Fig 2: Overview of datasets.Schematic representation of datasets used for overlap analysis of CESC and HNSCC integration sites with enhancers mapped in W12 cervical keratinocytes and FANCD2-associated common fragile sites mapped in C33-A and HeLa cervical carcinoma cell lines. APOT amplification of papillomavirus oncogene transcripts, WGS whole genome sequencing, WXS whole exome sequencing.

Fig 3: Integration hotspots are associated with cellular super-enhancers.H3K27ac- and Brd4-enriched regions were profiled in HPV16-positive cervical derived W12 keratinocyte subclones by ChIP-seq. Enhancer regions were defined as peaks that overlapped in both H3K27ac and Brd4 datasets, and that were identified across the four W12 subclones. a GREAT (Genomic Regions Enrichment of Annotations Tool) Gene Ontology analysis was performed using W12 enhancers that overlapped CESC integration breakpoints (±50 Kb flanks) as input and compared against all W12 enhancers, to identify putative target genes associated with these cis-regulatory regions based on enhancer frequency. Bars represent putative target genes plotted against their FDR (false discovery rate) adjusted p-values (q-value). Blue and gray bars represent genes that overlap integration hotspots and sites of non-recurrent integration, respectively. Enriched target genes within the same genomic locus were grouped (e.g., KLF5 and KLF12) and plotted using the most significant q-value. b Venn diagram showing the regions of overlap between integration loci, integration hotspots, and super-enhancers mapped in W12 subclones. c Bar chart showing the number of CESC integration loci that were grouped according to whether or not they are integration hotspots and plotted based on their overlap with super-enhancers, FANCD2-associated fragile sites, or both genomic features. Numbers above the graph indicate the total number of integration loci within each grouping. d Bar chart showing the number of CESC integration loci that are associated with super-enhancers (SE) that were grouped according to whether they are integration hotspots and plotted based on their viral transcription status. Numbers above the graph indicate the total number of integration loci that have associated viral transcription data within each grouping. e Alignment of Brd4 (blue) and H3K27ac (red) ChIP-seq signals mapped in W12 cervical keratinocytes at integration hotspots (top black bars; size indicated in Mb) in cervical carcinomas. Relative ChIP-seq peak heights are indicated in square parentheses. Gray bars represent amplified (AMP) host DNA in different CESC tumors from The Cancer Genome Atlas. Green, yellow, and black bars below the ChIP-seq signal tracks represent super-enhancers (SE) mapped in W12 subclones, FANCD2-associated fragile sites mapped in C33-A and HeLa cells, and CESC integration loci, respectively. Genes identified from GREAT Gene Ontology analysis53 and cancer driver genes56 are indicated by blue bars. Each integration hotspot is characterized in Supplementary Fig. 5 and Supplementary Data Table 4. f Bar chart showing the number of CESC integration loci that are associated with super-enhancers (SE) that were grouped according to whether they are integration hotspots and plotted based on their host somatic copy number alternation status. The number of integration loci per grouping was normal, n = 140, amplification (AMP), n = 240, and deletion (DEL), n = 17.

Fig 4: Cervical keratinocyte-specific fragile sites mapped by FANCD2 ChIP-seq.HPV-negative (C33-A) and HPV18-positive (HeLa) cervical carcinoma cells were treated for 24 h with 0.2 µM aphidicolin and FANCD2-enriched regions were identified by ChIP-seq. All analyses were performed using the combined C33-A and HeLa FANCD2 dataset. a Bar graph showing the genomic coverage of FANCD2-enriched regions relative to common fragile sites (FRA regions) and mitotic DNA synthesis (MDS) regions reported in the literature19,20. b Venn diagram showing the regions of overlap between fragile sites identified in the FANCD2, FRA, and MDS datasets. c Venn diagram showing the overlap of FANCD2-associated fragile sites with protein-coding genes longer or equal to 0.3 Mb. Red circle represents all long genes; blue circle represents long genes that are expressed in C33-A and/or HeLa cells. d Alignment of FANCD2-enriched regions in C33-A (red) and HeLa (yellow) cells with associated genes (blue bars represent genes >0.3 Mb; genes expressed in C33-A and/or HeLa cells are indicated by gene name), and FRA and MDS regions (black bars). Red and yellow bars below the FANCD2 ChIP-seq signal tracks represent peaks mapped by SICER analysis in the corresponding cell lines. Relative ChIP-seq peak heights are indicated in square parentheses.

Fig 5: FANCD2 Co-localizes with R-Loop-Enriched Foci at an Actively Transcribed Genomic Site(A) Schematic diagram of the DART system.(B) Immunostaining of FANCD2 in four cell lines expressing different fusion effector proteins in a DART assay. FANCD2 localizes to the KR foci in presence of ROS-induced DNA damage and active transcription.(C) Quantification of FANCD2 foci intensity at the TRE locus in (B).(D) Colocalization of FANCD2 and S9.6 foci at the TRE locus in TA-KR positive cells.(E) Numbers of TA-KR positive cells with or without S9.6 and FANCD2 foci at the TRE locus. Percentages of S9.6, FANCD2 foci double-positive cells over total examined cells and over S9.6 positive cells are shown.(F) Overexpression of wild-type or nuclease-dead D210N mutant of RNase H1 in TA-KR positive cells. Wild-type RNase H1 expression diminished FANCD2 signal.(G) Quantification of FANCD2 foci intensity at the TRE locus in (F).(H) siRNA depletion of FANCD2 or AQR increased S9.6 foci intensity in a DART assay.(I) Quantification of S9.6 foci intensity at the TRE locus in (H).For (C), (G), and (I), n = 50 cells, unpaired t test; error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001.