Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and cccDNA silencing in vivo without affecting posttranslational modifications of cccDNA-bound histones

We previously showed that treatments with siRNA targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) strongly reduce all HBV markers, including HBx levels, thus enabling the reappearance of the host restriction factor “structural maintenance of chromosome 5/6 complex (SMC5/6) (Allweiss/Giersch Gut 2021).

Aim Understanding the mechanism of SMC5/6-mediated transcriptional silencing by investigating the epigenetic landscape of cccDNA-bound histones after siRNA or peg-IFNα treatment.

Methods HBV-infected human liver chimeric mice received siRNA or peg-IFNα for 4 or 6 weeks, respectively, or were left untreated. Active and repressive posttranslational modifications (PTMs) of cccDNA-bound histones and SMC5/6 occupation were analysed by chromatin immunoprecipitation-qPCR (ChIP-qPCR) and compared with occupation on host genes.

Results In untreated mice with high HBV replicative activity, active PTMs (H3K27ac, H3K4me3) were highly enriched on the cccDNA minichromosome, while the repressive mark H3K27me3 was undetectable. Notably, these treatments did not provoke significant changes for the analysed PTMs, despite strong reduction of cccDNA transcription. Consistent with previous results, only NSE4 occupancy became detectable after siRNA and peg-IFNα treatment. Interestingly, the heterochromatin mark H3K9me3 displayed intermediate levels on the cccDNA as compared to host heterochromatin (MYT-1 promotor) regardless of the treatment.

Conclusion The pattern of cccDNA-bound histone PTMs is consistent with active cccDNA transcription. The role of H3K9me3 deserves further investigation as it is linked with constitutive heterochromatin. In line with the function of SMC5/6 as a DNA micro-compaction machinery (Serrano, Molecular Cell 2020), the data suggest that SMC5/6 mediates transcriptional silencing through physical cccDNA compaction rather than through epigenetic changes.

Publication History

Article published online:
26 January 2022