Objective The HBV HBx regulatory protein is required for transcription from the covalently closed circular DNA (cccDNA) minichromosome and affects the epigenetic control of both viral and host cellular chromatin.
Design We explored, in relevant cellular models of HBV replication, the functional consequences of HBx interaction with DLEU2, a long non-coding RNA (lncRNA) expressed in the liver and increased in human hepatocellular carcinoma (HCC), in the regulation of host target genes and the HBV cccDNA.
Results We show that HBx binds the promoter region, enhances the transcription and induces the accumulation of DLEU2 in infected hepatocytes. We found that nuclear DLEU2 directly binds HBx and the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic active subunit of the polycomb repressor complex 2 (PRC2) complex. Computational modelling and biochemical evidence suggest that HBx and EZH2 share two preferential binding sites in DLEU2 intron 1. HBx and DLEU2 co-recruitment on the cccDNA displaces EZH2 from the viral chromatin to boost transcription and viral replication. DLEU2-HBx association with target host promoters relieves EZH2 repression and leads to the transcriptional activation of a subset of EZH2/PRC2 target genes in HBV-infected cells and HBV-related HCCs.
Conclusions Our results highlight the ability of HBx to bind RNA to impact on the epigenetic control of both viral cccDNA and host genes and provide a new key to understand the role of DLEU2 and EZH2 overexpression in HBV-related HCCs and HBx contribution to hepatocytes transformation.
- hepatocellular carcinoma
- hepatitis B
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DS and LC are joint first authors.
Contributors DS and LC share co-first authorship. FG and ML conceived and designed the study. FG supervised the study. DS, VA, OF, AP, M-LP, MZ, SJ, LB and FG performed experiments and analysed data. LC and GC conducted computational analysis. M-LP performed bioinformatic analysis. LC, ML and FG wrote the manuscript.
Funding This work was supported by grants from Agence Nationale pour la Recherche sur le SIDA et les hepatites virales (ANRS) to ML (n° ECTZ8323; n. ECTZ27696; n. ECTZ66014); from the IDEX Package PALSE (Programme Avenir Lyon-St Etienne); from the Agence Nationale de la Recherche (ANR@TRACTION) to ML; from the EU project 667273 HEP-CAR to ML.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement The transcriptomic data used to generate the Venn diagram in Figure 5B ('genes whose expression is positively correlated with DLEU2 or EZH2') can be accessed on the TGCA-LIHC dataset and extracted using the R2 platform (https://r2.amc.nl) with the function 'Find correlated Genes with a Single Gene'. The ChIP-Seq dataset used in Figure 5B ('genes identified as HBx direct genes'; listed in online supplementary table 8) can be accessed at http://www.ebi.ac.uk/ena/data/view/PRJEB13227. All other raw data, materials and reagents are available on request from the corresponding authors (FG and ML).
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