### Transcriptional regulation of the murine PGK-1 gene.

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 Title: Transcriptional regulation of the murine PGK-1 gene. Authors: Sutherland, Leslie C. Date: 1994 Abstract: The gene encoding the glycolytic enzyme phosphoglycerate kinase is transcriptionally regulated at two levels. Expression of enzyme is related to the glycolytic activity of the cell, and is highest in transcriptionally active cells. Expression is also regulated by X chromosome inactivation, as the somatically expressed Pgk-1 gene is X-linked. The role of 5$\sp\prime$-flanking cis-acting DNA elements and trans-acting factors in the regulation of Pgk-1 expression was examined. The murine Pgk-1 gene contains an upstream activator sequence (UAS) in its 5$\sp\prime$-flanking region. This region was found to be responsible for elevating transcription levels at least ten-fold above basal Pgk-1 promoter levels in P19 embryonal carcinoma (EC) cells. Part of this activity was attributed to the R2 protein binding site, first identified by DNase 1 footprinting techniques. Mutation of the middle region of R2 resulted in a 5-fold reduction in expression of a Pgk-1 driven construct in stable transfection experiments into P19 cells. It was also determined that the R2 site was not important for transcription in P19 cells induced to differentiate with retinoic acid (RA). In undifferentiated P19 cells, another UAS protein binding site, R1, was identified by band shift analysis. R1 could not be detected by footprint analysis, suggesting that the affinity of binding at R1 was lower than at R2. The mutation of the R2 site did not abolish protein binding, which led to the hypothesis that multiple factors were binding the DNA at R2. R1 was also hypothesized to interact with multiple factors. However, fractionation of the P19 nuclear extract and use in band shift studies against the R1 DNA resulted in a single fraction with binding activity, suggesting a single R1 DNA binding protein and a non-DNA binding component. This non-DNA binding component at R1 was found to be tissue- or species-specific. Southwestern analysis in conjunction with fractionation experiments suggested that one of the R2 DNA binding proteins was approximately 70 kD and that the R1 DNA binding protein was 120 kD. Treatment of the P19 cells with RA led to a reduction in gene expression. Two days after exposure to the drug, the contribution to expression from the UAS was reduced by 50%, and four days after exposure the UAS no longer contributed to gene expression. Protein binding to the UAS was also altered after RA-treatment. A new site of protein interaction was detected in the distal region of the UAS, at R3, and binding at R1 was altered. There was, therefore, a correlation between protein interaction within the UAS and gene expression during differentiation. The results presented in this thesis demonstrate that the regulation of PGK-1 occurs, at least in part, at the level of gene expression, and that the UAS has an important role to play in regulating expression levels of the gene during differentiation. The results also suggest that the transcriptional stimulatory activity of the UAS depends on higher-order interactions between multiple low affinity DNA binding proteins which change upon differentiation. URL: http://hdl.handle.net/10393/10111http://dx.doi.org/10.20381/ruor-16663 Collection Thèses, 1910 - 2010 // Theses, 1910 - 2010