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

Area of Science:

• * Molecular Biology
• * Virology
• * Epigenetics

Background:

• * CCCTC-binding factor (CTCF) is a crucial host protein regulating transcription, epigenetic boundaries, and chromatin looping.
• * CTCF binds to the human papillomavirus (HPV) 18 E2 open reading frame (ORF), repressing viral oncogene expression in undifferentiated keratinocytes.
• * Keratinocyte differentiation disrupts CTCF-dependent HPV18 episome looping, leading to increased viral oncogene expression.

Purpose of the Study:

• * To investigate the role of CTCF in controlling HPV transcription, particularly during keratinocyte differentiation.
• * To characterize the structure and abundance of full-length HPV transcripts using Nanopore RNA-sequencing.
• * To identify novel viral transcripts and understand CTCF's regulation of early and late viral gene expression.

Main Methods:

• * Direct, long-read Nanopore RNA-sequencing of primary human keratinocytes with HPV18 episomes.
• * Analysis of viral transcript species before and after synchronous keratinocyte differentiation.
• * Comparison of transcripts from wild-type HPV18 cells versus CTCF-binding deficient HPV18 cells.

Main Results:

• * CTCF is identified as a key regulator of differentiation-dependent late promoter activation in HPV18.
• * CTCF is essential for efficient E1^E4 and L1 protein expression.
• * CTCF binding at the E2 ORF promotes the use of specific weak splice donor sites (SD3165, SD3284) to the E4 splice acceptor site (nt 3434).

Conclusions:

• * CTCF binding to the HPV18 E2 ORF facilitates both early and late viral transcription programs.
• * CTCF plays a dual role in HPV transcription, regulating oncogene expression and viral gene splicing.
• * Understanding CTCF's function in HPV transcription is critical for comprehending the viral life cycle.