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Transcriptional regulation of the human HFE gene indicates high liver expression and erythropoiesis coregulation

CATHERINE MURA^*,,1, GÉRALD LE GAC^*,, SANDRINE JACOLOT^*, and CLAUDE FÉREC^*,,

^* INSERM U613 Génétique Moléculaire et Génétique Épidémiologique,
UBO; and
EFS, Brest, France

¹ Correspondence: INSERM U613, Génétique Moléculaire, UBO, 46 rue Félix Le Dantec, Brest 29200, France. E-mail: catherine.mura{at}univ-brest.fr

SPECIFIC AIM

The human HFE gene is clearly involved in hereditary hemochromatosis, a common autosomal recessive genetic disorder of iron homeostasis, characterized by excessive intestinal iron absorption and progressive iron overload. HFE should be a key component of human iron homeostasis, but contrary to other genes involved in rare hemochromatosis cases, its precise role is still under investigation. To obtain new insight, we analyzed the transcriptional regulation of the HFE gene and defined the functional organization of the HFE promoter.

PRINCIPAL FINDINGS

1. Transcription initiation analysis of the HFE gene
Run off in vitro transcription using liver nuclear extract and 5' rapid amplification of cDNA ends revealed multiple transcriptional start sites, defining two strong sites clearly evidenced with liver extract and a window of initiation within the –120/–10 region. Sequence analysis of the 5'-flanking region of HFE revealed an AT-rich element (TTTAAA) around 35 bp upstream from both transcription start sites, these TATA-like sequences may be necessary to initiate high level of transcription from the HFE promoter. Moreover, the region contains several liver-specific C/EBP sites that would allow a specific high rate of transcription in liver cells. The transcription can also initiate from a –120/–10 window. The proximal –70 to –1 upstream region relative to translation initiation codon has a 68% (high) G+C content (containing CpG), whereas the G+C content of the farther upstream region is 46%. The functional analysis of HFE 5'-flanking region revealed that the –85/–8 region (containing the whole GC-rich sequence) is sufficient to promote low transcriptional activity, as stated for GC box core promoter (Fig. 1 ).

View larger version (41K): [in this window] [in a new window]   Figure 1. Nucleotide sequence and putative regulatory elements of the 5'-flanking region of the HFE gene. The first nucleotide of the translation start codon was assigned the +1 position and numbers on the left margin refer to this site. Consensus sequences for the binding of C/EBP, GATA-1, and Sp1 transcription factors are underlined above them, and bold letters indicate the location of the TATA binding protein (TBP) sites. Transcription initiation sites determined by runoff in vitro transcription are indicated by solid triangles, those determined by 5'RACE are indicated by opened triangles.

2. Functional analysis of the 5'-flanking region of the HFE gene
Functional analysis of the 5'-flanking sequence of the human HFE gene including 1485 bp of the upstream sequence from the first coding nucleotide, was carried out to identify the regions involved in the promoter activity of the HFE gene. Transient transfection using a series of luciferase reporter pGL3 plasmid constructs containing various HFE 5'-flanking sequences revealed a cell-line dependent activity of the HFE promoter, the activity was 2.8-fold higher in HepG2 cells and 1.7-fold higher in HT29-19A cells than in HeLa cells, which displayed almost no significant activity. These results should be the manifestation of the endogenous HFE expression in the tissues from which the cell lines were derived or nuclear extract prepared. Thus, liver-specific factors are required for maximal HFE transcription.

3. Cis control elements of the 5' -flanking region of the HFE gene and trans-factors
To identify the sites for interaction between DNA sequences and nuclear proteins involved in transcriptional regulation, DNase I footprint analyses with 5'-flanking regions of the HFE gene and electrophoretic mobility shift assay with synthetic double-stranded oligonucleotides were performed using mouse liver nuclear extract. The results allowed the identification of several liver-enriched C/EBP and erythropoietic GATA-1 (WGATAR) binding sites, as well as ubiquitous Sp1 sites. To test the functional roles of C/EBP, GATA-1, and Sp1 (transcription factors on the HFE promoter) cotransfection experiments were carried out with the heterologous promoter-driven plasmid expression vectors that encode these factors, and with the promoter-LUC reporter plasmids. Trans-activation of HFE was evidenced within the –1057 to –185 in agreement with the presence of C/EBP, GATA-1, and Sp1 sites and was consistent with the relative high binding affinity of the nuclear factors in these regions (Fig. 2 ).

View larger version (28K): [in this window] [in a new window]   Figure 2. Trans-activation of the HFE promoter by C/EBP, GATA-1 and Sp1. A series of promoter deletion-LUC constructs were cotransfected with C/EBP, GATA-1, and/or Sp1 cDNA expression clones (pMSV-C/EBP, pXM-GATA-1, and pPac-Sp1) in HepG2 cells, and luciferase activities were determined. The data are averages obtained from constructs tested in 5 independent experiments and luciferase activities were standardized relative to ß-galactosidase activity. The plasmid pGL3-Basic was included as control for trans-activation assigned a value of 1, and trans-activation of the plasmid constructs was corrected for background. The values indicate the -fold increase in activity on basal expression of each of the reporter gene construct when using one or two of pMSV-C/EBP, pXM-GATA-1, pPac-Sp1 plasmids.

CONCLUSIONS AND SIGNIFICANCE

Our results lead to the conclusion that liver-enriched C/EBP factor and GATA-1 factor that control erythropoiesis are both required to get the maximum in the transcriptional activation of the HFE gene. The C/EBP liver-enriched factor must contribute to the high level of HFE mRNA expression in the liver, and the GATA-1 factor may suggest the involvement of HFE in the pathway of iron requirement for erythropoiesis. Many promoters of genes involved in iron homeostasis (cellular iron uptake and storage (transferrin receptor, ferritin), heme biosynthesis (eALAS, ferrochelatase), and globin protein formation) contain cis elements recognized by the GATA-1 factor, which is essential for erythropoiesis process, and by other erythroid-specific factors such as NF-E2. Our results demonstrated that the activation of HFE expression is mediated by GATA-1 factor via multiple consensus GATA sites. As the largest pool of iron is used for heme formation in hemoglobin, the transcriptional regulation likely ensures a coordinated regulation of iron availability and hemoglobin synthesis. Our data bring some evidence for the coordinated expression of HFE with other genes involved in erythropoiesis process. Moreover, the association of the GATA-1 motif with either CCACC or GC motifs has been evidenced in most of the erythroid-expressed genes and iron homeostasis genes. By comparison, the ferrochelatase gene promoter contains GATA-1 motifs and sites for Sp1 transcription factors. It has been hypothesized that NF-E2 and GATA-1 are both involved in the induction of these genes during erythroid differentiation, whereas the GC box is responsible for maintaining the housekeeping expression of the gene. These transcription factors could play a similar role in the regulation of the HFE gene in vivo, thus allowing a differential tissue expression of HFE. TfR2 has been recently characterized and a mutated form has been found to be involved in some cases of hemochromatosis. TfR2 is mainly expressed in the liver, and its mRNA expression is regulated by liver-enriched C/EBP and erythroid-specific GATA-1 transcription factors. Both HFE and TfR2 genes, responsible for a hemochromatosis phenotype when mutated, are transcriptionally up-regulated by the same liver and erythropoietic factors and may thus be implicated in the same pathway of liver iron stock. According to the transcriptional regulation of HFE expression, it can be postulated that HFE signals iron status mainly at the hepatic level where iron is stocked, especially when iron is required for erythropoiesis.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2520fje;

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This Article Full Text (PDF) All Versions of this Article: 18/15/1922 04-2520fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MURA, C. Articles by FÉREC, C. Search for Related Content PubMed PubMed Citation Articles by MURA, C. Articles by FÉREC, C.