HPV8-E6 drives coordinated transcriptional and epigenetic reprogramming of keratinocytes.

Beta human papillomaviruses (betaHPVs) act as cofactors in keratinocyte carcinogenesis, but how they reprogram host gene regulation remains unclear. Here, we combined transcriptomic, small RNA and epigenomic profiling to dissect the impact of betaHPV8 oncogene expression on keratinocytes. The viral E6 protein emerged as the major driver of host gene regulatory reprogramming, repressing epidermal differentiation programs, whereas E7 induced fewer, complementary changes. Small RNA sequencing revealed E6-dependent remodeling of the microRNA (miRNA) landscape, and integrative analyses linked miRNA regulation to reciprocal changes in target gene expression, indicating a role for epigenetic post-transcriptional regulation in the E6-driven phenotype. Whole-genome bisulfite sequencing showed widespread E6-dependent DNA methylation changes at promoters, enhancers, and gene bodies. Motif enrichment analysis showed that hypermethylation silenced nuclear receptor and developmental regulators, while hypomethylation was associated with enrichment of AP1 and STAT3 binding motifs. Analysis of clinical specimens further revealed that betaHPV DNA was enriched in actinic keratoses and cutaneous squamous cell carcinomas displaying stem cell-like methylation profiles, linking viral activity to progenitor-associated tumorigenesis. Together, our data define a multilayered mechanism by which HPV8 destabilizes keratinocyte lineage identity which may create a cellular state permissive for malignant progression.