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Brazilian Journal of Medical and Biological Research

On-line version ISSN 1414-431X

Braz J Med Biol Res vol. 31 n. 11 Ribeirão Preto Nov. 1998

http://dx.doi.org/10.1590/S0100-879X1998001100006 

Braz J Med Biol Res, November 1998, Volume 31(11) 1405-1408 (Short Communication)

Association of hepatic nuclear factor-4 in the apolipoprotein B promoter: a preliminary report

E.M. Nóvak1, K.C. Dantas1, C.E. Charbel1 and S.P. Bydlowski1,2

1Divisão de Pesquisa e Biologia Molecular, Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, SP, Brasil
2Disciplina de Hematologia e Hemoterapia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil

Abstract
Text
References
Correspondence and Footnotes


Abstract

Previous studies have examined the arrangement of regulatory elements along the apolipoprotein B (apoB) promoter region (-3067 to +940) and a promoter fragment extending from nucleotides -150 to +124 has been demonstrated to be essential for transcriptional activation of the apoB gene in hepatic and intestinal cells. It has also been shown that transcriptional activation of apoB requires a synergistic interaction between hepatic nuclear factor-4 (HNF-4) and CCAAT/enhancer-binding protein a (C/EBPa) transcription factors. Here, we have examined the hypothesis that HNF-4 factor binding to DNA may induce a DNA helix bend, thus facilitating the communication with a C/EBPa factor located one helix turn from this HNF-4 factor in the apoB promoter. A gel electrophoretic mobility shift assay using wild type double-stranded oligonucleotides or modified wild type duplex oligonucleotides with 10 nucleotides inserted between HNF-4 and C/EBPa factor motifs showed similar retarded complexes, indicating that HNF-4 and C/EBPa factors interact independently of the distance between binding sites. However, when only one base, a thymidine, was inserted at the -71 position of the apoB promoter, the complex shift was completely abolished. In conclusion, these results regarding the study of the mechanisms involving the interaction between HNF-4 and C/EBPa factors in the apoB promoter suggest that the perfect 5'-CCCTTTGGA-3' motif is needed in order to facilitate the interaction between the two factors.

Key words: apolipoprotein B promoter, HNF-4, C/EBPa


Apolipoprotein B (apoB) is a constituent of several lipoproteins and acts as a ligand for the cellular recognition and catabolism of low density lipoprotein (LDL) by the LDL receptor (1,2). ApoB mRNA has been detected in the liver, intestine and placenta, indicating that apoB gene transcription is regulated in a tissue-specific manner (3,4). The regulatory elements of the human apoB gene are located in the proximal promoter region (5,6). Kardassis et al. (5) have demonstrated that four elements (named A, B, C and E) are recognized by nuclear factors. Three of these elements (A, B, and C) are spaced within the apoB promoter -36 to -118, and element E is located in the +35 to +53 region within the first exon of the apoB gene (5). Analysis of the interaction of nuclear proteins with element A (5'-GCGCCCTTTGGACCTTTTGCAATCC-3') localized in the -79 to -63 apoB promoter showed the existence of multiple sequence-specific DNA-complexes. Two liver-enriched transcription factors, hepatic nuclear factor-4 (HNF-4), a transcription factor that belongs to the steroid-thyroid hormone receptor superfamily (7), and CCAAT/enhancer-binding protein a (C/EBPa), a transcription factor that belongs to the bZip family of proteins (8), bind to the -81 to -52 region of the apoB promoter (6); both factors are critical for gene expression in hepatocytes. Furthermore, both transcription factors have an overlapping binding site in the apoB promoter and act synergistically in order to activate transcription (6). In this study, we have examined the role of the HNF-4 factor in the interaction between C/EBPa and HNF-4 proteins within the -81 to -52 region of the apoB promoter. The transcription factors HNF-4 and C/EBPa used in gel mobility shift assays were purified from rat liver nuclei using DNA-specific affinity chromatography, as described by Kadonaga and Tijan (9). HNF-4 protein was purified from crude rat liver nuclear extract by chromatography, using three different supports (Q-Sepharose, Bio-Rex 70 and S-Sepharose). The columns were washed with KCl gradients and then fractions containing HNF-4 protein activity were pooled and the solution was brought to 100 mM KCl and applied to affinity columns (10). The specific affinity column was prepared with a double-stranded oligonucleotide (5'-AGGCGCCCTTTGGACCTT-3') corresponding to the -81 to -64 region from the apoB promoter coupled to a cyanogen bromide-activated Sepharose 4B column. Twenty-five mg of protein from S-Sepharose fractions was loaded onto a 10-ml specific affinity column equilibrated with NDB buffer (25 mM HEPES, pH 7.6, 5 mM MgCl2, 0.1 mM EDTA, and 10% glycerol) containing 0.1 mM KCl. Active fractions were concentrated by Mono-Q column chromatography. Eluted HNF-4 protein was dialyzed in NDB solution containing 40 mM KCl, and stored at -70oC. C/EBPa protein was purified using heparin-agarose and DNA-cellulose columns, followed by heating for 6 min at 85oC and cooling immediately on ice. Fractions were centrifuged at 9,500 rpm for 15 min and a soluble material which contained the proteins was obtained. Twenty mg of protein was then passed through an affinity column. The affinity column for purification of C/EBPa protein was prepared with double-stranded oligonucleotide (5'-TGCAATCCTG G-3') corresponding to the -62 to -51 region from the apoB promoter coupled to a cyanogen bromide-activated Sepharose 4B column. The active protein C/EBPa was eluted from the affinity column with 0.8 M NaCl and stored at -70oC.

Gel mobility shift assays were prepared with 0.2-6.4 µg of purified nuclear protein. Samples were incubated for 30 min with wild type (5'-AGGCGCCCTTTGGACCTTTTGCAATCCTGG-3') or modified oligonucleotides (insertion of 1 or 10 nucleotides) were 32P labeled (sp. act. 5 x 108 cpm/µg) as described in Figure 2. The DNA binding reaction was carried out in a final volume of 20 µl solution containing 60 mM KCl, 20 mM HEPES, pH 7.9, 3% Ficoll, 0.5 mM dithiotreitol, and 1 mM MgCl2 and incubated at room temperature for 30 min. Two µg poly-(dI-dC) (Pharmacia, Uppsala, Sweden) was added as a nonspecific competitor. Then, the samples were submitted to 4% PAGE in 1 x TAE (10 x TAE: 67 mM Tris, 33 mM sodium acetate, 10 mm EDTA, pH 7.9), as described by Novak and Bydlowski (11). After drying, gels were exposed to Fuji RX X-ray film for at least 4 h at -70oC. Bands were visualized by autoradiography.

When both transcription factors were used together, as shown in Figure 1 (increasing concentrations of C/EBPa with a constant amount of HNF-4), a ternary complex was formed (slower migrating shift complex). However, when C/EBPa or HNF-4 proteins were used alone, no ternary complex was observed (Figure 1, lanes 1 and 8-13, and Figure 2, lanes 1-4 and 9).


Figure 1 - Synergistic effect of C/EBPa and HNF-4 in the apoB promoter. Transcription factors HNF-4 and C/EBPa were purified by affinity chromatography. The transcription factors were incubated with 32P-labeled double-stranded wild type oligonucleotides (5'-AGGCGCCCTTTGGACCTTTTGCAATCCTGG-3'). This sequence corresponds to the -81 to -52 region of the apoB promoter. Lane 1: 0.5 µg of HNF-4 protein. Lanes 2-7: increasing concentrations of C/EBPa factor (0.8, 3, 5 and 6.4 µg, respectively), and a constant concentration of HNF-4 protein (0.5 µg). Lanes 8-13: increasing amounts of C/EBPa factor (0.4, 0.8, 2, 4, 5 and 6.4 µg, respectively). The arrow shows the low mobility band derived from HNF-4 and C/EBPa specific complexes. High mobility bands correspond to unbound probes.

[View larger version of this image (30 K GIF file)]


Figure 2 - Effect of the insertion of nucleotides into the -81 to -52 region of the apoB promoter. Gel electrophoresis mobility shift assays were performed with HNF-4 and C/EBPa purified by affinity chromatography and each of the following 32P-labeled double-stranded oligonucleotides: A, Wild type probe (5'-AGGCGCCCTTTGGACCTTTTGCAATCCTGG-3') (lanes 1-4). Lane 1: 0.5 µg of HNF-4 and lanes 2-4: decreasing concentrations of C/EBPa (4, 2 and 0.8 µg, respectively). B, Probe modified by the insertion of ten nucleotides between the HNF-4 and C/EBPa motifs in the wild type sequence (5'-AGGCGCCCTTTGGAACCTTTCGGTCCTTTTGCAATCC-3') (lanes 5-8). Lane 5: 0.4 µg of C/EBPa and 0.2 µg of HNF-4. Lane 6: 0.8 µg of C/EBPa and 0.5 µg of HNF-4. Lane 7: 2 µg of C/EBPa and 0.5 µg of HNF-4. Lane 8: 4 µg of C/EBPa and 0.5 µg of HNF-4. C, Probe modified by the insertion of a thymidine nucleotide at the -71 position of the apoB promoter (5'-AGGCGCCCTTTTGGACCTTTTGCAATCCTGG-3') (lanes 9-13). Lane 9: 0.5 µg of HNF-4. Lane 10: 0.5 µg of HNF-4 and 1.5 µg of C/EBPa. Lane 11: 1 µg of HNF-4 and 0.8 µg of C/EBPa. Lane 12: 0.5 µg of HNF-4 and 2 µg of C/EBPa. Lane 13: 5 µg of C/EBPa and 2 µg of HNF-4. The arrow indicates the ternary complexes formed by interaction between HNF-4 and C/EBPa factors.

[View larger version of this image (30 K GIF file)]


Our results confirm previous data about the synergistic interaction between HNF-4 and C/EBPa in the apoB promoter (6). Surprisingly, when one helical turn was created by the insertion of 10-base pairs (5'-AGGCGCCCTTTGGAACCTTTCGGTCCTTTTGCAATCCTGG-3') between the HNF-4 and C/EBPa motifs in the -81 to -52 region of the apoB promoter, a ternary complex was also formed despite the distance between these factors (Figure 2, lines 5-8). These data suggest that the HNF-4 factor may be acting by direct communication with distant C/EBPa. Lymphoid enhancer-binding factor 1 (LEF-1) has been reported to have a similar effect regarding bent DNA (12). The LEF-1 factor recognizes the 5'-CCTTTGAA-3' sequence and induces a sharp bend in the DNA helix, facilitating an interaction between proteins bound at the distant 5' and 3' enhancer (12). In the LEF-1 DNA-complex, the A-T-rich sequences favor minor groove compression, whereas G-C-rich sequences favor major groove compression. Moreover, Erie et al. (13), using scanning force microscopy to visualize the DNA molecule conformation, have described bent DNA within specific complexes (12). The HNF-4 factor recognizes the 5'-CCTTTGGA-3' sequence in the -81 to -61 region of the apoB promoter. On the other hand, when a thymidine nucleotide was added at the -71 position, a 5'-CCTTTTGGA-3' sequence was created and the ternary complex was abolished (Figure 2, lanes 10-13). This insertion probably destroyed the perfect 5'-CCTTTGGA-3' motif. These results support the hypothesis that HNF-4 has an action similar to that of LEF-1 factors, probably inducing DNA bending by a minor groove through 5'-TTTGGA-3' and favoring binding of the C/EBPa to the -81 to -52 apoB promoter region. Therefore, the absence of the perfect motif 5'-CCTTTGGA-3' obstructed the DNA bend by the minor groove and consequently the communication between the HNF-4 and C/EBPa factors.

In summary, these data suggest that HNF-4 protein may be related to the structural changes which confer on the C/EBPa and HNF-4 factors the ability to form ternary complexes, as shown for other proteins, including LEF-1 (13-15). These interactions have an important role in the control of transcriptional regulation of the human apoB gene, as observed in cotransfection experiments with HeLa cells, where plasmids expressing C/EBPa and HNF-4 factors have increased the transcription of the reporter gene by 50- to 60-fold (6).


References

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6. Metzer S, Hallaas JL, Breslow JL & Sladek FM (1993). Orphan receptor HNF-4 and bZip protein C/EBPa bind to overlapping regions of the apolipoprotein B gene promoter and synergistically activate transcription. Journal of Biological Chemistry, 286: 16831-16838.        [ Links ]

7. Sladek FM, Zhong W, Lai E & Darnell Jr JE (1990). Liver enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes and Development, 23: 2353-2365.        [ Links ]

8. Landschulz WH, Johnson PF & McKnight S (1988). The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science, 240: 1759-1764.        [ Links ]

9. Kadonaga JT & Tijan J (1986). Affinity purification of sequence-specific DNA binding proteins. Proceedings of the National Academy of Sciences, USA, 83: 5889-5893.        [ Links ]

10. Kardassis D, Zannis V & Cladaras C (1990). Purification and characterization of the nuclear factor BA1. A transcriptional activator of the human apoB gene. Journal of Biological Chemistry, 265: 21733-21740.        [ Links ]

11. Novak EM & Bydlowski P (1997). NFY transcription factor binds to regulatory element AIC and transactivates the human apolipoprotein A-I promoter in HepG2 cells. Biochemical and Biophysical Research Communications, 231: 140-143.        [ Links ]

12. Giese K, Cox J & Grosschedl R (1992). The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures. Cell, 69: 185-195.        [ Links ]

13. Erie G, Yang HC, Schultz C & Bustamante C (1994). DNA bending by Cro protein in specific and nonspecific complexes: implications for protein site recognition and specificity. Science, 266: 1449-2062.        [ Links ]

14. Parekh B & Hatfield W (1996). Transcriptional activation by protein-induced DNA bending: Evidence for a DNA structural transmission model. Proceedings of the National Academy of Sciences, USA, 93: 1173-1177.        [ Links ]

15. Potthoff SJ, Lorene ER & Nardulli AM (1996). Effects of wild type and mutant estrogen receptors on DNA flexibility, DNA bending, and transcription activation. Molecular Endocrinology, 10: 1095-1105.        [ Links ]

Correspondence and Footnotes

Address for correspondence: E.M. Nóvak, Divisão de Pesquisa e Biologia Molecular, Fundação Pró-Sangue Hemocentro de São Paulo, Av. Enéias C. Aguiar, 155, 1º andar, 05403-000 São Paulo, SP, Brasil. Fax: +55-11-282-2398. E-mail: emnovak@br.homeshopping.com.br

Research supported by FAPESP (No. 97/12322-8) and Fundação Banco do Brasil (No.10/6186-7). Received March 11, 1998. Accepted August 14, 1998.