Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression

Abstract

Effective DNA replication is critical to the health and reproductive success of organisms. The six MCM2-7 proteins, which form the replicative helicase, are essential for high-fidelity replication of the genome. Many eukaryotes have a divergent paralog, MCM9, that was reported to be essential for loading MCM2-7 onto replication origins in the Xenopus oocyte extract system. To address the in vivo role of mammalian MCM9, we created and analyzed the phenotypes of mice with various mutations in Mcm9 and an intronic DNA replication-related gene Asf1a. Ablation of Mcm9 was compatible with cell proliferation and mouse viability, showing that it is nonessential for MCM2-7 loading or DNA replication. Mcm9 mutants underwent p53-independent embryonic germ-cell depletion in both sexes, with males also exhibiting defective spermatogonial stem-cell renewal. MCM9-deficient cells had elevated genomic instability and defective cell cycle reentry following replication stress, and mutant animals were prone to sex-specific cancers, most notably hepatocellular carcinoma in males. The phenotypes of mutant mice and cells suggest that MCM9 evolved a specialized but nonessential role in DNA replication or replication-linked quality-control mechanisms that are especially important for germ-line stem cells, and also for tumor suppression and genome maintenance in the soma.

Fig. 1.

Mcm9 locus structure and isoforms. (A) Mcm9 has 2 isoforms: Mcm9^s and Mcm9^L. The former uses an alternative splice site in exon 7 that results in termination. Triangles depict the insertion sites of the gene traps. XE518 created a deletion that includes Asf1a. Locations of polymorphic SNPs used for measuring deletion size in the Mcm9^XE518 allele are indicated. These are: (Left to Right) rs51772485, rs46087546, rs51382030, rs51176035, rs47659858, and rs49316622. The Asf1a gene trap is located 640-bp downstream of exon 2. (B) MCM9 isoforms and the gene-trap alleles. MCM9 contains a zinc finger, CDK site, and the MCM domain. (C) Tissue expression of Mcm9 and Asf1a by semi-qRT-PCR. Primer pairs used in the top four panels are sets 1 to 4, respectively, as shown in A. (D) qRT-PCR analysis of Mcm9 and Asf1a in mutant MEFs (n = 3). AWO, Mcm9^AWO655; XG, Mcm9^XG743. Error bars indicate SD.

Fig. 2.

Midgestation lethality in MCM9- and ASF1A-deficient embryos. (A–D) E9.5 embryos. (Scale bars, 500 μM.) (E) RT-PCR of E9.5 embryos. Primers for Mcm9^S and Mcm9^L are sets 2 and 3, respectively, from Fig. 1A. Primers (XE518WTF and XE518GTR) for Mcm9^XE are specific for the gene-trap fusion transcript. (F) RT-PCR/sequencing of polymorphic coding SNPs (rs51382030 for Asf1a; rs51772485 for Mcm9). The WT allele here is C3H, which is identical to the parental 129-based gene trap chromosome. XE, Mcm9^XE518 ; AL, Asf1a^AL0673.

Fig. 3.

Mcm9 mutations cause germ-cell depletion and loss of spermatogonial stem cells. (A and B) Quantification of testis weights and histological abnormalities. *Significantly different from WT; **significant decrease in mutant testis size over time (n = 10). Abbreviations in B for seminiferous tubule cross sections that contain or exihibit the following: GMC, giant multinucleated cells; MEI, meiotic arrest; MW, missing wave of spermatogenesis; GCD, germ-cell depletion; Norm, normal. (C and D) H&E-stained histological sections of testes from 12-wk-old mice of the indicated genotypes. WT, wild-type; XG, Mcm9^XG743 (magnification: 200×). Error bars indicate SD. Seminiferous tubules marked with an asterisk (*) are devoid of germ cells; those marked with a caret (^) exhibit meiotic arrest (Inset); and those indicated by arrows are depleted of spermatogonia but undergoing a final wave of spermatogenesis. Circled tubules are examples of normal spermatogenesis. (E and F) H&E-stained histological sections of ovaries from 24-wk-old mice of the indicated genotypes. (G–L) Immunofluorescence of 1-dpp testes (G and H) and ovaries (J and K) of the indicated genotypes (magnification: 200×). MVH (green) stains germ cells and DAPI stains nuclei (blue). (I and L) Germ-cell counts from MVH staining data at 1 dpp (n = 3). *Significant difference vs. WT (see Materials and Methods). XG, Mcm9^XG743/XG743; p53, Trp53^−/−; p21, Cdkn1a^−/−. Error bars indicate SD.

Fig. 4.

Loss of MCM9 does not alter MCM2-7 or CDT1 levels, however, leads to mild genomic instability and cell-cycle defects under replication stress. (A) Western blots of mutant and WT MEFS showing detergent soluble vs. chromatin bound levels of indicated proteins. (B) Quantification of data in A. (C) Micronucleus levels in erythrocytes. (D) Chromatid breaks in mutant MEFS. (E) Mcm9 mutant MEFs undergo premature senescence. The y-axis values were taken every 3 d upon passage. (F) Mutant MEFs exhibit a delay in cell-cycle entry following APH-induced replication stress. Primary data are in Fig. S3. MEFs were serum-starved to synchronize at G0/G1, serum was then added, then measurements were taken. AW, Mcm9^AWO655; XG. Mcm9^XG743. The P value is based on t testing. UN, measurements 20 h after serum addition, but untreated with APH; 20 hr APH, same as previous, but with APH treatement; 3hr Rel or 24hr Rel, same as previous, but 3 h or 24 h after APH was removed from media (n = 3). Error bars indicate SD.