CCCTC-binding factor recruitment to the early region of the human papillomavirus 18 genome regulates viral oncogene expression.
Bottom Line: Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts.In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing.These data highlight a novel virus-host interaction important for HPV pathogenicity.
Affiliation: University of St. Andrews, School of Medicine, St. Andrews, Fife, United Kingdom.Show MeSH
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Mentions: A diverse range of early transcripts is expressed from the HPV genome as a result of numerous alternative splicing events (14, 15). Alterations in the splicing events that are important in early gene expression in HPV infections could have a dramatic effect on the expression of early proteins and their truncated forms (E6*I, E6*II, and E6*III) (14). Given its previously described role in the control of RNA splicing (21), CTCF binding to the E2 ORF could affect splicing of the early transcripts and viral oncoprotein expression. To test this hypothesis, RNA was extracted from raft cultures harvested at day 14 and from early transcripts amplified by RT-PCR with primer pairs that were designed previously to identify the specific splicing events that occur within the early region of the HPV18 genome (14, 15). Amplification with a 5′ primer that anneals at nucleotide 121, upstream of the first splice donor site at nucleotide 233, and 3′ primer that anneals at nucleotide 3517, downstream of the five splice acceptor sites in the early region of HPV18 at nucleotides 416, 2779, 3434, 3465, and 3506 (14, 15), was used to detect any major splicing events that occur in the early region of the HPV18 genome. Amplification of RNA from WT HPV18 rafts resulted in two major products, with some minor products visible (Fig. 10A). As previously described (14), the two major products of 708 and 195 bp were identified by sequencing and shown to be spliced at 233^416 and 929^3434 and at 233^3434, respectively (Fig. 10B). Both of these products were consistently expressed in five raft cultures from each individual donor line of WT HPV18 HFKs. Of note, the 195-bp product, spliced between nucleotides 233 and 3434, was significantly reduced in and, in some cases, absent from the ΔCTCF HPV18 raft cultures (Fig. 10A and C). This is in contrast to the increase in unspliced transcript in the ΔCTCF HPV18 rafts (Fig. 9A and B). Therefore, a significant reduction in production of the short mRNA species (233^3434 spliced product) could result in the observed increase in unspliced E6E7 transcripts. Further analysis of viral transcripts revealed that splicing events at nucleotides 233^416 and 929^3434 were not altered by the loss of CTCF binding (Table 3). These experiments demonstrate that the loss of CTCF binding at position 2989 within the HPV18 genome results in a significant alteration in splice site usage, with the specific loss of 233^3434 spliced products in the early transcripts expressed.
Affiliation: University of St. Andrews, School of Medicine, St. Andrews, Fife, United Kingdom.