Role of the Liver-Enriched Transcription Factor Hepatocyte Nuclear Factor 1 in Transcriptional Regulation of the Factor VIII Gene

Abstract

Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5 untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) ϳ300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates ϳ1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide ؊44 results in a significant but small increase in promoter activity. The activity of each of the various 5 deletion constructs is significantly enhanced by cotransfection of C/EBP␣ and D-site-binding protein expression plasmids, while cotransfection of both C/EBP␣ and C/EBP␤ plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides ؊740 and ؊1002. Since the minimal promoter sequence (؊44 to ؉148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5 untranslated region.

Characterization of the Bovine Prion Protein Gene: The Expression Requires Interaction between the Promoter and Intron.

“…The downstream region of the transcription start site may contribute to tissue-specific positive or negative regulatory activity and increase or decrease synergistically the activity of the promoter in the 5'-flanking region of exon 1 [2,4,21,29,35]. Therefore, intron 1 may also contribute to the expression of the bovine PrP gene.…”

The Human and Mouse GATA-6 Genes Utilize Two Promoters and Two Initiation Codons

“…This is much greater than the homology between noncoding exons of most other mammalian genes (or GATA-6 exon Ib), suggesting that this region may have an evolutionarily conserved regulatory function. Although the promoters of cellular genes are classically assumed to be 5Ј to the transcriptional initiation site, there are several examples of regulatory DNA motifs, which bind transcription factors, within the transcription unit itself (43)(44)(45)(46). Moreover, sequences within noncoding exons have in a number of cases been shown to affect the rate of transcription initiation (47)(48)(49)(50).…”

An enhancer/promoter combination strengthens the expression of blood-coagulation factor VIII in non-viral expression vectors

“…The promoters were EF1-α, CMV, hAAT, and fviii. To better focus the experiments, we used truncated hAAT and fviii promoter sequences that retained the minimal transcription initiation function (Mendel et al, 1991;McGlynn et al, 1996;Kalsheker et al, 2002). We found that all four promoters were capable of directing FVIII expression in hepatocyte cell lines, as well as in Hek-293 and CHO cells.…”

“…However, FVIII is also expressed at moderate levels in the spleen, kidney, and lymph nodes (McGlynn et al, 1996). Transcriptional activity of both the minimal fviii promoter and the minimal hAAT promoter probably requires hepatocyte nuclear factor 1 (HNF1α), and it is enhanced by the absence of its homologous isoform HNF1β (Mendel et al, 1991;McGlynn et al, 1996).…”

“…For the fviii promoter, a minimal sequence consisting of nucleotides -44 to +148 was found to retain the wild-type transcriptional activity in human hepatocellular carcinoma (HepG2) cells (McGlynn et al, 1996). This promoter was used to form vector pcDNA-HCR-fviii-FVIIIDB.…”