Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Abstract

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

Figure 1.

Expression pattern of MLH3 and MLH1 in spermatocytes from wild-type mice and those with targeted mutations of the MutL homologues. (A) MLH1 (top; green, FITC) localizes to spermatocyte SCs (SYCP3: red, TRITC) from mid to late pachynema but is not present in early diplonema. MLH3 localizes to SCs earlier than MLH1, during early pachynema, and persists until late pachynema. Centromeres are labeled with CREST (blue, CY5). (B) Quantitation of MLH1 (black bars) and MLH3 (striped bars) on wild-type spermatocyte SCs confirms the earlier localization of MLH3. (C) Immunogold-EM localization of MLH3 (6 nm gold grains) and MLH1 (12 nm gold grains) on Pms2 ^{− / −} spermatocytes demonstrates colocalization to electron-dense MNs (inset) during mid-pachynema. Centromeres (cen) are labeled with CREST (18 nm gold grains). Bar, 200 nm. (D) Quantitation of MLH1 (black bars) and MLH3 (striped bars) localization indicates that MN frequency, as measured by MLH1/3 labeling, is unaffected, or slightly elevated, in Pms2 ^{− / −} spermatocytes. (E) Quantitation of MLH1 and MLH3 foci in spermatocytes from mice with targeted deletions of Mlh1, Mlh3, and Pms2 indicates that even though MLH3 can bind to SCs in the absence of MLH1, MLH1 is incapable of localizing in the absence of MLH3. Neither MLH1 nor MLH3 localization is reduced in the absence of PMS2. (F) Immunogold-EM showing MLH3 (12 nm gold grains) localization to MNs (inset) in the absence of MLH1. Centromeres (cen) are localized with CREST (18 nm gold grains). Bar, 500 nm. Error bars indicate SD.

Figure 2.

MLH3 immunolocalizes to a proportion of wild-type and Mlh1 ^−/− spermatocyte centromeres, and both MLH3 and MLH1 localize to the centromeres of Pms2 ^−/− spermatocytes but not Pms2 ^−/− oocytes. (A–D) Immunogold-EM localization of MLH1 (12 nm gold grains, red arrows), MLH3 (6 nm gold grains, green arrows), and CREST (18 nm gold grains, blue arrows) on meiotic chromosomes of Pms2 ^{− / −} (A), wild-type (B), Mlh1 ^{− / −} (C), and Mlh3 ^{− / −} (D) spermatocytes. Bars: (A, B, and D) 250 nm; (C) C 500 nm. All SCs are counterstained with phosphotungstic acid. (E) Quantitation of MMR protein localization to centromeres from Mlh1 ^{+ / +} , Mlh1 ^{− / −} , Mlh3 ^{+ / +}, Mlh3 ^{− / −}, Pms2 ^{+ / +}, and Pms2 ^{− / −} spermatocytes. Counts were obtained from immunogold-EM micrographs exemplified in A–D and represent those centromeres showing more than four gold grains for each antibody staining. At least 100 centromeres were counted for each genotype from three mice. Percentages represent pooled counts from all these mice. MLH3 (striped bars) immunolocalizes to 15–25% of wild-type and Mlh1 ^{− / −}, but not Mlh3 ^{− / −}, spermatocyte centromeres. MLH1 (black bars) does not localize in any appreciable amount to centromeres in wild-type, Mlh1 ^{− / −} or Mlh3 ^{− / −} spermatocytes. CREST (white bars) localizes to 100% of the centromeres. Pms2 ^{− / −} spermatocyte centromeres show a statistically significant increase in localization of both MLH1 and MLH3 in comparison with the Pms2 ^{+ / +} and all other mouse strains observed. (F) Quantitation of distal (noncentromeric) telomere localization (more than four gold grains) indicates that the localization of MutL homologues is specific to the centromere. (G) EM micrograph of MLH1 (6 nm gold grains) and MLH3 (12 nm gold grains) on SCs from Pms2 ^{− / −} oocytes indicates that, whereas MLH3 and MLH1 localize normally to MNs (bottom inset), these MutL homologues fail to localize to the centromere (cen), labeled with CREST (18 nm gold grains; top inset). Bar, 200 nm.

Figure 3.

Localization of MLH1 and MLH3 to Y chromosome centromere region. Immunogold-EM localization of MLH1 (6 nm gold grains, green arrows), MLH3 (12 nm gold grains, red arrows), and CREST (18 nm gold grains, blue arrows) on meiotic chromosome cores of Pms2 ^{− / −} sex chromosomes. The p-arm of the Y chromosome is cytologically distinct from the q-arm, which contains the PAR that synapses with the PAR of the X chromosome. Due to the length of the p-arm, the Y chromosome centromere (top inset) can be differentiated from the terminally located telomere and both MLH1 and MLH3 localize to both the X (bottom inset) and the Y chromosome centromere. SCs are counterstained with phosphotungstic acid. Bar, 500 nm.

Figure 4.

Chromatin immunoprecipitation of centromere-associated repeat sequences by antibodies against MMR proteins. (A) Representative slot blot analysis of major and minor satellite repeat sequences (CEN) performed on crude germ cell lysates immunoprecipitated with antibodies MLH3, CREST, or rabbit IgG (Mock control), compared with input DNA. The same slot blot membrane was stripped and reprobed with a B2 SINE probe as a loading control. (B) Quantitation of three centromere ChIP experiments, expressed as a percentage of input DNA + SD. Statistical analysis was performed using unpaired t-tests (***P < 0.0001). (C) ChIP assay followed by PCR using primers against minor and major satellite sequences performed on purified pachytene cells from Pms2 ^−/− testes. Lanes (from left to right): lane 1, molecular weight marker; lanes 2–5, minor satellite PCR; lanes 6–9, major satellite PCR. M, 100 bp marker; Input, input DNA. 10-fold dilutions of ChIP pull-down substrate DNA was used for minor satellite PCR, DNA for major satellite PCR was undiluted.

Figure 5.

Localization of MMR complexes on the mouse Y chromosome during prophase I. (A) Immunogold-EM micrograph of an XY bivalent from a Pms2 ^{− / −} spermatocyte showing the PAR, the entire Y chromosome and part of the X chromosome. MLH3 localization is marked by the presence of 12 nm gold beads, showing a large dense focus on the Y chromosome. The centromere (cen) is marked with CREST labeled with 18 nm gold beads, and the SC is counterstained with phosphotungstic acid. (B) Representative slot blot analysis of Y chromosome repeat sequences immunoprecipitated with antibodies against MutSβ (combined MSH2–MSH3 antibodies; Oncogene Research Products), MLH3, and rabbit IgG (Mock control). (C) Quantitation of ChIP experiment in B, expressed as a percentage of input DNA.

Figure 6.

Association of MMR proteins at centromere-associated and Y chromosome repeats is dependent on MutSβ (MSH2–MSH3). (A-C) chromosome localization of MSH2 (red, TRITC) on wild-type SCs, as marked by SYCP3 (green, FITC) in association with the centromere, as marked by CREST labeling (blue, CY5), and the XY bivalent (C, arrow). MSH2 localization occurs at centromeres and the Y chromosome in spermatocytes from wild-type males (A–C; same cell in each panel), and is also found at occasional sites along the chromosome and throughout the chromatin area (see arrowheads in A). (D–F) MSH3 localization follows a similar pattern to MSH2, as shown in D (MSH3 is red, TRITC) and localizes most strongly with the CREST-positive regions (blue, CY5) in E and F, and also localizes to the XY (indicated by the arrow in F). (G–I) Immunogold electron micrographs showing the centromere regions of chromosomes from Msh3 ^{− / −} (G), Msh4 ^{− / −} (H), and Msh5 ^{− / −} (I) spermatocytes in pachynema with MLH3 (12 nm gold beads, green), MSH2 (6 nm gold beads, red), and CREST (18 nm gold beads, blue) showing that the localization of MSH2 and MLH3 to this region is dependent on MSH3, but not on MSH4 and MSH5. (J) Quantitation of EM-gold localization of MLH3 (striped bars), MLH1 (filled bars), and CREST (empty bars) on spermatocyte centromeres from wild-type males, and males that are homozygous for mutations in Msh2, Msh3, Msh5, and Msh6. MLH3 localization is significantly decreased compared with wild type on centromeres from Msh2 ^{− / −} and Msh3 ^{− / −} spermatocytes, but not on centromeres from Msh5 ^{− / −} or Msh6 ^{− / −} spermatocytes. χ² analysis of MLH1 and MLH3 localization at spermatocyte centromeres reveals statistically significant differences between wild-type and both Msh2 ^{− / −} and Msh3 ^{− / −} males (***P < 0.0002), but not between wild-type and Msh5 ^{− / −} males (not significant, n.s., P = 0.81). MLH3 levels were slightly increased at the centromeres of Msh6 ^{− / −} spermatocytes (*P = 0.043).

Figure 7.

Localization of MSH2 to the centromere regions of meiotic chromosomes is dependent on MSH3 but not MSH6 and PMS2. (A–C) MSH2 localization to meiotic chromosome cores in a representative spermatocyte from a Pms2 ^{− / −} male. MSH2 is localized in red (TRITC), SYCP3 in green (FITC) and the centromere in blue (CY5-CREST). Panel C shows the superimposed channels from panels A and B. (D–F) MSH2 localization to meiotic chromosomes in two representative spermatocytes from a Msh3 ^{− / −} male. MSH2 is shown in red (TRITC), SYCP3 in green (FITC), and the centromere in blue (CY5-CREST). In the absence of MSH3, no MSH2 signal is apparent. (G–I) MSH2 localization to meiotic chromosomes in a representative spermatocyte from a Msh6 ^{− / −} male. MSH2 is localized in red (TRITC), SYCP3 in green (FITC), and the centromere in blue (CY5-CREST). In A, D, and G, white arrowheads indicate interstitial localization of MSH2 along chromosome cores. (J) For each genotype, 5–10 cells were quantitated and the percentage of centromeres showing fluorescent signal for MSH2 was scored.