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. 2020 Feb 29;69(11):2016–2024. doi: 10.1136/gutjnl-2019-319637

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© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

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DLEU2 controls HBV replication. (A) DLEU2-qChIRP (chromatin isolation by RNA purification) for covalently closed circular DNA (cccDNA) using a DLEU2-specific antisense biotinylated DNA probe (100 pmol) in mock-replicating and HBV-replicating HepG2 cells (48 hours). Affinity-purified material was analysed by real-time quantitative PCR (qPCR) with DLEU2 RNA-specific primer pairs and the results are expressed as % of input. (B) Heatmap of the expression levels of 34 long non-coding RNAs (lncRNAs) that are direct transcriptional targets of HBx analysed by Nanostring in HepG2 cells replicating wild-type HBV (HBV wt) or HBV with mutant HBx (HBV mt HBx) (48 hours). Colours in the heatmap indicate log2 counts normalised to GAPDH. (C) DLEU2-qChIRP for cccDNA using a DLEU2-specific antisense biotinylated DNA probe (100 pmol) in HBV or HBV HBx mt replicating HepG2 cells (48 hours). Affinity-purified material was analysed as in 4A. Results are expressed as % of input. (D) ddPCR quantification of HBV pregenomic RNA (pgRNA) (copies/cell) from HBV-infected primary human hepatocytes (PHHs) (4 days) transfected with scrambled Gapmer (CTL) or DLEU2-specific Gapmer pools. Results are expressed as copies for 10 ng of total RNA after normalisation to endogenous human β-globin. (E) ddPCR quantification of HBV cccDNA (copies/cell) from HBV-infected PHHs (4 days) transfected with scrambled Gapmer (CTL) or DLEU2-specific Gapmer pools. Results are expressed as copies for 10 ng of total RNA after normalisation to endogenous human β-globin. (F) cccDNA-bound histone H4 acetylation. Cross-linked chromatin from HBV-replicating HepG2 cells transfected with scrambled Gapmer (CTL) or DLEU2-specific Gapmer pools (48 hours) was subjected to immunoprecipitation with anti-AcH4 antibody or IgG controls, and then analysed by real-time qPCR using cccDNA-specific primer pairs. Chromatin immunoprecipitation (ChIP) results are expressed as % of input. (G) Enhancer of zeste homolog 2 (EZH2) occupancy on cccDNA in HBV wt-replicating or HBV mt HBx-replicating HepG2 cells (48 hours). ChIP results are expressed as % of input. Data in panels (A), (B), (C), (D) and (F) represent means±SD from at least three independent experiments. In (D), and (F), *p<0.05; Mann-Whitney U test.