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Antagonistic roles of ubiquitin ligase HEI10 and SUMO ligase RNF212 regulate meiotic recombination.

Qiao H, Prasada Rao HB, Yang Y, Fong JH, Cloutier JM, Deacon DC, Nagel KE, Swartz RK, Strong E, Holloway JK, Cohen PE, Schimenti J, Ward J, Hunter N - Nat. Genet. (2014)

Bottom Line: Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process.As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over.We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA. [2] Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA.

ABSTRACT
Crossover recombination facilitates the accurate segregation of homologous chromosomes during meiosis. In mammals, poorly characterized regulatory processes ensure that every pair of chromosomes obtains at least one crossover, even though most recombination sites yield non-crossovers. Designation of crossovers involves selective localization of the SUMO ligase RNF212 to a minority of recombination sites, where it stabilizes pertinent factors such as MutSγ (ref. 4). Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process. In HEI10-deficient mice, RNF212 localizes to most recombination sites, and dissociation of both RNF212 and MutSγ from chromosomes is blocked. Consequently, recombination is impeded, and crossing over fails. In wild-type mice, HEI10 accumulates at designated crossover sites, suggesting that it also has a late role in implementing crossing over. As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over. We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.

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HEI10 localization to synaptonemal complexes and crossover sites. (a–j) Representative SIM images of wild-type spermatocyte nuclei immunostained for HEI10 (green) and SYCP3 (red) at (a) leptonema, (b) late zygonema, (c,d) early pachynema, (e,f) mid pachynema, (g,h) late pachynema, and (i,j) early diplonema. (d, f, h, j) Magnified views of the chromosomes indicated by arrows in c, e, g, i, respectively. Arrowheads highlight HEI10 foci. (k) Numbers of HEI10 foci per nucleus at successive prophase stages (Z, zygonema; EP, early pachynema; MP, mid pachynema; LP, late pachynema; ED, early diplonema). Horizontal bars represent means ± s.d. Numbers of nuclei analyzed at Z, EP, MP, LP and ED, respectively: 13, 8, 22, 20 and 11. (l,m) Mid-pachytene spermatocyte immunostained for HEI10 (green), RNF212 (red), and SYCP3 (grey). (m) Magnified view of the chromosome indicated by the arrow in l. (n,o) Mid-pachytene spermatocyte immunostained for HEI10 (green), MSH4 (red) and synaptonemal complex central element protein, SYCE1 (grey). (o) Magnified view of the chromosome indicated by the arrow in n. (p,q) Mid-pachytene spermatocyte immunostained for HEI10 (green), CDK2 (red), and SYCE1 (grey). (q) Magnified view of the chromosome indicated by the arrow in p. (r,s) Mid-pachytene spermatocyte immunostained for HEI10 (green), MLH1 (red), and SYCE1 (grey). (s) Magnified view of the chromosome indicated by the arrow in r. The arrowhead highlights a small MLH1 focus that colocalizes with HEI10.Scale bars, 10 μm in a, b, c, e, g, i, l, n, p, r; 1 μm in d, f, h, j, m, o, q, s.
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Figure 4: HEI10 localization to synaptonemal complexes and crossover sites. (a–j) Representative SIM images of wild-type spermatocyte nuclei immunostained for HEI10 (green) and SYCP3 (red) at (a) leptonema, (b) late zygonema, (c,d) early pachynema, (e,f) mid pachynema, (g,h) late pachynema, and (i,j) early diplonema. (d, f, h, j) Magnified views of the chromosomes indicated by arrows in c, e, g, i, respectively. Arrowheads highlight HEI10 foci. (k) Numbers of HEI10 foci per nucleus at successive prophase stages (Z, zygonema; EP, early pachynema; MP, mid pachynema; LP, late pachynema; ED, early diplonema). Horizontal bars represent means ± s.d. Numbers of nuclei analyzed at Z, EP, MP, LP and ED, respectively: 13, 8, 22, 20 and 11. (l,m) Mid-pachytene spermatocyte immunostained for HEI10 (green), RNF212 (red), and SYCP3 (grey). (m) Magnified view of the chromosome indicated by the arrow in l. (n,o) Mid-pachytene spermatocyte immunostained for HEI10 (green), MSH4 (red) and synaptonemal complex central element protein, SYCE1 (grey). (o) Magnified view of the chromosome indicated by the arrow in n. (p,q) Mid-pachytene spermatocyte immunostained for HEI10 (green), CDK2 (red), and SYCE1 (grey). (q) Magnified view of the chromosome indicated by the arrow in p. (r,s) Mid-pachytene spermatocyte immunostained for HEI10 (green), MLH1 (red), and SYCE1 (grey). (s) Magnified view of the chromosome indicated by the arrow in r. The arrowhead highlights a small MLH1 focus that colocalizes with HEI10.Scale bars, 10 μm in a, b, c, e, g, i, l, n, p, r; 1 μm in d, f, h, j, m, o, q, s.

Mentions: Insights into this question, and how HEI10 regulates the dynamics of RNF212 and MutSγ, came from examining the chromosomal localization patterns of HEI10. Although previous attempts to localize mouse HEI10 to meiotic chromosomes failed6, we were able to show that mouse HEI10 associates with synaptonemal complexes to form distinct immunostaining foci (Fig. 4a–k and Supplementary Figs. 2 and 3). Unlike RNF212, distinct foci of HEI10 were rarely detected along nascent synaptonemal complexes during zygonema (Fig. 4a). Given that Hei10mei4/mei4 phenotypes are already apparent at this time (e.g. Fig. 2p), we infer that cytologically undetectable HEI10 regulates early-stage dynamics of RNF212 and MutSγ. Indeed, the possibility this function of HEI10 does not involve association with meiotic chromosomes cannot be ruled out. However, by early pachynema, HEI10 foci could be detected (Fig. 4c,d) and their numbers peaked during mid pachynema with an average of 27 foci per nucleus (27.2 ± 8.8 (s.d.); 22 nuclei; Fig. 4e,f,k). At this stage, HEI10 focus numbers were quite variable, with as few as 15 and as many as 50 foci per nucleus. In late pachytene nuclei, the average HEI10 focus number was lower and less variable (21.1 ± 3.6 (s.d.); 20 nuclei; Fig. 4g,h,k). At the onset of diplonema, HEI10 foci were no longer detected (Fig. 4i,j).


Antagonistic roles of ubiquitin ligase HEI10 and SUMO ligase RNF212 regulate meiotic recombination.

Qiao H, Prasada Rao HB, Yang Y, Fong JH, Cloutier JM, Deacon DC, Nagel KE, Swartz RK, Strong E, Holloway JK, Cohen PE, Schimenti J, Ward J, Hunter N - Nat. Genet. (2014)

HEI10 localization to synaptonemal complexes and crossover sites. (a–j) Representative SIM images of wild-type spermatocyte nuclei immunostained for HEI10 (green) and SYCP3 (red) at (a) leptonema, (b) late zygonema, (c,d) early pachynema, (e,f) mid pachynema, (g,h) late pachynema, and (i,j) early diplonema. (d, f, h, j) Magnified views of the chromosomes indicated by arrows in c, e, g, i, respectively. Arrowheads highlight HEI10 foci. (k) Numbers of HEI10 foci per nucleus at successive prophase stages (Z, zygonema; EP, early pachynema; MP, mid pachynema; LP, late pachynema; ED, early diplonema). Horizontal bars represent means ± s.d. Numbers of nuclei analyzed at Z, EP, MP, LP and ED, respectively: 13, 8, 22, 20 and 11. (l,m) Mid-pachytene spermatocyte immunostained for HEI10 (green), RNF212 (red), and SYCP3 (grey). (m) Magnified view of the chromosome indicated by the arrow in l. (n,o) Mid-pachytene spermatocyte immunostained for HEI10 (green), MSH4 (red) and synaptonemal complex central element protein, SYCE1 (grey). (o) Magnified view of the chromosome indicated by the arrow in n. (p,q) Mid-pachytene spermatocyte immunostained for HEI10 (green), CDK2 (red), and SYCE1 (grey). (q) Magnified view of the chromosome indicated by the arrow in p. (r,s) Mid-pachytene spermatocyte immunostained for HEI10 (green), MLH1 (red), and SYCE1 (grey). (s) Magnified view of the chromosome indicated by the arrow in r. The arrowhead highlights a small MLH1 focus that colocalizes with HEI10.Scale bars, 10 μm in a, b, c, e, g, i, l, n, p, r; 1 μm in d, f, h, j, m, o, q, s.
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Figure 4: HEI10 localization to synaptonemal complexes and crossover sites. (a–j) Representative SIM images of wild-type spermatocyte nuclei immunostained for HEI10 (green) and SYCP3 (red) at (a) leptonema, (b) late zygonema, (c,d) early pachynema, (e,f) mid pachynema, (g,h) late pachynema, and (i,j) early diplonema. (d, f, h, j) Magnified views of the chromosomes indicated by arrows in c, e, g, i, respectively. Arrowheads highlight HEI10 foci. (k) Numbers of HEI10 foci per nucleus at successive prophase stages (Z, zygonema; EP, early pachynema; MP, mid pachynema; LP, late pachynema; ED, early diplonema). Horizontal bars represent means ± s.d. Numbers of nuclei analyzed at Z, EP, MP, LP and ED, respectively: 13, 8, 22, 20 and 11. (l,m) Mid-pachytene spermatocyte immunostained for HEI10 (green), RNF212 (red), and SYCP3 (grey). (m) Magnified view of the chromosome indicated by the arrow in l. (n,o) Mid-pachytene spermatocyte immunostained for HEI10 (green), MSH4 (red) and synaptonemal complex central element protein, SYCE1 (grey). (o) Magnified view of the chromosome indicated by the arrow in n. (p,q) Mid-pachytene spermatocyte immunostained for HEI10 (green), CDK2 (red), and SYCE1 (grey). (q) Magnified view of the chromosome indicated by the arrow in p. (r,s) Mid-pachytene spermatocyte immunostained for HEI10 (green), MLH1 (red), and SYCE1 (grey). (s) Magnified view of the chromosome indicated by the arrow in r. The arrowhead highlights a small MLH1 focus that colocalizes with HEI10.Scale bars, 10 μm in a, b, c, e, g, i, l, n, p, r; 1 μm in d, f, h, j, m, o, q, s.
Mentions: Insights into this question, and how HEI10 regulates the dynamics of RNF212 and MutSγ, came from examining the chromosomal localization patterns of HEI10. Although previous attempts to localize mouse HEI10 to meiotic chromosomes failed6, we were able to show that mouse HEI10 associates with synaptonemal complexes to form distinct immunostaining foci (Fig. 4a–k and Supplementary Figs. 2 and 3). Unlike RNF212, distinct foci of HEI10 were rarely detected along nascent synaptonemal complexes during zygonema (Fig. 4a). Given that Hei10mei4/mei4 phenotypes are already apparent at this time (e.g. Fig. 2p), we infer that cytologically undetectable HEI10 regulates early-stage dynamics of RNF212 and MutSγ. Indeed, the possibility this function of HEI10 does not involve association with meiotic chromosomes cannot be ruled out. However, by early pachynema, HEI10 foci could be detected (Fig. 4c,d) and their numbers peaked during mid pachynema with an average of 27 foci per nucleus (27.2 ± 8.8 (s.d.); 22 nuclei; Fig. 4e,f,k). At this stage, HEI10 focus numbers were quite variable, with as few as 15 and as many as 50 foci per nucleus. In late pachytene nuclei, the average HEI10 focus number was lower and less variable (21.1 ± 3.6 (s.d.); 20 nuclei; Fig. 4g,h,k). At the onset of diplonema, HEI10 foci were no longer detected (Fig. 4i,j).

Bottom Line: Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process.As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over.We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, University of California, Davis, Davis, California, USA. [2] Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, California, USA.

ABSTRACT
Crossover recombination facilitates the accurate segregation of homologous chromosomes during meiosis. In mammals, poorly characterized regulatory processes ensure that every pair of chromosomes obtains at least one crossover, even though most recombination sites yield non-crossovers. Designation of crossovers involves selective localization of the SUMO ligase RNF212 to a minority of recombination sites, where it stabilizes pertinent factors such as MutSγ (ref. 4). Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process. In HEI10-deficient mice, RNF212 localizes to most recombination sites, and dissociation of both RNF212 and MutSγ from chromosomes is blocked. Consequently, recombination is impeded, and crossing over fails. In wild-type mice, HEI10 accumulates at designated crossover sites, suggesting that it also has a late role in implementing crossing over. As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over. We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.

Show MeSH
Related in: MedlinePlus