CTCF regulates hepatitis B virus cccDNA chromatin topology

Area of Science:

• Virology
• Epigenetics
• Molecular Biology

Background:

• Hepatitis B Virus (HBV) replicates using episomal covalently closed circular DNA (cccDNA) as a template.
• CCCTC-binding factor (CTCF) is a host protein regulating cellular transcription through epigenetic mechanisms.
• Previous studies showed CTCF represses HBV transcription, but mechanisms were unclear.

Purpose of the Study:

• To elucidate the mechanism by which CTCF regulates HBV transcription.
• To investigate the role of CTCF in HBV chromatin conformation and nucleosome positioning.
• To determine if CTCF regulation is specific to episomal cccDNA.

Main Methods:

• CTCF depletion in cells with cccDNA-like HBV molecules and de novo infected cells.
• Analysis of viral transcripts, including spliced forms.
• Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) to assess HBV chromatin topology.
• Mutation of CTCF binding sites in Enhancer I.

Main Results:

• CTCF depletion increased spliced HBV transcripts, particularly SP1, in cells with episomal cccDNA.
• CTCF depletion had no effect on integrated HBV DNA, and CTCF binding was limited to integrants.
• ATAC-Seq revealed an accessible region across HBV Enhancers I/II and BCP.
• Mutating CTCF sites disrupted chromatin accessibility and nucleosome phasing.

Conclusions:

• CTCF regulates HBV transcription specifically on episomal cccDNA.
• CTCF maintains HBV chromatin conformation and nucleosomal positioning.
• CTCF's role in chromatin regulation is crucial for controlling HBV gene expression.