Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro. We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK. Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis. Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.

Epigenetic alterations play a central role in cancer development and progression. The acetylation of histone 3 at lysine 27 (H3K27ac) specifically marks active genes. While chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) analyses are commonly performed in cell lines, only limited data are available from primary tumors. We therefore examined whether cancer-specific alterations in H3K27ac occupancy can be identified in primary rectal cancer. Tissue samples from primary rectal cancer and matched mucosa were obtained. ChIP-seq for H3K27ac was performed and differentially occupied regions were identified. The expression of selected genes displaying differential occupancy between tumor and mucosa were examined in gene expression data from an independent patient cohort. Differential expression of four proteins was further examined by immunohistochemistry. ChIP-seq for H3K27ac in primary rectal cancer and matched mucosa was successfully performed and revealed differential binding on 44 regions. This led to the identification of genes with increased H3K27ac, i.e., RIPK2, FOXQ1, KRT23, and EPHX4, which were also highly upregulated in primary rectal cancer in an independent dataset. The increased expression of these four proteins was confirmed by immunohistochemistry. This study demonstrates the feasibility of ChIP-seq-based epigenome mapping of primary rectal cancer and confirms the value of H3K27ac occupancy to predict gene expression differences.