Journal: Epigenetics & Chromatin
Article Title: Episomal HBV persistence within transcribed host nuclear chromatin compartments involves HBx
Figure Lengend Snippet: Several euchromatin markers are enriched at HBV cccDNA, and suppression of HBx-mRNA expression via FANA antisense oligonucleotides leads to a reduction in episomal HBV DNA in HepG2.2.15 cells. a This figure outlines (from top to bottom) RNA-seq data for cccDNA mRNA transcription (consensus data from HepaRG, HepG2.2.15 and HepG2 H1.3), as well as ChIP-seq derived enrichment of Pol2 (dark blue tracks), H3K4me3 (cyan), H3K36me3 (green), H3K27me3 (red) and HBx (gray). Enrichment peaks are indicated at the top, and read coverage is presented below. Results from RNA-seq suggest that remarkable amounts of mRNA are synthesized from ORF S and ORF X. ChIP-seq reveals that in these cells ‘active’ transcription markers, such as Pol2 and H3K4me3, are enriched at HBV cccDNA. The highest amount of H3K36me3 seemed to be present at the 3′-ends of ORF S and ORF X, whereas comparably low amounts of H3K27me3 were associated with cccDNA. b This diagram presents the localization of ORFs, CpG islands (major CpG islands: I–III; minor CpG islands not detected in all genotypes: IV–VI) and the GC content (adapted from Hensel et al. [ 66 ]). c This figure outlines a time course of episomal HBV DNA quantification by PCR in HepG2.2.15 cells under the influence of FANA oligonucleotide treatment targeting HBx-mRNA. Specifically, two different oligos (HBx-oligo1/HBx-oligo2) or an oligo mixture (HBx-oligo-mix) was used to suppress HBx-mRNA. For normalization, we performed similar experiments where a scrambled non-sense FANA oligonucleotide was used. This figure demonstrates the increasing attenuation of HBV DNA over time under the influence of FANA oligonucleotide anti-HBx treatment
Article Snippet: For mRNA enrichment, we used the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and then the NEBNext Ultra RNA Kit (NEB) for Illumina-compatible library preparation upon manufacturer’s recommendations.
Techniques: Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Derivative Assay, Synthesized, Polymerase Chain Reaction