Two Sp1-binding Sites Mediate cAMP-induced Transcription of the Bovine CYP11A Gene through the Protein Kinase A Signaling Pathway

Abstract

Two sequence elements located at ؊111 to ؊100 base pairs and ؊70 to ؊50 base pairs in the 5-flanking region of the bovine CYP11A gene and in closely related positions in CYP11A of other species contain G-rich regions that are similar to the consensus Sp1-binding site. These sequences bind the purified transcription factor Sp1 as well as nuclear proteins from mouse Y1 adrenal cells that interact with an antibody specific for Sp1. Both of these CYP11A sequences support basal and cAMP-dependent transcription of reporter gene plasmids transfected into Y1 cells, and mutations within the G-rich ؊111/؊100-base pair sequence that reduce or eliminate the binding of Sp1-related Y1 nuclear proteins also markedly reduce cAMP-induced transcription. cAMPdependent transcription supported by both CYP11A sequence elements is mediated by protein kinase A at levels comparable to that promoted by different cAMPresponse sequences and transcription factors in other genes involved in steroidogenesis. These results indicate that ACTH-dependent regulation of cholesterol side chain cleavage cytochrome P450 levels in the adrenal cortex which is mediated through cAMP involves the ubiquitous transcription factor Sp1.

Overlapping Sp1 and AP2 binding sites in a promoter element of the lens-specific MIP gene

“…Therefore, Sp1 is likely to play an important regulatory role in cell differentiation during development. In fact, Sp1 is involved in tissue-specific expression of a variety of genes and functions synergistically with other transcription factors (20)(21)(22)(23)(24)(25)30,41,42). Genes encoding Sp1-related proteins, Sp2, Sp3 and Sp4, have been cloned, indicating the existence of a Sp family of transcription factors (18,19).…”

“…Therefore, the domain -65 to -39, proximal to the TATA box, contains an element required for activity of the MIP promoter. Analysis of this sequence shows the presence of a CT box at positions -49/-56, which has been shown to bind the Sp1 transcription factor in the CYP2D5 and the CYP11A genes (22,24). A sequence with similarity to the AP2 consensus binding site (26) is observed at positions -45/-51.…”

“…Sp1 is able to interact with the CT box of several promoters (21,22,24,41). However, as other members of the Sp family also interact with this motif (18,19) it was important to determine which member was involved in interaction with the CT box present at positions -55/-49 of the functional MIP promoter.…”

“…The Sp1 footprinted domains S-3 and S-5 contain a GA (CT) box in opposite orientations. This box, which binds Sp1 with lower affinity than the GC box (39), has been characterized as an Sp1 binding site in several genes (21,22,24,41). The Sp1 footprinted regions S-1 and S-6 contain a GT (CA) box.…”

“…In the present study we have characterized a cis regulatory element required for MIP gene (18,19), is present in lens nuclear extracts and also interacts with this Sp1 binding site. Even though Sp1 is a ubiquitous factor, it regulates tissue-specific expression of a variety of genes (20)(21)(22)(23)(24)(25). AP2 is expressed in selected tissues and is involved in epidermis-specific gene expression (26)(27)(28).…”