Gender-associated gene expression and chromatin accessibility of human urothelium

Bladder cancer is 7th most common type of cancer in the UK, and presents up to 4 times more often men than in women, even when adjusting for environmental factors such as smoking and occupation. Bladder cancer also has the highest rates of mutations in chromatin modifier genes compared to any other cancer type. Despite this, studies regarding the epigenome of bladder and bladder cancer are lacking. This study considers the genome-wide transcriptional and chromatin accessibility landscape of healthy urothelium, and aims to identify gender-associated differences promoting the gender biases observed in bladder cancer. The study is the first to establish reliable protocols for chromatin immunoprecipitation (ChIP) of histone marks, and an assay for transposase-accessible chromatin followed by next generation sequencing (ATAC-seq) in normal urothelium, and the first to carry out transcriptional profiling on normal human urothelial cells (NHUC). Affymetrix HTA2.0 microarrays using three models of healthy urothelium [NHUC, immortalised NHUC (TERT-NHUC), and uncultured healthy urothelial cells (UHUC)], showed that although the majority of differentially expressed (DE) genes between genders are located on the sex chromosomes, five autosomal genes are upregulated in female NHUC that are associated with invasive bladder tumours, inflammation, and hypoxia. Furthermore, each gender showed different transcriptomic perturbations in response to common mutations in a cohort of 102 stage Ta grade 2 tumours, including in tumours with mutations in the X-linked histone demethylase KDM6A where females had DE of chromatin regulatory genes but males did not. ATAC-seq in two male and two female TERT-NHUC lines showed a genome-wide increase in chromatin accessibility in males, which could be seen by increased signal at individual loci and a greater number of overall peaks. Although this difference did not correlate with increased transcriptional activity, cell proliferation, or cell-cycle stage, it did correlate with a global increase of the activating histone marks H3K4me3 and H3K27ac, but not the heterochromatin marker H3K27me3. A reliable ChIP protocol with validated controls was developed for the histone marks H3K4me1, H3K4me3, H3K27ac, and H3K27me3, and provides a foundation for future epigenetic research in bladder cancer. The results of this study suggest that, although variation between individual donors is greater than between gender groups, future research in bladder should consider genders separately. For instance, therapeutic efforts aimed at targeting chromatin architecture, such as HDAC inhibition, may be more effective in females, particularly when they have acquired mutations in KDM6A.