1. Volume 142, Issue 4, Pages 989-998.e4 (April 2012)
Estrogen Receptor α Represses Transcription of HBV Genes via Interaction With Hepatocyte Nuclear Factor 4α 
Sheng–Han Wang, Shiou–Hwei Yeh, Wei–Hsiang Lin, Kun–Huei Yeh, Quan Yuan, Ning–Shao Xia, Ding–Shinn Chen, Pei–Jer Chen 
Gastroenterology 
Volume 142, Issue 4, Pages e4 (April 2012)
DOI: /j.gastro
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2. Figure 1
Estrogen down-regulates HBV titers but increases ER-α protein in HBV transgenic mice. (A) Serum HBV titers were compared between sham-operated mice (n = 12) and OVX (n = 7) groups of female mice, with the mean value for the sham-operated group set at 100%. (B) The concentrations of serum E2 before (set at 100%) and after surgery were compared in the OVX group of female mice. (C) Serum HBV titers were compared between male mice, either implanted with an E2 tablet (+E2, n = 8) or sham-treated (Non, n = 8), with the mean value for the control group set at 100%. (D) The serum E2 levels were measured in both groups of male mice, as described in C. (E) The expression levels of ER-α protein in the liver tissues of HBV transgenic mice were determined by Western blotting, normalized with β-actin, and shown at the bottom of each lane relative to that in lane 1. The graphic results represent the mean of each group with SE bars and were statistically analyzed with a t test. *P < .05, **P < .01, ***P < .001.
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3. Figure 2
Suppression of HBV mRNA transcripts by ER-α through HBV enhancer I. (A) RNA extracted from HepG2 cells cotransfected with the indicated plasmids and treated with 10 nmol/L E2 (or ethanol) was examined by Northern blotting. The amount of HBV mRNAs in each sample was determined by densitometry, normalized with the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal, and shown at the bottom of each lane relative to that in lane 1. (B) ERE-Luc reporter activity was examined in the cells, as shown in A. The results are the averages of 3 experiments. (C) The protein expression of ER-α, HBc, and HBx from the cells, as described in A, was analyzed by Western blotting. (D) Schematic illustration of the reporter constructs containing HBV enhancer I or II region 5′ upstream from the luciferase reporter. (E) The ER-α construct was cotransfected with the reporter plasmids as indicated, into HepG2 cells, followed by treatment with 10 nmol/L E2 (or ethanol) for subsequent reporter assay. The data are the averages of 3 experiments.
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4. Figure 3
Direct DNA binding and HDAC activity are not required for the repressive effect of ER-α on HBV transcription. (A) ERE-Luc (left panel) and Enh I-Xp-Luc (middle panel) reporter activities were determined in HepG2 cells cotransfected with the reporter and ER-α constructs and treated with 10 nmol/L E2 (or ethanol). The protein expression of ER-α by the cells was determined by Western blotting (right panel). (B) HepG2 cells cotransfected with the reporter and ER-α constructs were treated with 10 nmol/L E2 (or ethanol) and 0.53 μmol/L OSU-HDAC42 (or dimethyl sulfoxide) as indicated and then processed to assay the luciferase activity of ERE-Luc (left panel) and Enh I-Xp-Luc (middle panel). The levels of acetylated histone 3 (Ac-H3) in the cells were examined by Western blotting (right panel). Total H3, total forms of histone 3. The data are the averages of 3 independent experiments.
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5. Figure 4
The repressive effect of ER-α on HBV transcription can be rescued by the overexpression of HNF-4α. (A) HepG2 cells were cotransfected with the human ER-α and Enh I-Xp-Luc reporter plasmids and a STAT3-, HNF-3β–, or HNF-4α–expressing construct to screen their rescue effects on the suppression by ER-α of the reporter activity (left panel). The protein expression of the transfected genes was verified by Western blotting (right panel). (B) In the CL-48 cell line, which does not express endogenous HNF-4α, the HNF-4α construct and dose-titrated ER-α plasmids were cotransfected with Enh I-Xp-Luc for reporter assay (left panel). Western blotting was performed to verify the protein expression in the cells (right panel). The data are the averages of 3 independent experiments.
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6. Figure 5
HNF-4α binding site within HBV enhancer I is essential for the down-regulation of HBV transcripts by ER-α. (A) Schematic illustration of the wild-type (WT) and the mutant with a disrupted HNF-4α binding site (4A-Mut) of Enh I-Xp-Luc and I-4-Luc reporter constructs. The HNF-4α binding site is marked in blue, with the critical nucleotides mutated in the 4A-Mut construct marked in red. (B and C) HepG2 cells were cotransfected with the WT or 4A-Mut reporters and ER-α plasmids, treated with 10 nmol/L E2 (or ethanol), and then processed for reporter assay and Western blotting. The results are the averages of 3 independent experiments. (D) Schematic illustration of the HBV replicons containing either the WT (pHBV-48) or HNF-4α binding site–mutated (pHBV-48-4A-Mut) sequences, similar to those described in A. (E) HepG2 cells were cotransfected with ER-α constructs and the pHBV-48 or pHBV-48-4A-Mut replicons, treated with 10 nmol/L E2 (or ethanol), and processed for subsequent Northern blotting. The level of HBV transcripts in each sample was normalized to the GAPDH signal and is shown at the bottom of each lane relative to the amount in lane 1. The protein expression of ER-α by the cells was also confirmed by Western blotting.
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7. Figure 6
The hinge region of ER-α is required for the repression of HBV transcription through its sequestration of HNF-4α. (A) Schematic illustration of the expression constructs of WT and various deletion mutants of human ER-α, with the functional domains and amino acid residues as indicated. NTD, N-terminal transactivation domain; LBD, ligand-binding domain. (B) HepG2 cells were cotransfected with the ERE-Luc (upper panel) or Enh I-Xp-Luc reporter plasmid (lower panel) and individual ER-α constructs as indicated, treated with 10 nmol/L E2 (or ethanol), and processed for reporter assay. The data are the averages of 3 independent experiments. (C and D) CL-48 cells were cotransfected with protein-expressing plasmids (with the combinations indicated at the top of each lane), treated with 10 nmol/L E2 (or ethanol), and processed for coimmunoprecipitation and Western blotting with antibody as indicated. (E) HepG2 cells were cotransfected with the pCMV-ERα and the WT or mutant (4A-Mut) I-4-Luc reporter plasmids, treated with 10 nmol/L E2 (or ethanol), and processed for ChIP analysis. The plasmids pulled down by anti–HNF-4α antibody were used for polymerase chain reaction amplification, with the immunoglobulin G control included to verify the specificity of the anti–HNF-4α antibody (left panel). The luciferase activity of I-4-Luc and ER-α protein expression were determined by reporter assay and Western blotting (right panel).
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8. Figure 7
Proposed model of the suppressive mechanism of ER-α on the HBV life cycle by blocking HNF-4α binding to HBV enhancer I. (A) A schematic illustration of the proposed model. ER-α is translocated into the nucleus and binds to HNF-4α through the ER-α hinge region, causing molecular chelation, regardless of ligand treatment. The DNA-binding capacity of ER-α–squelched HNF-4α to HBV enhancer I is thus reduced, resulting in a decrease of enhancer I activity. This impairs HBV transcription and subsequently leads to a reduction in viral titer. Dotted lines indicate the events awaiting further studies. (B) Schematic illustration of the targets of transcriptional factors that bind within the HBV enhancer I region (in boxes), modified from the figure by Moolla et al.17 The nucleotide coordinated in HBV genome and the functional domains of enhancer I are indicated. The regions responsive to androgen receptor and ER-α axes are highlighted, with 2 androgen-responsive element sites at nucleotides 913–927 and 949–963,13 and the HNF-4α binding target site at nucleotides 1134–1146, respectively.
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