A PROMOTER WITHIN INTRON-35 OF THE HUMAN C4A GENE INITIATES ABUNDANT ADRENAL-SPECIFIC TRANSCRIPTION OF A 1 KB RNA - LOCATION OF A CRYPTIC CYP21 PROMOTER ELEMENT

Complement component C4 is encoded by two nearly identical genes, C4A and C4B, that encode a C4 precursor that is proteolytically cleaved in to the alpha, beta and gamma subunits of the mature protein. C4 is exp ressed primarily in liver and to a much lesser extent in immune cells. We have identified a unique 1 kb RNA transcript, termed Z, that arise s from a cryptic promoter lying in the intron between exons 35 and 36 of the C4 gene. Primer extension, RNase protection, and 5' RACE experi ments locate the cap site in intron 35, 55 bases upstream from exon 36 . Northern blotting and RNase protection assays show that expression o f this 1 kb Z RNA transcript is confined to the adrenal gland. Z RNA c ontains the same open reading frame as C4 which predicts a protein of 131 amino acids, but antisera to C4 do not interact with epitopes on t his protein when it is synthesized by cell-free translation, hence the presence or absence of a Z protein in vivo could not be determined. T ransfection of Z promoter/reporter constructs into human adrenal NCI-H 295 cells shows that most if not all of the sequences required for hig h-level adrenal expression lie within 235 bases upstream from the cap site, but that this region is inactive when transfected into COS-1, JE G-3 and Hep-G2 cells, suggesting it contains an adrenal-specific eleme nt. The 222 bases upstream from the cap site are 75% identical in the human C4A and mouse Slp genes, and contain a potential binding site fo r steroidogenic factor 1 (SF-1), an orphan zinc-finger nuclear recepto r. We propose that this region, like a nearby region in the mouse geno me, functions as an upstream element of the P450c21 promoter, and may be a component of an adrenal-specific locus-control region.