The Mash2 gene encodes a basic helix-loop-helix transcription factor, which is highly expressed in diploid trophoblast cells of the postimplantation mouse embryo and is required for development of the spongiotrophoblast in order to form a functional placenta. Genomic imprinting of Mash2 has been previously reported; transcriptional inactivation of the paternal wild-type allele in heterozygotes carrying a maternal null allele results in a null-equivalent embryonic lethal phenotype. In order to study the Mash2 imprinting mechanism, we have created a new allele at this locus carrying a targeted insertion of an IRES (internal ribosome entry site)-lacZ cassette within the 3' untranslated region of the gene (referred to as "Mash2-lacZ"). This new allele has made it feasible to monitor paternal Mash2 expression in a wild-type-equivalent background. Our data suggest that parental origin-specific expression of Mash2 begins in the early postimplantation conceptus (5.5 dpc) at the time when trophoblast-specific expression is observed. We also show that the paternal allele is continuously repressed up to 9.5 dpc in the developing ectoplacental cone (EPC) and early chorio-allantoic placenta, with some cells escaping paternal repression. When maternally inherited, lacZ expression from this allele reflects the expression pattern of endogenous Mash2 transcripts up to 8.5 dpc. Furthermore, we have addressed the question of a requirement for DNA methylation for the Mash2 imprinting mechanism by crossing our Mash2-lacZ mice with mice mutant for Dnmt1 (DNA-methyltransferase1). Our results show a partial loss of transcriptional repression of the paternal allele in Dnmt1 deficient background. Interestingly, however, this is not sufficient to eliminate the highly biased parental allele-specific expression of Mash2. Thus, the preferential maternal expression of the gene is still maintained in Dnmt1 null mutant embryos, although methylation analyses demonstrate that the Mash2 locus is highly demethylated in Dnmt1 null mutant embryos. The locus is also highly demythyled in wild-type EPCs. Our results suggest the possibility that a mechanism other than DNA methylation, such as allele-specific chromatin conformation, may be involved in maintenance of parental origin-specific expression of Mash2.
Dietary inadequacy of folate enhances and folate supplementation suppresses colorectal carcinogenesis in the dimethylhydrazine rat model. Folate is an essential factor for DNA methylation and the de novo biosynthesis of nucleotides, aberrations of which play important roles in mutagenesis. This study investigated whether the mutational hot spots of the Apc and p53 genes for human colorectal cancer are mutated in dimethylhydrazine-induced colorectal neoplasms and whether dietary folate can modulate mutations in these regions. Rats were fed diets containing 0, 2 (basal requirement), 8 or 40 mg folate/kg diet. Five weeks after diet initiation, dimethylhydrazine was injected weekly for 15 weeks. Mutations were determined by direct sequencing in 11 low and seven high grade dysplasias and 13 invasive adenocarcinomas. A total of six Apc mutations were found in four dysplastic and carcinomatous lesions: two in two low grade dysplasias, two in one high grade dysplasia and two in one adenocarcinoma. All mutations were single base substitutions, four of which were A:T-->G:C transitions. Five of the six mutations were located upstream from the region corresponding to the human APC mutation cluster region. Dietary folate had no effect on the frequency and type of Apc mutations. No mutations were detected in exons 5-9 of the p53 gene in neoplastic lesions. These data suggest that in the dimethylhydrazine rat model of colorectal cancer, the Apc gene is mutated in early stages, albeit to a lesser degree than observed in human colorectal cancer, whereas the mutational hot spot of the p53 gene for human colorectal cancer is not commonly mutated. Although the low frequency of Apc mutations and the small number of neoplasms studied in this study might have precluded our ability to observe modulatory effects of folate, dietary folate appears to have no significant effect on Apc and p53 mutations.
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