Distal control of the whey acidic protein (WAP) locus was studied using a transgenic approach. A series of pig genomic fragments encompassing increasing DNA lengths upstream of the mammary specific whey acidic protein (WAP) gene transcription start point (tsp) and 5 kb downstream were used for microinjection in mouse fertilized eggs. Our data pointed out three regions as potent regulators for WAP but not for RAMP3 gene expression (a non mammary-specific gene located 30 kb upstream of the WAP gene). WAP gene activating elements were present in the -80 kb to -30 kb and -145 kb to -130 kb regions whereas inhibitors were present in the -130 kb to -80 kb region. The stimulatory regions were characterized by peaks of histone H4 acetylation and a poor nucleosome occupancy in lactating sow mammary glands but not in liver. These data reveal for the first time the existence of several remote potent regulatory regions of the pig WAP gene.
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