CHARACTERIZATION OF THE COMPLETE GENOMIC STRUCTURE OF THE HUMAN WNT-5A GENE, FUNCTIONAL-ANALYSIS OF ITS PROMOTER, CHROMOSOMAL MAPPING, AND EXPRESSION IN EARLY HUMAN EMBRYOGENESIS

We report the complete genomic organization of the human WNT-5A gene, which encodes a cysteine-rich growth factor involved in cell-cell sign aling during growth and differentiation. The gene comprises five exons with the terminal exon coding for a large 3'-untranslated region of a pproximate to 6.5 kilobase pairs and utilizes multiple polyadenylation signals to generate at least four discrete transcripts, We discovered a new leader exon interrupted by a 411-base pair intron that was reta ined in our original cDNA cloning, The promoter region was located in a GpC-rich island and harbored numerous cis-acting elements including several GC boxes and Sp1, AP1, and AP2 binding motifs, It lacked TATA or CAAT boxes typical of housekeeping and growth factor genes, In supp ort of this, primer extension revealed two transcription start sites, Transient cell transfection assays showed functional promoter activity for the 3,g-kilobase pair 5'-flanking region, Interestingly, internal and 5' deletions revealed that the distal promoter was not required f or full transcriptional activity and that the first 631 base pairs of WNT-5A harbored the strongest promoter activity. Using a panel of rode nt-human hybrid DNAs carrying portions of chromosome 3p, we mapped the gene to 3p14.2-p21.1, between a constitutional and a familial renal c ell carcinoma-associated translocation, In situ hybridization analyses of early human embryos at 28-42 days of gestation revealed that WNT-5 A transcripts were not restricted to the developing brain and limbs bu t were also observed in the mesenchyme bordering the pharyngeal clefts and pouches and in the developing gonads and kidneys. The relatively high expression in the celomic epithelium and in the precursors of fol licles and seminiferous tubules suggest a novel role for WNT-5A in ger m-cell differentiation. This study provides the molecular basis for di scerning the regulation of the WNT-5A gene and offers the opportunity to investigate genetic disorders linked to this important gene.