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.     

Repression of enhancer activity by the adenovirus E1A tumor protein(s) is mediated through a novel HeLa cell nuclear factor.

We have examined the mechanism for the host cell-dependent repression of enhancer activity by the adenovirus early region 1A (E1A) proteins. The enhancer used in this study, from the human BK virus P2, functions efficiently in cis to activate expression from the adenovirus major late promoter in the human kidney cell line, 293, and in a monkey kidney cell line, MK2. In addition, enhancer activity can be stimulated by the E1A gene products in these cells. However, cis-enhancer activity is repressed in the HeLa cell line, and we demonstrate here that further repression can be induced by the E1A proteins. We show that the binding site for the negative regulatory factor involved in cis-repression, designated BK virus enhancer factor 1 (BEF-1), is also required for E1A-induced repression. Using gel mobility retardation assays, we demonstrated a 4-fold increase in active BEF-1 in nuclear extracts containing the E1A proteins. However, the E1A proteins did not change the binding pattern or the strength of binding of BEF-1 to its target sequence. BEF-1 was identified as a 98-kDa nuclear factor, and phosphorylation was shown to be important for DNA binding. Three potential nuclear factor 1 (NF-1) sites are present in the BEF-1-binding site. Using a known NF-1 site as competitor DNA in a gel mobility retardation assay, we provide evidence that BEF-1 may be a newly identified NF-1 family member. In addition, the sequence TGA present in the repressor-binding site was shown to be essential for high affinity binding of BEF-1. Overall, our data demonstrate that an enhancer can be repressed by the trans-activation of a negative regulatory factor.     

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.     

Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment

The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-κBα as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-κB were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-κB in TNF-induced VCAM-1 expression may be associated with its interaction with and phosphorylation by DNA-PK, which appears to be dominant over the requirement for p65 NF-κB activation. p50 NF-κB binding to its consensus sequence increased its susceptibility to phosphorylation by DNA-PK. Additionally, DNA-PK activity appeared to increase the association between p50/p50 and p50/p65 NF-κB dimers upon binding to DNA and after binding of p50 NF-κB to the VCAM-1 promoter. Analyses of the p50 NF-κB protein sequence revealed that both serine 20 and serine 227 at the amino terminus of the protein are putative sites for phosphorylation by DNA-PK. Mutation of serine 20 completely eliminated phosphorylation of p50 NF-κB by DNA-PK, suggesting that serine 20 is the only site in p50 NF-κB for phosphorylation by DNA-PK. Re-establishing wild-type p50 NF-κB, but not its serine 20/alanine mutant, in p50 NF-κB(-/-) fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Together, these results elucidate a novel mechanism for the involvement of DNA-PK in the positive regulation of p50 NF-κB to drive VCAM-1 expression.     

AML1, the target of chromosomal rearrangements in human leukemia, regulates the expression of human complement receptor type 1 (CR1) gene.

The human CR1 gene is expressed specifically in hematopoietic cells. It is suggested that some cell-type specific factors which involve in gene-specific activation or repression exist in cells according to the result that the gene expression varies differently depend on differentiation stage. Here, we demonstrate that the integrity of a polyomavirus enhancer core sequence, 5'-TGTGGT-3', is critical to the human CR1 promoter activity. AML1 is a site-specific DNA-binding protein that recognizes the enhancer core motif TGTGGT. We show that the AML1 binds specifically to this site and activates the human CR1 promoter. Furthermore, we demonstrate that the Ets binding site (GGAA) located 2 bp upstream of the AML1 site is also involved in the regulation of the human CR1 promoter activity. Point mutations of either the AML1 or the Ets binding site that abolish the binding of the respective factors result in significant decreases of the human CR1 promoter activity. These results suggest that AML1 and Ets proteins direct the expression of the human CR1 promoter.     

The mouse dystrophin enhancer is regulated by MyoD, E-box-binding factors, and by the serum response factor

In vivo studies in the mouse have revealed that the muscle promoter of the mouse dystrophin gene can target the right ventricle of the heart only, suggesting the need for other regulatory elements to target the skeletal muscle as well as other compartments of the heart. In this study we report the identification of the mouse dystrophin gene enhancer that is located approximately 8.5 kilobases downstream from the mouse dystrophin gene muscle promoter. The enhancer was tested in myogenic G8, H9-C2, and nonmyogenic 3T3 cell lines and is mostly active in G8 myotubes. Sequence analysis of the mouse dystrophin gene enhancer revealed the presence of four E-boxes numbered E1-E4, a putative mef-2 binding site, and a serum response element. Site-directed mutagenesis studies have shown that E-boxes 1, 2, and 3 as well as the serum response element are required for enhancer activity. Gel shift analysis revealed two binding activities at binding sites E1 and E3 which were specific to myotubes, and supershift assays confirmed that myoD binds at both these sites. Our study also shows that werum response factor binds the serum response element but in myoblasts and fibroblasts only, suggesting that serum response factor may repress enhancer function.