PMC

The fecundity is significantly reduced in the third generation of mSuv3 heterozygous mice. Designated female mice were housed together with C57/BL6 male since 8 weeks old; the number of pups of each delivery and litter were recorded to determine the fecundity of female mice.

Mitochondrial function is a dominant determinant for mSuv3 inheritance. (A) Tumor incidence and spectra of mSuv3+/+; mt-msuv3+/− offspring of mSuv3 heterozygote intercross. (B) Lifespan of heterozygous offsprings derived from either crossing a mSuv+/− female to a wild type C57/BL6 male or crossing a wild-type female and a mSuv+/− male. (C) Tumor incidence of mSuv3+/+ offspring from the cross of mSuv3 heterozygous female and wild type C57/BL6 male (mSuv3+/+; mt-msuv3+/−).

Reduced SUV3 expression in human tumors. (A) The relative expression level of SUV3 in tumor sample was compared to that of the normal adjacent tissue. Relative expression level of SUV3 gene normalized by β-actin. (B) Comparison of the expression level of SUV3 in normal breast tissue and breast tumor (Student's t-test was used to assess P value).

Lifespan and tumor spectra of the heterozygous offspring derived from mSuv3+/− intercross mice. (A) Shortened lifespan of mSuv3 heterozygotes at different intercross generations. (B) Tumor incidence and spectra of mSuv3+/− intercross mice. (C) H&E histology of mSuv3+/− mouse tumors: lung adenocarcinoma (a), high-grade lymphoma (b), squamous cell carcinoma (c), pituitary adenoma (d), metastasis – hepatocyte carcinoma (e) and papillary adenocarcinoma (f). (D) Microdissected tumors of mSuv3+/− mice. Micrographs showed paraffin-sections of lung adenocarcinoma (a & b) and lung metastatic HCC (c & d) before (a & c) and after (b & d) microdissection. (E) Genotyping of mSuv3 alleles in the tumor samples (wt: 149 bp: KO: 236 bp) (F) Immunostaining with anti-SUV3 antibodies demonstrated tumors derived from mSuv3+/− mice expressing SUV3.

Reduction of mtDNA copy number correlates with reduced MEF cell proliferation. (A) Genotyping of mSuv3 MEFs. (wt: 149bp, KO: 236bp): Cohort A (mSuv3+/+:mt-msuv3+/+) = A21 and A22; Cohort B (mSuv3+/−: mt-msuv3+/+) = B21 and B22; Cohort C (mSuv3+/+: mt-msuv3+/−) = C21 and C22; and Cohort D (mSuv3+/−: mt-msuv3+/−) = D21 and D22. (B) Proliferation capacity of MEFs. MEFs prepared from an embryo of each strain were serially passaged following 3T3 protocol. Each plot represents MEFs from a single embryo, with cell numbers on the Y-axis against passage numbers on the X-axis. Each data point was the mean of two calculations. Standard deviations were too small to be visible. (C) Quantification of relative mtDNA copy number of MEFs from each cohorts at forth passage (mean +/− s.e.m; n=8).

Defective mitochondria inherited from mSuv3+/− female leads to increased tumorigenicity and reduced lifespan regardless of nuclear genotype. The offspring from the cross of mSuv3+/− female with C57/BL6 male and mSuv3+/− male with C57/BL6 female were designated as four cohorts: Cohort A (mSuv3+/+; mt-msuv3+/+); Cohort B, (mSuv3+/−; mt-msuv3+/+); Cohort C, (mSuv3+/+; mt-msuv3+/−); and Cohort D (mSuv3+/−; mt-msuv3+/−). (A) The region surrounding the mtDNA L-strand origin was sequenced and mtDNA mutation rates were calculated from brain samples collected from ten-month-old mice in each cohort. (B) The mtDNA to nDNA copy number ratio was determined from seven to eight spleens of 15-month-old mice of each cohort by quantitative real-time PCR using mitochondrial 12S rDNA and nuclear 18S rDNA gene primers (Student's t-test was used to assess P value). (C) Enzyme activities of OXPHOS complexes containing mtDNA encoded subunits, complexes I, II +III, and IV, assessed on brain mitochondria of 10-month-old animals from each cohort. (D) Lifespan and tumor incidence of the offspring (the third generation) derived from mSuv3+/− backcross with C57/BL6 mice. (Student's t-test was used to assess P value).

Targeted disruption of the mouse mSuv3 locus leads to embryonic lethality. (A) Confirmation of the targeted disruption of the mSuv3 allele. DNA samples from parental El4.1 cells (lanes 1 & 4) and two candidate recombinant clones [mSuv3-ko 5 (lanes 2 & 5) and mSuv3 ko 149 (lanes 3 & 6)] were digested with SalI-BamHI and probed with either probe A or the neo probe. An additional fragment of the expected size of 11.6 kb was found in each of the recombinant clones. (B) Western blot analysis of SUV3 protein level. Whole-cell lysate of MEFs of wild type (lane 1) and heterozygote (lane 2) were separated by SDS-PAGE and probed with an antibody against SUV3. p48 was used as a loading control. (C) Genotypes of offspring from heterozygous mSuv3+/− mice intercross. Pregnant females of mSuv3+/− intercrosses were sacrificed and the fetuses examined at different gestation times from E3.5 to E11.5 days. (D) Developmental abnormalities of mSuv3 mutant embryos. Histological analysis was performed on embryo sections of wild type and mSuv3−/− conceptuses. The uteri of mSuv3+/− females were dissected between 5.0 and 8.0 days after intercross, and 4 µm sections were prepared. All uterine decidua were sectioned transversely and labeled as described (). The mesometrial to anti-mesometrial axis is from left to right. Abbreviations: (eer) extraembryonic region; (ve) viceral endoderm; (ep) epiblast; (pac) proamniotic cavity; (c) chorion; (a) amnion. Bar, 100 µm.