HepG2 human hepatocellular carcinoma cells and HBV-expressing HepG2.2.15 cells were obtained from Shanghai Xinyu Biotechnology Co., Ltd. (Shanghai, China). HepAD38 and HepG2 NTCP cell lines were provided by Prof. Deqiang Wang (Chongqing Medical University). All cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) at 37 °C in a humidified 5% CO₂ atmosphere. All cell lines were authenticated using short tandem repeat profiling, and they tested negative for Mycoplasma contamination.

The SIRT7-overexpression plasmid Flag-SIRT7 has been described previously [25]. DNA damage-binding protein 1 (DDB1)-overexpression plasmid was provided by Prof. Bing Ni (Third Military Medical University, Chongqing, China). The pcDNA3.1 vector served as an empty control. The sequences of siRNAs used in this study are detailed in Supplementary Table 1. Plasmids and siRNAs were transfected into cells with Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions.

HBV particles were prepared as previously described [29]. Briefly, the supernatant from HepAD38 cells was concentrated by adding 4% PEG8000 and incubating at 4 °C overnight. The precipitate was collected using centrifugation and resuspended in Opti-MEM to approximately 1% of the original volume. For infection, HepG2-NTCP cells were incubated with HBV at a multiplicity of infection of 1000 genome equivalents per cell in the presence of 4% PEG8000 for 24 h at 37 °C. Subsequently, the cells were washed three times with PBS and maintained in fresh medium for another 24 h.

Cells were lysed using RIPA buffer containing protease inhibitor cocktails (Roche; Basel, Switzerland). After centrifugation (12,000 $\times$g, 4 °C, 15 min), the protein concentration in the supernatants was quantified with a BCA assay kit (P0009; Beyotime Biotechnology, Shanghai, China). The protein samples were separated using 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore; Danvers, MA, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature, and then incubated overnight at 4 °C with the following primary antibodies diluted in 5% BSA: anti-SIRT7 (5360; Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti-DDB1 (ab109027; Abcam, Boston, MA, USA; 1:50,000), anti-histone H3 (17168-1-AP; Proteintech, Chicago, IL, USA; 1:2000) and anti-GAPDH (2118; Cell Signaling Technology, 1:1000). Subsequently, the membranes were probed with an HRP-conjugated secondary antibody (BL003A; Biosharp, Hefei, Anhui, China; 1:5000) for 1 h at room temperature. Protein bands were visualized using Pierce ECL substrate and captured with X-ray films (FujiFilm, Tokyo, Japan).

Total RNA was extracted from cells with TRIzol reagent (Life Technologies, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, cells were lysed with TRIzol, and then subjected to chloroform and phase separation via centrifugation. The RNA in the aqueous phase was precipitated with isopropanol and collected using centrifugation. The resulting RNA pellet was washed with 75% ethanol, briefly air-dried, and then dissolved in RNase-free water. The purified RNA was stored at –80 °C for subsequent analysis. RNA concentration and quality were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific), with an A260/A280 ratio of approximately 2.0 indicating high-purity RNA.

cDNA was generated from total RNA using the PrimeScript RT reagent kit (R323-01; Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) according to the manufacturer’s instructions. The resulting cDNA was diluted 1:5 with nuclease-free water and stored at –20 °C until use. Quantitative polymerase chain reaction (qPCR) was performed using SYBR qPCR Master Mix (Vazyme Biotech) on an ABI 7500 FAST system (Life Technologies). Primer sequences utilized in this study are provided in Supplementary Table 2.

Cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and the reaction was quenched by adding 0.125 M glycine for 5 min. After two washes with PBS, the cells were resuspended in SDS lysis buffer supplemented with protease inhibitors (100 µL per 1 $\times$ 10⁶ cells) and incubated at 4 °C for 10 min with intermittent vortexing. Chromatin was fragmented using a Branson Sonifier to generate DNA fragments ranging from 200 to 1000 bp. The lysate was centrifuged to remove insoluble debris, and a small aliquot of the supernatant was saved as the input control. The remaining chromatin was diluted 10-fold in ChIP dilution buffer and precleared with Protein A/G magnetic beads (Selleck Chemicals, Houston, TX, USA) at 4 °C for 1 h. The beads were subsequently removed using a magnetic rack. The precleared supernatant was then incubated overnight at 4 °C with anti-DDB1 (2 µg per immunoprecipitation; ab109027; Abcam), anti-RNA polymerase II (2 µg per immunoprecipitation; ab140509; Abcam), anti-H3K18Ac (2 µg per immunoprecipitation; 82832-1; Proteintech), anti-H3K36me3 (2 µg per immunoprecipitation; ab282596; Abcam), or control IgG (2 µg per immunoprecipitation; 30000-0-AP; Proteintech). Protein A/G magnetic beads were then added, and the samples were incubated for 1 h to capture the antibody–chromatin complexes. The beads were sequentially washed with low-salt, high-salt, LiCl, and TE buffers. After the final wash, the complexes were eluted and cross-linking was reversed by overnight incubation of samples at 65 °C in the presence of NaCl and proteinase K. DNA was purified using phenol–chloroform extraction, precipitated with isopropanol, and resuspended in nuclease-free water. Enrichment of target DNA was evaluated using qPCR and normalized to the levels of corresponding input samples.

Secreted levels of HBsAg and HBeAg in the culture supernatant were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (KeHua Biotech, Shanghai, China) according to the manufacturer’s instructions. Briefly, samples and standards were added to 96-well plates pre‑coated with capture antibodies specific to HBsAg or HBeAg and incubated for 1 h at 37 °C. After washing, HRP-conjugated detection antibodies were added, and the samples were incubated for 30 min at 37 °C. The color reaction was developed with 3,3^′,5,5^′-tetramethylbenzidine substrate and stopped by adding 2 M H₂SO₄. Absorbance at 450 nm was measured using a multifunctional microplate reader (Thermo Fisher Scientific). The concentrations of HBsAg and HBeAg were calculated using a standard curve.

The remaining supernatant was divided into 400-µL aliquots and precleared with Protein A/G magnetic beads (Selleck Chemicals) at 4 °C for 1 h. The beads were removed using a magnetic rack. The precleared supernatant was incubated overnight at 4 °C with anti-SIRT7, anti-DDB1, or control IgG. Protein A/G magnetic beads, pre-washed with IP buffer, were added to the antibody-treated supernatants, which were then rotated at 4 °C for 2–3 h. After three washes with IP buffer, the bead-bound proteins were eluted by heating in 1$\times$ loading buffer at 95 °C for 5 min. The eluate was collected after brief centrifugation at 500 $\times$g for 5 min at 4 °C. Input and immunoprecipitated samples were analyzed using western blotting with SIRT7 and DDB1 antibodies.

Protein–protein interaction networks were constructed using data from the BioGRID (https://thebiogrid.org/) and STRING (https://www.string-db.org/) databases. Gene expression correlation analysis was performed with the GEPIA2 web tool (http://gepia2.cancer-pku.cn/).

All experiments were independently repeated at least three times. Data are presented as mean $\pm$ SEM. For comparisons between two groups, a Student’s t-test was applied. For comparisons among more than two groups, one-way ANOVA followed by Tukey’s multiple-comparison post-hoc test was used. Results with a p-value of less than 0.05 were considered statistically significant.

To determine whether SIRT7 suppresses HBV transcription by catalyzing H3K18 deacetylation on the cccDNA minichromosome, we first knocked down SIRT7 in HepG2.2.15 cells and HBV-infected HepG2-NTCP cells (Fig. 1A). Western blotting showed that SIRT7 knockdown elevated global H3K18 acetylation levels (Fig. 1A). Similarly, the ChIP assay indicated that SIRT7 knockdown enhanced H3K18 acetylation, specifically on the cccDNA minichromosome (Fig. 1B). Conversely, SIRT7 overexpression reduced the acetylation of both global and cccDNA-bound H3K18 (Fig. 1C,D).

Fig. 1.

Sirtuin 7 (SIRT7) suppresses histone acetylation. (A,B) Changes in H3K18 acetylation levels in HepG2.2.15 cells and Hepatitis B virus (HBV)-infected HepG2-NTCP cells following SIRT7 knockdown, as detected using western blotting (A) and Chromatin Immunoprecipitation (ChIP) assay (B). (C,D) SIRT7 overexpression decreased H3K18 acetylation, as detected using western blotting (C) and ChIP assay (D). *p 0.05, **p 0.01.

Contrary to our initial hypothesis, ELISA showed that SIRT7 knockdown significantly reduced the secretion of both HBeAg and HBsAg into the culture supernatant (Fig. 2A). Correspondingly, the qPCR analysis indicated decreased expression of total HBV RNA and the 3.5-kb pre-core/pre-genome (pC/pg) RNA (Fig. 2B). Consistent with the loss-of-function results, SIRT7 overexpression increased the levels of viral antigens and RNAs (Fig. 2C,D). The ChIP assay revealed that SIRT7 knockdown and overexpression decreased and increased RNA polymerase II occupancy on cccDNA, respectively (Fig. 2E,F). Moreover, we found that SIRT7 overexpression increased the level of histone H3 lysine 36 trimethylation (H3K36me3)—a marker of transcriptional elongation—on cccDNA, whereas its knockdown decreased this modification (Fig. 2G,H). These findings suggest that SIRT7 facilitates HBV transcription.

Fig. 2.

SIRT7 promotes HBV transcription. (A,B) Following siRNA-mediated SIRT7 depletion, HBeAg and HBsAg secretion in culture supernatants was quantified using ELISA (A). qPCR analysis of total HBV RNA and 3.5-kb pC/pg RNA (B). (C,D) ELISA-based analysis of secreted HBeAg/HBsAg (C) and qPCR analysis of total HBV RNA and 3.5-kb pC/pg RNA levels (D), which revealed the functional consequences of SIRT7 overexpression. (E,F) ChIP assay of RNA polymerase II occupancy on cccDNA in SIRT7-knockdown (E) or SIRT7-overexpressing HBV-infected HepG2-NTCP cells (F). (G,H) ChIP assay of the levels of H3K36me3 on cccDNA in SIRT7-knockdown (G) or SIRT7-overexpressing HBV-infected HepG2-NTCP cells (H). *p 0.05, **p 0.01.

To elucidate the molecular mechanism by which SIRT7 promotes HBV transcription, we first identified potential SIRT7-interacting proteins by screening the BioGRID and STRING databases. Preliminary data suggested that SIRT7 physically interacts with DDB1, an adaptor protein of the CUL4 E3 ubiquitin ligase complex (Fig. 3A). The co-immunoprecipitation analysis revealed an interaction between SIRT7 and DDB1 (Fig. 3B,C). The bioinformatic analysis using the GEPIA2 tool demonstrated that the mRNA levels of SIRT7 positively correlated with those of DDB1 in various human tissues, including the liver tissue (Fig. 3D and Supplementary Fig. 1). SIRT7 knockdown led to reduced DDB1 expression at both the protein (Fig. 3E) and mRNA levels (Supplementary Fig. 2A), whereas SIRT7 overexpression had the opposite effect (Fig. 3F and Supplementary Fig. 2B). The ChIP assay revealed the binding of DDB1 to cccDNA, as well as attenuation of this interaction upon SIRT7 knockdown (Fig. 3G).

Fig. 3.

SIRT7 interacts with DDB1 to regulate HBV transcription. (A) Bioinformatic analysis (BioGRID/STRING databases) identified DNA damage-binding protein 1 (DDB1) (indicated by red arrow) as a potential SIRT7-interacting protein. (B,C) Reciprocal co-immunoprecipitation assays using anti-SIRT7 (B) and anti-DDB1 (C) antibodies confirmed the interaction between SIRT7 and DDB1 in HepG2 and HepG2.2.15 cells. (D) GEPIA2 revealed a significant positive correlation between SIRT7 and DDB1 expression in liver tissues. (E,F) Modulation of SIRT7 expression regulates DDB1 protein levels in HepG2 (left) and HepG2.2.15 (right) cells. (G) ChIP demonstrated DDB1 binding to HBV cccDNA, which was attenuated upon SIRT7 knockdown. *p 0.05, **p 0.01.

Consistent with our previous study results [30], DDB1 knockdown led to repressed HBV transcription (Fig. 4A,B), whereas DDB1 overexpression had the opposite effect (Fig. 4C,D). To investigate whether SIRT7 facilitates HBV transcription by promoting the interaction between DDB1 and cccDNA, we overexpressed DDB1 in SIRT7-knockdown cells (Fig. 5A). The ELISA showed that DDB1 overexpression partially reversed the inhibitory effect of SIRT7 knockdown on HBeAg and HBsAg secretion (Fig. 5B), whereas the qPCR analysis indicated that DDB1 overexpression alleviated the inhibitory effect of SIRT7 knockdown on total HBV RNA and 3.5-kb pC/pg RNA expression (Fig. 5C).

Fig. 4.

DDB1 mediates the SIRT7-dependent regulation of HBV transcription. (A) Effective knockdown of DDB1 in HepG2.2.15 cells was confirmed using western blotting and qPCR analysis. (B) DDB1 depletion significantly reduced HBV transcription, as evidenced by decreased HBeAg/HBsAg secretion and viral RNA levels. (C) Successful ectopic expression of DDB1 was verified using western blotting. (D) DDB1 overexpression enhanced HBV transcription, increasing both viral antigen and RNA levels. *p 0.05, **p 0.01.

Fig. 5.

DDB1 overexpression rescues HBV transcription suppression caused by SIRT7 knockdown. (A) Western blotting verified simultaneous SIRT7 knockdown and DDB1 overexpression in HepG2.2.15 cells. (B) ELISA showed that DDB1 overexpression rescued SIRT7 knockdown-mediated reduction in HBeAg and HBsAg secretion. (C) qPCR analysis demonstrated that DDB1 overexpression reversed SIRT7 knockdown-induced decrease in total HBV RNA and 3.5-kb pC/pg RNA levels. *p 0.05; **p 0.01.

To confirm that SIRT7 facilitated HBV transcription by promoting DDB1–cccDNA interaction, we knocked down DDB1 in SIRT7-overexpressing cells (Fig. 6A). The ELISA revealed that DDB1 knockdown repressed the SIRT7 overexpression-induced increase in HBeAg and HBsAg secretion (Fig. 6B), whereas qPCR analysis indicated that DDB1 knockdown blocked the SIRT7 overexpression-induced increase in total HBV RNA and 3.5-kb pC/pg RNA expression (Fig. 6C).

Fig. 6.

DDB1 knockdown reverses SIRT7 overexpression-induced promotion of HBV transcription. (A) Western blot showing the levels of the indicated protein in HepG2.2.15 cells co-transfected with SIRT7-overexpression plasmid and DDB1-specific siRNA. (B) ELISA-based analysis showed that DDB1 depletion abolished SIRT7-mediated increase in HBeAg and HBsAg secretion. (C) qPCR results demonstrating that DDB1 knockdown reversed SIRT7-induced increase in total HBV RNA and 3.5-kb pC/pg RNA levels. *p 0.05, **p 0.01; ns, not significant.