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HAT1 signaling confers to assembly and epigenetic regulation of HBV cccDNA minichromosome - PubMed

  • ️Tue Jan 01 2019

. 2019 Sep 25;9(24):7345-7358.

doi: 10.7150/thno.37173. eCollection 2019.

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HAT1 signaling confers to assembly and epigenetic regulation of HBV cccDNA minichromosome

Guang Yang et al. Theranostics. 2019.

Abstract

Rationale: Hepatitis B virus (HBV) is a leading cause of liver diseases. HBV covalently closed circular DNA (cccDNA) is a critical obstacle of complete elimination by anti-HBV therapy. HBV cccDNA accumulates in nucleus as a chromatin-like cccDNA minichromosome assembled by histones and non-histones. However, the underlying mechanism of modulation of cccDNA minichromosome in hepatocytes is poorly understood. Methods: A human liver-chimeric mouse model was established. The cccDNA-ChIP, Southern blot analysis, confocal assays, RIP assays and RNA pull-down assays, et al. were performed to assess the mechanism of assembly and epigenetic regulation of cccDNA minichromosome in human liver-chimeric mouse model, human primary hepatocytes (PHH), dHepaRG, HepG2-NTCP cell lines and clinical liver tissues. Results: Importantly, the expression levels of HAT1, CAF-1 and lncRNA HULC were significantly elevated in the liver from HBV-infected human liver-chimeric mice. Strikingly, the depletion of HAT1 reduced HBV replication and cccDNA accumulation, and impaired the assembly of histone H3/H4 and the deposition of HBx and p300 onto cccDNA to form cccDNA minichromosome in the cells. Mechanically, chromatin assembly factor-1 (CAF-1) was involved in the events. Interestingly, HAT1 modified the acetylation of histone H3K27/H4K5/H4K12 on cccDNA minichromosome. Moreover, lncRNA HULC-scaffold HAT1/HULC/HBc complex was responsible for the modification on cccDNA minichromosome. Additionally, HBV activated HAT1 through HBx-co-activated transcriptional factor Sp1 in a positive feedback manner. Conclusion: HAT1 signaling contributes to assembly and epigenetic regulation of HBV cccDNA minichromosome.

Keywords: HAT1 signaling; HBV cccDNA minichromosome; assembly; epigenetic modification.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1

HAT1 contributes to HBV replication and cccDNA accumulation. (A) A model of the establishment of HBV-infected human liver-chimeric mice. (B and C) The mRNA and protein levels of HAT1 were examined by RT-qPCR and Western blot analysis in the liver of human liver-chimeric mice (n=3) and HBV-infected human liver-chimeric mice (n=3), respectively. (D-G) The PHH, dHepaRG and HepG2-NTCP cells were infected at MOI of 600 vp/cell with HBV and were continuously transfected with siRNA of HAT1 at -4, 0, and 4 dpi (days post-infection). (D) The intracellular HBV-RNA and intracellular HBV-DNA were detected 8 dpi by qPCR in the cells. (E) The levels of HBV DNA, HBeAg and HBsAg in the medium were measured by qPCR and ELISA in the cells. (F) The levels of HBcAg were assessed by immunofluorescence assays in the cells. (G) HBV cccDNA was analyzed 8 dpi by selective qPCR in the cells. (H) The dHepaRG cells were infected with HBV at MOI of 600 vp/cell and were continuously transfected with siHAT1 (0, 50 or 100 nM) at -4, 0, 4 and 8 dpi. HBV cccDNA was analyzed 4, 8 and 12 dpi by Southern blot analysis in the cells. (I) Relative mRNA levels of HAT1 were detected by RT-qPCR in cccDNA-positive non-tumorous liver tissues (n=24) and cccDNA-negative non-tumorous liver tissues (n=15). Mean ± SD of at least three experiments are shown, in which each experiment was designed by three replicates. Statistical significant differences are indicated: *P<0.05; **P<0.01; ***P<0.001.

Figure 2
Figure 2

HAT1/CAF-1 signaling confers to the assembly of HBV cccDNA minichromosome. (A and B) The assembly of histone H3/H4 onto cccDNA was examined by ChIP-qPCR at indicated dpi in HBV-infected dHepaRG cells transfected with siHAT1. (C) The deposition of HBc, HBx and p300 onto cccDNA minichromosome or the promoter of CCNA2 was verified 8 dpi by ChIP-qPCR in HBV-infected dHepaRG cells continuously transfected with siHAT1. (D and E) The assembly of histone H3/H4 onto cccDNA was determined by ChIP-qPCR at indicated dpi in HBV-infected dHepaRG cells continuously transfected with siCAF-1 (p150) (100 nM) at -4, 0, 4 and 8 dpi. (F) The mRNA of CAF-1 were examined by RT-qPCR in the liver of human liver-chimeric mice (n=3) and HBV-infected human liver-chimeric mice (n=3). Mean ± SD of at least three experiments are shown, in which each experiment was designed by three replicates. Statistical significant differences are indicated: **P<0.01; ***P<0.001; ns, no significance.

Figure 3
Figure 3

HAT1 promotes histone acetylation on HBV cccDNA minichromosome. (A) The deposition of HAT1 on cccDNA was measured by ChIP-qPCR in the HBV-infected dHepaRG and HepG2-NTCP cells. (B) The deposition of HAT1 and HBc on cccDNA was analyzed by ChIP-qPCR at indicated time in the HBV-infected dHepaRG cells. (C) The colocalization of HAT1 and HBc was assessed 8 dpi by confocal microscopy in the dHepaRG cells. (D-G) The acetylation of histone, including AcH3, AcH4, H3K27ac, H4K5ac and H4K12ac, associated to cccDNA minichromosome or the promoter of CCNA2 was verified 8 dpi by ChIP-qPCR in the dHepaRG and HepG2-NTCP cells. (H) The acetylation of histone H3K27, H4K5 and H4K12 were examined by Western blot analysis in the liver of human liver-chimeric mice (n=3) and HBV-infected human liver-chimeric mice (n=3). (I) A model for the function of HAT1 in promoting histone acetylation of cccDNA minichromosome was shown. Mean ± SD of at least three experiments are shown, in which each experiment was designed by three replicates. Statistical significant differences are indicated: **P<0.01; ***P<0.001; ns, no significance.

Figure 4
Figure 4

HAT1 is recruited to cccDNA minichromosome by lncRNA HULC-scaffold HBc. (A) The combination of HAT1 and HBc was measured 8 dpi by immunoprecipitation assays in the HBV-infected dHepaRG cells. (B) The deposition of HAT1 and acetylation of histone including AcH3 and AcH4 on cccDNA minichromosome or the promoter of CCNA2 were verified 8 dpi by ChIP-qPCR in HBV-infected dHepaRG cells continuously transfected with pcDNA 3.1-HBC (2 μg/well) at -4, 0, and 4 dpi. (C) The interaction of HULC with HAT1 was examined by RIP-qPCR assays in the indicated cells. (D) The deposition of HAT1 and acetylation of histone including AcH3 and AcH4 on cccDNA minichromosome or the promoter of CCNA2 were analyzed 8 dpi by ChIP-qPCR in HBV-infected dHepaRG cells continuously transfected with siHULC (100 nM) at -4, 0, and 4 dpi. (E) The combination of HULC with HBc or HBx was examined by RIP-qPCR assays 8 dpi in HBV-infected dHepaRG cells. (F) The interaction of HULC with HBc or HAT1 was assessed 8 dpi by RNA pull-down assays followed by Western blot analysis in HBV-infected dHepaRG cells. (G) The combination of HAT1 and HBc was analyzed 8 dpi by immunoprecipitation assays in HBV-infected dHepaRG cells transfected with siHULC. (H) The levels of lncRNA HULC were examined in the liver of human liver-chimeric mice (n=3) and HBV-infected human liver-chimeric mice (n=3) by RT-qPCR. Mean ± SD of at least three experiments are shown, in which each experiment was designed by three replicates. Statistical significant differences are indicated: **P<0.01; ***P<0.001; ns, no significance.

Figure 5
Figure 5

HBV stimulates HAT1 promoter through HBx-co-activated transcriptional factor Sp1. (A) Correlation between HAT1 and HBX/pgRNA was examined by RT-qPCR in HBV-positive clinical HCC tissues. (B) The expression levels of HAT1 were assessed by RT-qPCR and Western blot analysis in HepG2 cells transfected with pcDNA3.1-HBX (0, 1 or 2 μg/well). (C) The expression levels of HAT1 were tested by RT-qPCR and Western blot analysis in HepG2 cells transfected with wild-type pCH-9/3091 (HBV-wt) or HBX deficient pCH-9/3091 (HBV-HBX-). (D) Luciferase activities of HAT1 promoter were analyzed in the cells. HepG2 and Huh7 cells were transfected with pGL3-Basic or reporter constructs containing various lengths of the 5'-flanking region of the HAT1 gene, as indicated (pGL3-P1, pGL3-P2, pGL3-P3, pGL3-P4, pGL3-P5 and pGL3-P6, respectively). (E) Luciferase activities of HAT1 P6 promoter were analyzed in HepG2 and Huh7 cells transfected with control vector or pcDNA3.1-HBX. (F) Luciferase activities of HAT1 P6 wild type or HAT1 P6 Sp1 binding site mutant type were determined in HepG2 cells transfected with pcDNA3.1-HBX. The illustration of the responsive region to Sp1 in HAT1 promoter was shown. Mean ± SD of at least three experiments are shown, in which each experiment was designed by three replicates. Statistical significant differences are indicated: **P<0.01; ***P<0.001.

Figure 6
Figure 6

HAT1 signaling confers to the regulation of HBV cccDNA minichromosome. In this model, HAT1/CAF-1 signaling, which is the host nucleosome assembly machinery, contributes to the assembly of cccDNA minichromosome by acetylating histone H4 at the sites of K5 and K12, leading to the accumulation of cccDNA. HAT1 is recruited to the cccDNA minichromosome through interacting with HBc, in which lncRNA HULC serves as a scaffold in the complex of HAT1/HULC/HBc for acetylation of histones on cccDNA minichromosome to activate HBV transcription.

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