Effects of FoxA deletion on liver TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, αSMA, Col1A1, CK19 and CK20 transcript levels in adult HBV transgenic mice.

<p>Quantitative analysis of the (A) TNFα, (B) 2OAS, (C) IL6, (D) TGFβ1, (E) TGFβ2, (F) TGFβ3, (G) αSMA, (H) Col1A1, (I) CK19 and (J) CK20 transcripts by RT-qPCR in the HBV transgenic mice. The GAPDH transcript was used as an internal control for the quantitation of the TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, αSMA, Col1A1, CK19 and CK20 RNAs. The mean relative TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, αSMA, Col1A1, CK19 and CK20 transcript levels plus standard deviations derived from male and female FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice is shown. The levels of the transcripts which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*). Average number of mice per group was 6.5±1.8 (Range: 4–9).</p

Model for the tissue-specific and developmental regulation of HBV DNA methylation and transcription by the pioneer transcription factor, FoxA.

<p>(A) In the neonatal (P0.5) wild-type HBV transgenic mice, the HBV transgene DNA is extensively methylated but FoxA is expressed and marks the HBV genome for later developmental expression upon recruitment of additional transcription factors to the viral promoters. In the adult wild-type HBV transgenic mice, the HBV transgene DNA is unmethylated, FoxA plus additional transcription factors are recruited to the viral promoters and HBV RNA and DNA synthesis is observed. (B) In the neonatal (P0.5) FoxA-deficient HBV transgenic mice, the HBV transgene DNA is extensively methylated while limiting levels of FoxA fail to mark the HBV genome for later developmental DNA demethylation (due to the failure to recruit TET for active DNA demethylation and/or inhibition of DNA methylation during replication), and hence subsequent recruitment of additional transcription factors to the viral promoters with concomitant viral gene expression. In the adult FoxA-deficient HBV transgenic mice, the HBV genome remains extensively methylated because the limiting abundance of FoxA throughout development fails to mark the viral transgene DNA for demethylation which is essential for HBV RNA and DNA synthesis. (C) In the neonatal (P0.5) wild-type HBV transgenic mice, the essential liver-specific genes which are dependent on FoxA for their expression at this stage of development have presumably been marked by this pioneer transcription factor leading to the recruitment of additional transcription factors necessary for the demethylation and subsequent expression of these genes. Increasing levels of liver-specific transcription factors associated with liver maturation may be associated with increased levels of gene expression in the adult mice. (D) In contrast to the effect of limiting FoxA abundance on HBV DNA methylation and transcription, limiting postnatal FoxA abundance in the hepatocytes of these mice must be sufficient to support gene expression levels consistent with host viability presumably by marking these genes for DNA demethylation and transcription at the neonatal stage of development. The size of the transcription factors (FoxA and TFs) reflects the number of hepatocytes expressing HBV or essential liver-specific genes and/or the overall level of gene expression. The size of the arrow reflects the level of gene transcription. 5MeC, 5-methylcytosine; C, cytosine.</p

Effects of FoxA deletion on liver FoxA1, FoxA2, FoxA3, TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, Col1A1 and CK19 transcript levels in 1, 2, 3, 4 and 7 week old HBV transgenic mice.

<p>Quantitative analysis of the (A) FoxA1, (B) FoxA2, (C) FoxA3, (D) TNFα, (E) 2OAS, (F) IL6, (G) TGFβ1, (H) TGFβ2, (I) TGFβ3, (J) Col1A1 and (K) CK19 transcripts by RT-qPCR in the HBV transgenic mice. The GAPDH transcript was used as an internal control for the quantitation of the FoxA1, FoxA2, FoxA3, TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, Col1A1 and CK19 RNAs. The mean relative FoxA1, FoxA2, FoxA3, TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, Col1A1 and CK19 transcript levels plus standard deviations derived from FoxA-expressing (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice is shown. The levels of the transcripts which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*). Average number of mice per group was 3.5±1.7 (Range: 2–7).</p

DNA (Southern) filter hybridization analysis of HBV DNA replication intermediates in the livers of adult HBV transgenic mice.

<p>(A) DNA (Southern) filter hybridization analysis of representative mice of each sex and genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probe used was HBVayw genomic DNA. FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice are indicated (Genotype A2, A1A2 and A1A2A3, respectively). The HBV transgene (Tg) was used as an internal control for the quantitation of the HBV replication intermediates. Tg = HBV transgene; RC = HBV relaxed circular replication intermediates; SS = HBV single stranded replication intermediates. (B) Quantitative analysis of the HBV DNA replication intermediate (RI) levels in HBV transgenic mice. The mean DNA replication intermediate levels plus standard deviations are indicated. Average number of mice per group was 6.7±1.8 (Range: 4–9). The levels of replication intermediates which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*).</p

RNA (Northern) filter hybridization and RT-qPCR analysis of HBV transcripts in the livers of adult HBV transgenic mice.

<p>(A) RNA (Northern) filter hybridization analysis of representative mice of each sex and genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probes used were HBV<i>ayw</i> genomic DNA plus GAPDH cDNA. FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice are indicated (Genotype A2, A1A2 and A1A2A3, respectively). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for the quantitation of the HBV 3.5kb RNA. (B) Quantitative analysis by RNA (Northern) filter hybridization of the HBV 3.5kb transcript in the HBV transgenic mice. The mean HBV 3.5kb transcript levels plus standard deviations are indicated. Average number of mice per group was 6.6±1.9 (Range: 4–9). The levels of the HBV 3.5kb transcript which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*). (C) Quantitative analysis by RT-qPCR of the HBV 3.5kb transcript in the HBV transgenic mice. The mean HBV 3.5kb transcript levels plus standard deviations are indicated. Average number of mice per group was 6.5±1.8 (Range: 4–9). The levels of the HBV 3.5kb transcript which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*).</p

Tissue-specific and developmental regulation of HBV DNA methylation distribution.

<p>The CpG DNA methylation frequency distribution across the 11, 38 and 13 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2264–2474, respectively, from male (A-C) and female (D-F) wild-type HBV transgenic mouse liver, kidney, muscle, spleen, lung and brain DNA is shown. The CpG DNA methylation frequency distribution across the 11, 38 and 13 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2264–2474, respectively, from three independent 0.5 day old neonatal (G-I) wild-type HBV transgenic mouse liver DNA is shown.</p

Effect of FoxA-deletion on HBV DNA methylation in adult mouse liver.

<p>The percentage of CpG DNA methylation at each position within the HBV genome from male (A) and female (B) FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice is shown. The positions of the viral transcription initiation sites for the X-gene (X RNA), the precore/pregenomic transcripts (C RNA), the large surface antigen transcript (PS RNA) and the middle/major surface antigen transcript (S RNA) are shown. The locations of the FoxA binding sites and CpG island within the HBV genome are also indicated. (C) The average percent methylation of the CpG sites spanning nucleotide coordinate 1–706 is correlated with the level of serum HBeAg.</p

Effects of FoxA deletion on liver FoxA1, FoxA2, FoxA3 and FoxO1 transcript levels, liver size and serum HBeAg in adult HBV transgenic mice.

<p>Quantitative analysis of the (A) FoxA1, (B) FoxA2, (C) FoxA3 and (D) FoxO1 transcripts by RT-qPCR in the HBV transgenic mice. The GAPDH transcript was used as an internal control for the quantitation of the FoxA1, FoxA2, FoxA3 and FoxO1 RNAs. The mean relative FoxA1, FoxA2, FoxA3 and FoxO1 transcript levels plus standard deviations derived from male and female FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice is shown. The levels of the transcripts which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*). Similar quantitative analysis of (E) liver size and (F) serum HBeAg levels is also shown. Average number of mice per group was 6.5±1.9 (Range: 3–9).</p

Histological analysis of liver samples from adult HBV transgenic mice.

<p>Control FoxA-expressing (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-), panels A-C) and FoxA-deleted (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+), panels D-F) HBV transgenic mice are indicated. Immunohistochemical staining indicates the presence of nuclear HBcAg throughout the liver whereas cytoplasmic staining is located primarily in the centrolobular hepatocytes in control mice (panel A) whereas HBcAg is minimally detectable in the FoxA-deleted mice (panel D). Hematoxylin and eosin (H&E) staining indicates biliary epithelial proliferation in the FoxA-deleted mice (panel E) which is absent in the control mice (panel B). Trichrome (TC) staining indicates bridging portal fibrosis in the FoxA-deleted mice (panel F) which is absent in the control mice (panel C). The black size bar is 100 μm.</p

Effect of FoxA-deletion on HBV DNA methylation distribution in adult mouse liver.

<p>The CpG DNA methylation frequency distribution across the 11, 38 and 14 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2131–2441, respectively, from male (A, C and E) and female (B, D and F) FoxA-expressing (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-); M A1A2A3Cre(-) and F A1A2A3Cre(-)) and FoxA-deleted (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+); M A1A2A3Cre(+) and F A1A2A3Cre(+)) HBV transgenic mice is shown.</p