Parental origin-specific expression of Mash2 is established at the time ofimplantation with its imprinting mechanism highly resistant to genome-widedemethylation

The Mash2 gene encodes a basic helix-loop-helix transcription factor, which is highly expressed in diploid trophoblast cells of the postimplantation m ouse embryo and is required for development of the spongiotrophoblast in or der to form a functional placenta. Genomic imprinting of Mash2 has been pre viously reported; transcriptional inactivation of the paternal wild-type al lele in heterozygotes carrying a maternal null allele results in a null-equ ivalent embryonic lethal phenotype. In order to study the Mash2 imprinting mechanism, we have created a new allele at this locus carrying a targeted i nsertion of an IRES (internal ribosome entry site)-lacZ cassette within the 3' untranslated region of the gene (referred to as ''Mash2-lacZ"). This ne w allele has made it feasible to monitor paternal Mash2 expression in a wil d-type-equivalent background. Our data suggest that parental origin-specifi c expression of Mash2 begins in the early postimplantation conceptus (5.5 d pc) at the time when trophoblast-specific expression is observed. We also s how that the paternal allele is continuously repressed up to 9.5 dpc in the developing ectoplacental cone (EPC) and early chorio-allantoic placenta, w ith some cells escaping paternal repression. When maternally inherited, lac Z expression from this allele reflects the expression pattern of endogenous MashZ transcripts up to 8.5 dpc. Furthermore, we have addressed the questi on of a requirement for DNA methylation for the Mash2 imprinting mechanism by crossing our Mash2-lacZ mice with mice mutant for Dnmt1 (DNA-methyltrans ferase1). 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-speci fic 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 MashZ locus is highly demethylated in Dnqmt1 null mutant embryos. The locus is also highly demythyled in wild-type EPCs . Our results suggest the possibility that a mechanism other than DNA methy lation, such as allele-specific chromatin conformation, may be involved in maintenance of parental origin-specific expression of Mash2. (C) 1999 Elsev ier Science Ireland Ltd. All rights reserved.