Fig 2: APOBEC3A and APOBEC3B are expressed in different subsets of tonsillar epithelial cells.(A) UMAP projection of 22,595 epithelial cells from oropharyngeal squamous cell carcinoma samples (n = 10; 18,619 malignant cells (red), 695 non-malignant cells (purple)), and matched normal tonsil (n = 7; 3281 cells (blue)). (B) GOBP terms enriched amongst the sets of 100 genes that were co-expressed with either APOBEC3A or APOBEC3B in 2649 (after QC) epithelial cells from normal tonsil. Terms ranked by P value calculated by EnrichR package (Fisher Exact test bias corrected using the z-score of the deviation from the expected rank). (C) UMAP projection depicting four phenotypes (basal, proliferating, differentiating, terminally differentiated) displayed by the normal tonsillar epithelial cells in our dataset. (D) Marker genes are used to identify the four epithelial phenotypes represented in (C). (E) Violin plots of gene expression in individual tonsillar epithelial cells, and UMAP projections of the density of gene expression in the tonsillar epithelial subtypes. (****P < 0.0001, Wilcoxon’s rank-sum test; Basal, n = 1047; Proliferating, n = 353; Differentiating, n = 1091; Terminally differentiated, n = 158).

Fig 3: Keratinocyte cell cycle exit and initiation of differentiation is marked by a switch from APOBEC3B to APOBEC3A expression.(A) qRT-PCR-based gene expression measurements (n = 3) for APOBEC3A (P = 0.0002), KRT10 (P = 0.0002), IVL (P < 0.0001), APOBEC3B (P = 0.0001), MKI67 (P < 0.0001) and MCM7 (P = 0.0033) in proliferating NIKS (Async) or following 48 h of growth factor deprivation (Starved). (B) Representative cell cycle profiles of Async and starved NIKS measured by PI staining and flow cytometry. (C) qRT-PCR-based gene expression measurements (n = 3) for APOBEC3A (P = 0.0036), KRT10 (P = 0.0004), IVL (P = 0.0007), APOBEC3B (P = 0.9445), MKI67 (P = 0.0032) and MCM7 (P = 0.0001) following 24 h of vehicle control (DMSO) or 100 nM afatinib treatment (EGFRi). (D) Representative cell cycle profiles of DMSO and afatinib-treated NIKS measured by PI staining and flow cytometry. (E) qRT-PCR-based gene expression measurements (n = 3) for APOBEC3A (adjusted P values versus day 3 measurement: day 5 = 0.9960; day 7 = 0.0009; day 9 <0.0001), KRT10 (adjusted P values versus day 3 measurement: day 5 = 0.9518; day 7 = 0.0005; day 9 <0.0001), IVL (adjusted P values versus day 3 measurement: day 5 = 0.9983; day 7 = 0.0035; day 9 <0.0001), APOBEC3B (adjusted P values versus day 3 measurement: day 5 <0.0001; day 7 <0.0001; day 9 <0.0001), MKI67 (adjusted P values versus day 3 measurement: day 5 = 0.0001; day 7 <0.0001; day 9 <0.0001) and MCM7 (adjusted P values versus day 3 measurement: day 5 <0.0001; day 7 <0.0001; day 9 <0.0001) in NIKS collected 3, 5, 7, or 9 days after plating. (F) Representative cell cycle profiles of NIKS collected 3, 5, 7, or 9 days after plating were measured by PI staining and flow cytometry. (G) qRT-PCR measurements of APOBEC3A expression in primary human epidermal keratinocytes (NHEK) growing in FC medium and treated for 24 h with vehicle control (DMSO) or 100 nM afatinib (EGFRi, P < 0.0001) or following growth factor deprivation for 48 h (Starved, P = 0.0003). (H) Percentage of DDOST transcripts that were C > U edited at c558 in asynchronous growing NIKS (Async) and following 48 h of growth factor withdrawal (starved) measured by digital PCR assay (n = 3; P < 0.0001). Error bars = SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Pairwise comparisons were performed using unpaired two-tailed t-tests in (A, C, H). Comparisons of mRNA levels on days 5, 7 and 9 to day 3 in (E), and of mRNA levels in starved and afatinib-treated cells to DMSO-treated cells in (G) were performed using one-way ANOVA with Dunnett’s multiple comparisons test. Source data are available online for this figure.

Fig 4: GRHL3 regulates APOBEC3A expression in squamous cell carcinoma.(A) UMAPs heatmap showing gene expression of APOBEC3A and IVL and predicted activity of GRHL3 in scRNA-seq data from four independent tumour cohorts (three HNSCC and one ESCC). (B) qRT-PCR-based gene expression measurements of APOBEC3A and GRHL3 in sub-confluent BICR6 (top row) and BICR22 (bottom row) HNSCC cells transfected with control (NC#1) or GRHL3-specific siRNAs as indicated. Adjusted P values: NC#1 vs GRHL3 siRNA #1 (APOBEC3A: BICR6 <0.0001; BICR22 = 0.0145; GRHL3: BICR6 <0.0001; BICR22 <0.0001); NC#1 vs GRHL3 siRNA #2 (APOBEC3A: BICR6 <0.0001; BICR22 = 0.0035; GRHL3: BICR6 <0.0001; BICR22 <0.0001). Gene expression in GRHL3 siRNA-transfected cells was compared with control siRNA-transfected cells using one-way ANOVA with Tukey’s multiple comparisons test (n = 3; error bars represent SEM; ****P < 0.0001; **P < 0.01 and *P < 0.05). (C) matrix showing the relationship between expression of the indicated genes in spatial transcriptomics data from the Southampton HNSCC cohort obtained using the Visium platform (10X Genomics). (D) Images displaying expression levels (Visium spot intensities) of selected genes in HN485, an HPV+ve HNSCC case from the Southampton cohort. (E) Immunohistochemistry with an antibody specific for APOBEC3A (left) and with an antibody that cross-reacts with APOBEC3A, APOBEC3B and APOBEC3G (right) in sections from the same tissue block from HN485 used for the Visium profiling displayed in part D. (F) Boxplot showing expression of APOBEC3A in those cells predicted to be in S-phase in normal tonsil and HNSCC, stratified by binary GRHL3 activity score (on/off). Cells shown in black are outliers relative to the distribution of expression in the cells from healthy tonsils. Inset: UMAP showing the predicted cell cycle phase for each cell in the Southampton HNSCC scRNA-seq dataset. The top, middle and bottom lines of the boxplot represent the upper quartile (Q3), median, and lower quartile (Q1), respectively. The maximum value represented by the top whisker represents the highest observed data point within Q3 + (1.5 × (Q3 − Q1)), and the minimum value represented by the bottom whisker represents the lowest situated point within Q1 − (1.5 × (Q3 − Q1)). Healthy GRHL3 ‘OFF’, n = 19; Healthy GRHL3 ‘ON’, n = 173; Tumour GRHL3 ‘OFF’, n = 98; Tumour GRHL3 ‘ON’, n = 620. Source data are available online for this figure.

Fig 5: APOBEC3B is predominantly co-expressed with proliferation markers MKI67 and MCM7 in cycling basal and suprabasal cells of the healthy human airway.Stacked barplot showing the proportion of each cell type in the single cell atlas of healthy human airways from Deprez et al that express each selected gene.