Biniossek, M. L. | 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

Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity

Loss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors. After in silico screening of 253 disease-related MEN1 missense mutations, we selected a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the interaction with MLL1 compared to JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites. These findings support the general importance of the menin-MLL1 and menin-JunD interactions in MEN1 gene-associated pathogenic conditions.

molecular biology↗