatal day 40) testes immunostained for MLH1 (green) and SYCP3 (red) are shown for WT (A–D) and Cdk2^T160A (E–F) for selected stages of meiotic prophase I. Positivity for MLH1 is seen in mid-pachytene through diplotene in WT (C, D) but could not be detected in any Cdk2^T160A stage. Images are representative of at least 20 images taken for equivalent stages. Identical staining patterns were confirmed in at least 3 biological replicates. Scale bars are representative of 5 μm. MLH1 foci were quantified specifically for mid-pachytene, late-pachytene, and diplotene/diplotene-like stages (G) by counting the average numbers of MLH1 foci per nucleus. Data are presented as individual foci counts for WT (orange bars, N = 90 for mid-pachytene, late-pachytene, and diplotene stages) and Cdk2^T160A (blue bars, N = 72 for mid-pachytene and N = 60 for diplotene-like stages; late-pachytene stages were not detected in this genotype). Error bars are indicative of the mean and SD. All data were assumed to be non-normally distributed. Statistical significance between genotypes was determined by unpaired t test. Significance and P-values are reported directly over each comparison. The underlying data for (G) can be found in S1 Data. CDK2, cyclin-dependent kinase 2; MLH1, MutL homolog 1; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type.

Fig 7. Analysis of MLH1 dynamics in Cdk2^Y15S and Cdk2^Y15S/T160A spermatocytes.

Chromosome spread preparations from pubertal (postnatal day 25) testes immunostained for CDK2 or MLH1 (green) and SYCP3 (red) are shown for WT (A,D), Cdk2^Y15S (B,E), and Cdk2^Y15S/T160A (C,F) pachytene spermatocytes. Interstitial CDK2 and MLH1 foci can be detected in Cdk2^Y15S, but not Cdk2^Y15S/T160A, spermatocytes, which do not reach the equivalent meiotic stage. Images are representative of at least 20 nuclei imaged at equivalent stages. Identical staining patterns were confirmed in at least 3 biological replicates. Scale bars = 5 μm. Quantification of MLH1 foci, CDK2 foci, and frequency of homologs with 2 or 3 foci in indicated genotypes are shown in panels G, H, and I, respectively. Gamma distribution analysis of MLH1 foci along bivalents (J). Giemsa staining of diakinesis preparations from WT and Cdk2^Y15S/Y15S gonads showing comparable chiasmata with 20 bivalent chromosomes in either genotype (K). Chiasmata numbers for each cell as well as average counts (+SD) for WT and Y15S are shown in (L). Data from age-matched WT animals are shown for comparison. Individual points are pooled from at least 3 biological replicates; error bars are representative of SD. Interfocus distances between MLH1 foci on (chromosomes with >1 MLH1 focus) are quantified for each genotype in panel J for WT and Cdk2^Y15S spermatocytes. The underlying data for (G, H, I, J, L) can be found in S1 Data. CDK2, cyclin-dependent kinase 2; MLH1, MutL homolog 1; SD, standard deviation; SYCP, synaptonemal complex protein; WT, wild-type; YS, CDK2^Y15S genotype; YS-TA, CDK2^Y15S/T160A genotype.