Specific methylation of cytosine residues at the CpG sites in mammalian DNA appears to be involved in inactivation of gene expression and to play a role in tissue-and developmental stage-specific gene expression by providing additional genetic regulation. However, the mechanisms by which methylation influences gene expression and the mechanisms that control methylation itself are unknown. We have isolated the intracisternal A-particle (IAP) gene, the genome of an endogenous retrovirus, whose expression is activated during early murine embryogenesis and during differentiation of F9 embryonal carcinoma (EC) cells. The latter resembles embryonic ectoderm and upon induction with retinoic acid (RA) and dibutyryl cyclic AMP (cAMP), differentiate into cells that resemble parietal endoderm (PE). Using the well established EC cell model system, we have identified an IAP upstream enhancer (IUE) located at the -186/-167 region which is active in both undifferentiated and differentiated cells. A 65 kD IUE binding protein (IUEB) present in both undifferentiated and differentiated cells was also identified. Site-specific in vitro methylation within the IUEB binding site strongly inhibited both IUEB binding and IUE transcriptional activity, suggesting the IUEB is a transcriptional activator and that methylation plays a role in specifically regulating IAP expression. In this application, we will extend our findings to investigate mechanisms of methylation- mediated transcriptional repression and IUEB-mediated transcriptional activation by: (1) deriving F9 EC cell lines that stably express the IAP promotor-driven beta-galactosidase (gal) gene linked to a neomycin- resistant (neo) gene and using them to investigate (by beta- galactosidase enzyme assays and by polymerase chain reaction (PCR) and genomic sequencing) changes in the patterns of in vivo methylation states and protein binding profiles associated with transcriptional activation of the IAP promoter during EC cell differentiation into PE- like cells; (2) mapping the cis-acting elements regulating demethylation by deletion analysis to elucidate the mechanisms that control methylation; (3) isolating IUEB-encoding cDNA clones from lambda gt11 (lambda gt11) expression libraries using IUE as a probe and characterizing the cDNA clones for their DNA binding specificity in binding assays, binding activity to methylated and hemimethylated IUE by bandshift assays, the mRNA expression patterns by Northern hybridization to assess the possible role of IUEB in development, the DNA sequence for its possible function, transcriptional activity and the activation domain by co-transfection of the IUEB fusion gene expression plasmids and a reporter gene, and the DNA binding domain by binding deletion polypeptide mutants to IUE; (4) testing whether IUEB is phosphorylated and/or dimerized by immunoprecipitating the metabolically-labeled proteins with antisera produced from the cloned proteins to investigate the mechanism by which IUEB mediates transcriptional activation. The proposed studies should contribute to an understanding of the role of DNA methylation in gene expression and thus development.
