Functional Characterization of the HumanPAX3Gene Regulatory Region

Abstract

Spatiotemporal expression of thePAX3gene is tightly regulated during development. We have isolated and sequenced the 5′-flanking regulatory region of humanPAX3.Primer extension and ribonuclease protection mapping revealed that transcription is initiated from a single start site downstream of a TATA-like motif in human brain and peripheral tissues. Functional dissection of the gene's 5′-flanking region, which had been fused to a luciferase reporter gene and transiently expressed in rhabdomyosarcoma (RD) and cos-7 cells, indicated that the upstream region ofPAX3contains multiple positive and negativecis-acting regulatory elements. While the basal promoter is likely to be driven by two CCAAT boxes located at nucleotide positions −90 and −135, a cluster of regulatory elements acting as a strong repressor was detected between nucleotides −1200 and −650. Comparison of human and murine sequences revealed more than 90% identity in this segment. A polymorphic (CA)nrepeat sequence and a G/C substitution are located 337 bp and 328 bp upstream of the transcription start site, respectively. PCR-based systematic screening for length variations in 225 unrelated individuals of a Caucasian population showed a bimodal distribution of multiple alleles containing between 13 and 30 repeat units. Although the (CA)25variant of thisPAX3gene-linked polymorphic region (PAX3LPR) conferred lower transcriptional efficiency on thePAX3promoter, a regulatory impact of the PAX3LPR on PAX3 expression related to brain plasticity and function remains to be demonstrated.