Zac1 (Lot1), a potential tumor suppressor gene, and the gene for epsilon-sarcoglycan are maternally imprinted genes: identification by a subtractive screen of novel uniparental fibroblast lines

Abstract

Imprinted genes are expressed from one allele according to their parent of origin, and many are essential to mammalian embryogenesis. Here we show that the epsilon-sarcoglycan gene (Sgce) and Zac1 (Lot1) are both paternally expressed imprinted genes. They were identified in a subtractive screen for imprinted genes using a cDNA library made from novel parthenogenetic and wild-type fibroblast lines. Sgce is a component of the dystrophin-sarcoglycan complex, Zac1 is a nuclear protein inducing growth arrest and/or apoptosis, and Zac1 is a potential tumor suppressor gene. Sgce and Zac1 are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1 is biallelic in the liver and Sgce is weakly expressed from the maternal allele in the brain. Sgce and Zac1 are broadly expressed in embryos, with Zac1 being highly expressed in the liver primordium, the umbilical region, and the neural tube. Sgce, however, is strongly expressed in the allantoic region on day 9.5 but becomes more widely expressed throughout the embryo by day 11.5. Sgce is located at the proximal end of mouse chromosome 6 and is a candidate gene for embryonic lethality associated with uniparental maternal inheritance of this region. Zac1 maps to the proximal region of chromosome 10, identifying a new imprinted locus in the mouse, homologous with human chromosome 6q24-q25. In humans, unipaternal disomy for this region is associated with fetal growth retardation and transient neonatal diabetes mellitus. In addition, loss of expression of ZAC has been described for a number of breast and ovarian carcinomas, suggesting that ZAC is a potential tumor suppressor gene.

FIG. 1

Appropriate expression of known imprinted genes in PG MEFs. Total RNA isolated from PG and WT MEFs was analyzed by RT-PCR (lanes 1, 3, 5, and 7 are WT MEFs, and lanes 2, 4, 6, and 8 are PG MEFs).

FIG. 2

Differential expression of clones isolated from the screen by virtual Northern blotting (A) and RT-PCR (B). Rpl19 probe was used as a control for both the virtual Northern blotting and RT-PCR. Both Sgce and Zac1 are expressed only in the AG and WT MEFs by both virtual Northern blotting and RT-PCR. Other genes such as Gas and Igfbp5 are biallelically expressed. The maternally expressed H19 was analyzed by RT-PCR as a control for AG MEFs not expressing maternally expressed imprinted genes (abbreviations: M, markers; N, WT MEFs; P, PG MEFs; A, AG MEF cDNA).

FIG. 3

(A) Monoallelic expression of Sgce in interspecific embryos by RFLP analysis. A PCR fragment was amplified from both C57BL/6J and M. spretus and sequenced. In lanes 1 to 4, the 404-bp fragment is amplified in both species. In lanes 5 to 7, only the C57BL/6J product is cut with RsaI. In embryonic cDNAs from the interspecific cross, the 404-bp fragment is undigested by RsaI, showing exclusive paternal (M. spretus) expression (lane 8), as seen also for adult tissues (lanes 10 to 12). Only in the adult interspecific brain is weak expression of the maternal allele detected (lane 9). Lane 13 is a control showing coamplification of M. musculus and M. spretus alleles after they are mixed in the same reaction. (B) Paternal expression of Zac1 in interspecific embryos by RFLP analysis. A 465-bp PCR fragment was amplified from both C57BL/6J and M. m. castaneus and digested with BstNI. M. m. castaneus produces three fragments, of 311, 124, and 30 bp (lane 1), whereas the C57BL/6 allele produces two fragments, of 311 and 154 bp (lane 4). Embryo and MEF interspecific cDNA from reciprocal crosses revealed that only the paternal allele is expressed (lanes 2, 4, 13, and 14). In adult tissues, biparental expression is most apparent in the liver (lane 9) and to a lesser extent in kidney and skeletal muscle (lanes 7 and 8).

FIG. 4

Detection of Zac1 and Sgce transcripts in mouse embryos by in situ hybridization. (A) Zac1 is highly expressed in the liver primordium and body wall of the umbilical region (arrow) of a d9.5 embryo. (B) At d11, Zac1 expression is observed in neural tube (nt), somites (s), sympathetic ganglia (sg), distal second branchial arch (ba), and telencephalic vesicles (te). (C) Zac1 showed a strong expression in the neural tube (nt) at d10.5. (D and E) Sgce expression is restricted to the allantoic region (al) at d9.0 (D, right), whereas at d12 (E, right) this transcript is widely distributed (embryos hybridized with a sense probe are shown on the left of each panel).

FIG. 5

Murine chromosomal location of Sgce and Zac1. Partial chromosome 6 and 10 linkage maps showing the location of Sgce and Zac1 in relation to linked genes are shown. The number of recombinant N₂ animals over the total of N₂ animals typed together with the recombination frequency (genetic distance in centimorgans ± standard error) is shown for each pair of loci on the left of the map. Where no recombination was detected between loci, the upper 95% confidence limit of the recombination distance is shown in parentheses. The positions of homologous loci on human chromosomes are shown to the right.