The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression

Abstract

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF.

Epigenetic and Transcriptomic Regulation Landscape in HPV+ Cancers: Biological and Clinical Implications

“…Initially, when HPV infects undifferentiated cells of the basal layer of the epithelium, the viruses remain in an epigenetically repressed episomal state, with enrichment of H3K27Me3 and low levels of H3K4Me3. Consequently, there is a low viral load (between 50 and 100 viral copies per cell), which favors a deficient activation of the immune response ( Ferguson et al, 2021 ).…”

“…Epithelial differentiation disrupts the CTCF-dependent chromatin loop ( Figure 2 ). Studies in HPV18 showed that abrogation of CTCF binding to the E2 ORF increases the transcriptional activity of the p102 early promoter, increasing the expression of E6/E7 proteins ( Ferguson et al, 2021 ).…”

“…Interestingly, CTCF is a key regulator in these processes, since mutations that disrupt the binding sites as well as the introduction of new sites for CTCF can reorganize chromatin and modify transcriptional regulation, allowing tumorigenesis beyond the transcriptional regulation of E6/E7 ( Karimzadeh et al, 2022 ). In addition, sequencing studies elucidated that CTCF may play an important role in the regulation of alternative splicing of viral genes ( Ferguson et al, 2021 ).…”

Synonymous nucleotide changes drive papillomavirus evolution

“…Temporal expression of the viral genome is associated with tissue differentiation and is tightly regulated at the level of transcription and RNA processing [ [10] , [11] , [12] ]. A typical mammalian papillomavirus encodes six to nine proteins ( Fig.…”

Revisiting Papillomavirus Taxonomy: A Proposal for Updating the Current Classification in Line with Evolutionary Evidence

“…Expression of viral mRNA is temporally synced with tissue differentiation and is tightly regulated at the level of transcription and RNA processing [ 15 , 16 , 17 ]. In addition to these viral proteins, most papillomavirus types also express an E1^E4 and E8^E2 gene product [ 18 , 19 ].…”