The function of chromatin architectural protein CTCF in HBV transcription

Abstract

Hepatitis B virus (HBV) is a small DNA virus with an envelope, currently still posing a great threat to human health. Its genome is a minichromosome in the nucleus decorated with histones and other host factors. It is still not well elucidated as to how HBV transcription is regulated. CCCTC-binding factor (CTCF) is a multi-functional host chromatin architectural protein that regulates gene expression. Depending on the partners recruited, CTCF up-regulates the transcription of some genes but represses the expression of others. Previous investigations suggest that CTCF represses gene transcription of viruses such as human papillomavirus (HPV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV), but promotes adenoviral gene expression. However, the roles of CTCF in HBV transcription remain obscure. Thus, the research goal in this thesis is to examine the function of CTCF in HBV transcription. Knocking down CTCF expression repressed HBV transcription, whereas overexpression of CTCF facilitated HBV transcription. In addition, both full-length CTCF and truncated CTCF NZ without C-terminal domain of CTCF but not truncated CTCF Z or CTCF ZC lacking N-terminal domain of CTCF rescued the phenotypes caused by knocking down CTCF expression, suggesting that CTCF promotes HBV transcription and that its N-terminal domain is required for CTCF promotion of HBV transcription. Overexpressing only the CTCF N-terminal domain exhibited an inhibitory effect on HBV transcription possibly by squelching some proteins, but the mutant of the CTCT N-terminal domain that cannot bind to Rad21 (one component of the cohesin complex) lost the inhibitory effect on HBV transcription, indicating that CTCF plays a positive role in HBV transcription plausibly by recruiting the cohesin complex via the N-terminal domain of CTCF. The data from high-throughput chromosome conformation capture (Hi-C) coupled to chromatin immunoprecipitation (HiChIP) suggested that the Enhancer II of The HBV genome contacted the PreS2 promoter of the HBV genome via CTCF-mediated DNA looping that enabled Enhancer II to stimulate the activity of the PreS2 promoter. Such CTCF-mediated DNA looping was further validated by chromosome conformation capture coupled to chromatin immunoprecipitation (3C-ChIP), which indicated that anti-CTCF antibodies rather than irrelevant IgG enriched the CTCF-mediated DNA loop and that knocking down CTCF decreased the CTCF-mediated DNA loop. Furthermore, knocking down CTCF triggered less HBx, less WDR5, and less RNA polymerase II occupancy to the HBV genome, and also triggered less H3K4Me3 and more H3K9Me3 occupancy to the HBV genome. In conclusion, CTCF promoted HBV transcription. The CTCF-mediated DNA looping between the Enhancer II and the preS2 promoter might explain why Enhancer II increases the activity of the PreS2 promoter whereas Enhancer I had subtle effect on this promoter. CTCF also facilitated HBx binding to the HBV genome. Our results documented new roles of CTCF in HBV transcription and had implications in designing and developing new anti-HBV drugs. (An abstract of 455 words)