Determination of transcription factors and their possible roles in the regulation of Pax3 gene expression in the mouse B16 F1 melanoma cell line

Abstract

The objective of this study was to determine which transcription factors regulate the expression of the Pax3 gene in the mouse B16 F1 melanoma cell line. The results showed that the -14 kilobase pair (kbp) Pax3 promoter, but not the -1.6 kbp Pax3 promoter, promoted Pax3 gene expression in B16 cells. Comparison of the sequence of the -14 kbp human Pax3 promoter with mouse Pax3 promoters indicated that homology sequences were located between -6.9 and -5.8 kbp, and also that the 1.1 kbp fragment (between -6.9 and -5.8 kbp), linked -1.6 kbp proximal to the Pax3 promoter [plasmid PGPax3PIV (N6.9/5.8) delta SST Lacz], could mimic the functions of plasmid PGPax3 -14(N-1.6) Lacz. Mutations of the core binding elements of either Pax3 site I or II or both sites I and II reduced significantly the beta-galactosidase (beta-gal) activity in the cells. However, mutations of the core binding sequences of site A or B increased significantly the beta-gal activity in the cells. Biochemistry analysis demonstrated that POU transcription factors (Oct-1 and Brn-2) bind to the specific binding elements of both sites I and II to stimulate Pax3 gene expression, whereas the TALE homeodomain-containing proteins (Pbx and Prep1) bind with the core binding sequences of sites A and B to repress the expression of the Pax3 gene in B16 cells.