Abstract
Previous studies of bovine CYP11B1 gene regulation revealed six cis-acting elements, Ad1, Ad2, Ad3, Ad4, Ad5, and Ad6, in the 5' upstream region of the gene. Ad4 site was a positive transcription element in the stimulation by cAMP. Ad4-binding protein (Ad4BP) was purified from the nuclear extract of bovine adrenal cortex using affinity latex particles conjugated with polymerized Ad4 sequences. The molecular mass of the purified Ad4BP estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa. To characterize the binding specificity of Ad4BP, oligonucleotides homologous to Ad4 sequence and AGGTCA containing sequences in the promoter regions of steroidogenic P-450s were synthesized and used for gel shift analyses as competitors. The competition experiments revealed that Ad4BP bound not only to (C/T)CAAGG(T/C)(C/T), which was originally identified as the Ad4 binding site, but also to (Pu)PuPuAGGTCA. All the steroidogenic P-450 genes examined had at least one Ad4BP binding sequence. Experiments with model sequences containing various nucleotide substitutions established that (C/T)CAAGG(T/C)CA is the strongest binding sequence for Ad4BP. The expression of Ad4BP was examined with adrenal cortex cells and several other steroidogenic and nonsteroidogenic cells. Only the steroidogenic cells, the granulosa cells of bovine ovary, and I-10 cells derived from mouse Leydig cells, expressed the binding activity to Ad4 site. The presence of Ad4 site as a common cis-acting element in the genes of all the steroidogenic P-450s and the steroidogenic tissue-specific expression of Ad4BP strongly suggests that Ad4BP is an indispensable transcription factor for the expression of all the steroidogenic P-450 genes.
Highlights
The was purified to homogeneity by the latex particlesconjugated sequences mouse CypllA (MSC-a) and MSC-b examined in this report coinwith polymerized Ad4 site, which was originally identified in cide with SF-1 and SF-2 sequences, respectively
Since the bovine CYPIIBI gene promoter,thepurifiedprotein was sequences correspondingto MSC-a are highly conserved shown to have binding activity for Ad5 as well as Ad4 site. among theCYPIIA genes of mouse, rat, bovine, and human, Since we previously observed that anoligonucleotide contain- the sequence is supposed to have a significant role for traning Ad4 site inhibited the binding of a factor in the adrenal scription
F1, and the common core sequence shared by Ad4BP and hormone receptors suggest that Ad4BP might be one of zincfinger type DNA-binding proteins
Summary
Purification of Ad4BP-In previous papers (19-21), we nucleotidescontaining 5' protrudingend dCTP (110 TBq/mmol, Amersham, United fragment (Takara Shuzo Co., Ltd., Kyoto, were labeled by [ C U - ~ Kingdom) with Klenow Japan) and used for gel. A DNA fragment (-145 to -407) prepared from bovine CYPllBl gene promoter was labeled by kinase reaction with [y-32P]ATP(222 TBq/mmol, Amersham, United Kingdom) and used as the probe. Purification of Ad4-binding Protein (Ad4BP)"Bovine adrenal cortex (1.0-1.5 kg) was homogenized with homogenization buffer (10 mM HEPES-NaOH (pH7.6), 10 mM KCl, 1.0 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, 1.0 mM EDTA, 10%glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT, and 0.5 pg/ml each leupeptin terns of the proteins in the eluates from a heparin-agarose column and two cycles of the affinity steps areshown in Fig. 1.After twocycles of the affinity purificationa,single protein band was observedon SDS-PAGE, whose molecular masswas estimated as 53 kDa from thme obility on the gel. The washing of the nuclear pellet as described above was performed at least three the sixcis-acting elements of CYPllBl gene promoter clearly differed from one another. The Ad5 sequence conserved among bovine (-209 to -2051, human (-118 to -114), and mouse (-104 to -100) CYPllBl
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