435-bp liver regulatory sequence in the liver fatty acid binding protein (L-FABP) gene is sufficient to modulate liver regional expression in transgenic zebrafish.

Liver fatty acid binding protein (L-FABP) is a small protein that is thought to play an important role in the intracellular binding and trafficking of long chain fatty acids in the liver. Expression of the gene encoding the zebrafish liver fatty acid binding protein is regulated by a 435-bp distal region (-1944 to -1510) of the L-FABP promoter. The 435-bp sequence is sufficient for gene activation in the liver primordia (or bud) and continues to be active in the adult liver when positioned adjacent to the SV40 basal promoter and linked directly to green fluorescent protein. The 435-bp sequence region has two distinct liver regulatory elements, A (-1944 to -1623) and B (-1622 to -1510), and contains multiple putative consensus binding sites. The element A sequence includes two consensus HFH and one HNF-1alpha site and the element B sequence includes one consensus HNF-3beta site. Deletion of an internal 435-bp fragment (-1944 to -1510) including the A and B elements totally ablated the liver-specific activity of the zebrafish L-FABP gene promoter. Deletion of either of the two elements reduces the liver activity. Mutation of the HNF-1alpha site or either of the two HFH sites in the A element or the HNF-3beta site in the B element significantly altered specificity in the liver primordia of transient expression embryos. The importance of the HNF-1alpha consensus binding site in the A element and the HNF-3beta consensus binding site in the B element within the 435-bp distal region of the L-FABP promoter region suggests that combinatorial interactions between multiple regulatory factors are responsible for the gene expression of L-FABP in the liver.     

Effect of peroxisome proliferator-activated receptor alpha on human angiotensinogen promoter

The renin-angiotensin system plays a key role in the regulation of blood pressure. Angiotensinogen (ANG), mainly synthesized in the liver, is the first substrate of renin-angiotensin system. We had previously found that hepatocyte nuclear factor 4 (HNF-4) dramatically activates the human ANG promoter. It is generally known that HNF-4 and peroxisome proliferator-activated receptor alpha (PPARalpha) bind to response elements composed of two core motifs, RG(G/T)TCA, or a closely related sequence separated by 1 nucleotide (DR1 element). To examine whether or not PPARalpha activates the human ANG promoter, we used the reporter gene containing the sequence from -1222 to +44 of the human ANG gene promoter. PPARalpha and RXR heterodimer activated this promoter, and the PPARalpha responsive region was the same site that we had previously mapped as a binding site for HNF-4. Although the human ANG promoter was not induced by PPARalpha ligand bezafibrate in HepG2 cells, this reporter gene was inducible by bezafibrate treatment in HeLa cells, which do not express endogenous HNF-4. We suspected that the high level expression of HNF-4 in HepG2 cells might interfere with the effect of bezafibrate on the human ANG promoter. To confirm this model, we cotransfected HNF-4 expression vector with PPARalpha expression vector into HeLa cells. The bezafibrate-dependent activation of the ANG promoter was inhibited by HNF-4. These results suggest that PPARalpha and HNF-4 competitively affect the human ANG promoter through the C region.     

Germline hMLH1 promoter mutation in a Newfoundland HNPCC kindred

Hereditary non-polyposis colon cancer (HNPCC) is an autosomal dominant form of inherited predisposition to colorectal and other malignancies. It is associated with mutations in DNA mismatch-repair genes, especially hMSH2 and hMLH1. Management of HNPCC families is improved if the underlying mutation in each family can be discovered. We describe a Newfoundland kindred, meeting the Amsterdam Criteria for HNPCC, in which a mutation in the promoter region of the hMLH1 gene co-segregates with the disease phenotype. The -42C > T mutation is within a putative Myb proto-oncogene binding site. Using electrophoretic mobility shift assays, we demonstrated that the mutated Myb binding sequence is less effective in binding nuclear proteins than the wild-type promoter sequence. Using in vivo transfection experiments in HeLa cells, we further demonstrated that the mutated promoter has only 37% of the activity of the wild-type promoter in driving the expression of a reporter gene. The average age of onset in six family members affected with colorectal cancer is 62 years, which is substantially later than the typical age of onset in HNPCC families. This is consistent with a substantial decrease, but not total elimination, of mismatch repair function in affected members of this family. This is the first report of a heritable hMLH1 promoter mutation in any HNPCC family.     

Properties of the adenovirus type 40 E1B promoter that contribute to its low transcriptional activity

The adenovirus type 5 (Ad5) E1B promoter contains two elements essential for maximal activity, a TATA box and a GC box. The enteric adenovirus type 40 (Ad40) E1B promoter has a TATA box sequence identical to that of Ad5 and a GC box that fits the Sp1 binding site consensus. Nevertheless, Ad40 E1B RNA synthesis is severely impaired in HeLa cells, attributable in part at least to the weak transactivating activity of Ad40 E1A. However, the responsiveness of Ad40 early promoters to E1A transactivation has not been directly demonstrated. Using a transient expression assay with a chloramphenicol acetyl transferase (CAT) reporter gene, the Ad40 E1B promoter was very poorly transactivated by E1A of both Ad40 and Ad5 and showed only a limited response to the promiscuous varicella zoster virus transactivator p140. Construction of Ad5 recombinant viruses expressing the CAT gene under the control of the Ad5 or Ad40 E1B promoter allowed detection and measurement of expression from the Ad40 E1B promoter in a well-defined background and showed that overall activity is some 100-fold lower than for the Ad5 E1B promoter. Deletion analysis revealed that sequences upstream of the Sp1 binding site down-modulated Ad40 E1B promoter responsiveness, and two protein binding sites, identified by DNase footprinting and gel retardation assay, may be implicated in this effect. Gel shift analysis also showed that the Ad40 Sp1 binding site had a reduced affinity for Sp1 protein, relative to the Ad5 site, and that the context as well as the core sequence had an influence on Sp1 recognition.