CTCF regulates hepatitis B virus cccDNA chromatin topology

Abstract

Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcription, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation.

Co-Transcriptional Regulation of HBV Replication: RNA Quality Also Matters

“…The host genome organizer CCCTC-binding factor (CTCF) was recently shown to repress HBV transcription by binding to two CTCF sites located in the EnhI region [ 48 ]. Moreover, CTCF binding within this region was reported to regulate the specific phasing of nucleosomes to maintain an open chromatin conformation and regulate HBV transcription [ 49 ]. The identification of CTCF as a regulator of cccDNA nucleosome positioning and transcription suggests that cccDNA may adopt a tri-dimensional conformation depending on its transcriptional activity.…”

“… Regulators of HBV cccDNA transcription. Examples of host or viral factors activating (green) or silencing (red) cccDNA transcriptional activity associated with an active (green nucleosomes) or a silent (red nucleosomes) chromatin [ 37 , 38 , 39 , 40 , 43 , 47 , 48 , 49 , 50 , 52 , 53 , 54 , 56 , 57 , 58 , 59 , 60 , 62 , 63 , 65 , 66 , 67 , 68 , 70 , 74 , 75 , 76 , 77 ]. The image was created using Biorender.com.…”

A nucleosome switch primes Hepatitis B Virus infection

“…78 Likewise, CBL137-mediated disruption of spatial genome organization (e.g., looping by CTCF or torsional strain) are also potential mechanisms of inhibition that remain to be more thoroughly addressed. 66,[79][80][81]…”