The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28

The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.     

Characterization of the human aldose reductase gene promoter

The promoter region of the human aldose reductase gene has been identified upstream of the translation start ATG codon. The promoter contains a TATA box, a CCAAT promoter element, and three Sp1 protein binding consensus sequences upstream of the capsite. A 640-base pair insert spanning +31 to -609 directs expression of the reporter gene chloramphenicol acetyltransferase in an orientation-specific manner in transfected Hep G2 cells. The promoter activity remained constant with deletions from base pairs -609 to -186. The TATA and the CCAAT consensus sequences show significant promoter activity, whereas the three Sp1 binding consensus sequences, individually, have no significant promoter activity. A GA-rich region (-186 to -146) contains two CGGAAA/G motifs, which show promoter activity and interaction with Hep G2 nuclear extract and GA-binding proteins (GABP alpha and GABP beta 1) as shown by mobility shift assays and DNase I footprinting. Similar cis-elements in herpes simplex virus type 1 interact with rat liver GABP and the viral VP16 protein to mediate the induction of immediate early viral genes. A GC-rich region (-87 to -31) is identified by mobility shift assay, and a consensus sequence of an androgen response element is present at -396 to -382. The human aldose reductase promoter, thus, has regulatory response elements that may be important during early development and puberty. These regulatory elements may play a significant role in the development of certain diabetic complications.     

An NFAT-dependent enhancer is necessary for anti-IgM-mediated induction of murine CD5 expression in primary splenic B cells

CD5 is a 67-kDa membrane glycoprotein the expression of which in murine splenic B cells is induced by surface IgM cross-linking. To analyze this induction, we transiently transfected primary splenic B cells with luciferase reporter constructs driven by various wild-type and mutated CD5 5'-flanking sequences. The transfected cells were subsequently cultured in medium with or without F(ab')2 anti-IgM (anti-IgM), and luciferase expression was assayed. Using this approach, we identified a 122-bp enhancer element necessary for anti-IgM-mediated induction of the CD5 promoter. Electrophoretic mobility shift assays indicated that four inducible and four constitutive complexes form on the enhancer fragment in nuclear extracts of primary B cells. Supershift assays revealed that two of the inducible complexes contained NFATc. Point mutations that abolished NFAT binding severely impaired enhancer function. Thus, CD5 is a target of NFAT in B cells. A third inducible complex required an intact H4TF-1 site. One of several constitutive complexes required an intact Ebox site while a second required an intact putative ets binding site. Mutation of the H4TF-1, Ebox, and Ets sites, in the presence of wild-type NFAT sites, significantly reduced the activity of the enhancer. Therefore, the induction of B cell CD5 expression requires NFAT binding and binding to at least one of three additional sites in the CD5 enhancer.