H3K36me3 is required for productive viral replication.

(A) CIN612 cells were left untreated (UT) or transduced with lentivirus expressing either wild-type (WT) H3.3 or the H3.3K36M mutant. 72hr post-transduction, cells were harvested as a T0 (undifferentiated) or differentiated in high calcium medium for 72hr. (B) CIN612 cells were transduced with either pLenti-control (CTR) or pLenti-FLAG-KDM4A. Following selection in puromycin, cells were harvested as a T0 (undifferentiated) or were induced in high calcium for 72hr. For (A) and (B) DNA and protein were harvested at the indicated time points. DNA was digested with BamHI (non-cutter) and Southern blotting analysis was performed to analyze episome copy number using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of H3K36me3, with H3.1 serving as a loading control. Involucrin and K10 were used as markers of differentiation and GAPDH as loading control. For (B) western blot analysis was performed using an antibody to FLAG to detect KDM4A. For (A) and (B), fold change in episome copy number was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Graphed is the average fold change relative to (A) UT T0 and (B) pLenti-CTR, which are set to one. Error bars represent means ± standard errors. Statistics were assayed using a student’s t test. * p < 0.05. Ca = calcium.