Knuechel-Clarke, R. | bioRxiv Science

bioRxiv2023

Alternative mRNA splicing controls the functions of the histone H3K27 demethylase UTX/KDM6A

BackgroundThe UTX/KDM6A histone H3K27 demethylase plays an important role in development and is frequently mutated in cancers such as urothelial cancer. Despite many studies on UTX proteins, variations in mRNA splicing have been overlooked. MethodsUsing Nanopore sequencing, we present a comprehensive analysis of UTX/KDM6A splicing events in human cell lines and in tissue samples from bladder cancer and normal epithelium. ResultsThe central region of UTX mRNAs encoded by exons 12 to 17 undergoes extensive alternative splicing. Up to half of all stable mRNAs (8-48% in bladder tissues and 18-58% in cell lines) are represented by the UTX canonical isoform lacking exon 14 encoding a nuclear localization sequence, hence localize to the nucleus, unlike cytonuclear localization of the canonical isoform. Chromatin association was also higher for exon 14-containing isoform compared to the canonical UTX. Using quantitative mass spectrometry, we found that all UTX isoforms integrated into the MLL3 and MLL4, PR-DUB and MiDAC complexes. Interestingly, one of the novel UTX isoforms, which lacks exons 14 and 16, fails to interact with PR-DUB and MiDAC complex members. ConclusionUTX mRNAs undergo extensive alternative splicing that controls the subcellular localization of UTX and its interactions with other chromatin regulatory complexes. Simple SummaryUTX/KDM6A is a histone H3K27 demethylase and plays an important role in mammalian development and human diseases such as urothelial cancer. We identified a region encompassing exons 12-17 of UTX that undergoes extensive splicing events. As a result, a nuclear localization sequence located in exon14 is missing in a considerable part of UTX transcripts in different cell lines and tissues from normal bladder epithelium and bladder cancer. Mass spectrometry analysis showed a role for this region in binding to the epigenetic PR-DUB and MiDAC complexes. UTX was also more extensively bound to chromatin when the alternative splicing region presented. Our study showed that alternative splicing of UTX transcripts plays an important role in its functions.

biochemistry↗

bioRxiv2022

SPAG6 promotes cell migration and induces epithelial-to-mesenchymal transition in luminal breast cancer cells

Understanding the involvement of promoter DNA methylation changes in the development of breast cancer may be highly informative for designing more effective therapeutic treatments. We recently characterized the Sperm Associated Antigen 6 (SPAG6) gene, encoding a flagellar motility protein, as a potential DNA methylation biomarker for blood-based early breast cancer detection. Here we present the first study to evaluate the functional role of SPAG6 in human breast cancer. In silico analysis of the HumanMethylation450 BeadChip and Illumina HiSeq data of The Cancer Genome Atlas (TCGA) was performed in both normal (n=114) and breast cancer patient tissues (n=1104) to determine SPAG6 DNA methylation and expression. Stable SPAG6 overexpressing cancer models for in vitro analysis were obtained by lentivirus-mediated gene delivery in T-47D, MCF-7, MDA-MB-231 and BT-549 breast cancer cells. Subsequently stable mock and SPAG6 cell lines were compared in cellular assays. In addition, involvement of SPAG6 in EMT was analysed by qPCR and immunolabeling experiments. All major molecular subtypes of breast cancer (luminal A, luminal B, basal-type, HER2-enriched) revealed a tumor-specific increased SPAG6 promoter hypermethylation that correlated with strong reduction in SPAG6 mRNA expression. Interestingly, a small group of luminal breast tumors exhibited SPAG6 mRNA overexpression compared to normal breast tissue. SPAG6 overexpression caused a significant reduction (p<0.05) in colony formation in basal MDA-MB-231 and BT-549 cells. In turn, luminal T-47D cells overexpressing SPAG6 showed a significant increase in colony formation (p=0.0004) and both T-47D-and MCF-7 cells overexpressing SPAG6 exhibited a robust increase in migration speed (p<0.0001). In SPAG6-positive T-47D cells SNAIL, TWIST1 and Vimentin expression was found to be significantly upregulated, while E-Cadherin expression was supressed. SPAG6 overexpressing T47D cells showed a typical epithelial-mesenchymal transition (EMT). This was accompanied by a nearly complete displacement of both actin and E-cadherin from cell-cell junctions. Our in vitro analyses give functional evidence that SPAG6 has a profound effect on colony formation, migration and intercellular junction composition in breast cancer cells. Our study is the first to show opposing SPAG6 effects in a single tumour entity depending on the molecular subtype. We propose that SPAG6 might be a key player for inducing the EMT program in luminal-type breast cancers, driving tumour progression and metastasis.

cell biology↗