The CCCTC-binding factor CTCF represses hepatitis B virus enhancer I and regulates viral transcription.

Valentina D'Arienzo,Fleur Chapus,Barbara Testoni,Peter Balfe,Joanna L Parish,Adam Claydon,Peter A C Wing,Guillaume Giraud,Jane A Mckeating,Sophia Begum,James M Harris,Xiaodong Zhuang,Jack Ferguson

doi:10.1111/cmi.13274

Valentina D'Arienzo, Fleur Chapus + Show 11 more

Open Access

https://doi.org/10.1111/cmi.13274

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Abstract

Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the Hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin-organising transcriptional insulator protein, CCCTC-binding factor (CTCF), has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or epi-somal copies of the viral genome. siRNA knock-down of CTCF results in a significant increase in pre-genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre-genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription.

Highlights

Hepatitis B virus (HBV) infection is one of the world's unconquered infections with an estimated 2 billion people exposed to the virus in their lifetime
We identified two CCCTC-binding factor (CTCF)-binding motifs within transcription regulatory elements, Enhancer I (EnhI) and X promoter (Xp), of the HBV genome
We demonstrate CTCF binding to HBV DNA by chromatin immunoprecipitation (ChIP)-qPCR in the region of these binding sites using various model systems that bear both integrated genomes and a closed circular DNA (cccDNA) pool, or cells exclusively expressing

Summary

| INTRODUCTION

Hepatitis B virus (HBV) infection is one of the world's unconquered infections with an estimated 2 billion people exposed to the virus in their lifetime. The HBV genome is transcribed by the host RNA polymerase II (RNA pol II) complex from four promoters (basal core promoter, BCP, Sp1, Sp2 and Xp) (Hong et al, 2017) that results in six major viral RNAs of increasing length with heterogeneous 50 ends and a common polyadenylation signal (Stadelmayer et al, 2020) These RNAs include: pre-core (preC) that encodes e antigen (HBeAg); pre-genomic (pgRNA) that is translated to yield core protein (HBcAg) and polymerase; preS1, preS2 and S RNAs encoding the surface envelope glycoproteins and X transcript for the multi-functional x protein (HBx). TADs are separated by regions enriched in binding sites of the ubiquitously expressed CCCTC-binding factor (CTCF), which stabilises chromatin loops by anchoring cohesin rings at the base of the loops (Rowley & Corces, 2018) Such spatial organisation can create epigenetic boundaries that separate transcriptionally active and inactive chromatin domains and control cis-regulatory elements, such as transcriptional enhancers. A role in HBV transcription regulation has not yet been reported, we show that CTCF binds HBV DNA and acts as a repressor of viral transcription

| RESULTS

Findings

| DISCUSSION