Fig 1.
Levels of APOBEC3B are elevated within HPV positive cells and are critical for maintenance of viral genomes and gene expression.
(A, left) Western blot analysis of whole cell lysates from HFK, HFK 31, and CIN 612 cells using a previously published A3B antibody [48]. Predicted molecular weight of A3B is ~ 46 kDa, but is observed to resolve ~37 kDa. RT-qPCR analysis of RNA extracts from HFK and CIN 612 cells (A, right). (B, left) Western blot analysis of CIN 612 cells depleted of A3B through stable expression of shRNAs. RT-qPCR analysis of RNA extracts from scramble control and two cell lines stably expressing shRNAs targeting A3B (B, right). (C) Analysis of expression levels of other A3 family members in scramble control and shA3B CIN 612 cells from total RNA sequencing (n = 2, n.s. = not significant). (D) RT-qPCR analysis of RNA extracts from scramble control and shA3B CIN 612 cells examining HPV 31 E6 and E7 expression. (E) Southern blot analysis of HPV genome levels from scramble control and shA3B CIN 612 cells. Quantification depicted on the right.
Fig 2.
APOBEC3B remains nuclear in HPV positive cells and interacts with viral and cellular chromatin on regions with high R-loop formation.
(A) Immunofluorescence analysis of HFK and CIN 612 cells using anti-A3B and DAPI. Scale bars = 10μm. (B) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis for A3B binding to the HPV URR and early polyA for HPV 16, 18, and 31. The percentage input was plotted: Input% = 100/2(ΔCt) normalized to input controls. (C) ChIP-qPCR of HFK 31 and CIN 612 for A3B interaction sites on the HPV 31 genome. The percentage input was plotted. (D) ChIP-qPCR of CIN 612 cells for A3B binding to previously identified cellular genes with high R-loop levels in HPV positive cells in comparison to normal (MYADM & RPL13a) and at genes with similar levels of R-loops in HPV positive and normal cells (EGR1 & SNRPN). The percentage input was plotted.
Fig 3.
APOBEC3B’s binding to chromatin is facilitated through R-loop formation and is responsible for the induction of~50% of the R-loops present in HPV positive cells.
(A) DRIP-qPCR analysis of scramble control and RNase H1 overexpressing CIN 612 cells. RNase H treatment was performed post nucleic acid extraction from each condition. R-loop levels were plotted normalized to the scramble control: (S9.6x/IgGx)/ (S9.6Scrm/IgGScrm), where x is Ct values from RNase H treated scramble control, overexpressing RNase H1 vehicle, or RNase H treated. (B) APOBEC3B ChIP-qPCR of scramble control and RNase H1 overexpressing CIN 612 cells on R-loop forming regions in viral (left) and cellular (right) chromatin. The percentage input is plotted. (C) S9.6 dot blot analysis of nucleic acid extracts from scramble control and A3B depleted CIN 612 cells. Nucleic acid retention on the membrane was normalized to methylene blue staining. (D) DRIP-qPCR analysis of R-loop forming regions on the viral chromatin from scramble control and shA3B depleted CIN 612 cells. R-loop levels were plotted normalized to the scramble control: (S9.6x/IgGx)/ (S9.6Scrm/IgGScrm), where x is Ct values from the A3B depleted cell lines.
Fig 4.
APOBEC3B preferentially regulates R-loop formation at the transcriptional start sites as well as termination sites in HPV positive cells.
(A) R-loop forming sites identified in the scramble control CIN 612 cells, A3B depleted CIN 612 cells, or both by DRIP-sequencing. (B) MA plot analysis of shared R-loop forming sites, where the base mean is equal to the average R-loop reads across all conditions. Median and interquartile range are provided. (C) Genomic annotation of R-loop sequences as to whether they form at 3’ UTR, 5’ UTR, gene bodies, intergenic regions, transcriptional start sites, or transcriptional termination sites in scramble control CIN 612 cells (left) or A3B depleted CIN 612 cells (right). (D) Venn diagrams depicting genes with R-loops present on their transcriptional start site (left) or termination site (right) in scramble control CIN 612 cells, A3B depleted CIN 612 cells, or both. (E) KEGG analysis of genes containing R-loops at their TSS or TTS in scramble control but not A3B depleted CIN 612 cells.
Fig 5.
APOBEC3B induces DNA damage and regulates the expression of cellular immune genes in HPV positive cells.
(A) Volcano plot of differentially expressed genes in scramble control or A3B depleted CIN 612 cells. Red dots represent upregulated and blue dots downregulated genes in A3B depleted CIN 612 cells compared to the scramble control. (B) A representative list of upregulated immune genes that no longer contained R-loops at their TSS/TTS in A3B depleted CIN 612 cells compared to the scramble control. (C) Western blot analysis of scramble control and A3B depleted CIN 612 cells for DNA damage repair factor, together with quantification of pChk1, tChk1, and γH2AX levels from three biological replicates (right). (D) Neutral and (E) alkaline COMET assays measuring dsDNA and ssDNA breaks, respectively, in scramble control and A3B depleted CIN 612 cells. Representative COMETs are displayed.