Structural and functional characterization of the human PAX7 5'-flanking regulatory region

The human PAX7 gene is a member of the paired box containing gene family of transcription factors implicated in development of the skeletal muscle of the trunk and limbs as well as elements of the central nervous system. To understand the molecular mechanisms involved in its expression, we have localized the transcription start sites in adult skeletal muscle and functionally characterized the 5'-flanking regulatory region responsible for PAX7 expression in this tissue. The major transcription start was identified 664 bp upstream from the ATG codon using primer extension and 5'-rapid amplification of cDNA ends (5'-RACE). Analysis of the 5'-flanking sequence revealed the absence of a TATA-box and the presence of an inverted CCAAT-box. Several consensus sites for common transcriptional regulators including Oct-1, NF1, AP2, AP4, CREB, Sp1, Nkx2.5, and MyoD are present in the promoter region. To determine the sites critical for the function of the PAX7 promoter, a series of deletion fragments of the 5'-flanking region were cloned adjacent to luciferase reporter gene and expressed in RD, Cos-7 and JAR cell lines. The maximal promoter activity was achieved by a fragment extending from the position -403 to +373. No strong positive or negative regulatory elements were discovered by adding of further sequences (up to 2.97 kb). A polymorphic (CCT)(n) repeat sequence was found 107 bp upstream of the transcription initiation site. PCR-based systematic screening for length variations in 227 unrelated individuals of a Caucasian population showed a bimodal distribution of three alleles containing 8, 10 or 11 repeat units. When different variants of this PAX7 gene-linked polymorphic region (PAX7-LPR) were fused to a luciferase reporter gene and transfected into RD cells, the variant with 11 repeat units revealed higher transcriptional efficiency compared to the 8 or 10 repeat alleles.