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Identification of multiple transcription factors, HLF, FTF, and E4BP4, controlling hepatitis B virus enhancer II - PubMed

Identification of multiple transcription factors, HLF, FTF, and E4BP4, controlling hepatitis B virus enhancer II

H Ishida et al. J Virol. 2000 Feb.

Abstract

Hepatitis B virus (HBV) enhancer II (EnII) is a hepatotropic cis element which is responsible for the hepatocyte-specific gene expression of HBV. Multiple transcription factors have been demonstrated to interact with this region. In this study, the region from HBV nucleotides (nt) 1640 to 1663 in EnII was demonstrated to be essential for enhancer activity and to be another target sequence of putative transcription factors. To elucidate the factors which bind to this region, we used a yeast one-hybrid screening system and cloned three transcription factors, HLF, FTF, and E4BP4, from a human adult liver cDNA library. All of these factors had binding affinity to the sequence from nt 1640 to 1663. Investigation of the effects of these factors on transcriptional regulation revealed that HLF and FTF had stimulatory activity on nt 1640 to 1663, whereas E4BP4 had a suppressing effect. FTF coordinately activated both 3. 5-kb RNA and 2.4/2.1-kb RNA transcription in a transient transfection assay with an HBV expression vector. HLF, however, activated only 3.5-kb RNA transcription, and in primer extension analysis, HLF strongly stimulated the synthesis of pregenome RNA compared to precore RNA. Thus, FTF stimulated the activity of the second enhancer, while HLF stimulated the activity of the core upstream regulatory sequence, which affects only the core promoter, and had a dominant effect on the pregenome RNA synthesis.

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Figures

FIG. 1
FIG. 1

Nucleotide sequences of HBV between nt 1570 to 1771 including the EnII region. The underlined sequences represent Sp1, HNF1, HNF3, HNF4, and FTF (hB1F) recognition sequences, as labeled. Box α (nt 1645 to 1669) and box β (nt 1705 to 1716) in the CURS are shown in italics. The open box indicates the sequence from nt 1640 to 1663 which was used in gel retardation analysis. This region includes the extended C/EBP consensus sequence (T[T/G]NNG[C/T]AA[T/G]).

FIG. 2
FIG. 2

Deletion analysis of HBV EnII. The deletion mutants are shown at the left. Open boxes represent the EnII sequences, and the numerals indicate nucleotides of the HBV genome. The fragment containing the EnII region was cloned into pTATALUC just upstream of the E1b minimal promoter in the antisense orientation. Plasmid DNA was transfected into HepG2 cells, and luciferase activity was determined for the lysate of each sample. Fold stimulation of luciferase activity (mean ± standard deviation) is shown on the right.

FIG. 3
FIG. 3

Transcriptional regulation of HBV nt 1640 to 1663 or mutant sequence m4, which has four nucleotide mutations within nt 1640 to 1663. Five tandem repeats of the sequences were inserted upstream of the E1b minimal promoter in the sense or antisense orientation. Fold stimulation of luciferase activity (mean ± standard deviation) is shown on the right.

FIG. 4
FIG. 4

Deletion analysis of HBV nt 1640 to 1771 to determine the correlation between nt 1640 to 1663 and the region from nt 1664 to 1771. The sequences are shown at the left as open boxes. Fold stimulation of luciferase activity (mean ± standard deviation) is shown on the right.

FIG. 5
FIG. 5

Gel retardation analysis of HBV nt 1640 to 1663. The 32P-labeled double-stranded oligonucleotide containing the sequence from nt 1640 to 1663 (lane 1 to 7) or 32P-labeled HNF1 consensus sequence (lanes 8 to 12) was incubated with HepG2 nuclear extract at room temperature for 30 min. The mixture was resolved on a 4% polyacrylamide gel. Lane 1, probe only; lanes 2 to 7, probe and 10 μg of nuclear extract with no competitor (lane 2), with 10-fold molar excess of unlabeled specific oligonucleotide of nt 1640 to 1663 (lane 3), with 10- and 50-fold molar excess of unlabeled nonspecific HNF1 oligonucleotide (lanes 4 and 5), and with 10- and 50-fold molar excess of unlabeled mutant sequence of nt 1640 to 1663, m4 (lanes 6 and 7), respectively; lane 8, probe only; lanes 8 to 12, probe and 10 μg of nuclear extract with no competitor (lane 9), with 10-fold molar excess of unlabeled HNF1 consensus sequence (lane 10), and with 10- and 50-fold molar excess of unlabeled nonspecific oligonucleotide of nt 1640 to 1663 (lanes 11 and 12). The specifically shifted band of nt 1640 to 1663 is indicated by a bracket, and the specifically shifted band of HNF1 is indicated by arrowheads.

FIG. 6
FIG. 6

Nucleotide sequence of HBV nt 1640 to 1663. The underlined sequences represent the regions homologous to FTF and HLF/E4BP4 recognition sequences. Comparison of the HLF, FTF, and E4BP4 binding sequences in HBV EnII and their consensus binding sequences is shown below (R = A or G; K = G or T; Y = C or T; W = A or T). The nucleotide sequence of HBV compared to the E4BP4 consensus is shown in the antisense orientation.

FIG. 7
FIG. 7

Gel retardation analyses detecting complex formation of in vitro-translated protein or HepG2 nuclear extract and the probe containing the sequence from nt 1640 to 1663 or m4, the mutant sequence of nt 1640 to 1663. (A) Lane 1, probe only; lane 2, negative control [NC; probe and 0.5 μl of in vitro translation reaction of pGEM-3Zf(+)]; lanes 3 to 6, probe and 0.5 μl of in vitro translation reaction of HLF without competitor (lane 3), with 10- and 50-fold molar excess of unlabeled specific competitor of nt 1640 to 1663 (lanes 4 and 5), and with 50-fold molar excess of m4 (lane 6); lanes 7 to 10, probe and 0.5 μl of in vitro translation reaction of FTF without competitor (lane 7), with 10- and 50-fold molar excess of unlabeled specific competitor of nt 1640 to 1663 (lanes 8 and 9), and with 50-fold molar excess of m4 (lane 10); lanes 11 to 14, probe and 0.5 μl of in vitro translation reaction of E4BP4 without competitor (lane 11), with 10- and 50-fold molar excess of unlabeled specific competitor of nt 1640 to 1663 (lanes 12 and 13), and with 50-fold molar excess of m4 (lane 14). (B) Probes used in the assays were the sequence from nt 1640 to 1663 (lane 1, 3, 5, 7, and 9) and m4 (lane 2, 4, 6, 8, and 10). Lanes 1 and 2, probe only; lanes 3 and 4, probe and 0.5 μl of in vitro translation reaction of HLF; lanes 5 and 6, probe and 0.5 μl of in vitro translation reaction of FTF; lanes 7 and 8, probe and 0.5 μl of in vitro translation reaction of E4BP4; lanes 9 and 10, probe and 10 μg of HepG2 nuclear extract.

FIG. 8
FIG. 8

Transcriptional regulation of nt 1640 to 1663 by HLF, FTF, and E4BP4 in HuH7 cells (A) and HepG2 cells (B). pTATALUC, pTATALUC-E5, which contains five iterations of nt 1640 to 1663 in tandem, pTATALUC-EII2, which contains HBV nt 1640 to 1771, or pTATALUC-EII3, which contains HBV nt 1660 to 1771, was cotransfected with pFLAG-CMV-2, pCMVHLF, pCMVFTF, or pCMVE4BP4, and the cell lysate was assayed for luciferase activity. The fold activity of each transfectant relative to the cells transfected with pTATALUC and pFLAG-CMV-2 was calculated.

FIG. 9
FIG. 9

Regulation of HBV transcripts and of HBeAg and HBsAg by HLF, FTF, E4BP4. HuH7 cells (3 × 106 per 10-cm-diameter dish) were transfected with 2.5 μg of pHBV1.5 and 7.5 μg of pFLAG-CMV-2, pCMVHLF, pCMVFTF, or pCMVE4BP4. (A) Northern blot analysis of HBV transcripts. Total RNA (20 μg) was hybridized with an HBV adw2 probe. Arrows indicate the 3.5- and 2.4/2.1-kb transcripts. The band intensity of the HBV transcripts was measured with an image analyzer. The band intensity of the reaction using pFLAG-CMV-2 was considered 100%, and the band intensity relative to this value was calculated. (B) Quantitation of the degree of expression of HBeAg and HBsAg measured by RIA. The expression level of the reaction using pFLAG-CMV-2 was considered 100%, and percent expression relative to this value was calculated. (C) Synthesis of pre-C and pregenome RNAs. mRNAs selected from 100 μg of total RNAs with oligo(dT) were used for primer extension. The products corresponding to pre-C and pregenome RNAs are indicated by arrows.

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