Core binding factor (CBF) is required for Epstein-Barr virus EBNA3 proteins to regulate target gene expression

Area of Science:

• Virology
• Molecular Biology
• Epigenetics

Background:

• Epstein-Barr virus (EBV) nuclear antigens (EBNA3s) play critical roles in B cell transformation and latency.
• Understanding EBNA3 protein binding to chromatin and their regulatory mechanisms is crucial for comprehending EBV pathogenesis.

Purpose of the Study:

• To investigate the chromatin binding principles of EBNA3 proteins.
• To elucidate the relationship between EBNA3 binding sites and gene regulation (activation/repression).
• To determine the role of RUNX3 and core binding factor beta (CBFβ) in EBNA3 recruitment and function.

Main Methods:

• Chromatin immunoprecipitation sequencing (ChIP-seq) on lymphoblastoid cell lines (LCLs) expressing epitope-tagged EBNA3 proteins.
• Global chromatin looping analysis.
• Comparison of EBNA3 binding sites with known transcription factor binding sites.
• Co-immunoprecipitation assays.
• Lentiviral shRNA-mediated depletion of RUNX3 and CBFβ.

Main Results:

• EBNA3-bound loci were uniquely associated with either EBNA3-repressed or EBNA3-activated genes, not both.
• EBNA3A and EBNA3C associated with both activated and repressed genes, while EBNA3B primarily associated with repressed genes.
• Substantial co-localization of EBNA3s with RUNX3 was observed.
• CBFβ robustly co-immunoprecipitated with EBNA3B and EBNA3C, but weakly with EBNA3A.
• Depletion of RUNX3 or CBFβ impaired EBNA3B and EBNA3C binding, confirming their requirement for EBNA3 recruitment.

Conclusions:

• RUNX3/CBFβ complexes are essential for the recruitment of EBNA3B and EBNA3C to target genes.
• RUNX3/CBFβ play a significant, previously unrecognized role in EBV-mediated B cell transformation and latency.
• These findings reveal a novel mechanism of EBV gene regulation involving RUNX3/CBFβ and EBNA3 proteins.