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Untimely expression of gametogenic genes in vegetative cells causes uniparental disomy

Nature volume 543, pages 126–130 (2017)Cite this article

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Abstract

Uniparental disomy (UPD), in which an individual contains a pair of homologous chromosomes originating from only one parent, is a frequent phenomenon that is linked to congenital disorders and various cancers1,2. UPD is thought to result mostly from pre- or post-zygotic chromosome missegregation2. However, the factors that drive UPD remain unknown. Here we use the fission yeast Schizosaccharomyces pombe as a model to investigate UPD, and show that defects in the RNA interference (RNAi) machinery or in the YTH domain-containing RNA elimination factor Mmi1 cause high levels of UPD in vegetative diploid cells. This phenomenon is not due to defects in heterochromatin assembly at centromeres. Notably, in cells lacking RNAi components or Mmi1, UPD is associated with the untimely expression of gametogenic genes. Deletion of the upregulated gene encoding the meiotic cohesin Rec8 or the cyclin Crs1 suppresses UPD in both RNAi and mmi1 mutants. Moreover, overexpression of Rec8 is sufficient to trigger UPD in wild-type cells. Rec8 expressed in vegetative cells localizes to chromosomal arms and to the centromere core, where it is required for localization of the cohesin subunit Psc3. The centromeric localization of Rec8 and Psc3 promotes UPD by uniquely affecting chromosome segregation, causing a reductional segregation of one homologue. Together, these findings establish the untimely vegetative expression of gametogenic genes as a causative factor of UPD, and provide a solid foundation for understanding this phenomenon, which is linked to diverse human diseases.

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Figure 1: Defective RNAi machinery triggers UPD.
Figure 2: Untimely expression of gametogenic genes triggers UPD.
Figure 3: Deletion of the meiotic gene encoding Rec8 or Crs1 suppresses UPD in cells lacking Mmi1 or RNAi machinery.
Figure 4: Rec8 associated with Psc3 at centromeres drives UPD.

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Acknowledgements

We thank Y. Watanabe, M. Yamamoto, Y. Hiraoka, F. Ishikawa, G. Smith and the National BioResource Project (NBRP) Japan for reagents, J. Barrowman for editing the manuscript, N. Taneja and A. Fernandez-Alvarez for technical assistance and members of the Laboratory of Biochemistry and Molecular Biology, in particular the Grewal laboratory, for discussions. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

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  1. Takeshi Mizuguchi

    Present address: †Present address: Department of Human Genetics, Yokohama City University Graduate School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan.,

Authors and Affiliations

  1. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    H. Diego Folco, Venkata R. Chalamcharla, Tomoyasu Sugiyama, Gobi Thillainadesan, Martin Zofall, Vanivilasini Balachandran, Jothy Dhakshnamoorthy, Takeshi Mizuguchi & Shiv I. S. Grewal

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Contributions

H.D.F. and S.I.S.G. conceived the project and designed experiments. H.D.F. performed live cell imaging, UPD and other genetic assays, V.R.C. and H.D.F. conducted ChIP–chip and ChIP–qPCR, T.S. performed western blots, G.T. conducted bioinformatics analyses, M.Z. and V.C. performed RNA-seq, and V.B., J.D., H.D.F., T.S. and T.M. constructed strains. All authors contributed to data interpretation. H.D.F. and S.I.S.G. wrote the manuscript with input from all authors.

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Correspondence to Shiv I. S. Grewal.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks Y. Watanabe, M. Zaratiegui and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Additional validation of LOH and schematic of UPD assays.

a, Sporulation efficiency of the resulting diploid colonies from LOH assays. Resulting diploid colonies from starting heterozygous diploids were scored for sporulation by iodine staining (iodine stains the starch-like compound produced by sporulating cells). Note that for each dcr1∆ diploid, the fraction of iodine positive colonies is similar in both the resulting LOH and heterozygous diploid populations, indicating that meiosis efficiency is independent of LOH (resulting heterozygous diploids n > 100; resulting LOH diploids n ≥ 7). b, Resulting dcr1∆ heterozygous diploids (AB) or progeny homozygosed for ChrI (AA and BB) were obtained from LOH assays and subjected to random spore analysis. Six diploids from each class were sporulated and plated onto rich medium for germination. Subsequently, colonies were genotyped by replica plating. Note that markers on ChrI were homozygosed in LOH diploids whereas markers on ChrIII show normal segregation. Over 80 haploid colonies were counted per diploid. c, Haploid parental S. pombe chromosomes showing relevant markers for UPD assays. Starting heterozygous diploids obtained by mating or fusing parental strains A and B were used to quantify UPD frequency. Diploids were maintained by selecting for heterozygosis of ChrI (assays A and B) or ChrII (assay C), whereas ChrIII was used as a reporter for UPD. The chromosome containing no relevant markers for the respective assay is shaded. UPD frequencies were calculated using the indicated formula and were adjusted using the validation coefficient (that is, the fraction of candidates exhibiting the expected homozygous markers on both arms of ChrIII; see Supplementary Table 3). Marker denotes various genetic markers at ura4 and mmi1 loci for assays A–B and C, respectively. Detailed information is provided in the Methods.

Extended Data Figure 2 Characterization and additional validation of UPD in dcr1∆ mutants.

a, Left, distribution of resulting UPD diploids homozygosed for ChrIII (AA and BB) per starting heterozygous sporulating diploid. Right, validation coefficients per single starting diploid used for adjustment of UPD frequencies. More than 12 UPD candidates from each starting diploid were validated. b, Tetrad dissection analyses of dcr1∆ diploids (AA and BB) homozygosed for ChrIII, obtained from UPD assays. A schematic of the S. pombe chromosomes in the haploid parental strains A and B, indicating the location of the relevant markers, is depicted at the top. Note the asymmetric (4:0 or 0:4) segregation at ChrIII, in contrast to the (2:2) segregation at ChrI and ChrII. The yellow arrowhead denotes dead cells carried over from the master plate during replica plating. c, d, Quantification of UPD in the indicated mat2-102/mat1M-smt0 (c) and h−/h− (d) nonsporulating diploids obtained by mating (c) and protoplast fusion (d). Note that clr4∆ caused only a modest increase in UPD compared to RNAi mutant dcr1Δ as observed in Fig. 1c, f. These results are explained by the fact that RNAi mutants show more penetrance than clr4∆ in meiotic gene misregulation. Each filled red circle represents the UPD frequency of an independent starting heterozygous diploid. More than 100 colonies were scored for each diploid. Filled bars and error bars are mean and s.d. **P < 0.01; ****P < 0.0001 (Mann–Whitney U test).

Source data

Extended Data Figure 3 Pericentromeric cohesin is reduced but not abolished in the dcr1∆ mutant.

a, Distribution of the Psc3 subunit of cohesin as determined by ChIP–chip. Psc3–GFP localization along ChrI is shown for the indicated strains. Note that Psc3 localization in clr4∆ was specifically affected at heterochromatic regions (grey shaded), but not at chromosome arm regions. On the other hand, Psc3 centromeric localization was reduced but not abolished in dcr1∆. b, c, Psc3–GFP localization to heterochromatin coated centromere 2 (cen2; b) and to telomere 1 left (tel1L; c). d, Psc3–GFP localization to euchromatic chromosome arm regions. Enrichments along a 200-kb region of the right arm of ChrII are shown. Green bars represent open reading frames according to the 2007 S. pombe genome assembly. The fold enrichment of Psc3–GFP (y axis) is plotted at the indicated chromosome positions (x axis).

Extended Data Figure 4 Centromeric heterochromatin is maintained in mmi1∆.

a, Tenfold serial dilutions of each strain were plated on YEA rich media containing the indicated concentrations of the spindle poison TBZ, and were grown at 33 °C. b, H3K9me2 enrichments in the indicated strains were determined by ChIP–chip analysis. The fold enrichment of H3K9me2 (y axis) is plotted at the indicated chromosome position (shown at top). H3K9me2 distribution at the mat locus is shown in addition to cen1L. c, Tenfold serial dilutions of strains containing a ura4+ insertion at the outer repeats of centromere 1 were plated on the indicated PMG minimal media and grown at 33 °C. Note that mmi1∆ is lethal but can be rescued by loss of function of Mei4, a meiotic transcription factor. mei4 mmi∆ is compared to appropriate mei4 and wild-type controls.

Extended Data Figure 5 Rec8 is enriched at centromeres and colocalizes with Rec11 on chromosome arms in mmi1∆.

a–c, Distribution of the Rec8 and Rec11 subunits of cohesin as determined by ChIP–chip. HA–Rec8 and Rec11–GFP localization along the S. pombe genome (a, b) and centromere 2 (cen2) (c) is shown for the indicated strains. Note that Rec8 localization is highly enriched at centromeres (grey shaded) and colocalizes with Rec11 at chromosomal arms in mmi1∆. An enhanced view of the left arm of ChrI (indicated by the dotted lines) is shown. d, Increased localization of Rec8 and Rec11 to chromosomal arms correlates with known mitotic cohesin peaks. Rec8, Rec11 and Psc3 enrichment along a 200-kb region of the left arm of ChrI is shown. ChIP–chip analysis of Psc3 (brown), Rec8 (green) and Rec11 (grey) was performed in wild-type or mmi1∆ strains as indicated. The fold enrichment of Psc3–GFP, HA–Rec8 and Rec11–GFP (y axis) is plotted at the indicated chromosome position (x axis). Note that the regions displaying higher enrichment of Rec8 (shaded grey) are correspondingly enriched for Rec11 and Psc3. e, Rec11 enrichment at the indicated chromosomal arm locations and centromere central core (cc1/3) were determined by ChIP–qPCR. The mmi1∆ strain used in this study carries a truncated non-functional allele of mei4.

Extended Data Figure 6 Chromosomal localization of Psc3 and Rad21 in mmi1∆.

a, b, Distribution of Rad21 and the Psc3 subunit of cohesin as determined by ChIP–chip. Rad21–GFP (a) or Psc3–GFP (b) localization along the S. pombe genome is shown for the indicated strains. Enrichments at mae1 and mae2, marked by asterisks, reflect cross-hybridization of these loci to subtelomeric sequences. c, d, Rad21 (c) or Psc3 (d) localization to cen2. The fold enrichment of Rad21–GFP or Psc3–GFP (y axis) is plotted at the indicated chromosome position (x axis). Note the abnormally high enrichment of Psc3 but not Rad21 at the central core in mmi1∆ (blue arrow). The mmi1∆ strain used in this study carries a truncated non-functional allele of mei4.

Extended Data Figure 7 Increased localization of Rec8 and Rec11 to chromosomal arms results in decreased Psc3 and Rad21.

a, Distribution of Rec8 (green), Rec11 (grey), Psc3 (brown) and Rad21 (blue) subunits of cohesin as determined by ChIP–chip in mmi1∆ and wild-type strains. Enrichments along two 200-kb regions of ChrI (left arm) and ChrII (right arm) are shown. The fold enrichment of the indicated proteins in mmi1∆, calculated by subtraction of wild-type, is plotted (y axis) at the indicated chromosome positions (x axis). Note that the regions showing high Rec8 enrichment in mmi1∆ (grey shaded) are also enriched for Rec11, but are depleted of Psc3 and Rad21. b, Boxplots showing ChIP enrichments of the indicated proteins at 133 Rec8-enriched chromosomal arm locations in wild-type and mmi1∆ strains. **P < 0.01; ****P < 0.0001 (one-way ANOVA plus Bonferroni post-tests). The mmi1∆ strain used in this study carries a truncated non-functional allele of mei4.

Extended Data Figure 8 Additional characterization of rec8-OE diploids from UPD assays.

a, Serial dilution growth assay of the indicated strains. Tenfold serial dilutions were spotted. Cells were grown for 3 days at 32 °C in YEA rich medium with or without TBZ (15 μg ml−1). Note that like mei4 mmi1∆, rec8-OE exhibits TBZ sensitivity. b, Distribution of resulting UPD diploids (AA and BB) and validation coefficients used for adjustment of UPD frequencies in rec8-OE diploids. At least 12 UPD candidates per starting diploid were validated. c, Tetrad dissection analyses were performed with rec8-OE diploids obtained from UPD assays that were heterozygous (AB) or homozygosed for ChrIII (AA and BB). A schematic of the S. pombe chromosomes in haploid parental strains A and B, indicating the location of the relevant markers, is depicted at the top. Note the asymmetric (4:0 or 0:4) segregations observed for ChrIII, in contrast to the normal (2:2) segregations observed for ChrI and ChrII. d, Left, quantification of asci based on the number of viable spores. Right, spore viability quantified by tetrad dissection analysis of rec8-OE diploids shown in c. In each set, data correspond to a total of ≥59 asci from ≥7 independent diploids. NS, P > 0.1 (multiple t-tests).

Extended Data Figure 9 Overexpression of Rec8 (rec8-OE) in wild-type cells phenocopies mmi1∆.

a, Examples of UPD in rec8-OE strains. Schematic of fission yeast chromosomes of haploid parental strains A and B, indicating the locations of the relevant markers, is depicted at the top. Haploid parental and the indicated diploid strains are shown. Note that the genetic markers on both arms of ChrIII were homozygosed in AA and BB diploids, which indicates UPD. b, Quantification of UPD in the indicated mat2-102/mat1M-smt0 non-sporulating diploid strains as depicted in Extended Data Fig. 1c (assay A). The data for wild-type were replotted from Extended Data Fig. 2c. Each filled red circle represents the UPD frequency of an independent starting heterozygous diploid. Over 100 colonies were scored for each diploid. Filled bars and error bars are mean and s.d. ****P < 0.0001 (Mann–Whitney U test). c, d, Distribution of the Rec8 subunit of cohesin along the S. pombe genome (c) as determined by HA–Rec8 ChIP–chip. Note that Rec8 is highly enriched at centromeres (shaded grey) and at chromosome arms in rec8-OE. An enhanced view of the left arm of ChrI (indicated by the dotted lines) is also shown (d). e, Rec8 localization to centromere 1 (cen1) is shown for the indicated strains. The fold enrichment of HA–Rec8 is plotted (y axis) at the indicated chromosome positions (x axis). Green bars represent open reading frames according to the 2007 S. pombe genome assembly.

Source data

Extended Data Figure 10 UPD is likely to be caused by a reductional event affecting one homologue, whereas the other segregates equationally.

a, Schematic of fission yeast chromosomes of haploid parental strains A and B, indicating the locations of relevant markers (top). Quantification of rec8-OE half-sectored resulting diploids from UPD assays shown in Fig. 4f, g. b, Random spore analysis of half-sectored resulting diploids. Eight half-sectored diploids, white/red (1–4) and white/pink (5–8), were sporulated. Note that the colonies formed by spores from the white sectors show normal distribution of markers, spores from red or pink sectors show normal segregation of ChrI-based markers but homozygosis of ChrIII-based markers. Over 50 haploid colonies were counted per sectored diploid. c, d, Quantification of UPD in the indicated diploid strains as depicted in Extended Data Fig. 1c. Note that, as observed in mmi1∆ (Fig. 4d), rec11∆ does not suppress UPD in rec8-OE confirming that the Rec8 along with interaction partner Psc3 at centromeres is the main driver of UPD. The data for wild type, dcr1∆, mei4 mmi1∆ and rec8-OE were replotted from Figs 1f, 2e and Extended Data Figs 2c and 9b. Each filled red circle represents the UPD frequency of an independent starting heterozygous diploid. Over 100 colonies were scored for each diploid. Filled bars and error bars are mean and s.d. ****P < 0.0001 (Mann–Whitney U test).

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Supplementary information

Supplementary Table 1

Candidate genes tested for suppression of minichromosome loss in mei4 mmi1δ. (XLSX 32 kb)

Supplementary Table 2

This table contains the strains used in this study. (XLSX 33 kb)

Supplementary Table 3

Validation coefficients calculated for each set of diploids. (XLSX 56 kb)

Supplementary Figure 1

This file contains uncropped scans from immunoblots displayed in Figures 3e, f and 4e, with size marker indications. (PDF 509 kb)

UPD is caused by a reductional event affecting only one of the homologs

A rec8-OE/rec8-OE cen2-tetO-Tomato/cen2-lacO-GFP vegetative diploid was grown at 30°C on a PMG-agarose pad and imaged by time-lapse microscopy. Note that both decorated cen2s are detected as bright single dots and nuclei can also be visualized due to unbound lacI-GFP and tetR-Tomato proteins. Two mitotic segregations are reductional for cen2-Tomato and equational for cen2-GFP, indicated by arrows, whereas the other segregations are normal. Images were taken every 40 min for 8 h and acquired over 11 focal planes at a 0.60-μm step size. Scale bar represents 15 μm. (MP4 4466 kb)

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Folco, H., Chalamcharla, V., Sugiyama, T. et al. Untimely expression of gametogenic genes in vegetative cells causes uniparental disomy. Nature 543, 126–130 (2017). https://doi.org/10.1038/nature21372

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