Functional analysis of the trans-acting factor binding sites of the mouse alpha-fetoprotein proximal promoter by site-directed mutagenesis.

The trans-acting factors of the mouse alpha-fetoprotein proximal promoter (-202 base pairs) are aligned as follows: regions Ia (HNF-1), Ib (C/EBP), II (NF-1 or C/EBP), II' (NF-1 or HNF-1), III (NP-III), IV (NP-IV), Va (NP-Va), and Vb (C/EBP). Site-specific mutation abolished protein binding to the corresponding mutated site with the exception of the NF-1 site, in which mutation causes partial protection. Transient expression analyses indicate that chloramphenicol acetyl-transferase (CAT) activity is reduced by mutations in regions Ia, II', Ib, II, and IV. Mutation of region III causes an increased activity and mutation of regions Va and Vb shows a slight inhibitory effect. Linking alpha-fetoprotein enhancer I to the wild type promoter resulted in a 12-fold stimulation of CAT activity. The activity of promoters with mutated C/EBP-binding sites (Ib, II, and Vb), was slightly above controls, indicating that enhancer I can reverse the effect of these mutations. Inhibition or stimulation of promoter activity resulting from mutations of the HNF-1 or NP-III binding sites, respectively, persisted when enhancer I was linked to the promoters, indicating that enhancer I cannot rescue these mutations. Mutation of both HNF-1-binding sites resulted in greater than 90% inhibition of CAT expression with and without enhancer I, indicating these sites are essential for promoter activity. The stimulation of promoter activity by mutation of the NP-III site suggests that this site may be essential for repression or attenuation of the alpha-fetoprotein gene. Our studies indicate that regulation of the alpha-fetoprotein gene requires the combinatorial effect of multiple cis- and trans-acting elements in the proximal promoter and that enhancer I may provide a factor(s) that specifically rescue the promoter from the inhibitory effect of mutation in the C/EBP-binding sites.     

Analysis of basal regulatory elements in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene promoter

5'-Deletion analysis of the 1.7-kb mouse 1alpha-hydroxylase gene promoter reveals that the minimal promoter region for basal activity is -85/+22 and requires a functional CCAAT element. Mutational analysis also demonstrates that deletion of the internal promoter region from nucleotides -1125 to -86 leads to a 25- to 30-fold increase in basal promoter activity. The increased activity is not the result of positional effects, but is caused in part by the removal of an AC repeat. Further analysis of the promoter revealed an enhancer element localizes to an upstream region -1385 to -1125, which contains three consensus AP-1 sites. Deletion of the most proximal AP-1 site causes a 60% loss of enhancer activity. The data suggest the presence of the AC repeat prevents the full potential activation of the 1alpha-hydroxylase promoter by factors binding to AP-1 sites.     

Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells.

The proximal rat prolactin (rPRL) promoter contains three cell-specific elements, designated footprints I, III, and IV, which restrict rPRL gene expression to anterior pituitary lactotroph cells. Footprint II (-130 to -120) binds a factor, which we have termed F2F, present in pituitary and nonpituitary cell types. Here we demonstrate that a key role of the footprint II site is to inhibit rPRL promoter activity in nonpituitary cells, specifically, by interfering with the basal activating function of a vicinal element. Gene transfer analysis revealed 20-fold activation of the rPRL promoter in nonpituitary cell types when footprint II was either deleted or specifically mutated. Similar activation of the intact rPRL promoter was obtained by in vivo F2F titration studies. In GH4 rat pituitary cells, the footprint II inhibitory activity was masked by the redundant, positively acting cell-specific elements and was inhibitory only if the two upstream sites, footprints III and IV, were deleted. Deletion of the -112 to -80 region in the footprint II site-specific mutant background resulted in complete loss of rPRL promoter activity in both pituitary and nonpituitary cell types, mapping a basal activating element that is operative irrespective of cell type to this region. While the basal activating element imparted an activating function in a heterologous promoter assay, the footprint II sequence did not display any inherent repressor function and actually induced several minimal heterologous promoters. However, the inhibitory activity of the footprint II site was detected only if it was in context with the basal activating element. These data underscore the importance of ubiquitous activating and inhibitory factors in establishing cell-specific gene expression and further emphasize the complexity of the molecular mechanisms which restrict gene expression to specific cell types. We provide a novel paradigm to study rPRL promoter function and hormone responsiveness independently of lactotroph cell-specific requirements.     

Regulation of human microsomal prostaglandin E synthase-1 by IL-1β requires a distal enhancer element with a unique role for C/EBPβ

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1β (interleukin 1β) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPβ (CCAAT/enhancer-binding protein β) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPβ binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPβ in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPβ highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.     

Functional interaction of nuclear factors EF-C, HNF-4, and RXR alpha with hepatitis B virus enhancer I.

Hepatitis B virus (HBV) enhancer I contains cis-acting elements that are both sufficient and essential for liver-specific enhancer function. The EF-C binding site was previously shown to be a key element in enhancer I. EF-C binding activity is evident in hepatic and nonhepatic cells. Although the EF-C binding site is required for efficient HBV enhancer I function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. We have defined a novel region in HBV enhancer I, termed the GB element, that is adjacent to and functions in conjunction with the EF-C binding site. The GB element and EF-C site confer interdependent liver-specific enhancer activity in the absence of flanking HBV enhancer sequences. The nucleotide sequence of the GB element is similar to sequences of the DNA binding sites for members of the steroid receptor superfamily. Among these proteins, we demonstrate that HNF-4, RXR (retinoid X receptor), and COUP-TF bind to the GB element in vitro. HNF-4 transactivates a promoter linked to a multimerized GB/EF-C domain via the GB element in vivo in a manner that is dependent on the integrity of the adjacent EF-C binding site. RXR alpha also transactivates promoter expression via the GB element in vivo in response to retinoic acid but in a largely EF-C-independent manner. Finally, we show that COUP-TF antagonizes the activity of the GB element in human liver cells.     

A novel E box/AT-rich element is required for muscle-specific expression of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene.

The cardiac/slow twitch sarcoplasmic reticulum (SR) Ca2+-ATPase gene (SERCA2 ) encodes a calcium transport pump whose expression is regulated in a tissue- and development-specific manner. Previously we have identified two distinct positive regulatory regions (bp -284 to -72 and -1815 to -1105) as important for SERCA2 promoter activity. Here we demonstrate that the SERCA2 distal promoter region functions like an enhancer by activating a heterologous promoter (TK) in a muscle cell-specific manner. Through deletion analysis a core enhancer region was delimited to the -1467 to -1105 bp fragment. We identified the E box/AT-rich element located at -1115 bp as critical for maximal enhancer activity. Gel mobility shift studies revealed that this E box/AT-rich element specifically binds a protein which is induced during Sol8 myogenesis. This region includes two other cis -acting elements, CArG and MCAT, which also bind specific nuclear protein complexes from Sol8 myotubes. Mutagenesis of each of these sites resulted in decreased SERCA/TK-CAT promoter activity. Based on these data, we propose that the E box/AT-rich element may contribute along with CArG and MCAT elements to the overall activation and regulation of the SERCA2 gene promoter.     

The role of proximal-enhancer elements in the glucocorticoid regulation of carbamoylphosphate synthetase gene transcription from the upstream response unit

As part of the urea cycle, carbamoylphosphate synthetase (CPS) converts toxic ammonia resulting from amino-acid catabolism into urea. Liver-specific and glucocorticoid-dependent expression of the gene involves a distal enhancer, a promoter-proximal enhancer, and the minimal promoter itself. When challenged with glucocorticoids, the glucocorticoid-responsive unit (GRU) in the distal enhancer of the carbamoylphosphate-synthetase gene can only activate gene expression if, in addition to the minimal promoter, the proximal enhancer is present. Here, we identify and characterise two elements in the proximal CPS enhancer that are involved in glucocorticoid-dependent gene activation mediated by the GRU. A purine-rich stretch forming a so-called GAGA-box and a glucocorticoid-response element (GRE) are both crucial for the efficacy of the GRU and appear to constitute a promoter-proximal response unit that activates the promoter. The glucocorticoid response of the CPS gene is, therefore, dependent on the combined action of a distal and a promoter-proximal response unit.