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.
