A goal of this research project is to isolate genes undergoing unique epigenetic regulation in monoallelic expression. In general, epigenetic phenomena that achieve dosage control fall into three categories: 1) random X chromosome inactivation in female, 2) parental specific imprinting of selected autosomal genes, and 3) random autosomal inactivation, e.g. immunoglobulin and olfactory receptor genes. Based on the hypothesis that a significant fraction of genes expressed in extraembryonic tissues is dosage-controlled, we constructed cDNA libraries from E7.5 extraembryonic tissues, sequenced 3,000 cDNA clones, and mapped 155 new genes from this collection. We particularly focused on genes clustered in established imprinted regions in the mouse genomes. One of the genes we have studied is Nucleotide binding protein 2 (Nubp2), which is mapped to the developmentally important t- complex on chromosome 17 along with other nine genes from the same cDNA collection. Southern blot analyses of this gene using 129SV/J inbred mouse genomic DNA with the methylation-sensitive HpaII enzyme indicates the presence of two hemizygously methylated CpG sites in this gene. Allele-specific RT-PCR analyses of F1 hybrid animals revealed that the gene shows random monoallelic expression in single cells from hepatocytes and fibroblast cells. Thus, the gene belongs to an emerging category of epigenetic phenomena: random monoallelic expression of autosomal genes. Unlike five other genes so far enrolled in this category, the Nubp2 has no apparent physiological reason for monoallelic expression; but this finding may initiate an approach to understand both the mechanism of monoallelic gene expression and the unique features of the t-complex. - allele-specific expression; dosage control; maternal alleles; paternal alleles; allelic exclusion
