MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination

Abstract

Meiotic recombination enables the reciprocal exchange of genetic material between parental homologous chromosomes, and ensures faithful chromosome segregation during meiosis in sexually reproducing organisms. This process relies on the complex interaction of DNA repair factors and many steps remain poorly understood in mammals. Here we report the identification of MEIOB, a meiosis-specific protein, in a proteomics screen for novel meiotic chromatin-associated proteins in mice. MEIOB contains an OB domain with homology to one of the RPA1 OB folds. MEIOB binds to single-stranded DNA and exhibits 3'-5' exonuclease activity. MEIOB forms a complex with RPA and with SPATA22, and these three proteins co-localize in foci that are associated with meiotic chromosomes. Strikingly, chromatin localization and stability of MEIOB depends on SPATA22 and vice versa. Meiob-null mouse mutants exhibit a failure in meiosis and sterility in both sexes. Our results suggest that MEIOB is required for meiotic recombination and chromosomal synapsis.

Figure 1. Proteomic identification of meiotic chromatin-associated proteins from mouse testes

(a) Biochemical purification of chromatin from P18 testes. (b) Purity assessment of isolated meiotic chromatin by western blotting. SYCP2 is known to associate with meiotic chromatin. TEX19 is a cytoplasmic protein control. Histone H3 is a ubiquitous chromatin-associated protein. (c) Identification of chromatin-associated proteins by tandem mass spectrometry (MS/MS). The gel was cut into 10 slices, separating bands with abundant proteins from those with lower protein content. (d) Systematic identification of meiotic chromatin-associated proteins (MCAPs) by subtractive analysis of three proteomic datasets. Proteins represented with at least 3 unique peptides in P18 testes but absent from both P6 and XX^Y* testes were considered candidate meiotic chromatin-associated proteins. (e) Association of MEIOB with chromatin in P18 testes. Western blot analysis was performed using 20 µg protein from cytoplasmic extract, nuclear extract, and chromatin. TEX19 and histone H3 serve as cytoplasmic and chromatin controls, respectively. (f) MEIOB protein expression in adult mouse tissues. ACTB serves as a loading control. Note that heart and skeletal muscle contain little ACTB. Protein molecular mass standards are shown in kilodaltons.

Figure 2. MEIOB colocalizes with RPA in foci on meiotic chromosomes

(a–e) Distribution pattern of MEIOB foci on chromatin of spermatocytes from early leptotene through late pachytene stages. (f–j) the same cells in panels a–e are shown with MEIOB labeling only. (k) Number of MEIOB foci in spermatocytes at successive stages of meiotic prophase I. Stages were determined by SYCP2 staining. l-Le, late leptotene; Zy, zygotene; e-Pa, early pachytene; n indicates the number of cells counted. The line indicates the average. (l–p) MEIOB colocalizes with RPA1 on the meiotic chromosomes of spermatocytes. The same spermatocyte is shown with MEIOB labeling only (l), RPA1 labeling only (m), and in a merged image (n). Enlarged views of the marked chromosome in panel n are shown in panels o and p (without or with offset channels). (q–u) MEIOB colocalizes with RPA2 on the meiotic chromosomes of spermatocytes. The same spermatocyte is shown with MEIOB labeling only (q), RPA2 labeling only (r), and in a merged image (s). Enlarged views of the marked chromosome in panel s are shown in panels t and u (without or with offset channels). (v) Electron microscopy with immunogold (6 nm particles, arrowhead) -labeled MEIOB reveals signal between the lateral elements (LE) in spermatocytes. Scale bars: a–s, 10 µm; v, 0.2 µm.

Figure 3. MEIOB binds to ssDNA but not dsDNA

(a) MEIOB contains an OB fold. All MEIOB constructs were expressed as GST fusion proteins. Only construct A (MEIOB central region) (aa 136–307) was used in experiments illustrated in panels b through e. The summary of ssDNA-binding activity is based on the results from panel f: +++, strong; ++, intermediate; +, weak; -, no binding. The Kd values are shown in panel f. (b) Truncated MEIOB binds to ssDNA. The ssDNA (d36GT) was 5’-labeled with ³²P (indicated by asterisk). (c) Binding curve for truncated MEIOB-ssDNA (d36GT) from triplicate EMSA experiments (mean ± sd). (d) Binding of truncated MEIOB to ssDNA oligonucleotides of various lengths. All oligonucleotides [d(GT)n or d(GT)nG] were 5’ ³²P-labeled. (e) Truncated MEIOB binds to 5’ and 3’ ssDNA flap (30-nt) but not dsDNA. One strand of the dsDNA and the 5’ flap DNA were 5’ ³²P-labeled. The long strand in the 3’ flap DNA was 3’ ³²P-labeled. (f) EMSA analysis of various MEIOB constructs depicted in panel A. GST was used as negative controls.

Figure 4. MEIOB exhibits ssDNA-specific 3’ exonuclease activity

Construct A (MEIOB central region, Fig. 3a) (aa 136–307) was used in experiments illustrated in panels a through h. (a) Truncated MEIOB exhibits nuclease activity in addition to its ssDNA-binding activity. (b) Size determination of 5’-labeled products generated by MEIOB. An 8-nt product was predominant. All products (panels b through i) were resolved on denaturing acrylamide gels. (c) Truncated MEIOB processes 5’ labeled oligonucleotides of various lengths. ExoI digests ssDNA completely. (d) Truncated MEIOB lacks nuclease activity on dsDNA and 5’ flap (30 nt) DNA substrates. (e) Truncated MEIOB exhibits 3’ exonuclease activity on 3’ labeled oligonucleotides of various lengths. EXO1 serves as a positive control. (f) Truncated MEIOB efficiently processes oligonucleotides with a 3’ flap (30 nt). (g) Extent of 3’ flap digestion by truncated MEIOB. (h) Time course and gradient analyses of truncated MEIOB nuclease activity. Note that MEIOB at higher concentrations (2 to 8 µM) produced shorter end products (<8 nt). (i) Nuclease activities of various MEIOB mutants shown in Fig. 3A. Recombinant GST-RPA2 and -RPA3 were used as negative controls. Percent (%) cleavage represents the average from three independent experiments.

Figure 5. MEIOB is required for meiosis and fertility in mice of both sexes

(a) Targeted inactivation of the Meiob gene. The mouse Meiob gene maps to Chromosome 17 and has 14 exons. Deletion of exons 6–10 (aa 111–293) is expected to cause a frame shift in the resulting transcript. (b) Absence of MEIOB protein in P18 Meiob^−/− testis. (c) Significant size reduction in 8-wk-old Meiob^−/− testis. (d, e) Histological analysis of 8-wk-old wild type and Meiob^−/− testes. Abbreviations: Zyg, zygotene spermatocytes; Pa, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. (f, g) TUNEL analysis of wild type and Meiob^−/− seminiferous tubules. Note the large number of apoptotic cells (green) in Meiob^−/− tubules. Scale bar (e, g), 25 µm. (h) Histological analysis of ovaries from adult wild type and Meiob^−/− mice. Scale bar, 50 µm. (i–k) Progressive loss of oocytes in Meiob^−/− ovaries. Frozen sections prepared from postnatal day 0, 1, and 2 wild type and Meiob^−/− ovaries were immunolabeled with anti-YBX2 antibodies. (l) TUNEL analysis of P1 ovaries. Nuclear DNA (i–l) was counterstained with DAPI (blue). Scale bars (i–l), 50 µm.

Figure 6. MEIOB is essential for meiotic recombination

Immunolabeling for proteins of the synaptonemal complex (SYCP2, lateral elements) and recombination nodules was performed on spread nuclei of spermatocytes from wild type and Meiob^−/− testes at postnatal day 16. The meiotic stages of spermatocytes were determined based on the morphology of the synaptonemal complexes. Spermatocytes were categorized into the following groups: leptotene (Le), early-mid zygotene (e-Zy), late zygotene (l-Zy), and pachytene (Pa). Pachytene-like (Pa-like) spermatocytes contained prominent but short lateral elements (See supplementary Fig. S8f). Pa-like spermatocytes were present in Meiob^−/− testes but absent in wild type testes. Dot plots for each DNA repair protein show the number of foci per cell; solid lines designate the average for each spermatocyte category. n, number of cells counted. Representative images of spermatocytes at early-mid zygotene stages are shown in separate channels and as merged images. (a) RAD51 foci. (b) DMC1 foci. (c) RPA foci. (d) TEX11 foci. Scale bars, 10 µm.

Figure 7. MEIOB forms complexes with RPA2 and SPATA22

(a) Identification of MEIOB-associated proteins from P18 testes by immunoprecipitation and mass spectrometry. The gel was stained with SYPRO Ruby. (b) Co-immunoprecipitation of MEIOB with RPA2 and SPATA22 from testicular protein extracts. The asterisk indicates a non-specific band. (c) Inter-dependent localization of MEIOB and SPATA22 on meiotic chromosomes from spermatocytes. Spata22^−/− refers to Spata22^{repro42/repro42} mice. Scale bar, 10 µm. (d) Western blot analysis of MEIOB and SPATA22 proteins in testes from wild type, Meiob^−/−, and Spata22^−/− mutant mice.

Figure 8. Proposed model for MEIOB function during meiotic recombination

Key steps of meiotic recombination are illustrated. We propose that the RPA-MEIOB-SPATA22 complex coats both the D-loop and the ssDNA of the second end. In this model, the interaction between RPA and MEIOB-SPATA22 mediates second end capture. Most DSBs are repaired through the SDSA (synthesis dependent strand annealing) pathway, in which the D-loop collapses back to its sister chromatid. After second end capture, intermediates continue to form double Holliday junctions, which are resolved into either crossovers or non-crossovers. We propose that MEIOB removes the 3’ flaps resulting from the first end DNA synthesis in both dHJ and SDSA pathways.