| The mechanisms controlling the stage-specific and tissue-specific expression of the autosomal phosphoglycerate kinase gene (Pgk2) in eutherian mammals are not completely understood. Previous studies have shown that initial transcription of Pgk2 is known to be preceded by several events that occur in the following order: demethylation, appearance of general nuclease sensitivity, appearance of DNase I hypersensitive sites, and binding of tissue-specific transcription factors (Gebara and McCarrey, 1992; Ariel et al., 1994; Kumari et al., 1996; Kramer et al., 1998). Using a reporter construct in transgenic mice, it was originally shown that 515 bp of sequence upstream of the translational start site of the human Pgk2 gene is sufficient to direct tissue-specific and temporal expression similar to that seen for the endogenous mouse Pgk2 gene (Robinson et al., 1989). Therefore, all of the elements necessary for the aforementioned potentiation events (demethylation, chromatin decondensation, and transcription factor-binding) are presumably directed by signals encoded in this region. In later work, it was shown that 232 bp of 5' sequence is sufficient to direct appropriate testis-specific expression in transgenic mice (Zhang et al., 1999) and that 188 bp, containing only the core promoter of the Pgk2 gene, is capable of signaling the testis-specific demethylation event, despite being unable to drive transcription of a transgene in vivo (Zhang et al., 1998). Therefore, sequence(s) within the 188 bp core promoter are responsible for initiating the demethylation event that precedes transcription, but this potentiation, which may be a necessary prerequisite, is not, by itself, sufficient for transcriptional activation. The focus of this dissertation is to further study the demethylation that takes place over the Pgk2 promoter in vivo, delimit the sequence(s) responsible for this demethylation, and to investigate protein-DNA interactions that take place at the endogenous Pgk2 promoter involved in transcriptional activation. |
