Biology subjects

Timmers, H. T. M.

Publications and source records attributed to Timmers, H. T. M..

6 recordsLinked to original sources

bioRxiv2023

ATAC and SAGA coactivator complexes utilize co-translational assembly, but their cellular localization properties and functions are distinct

To understand the function of multisubunit complexes it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here we demonstrate that the core modules of ATAC (ADA-Two-A-Containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription coactivator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histones proteins. In contrast, fully assembled ATAC complex cannot be detected in the cytoplasm of mammalian cells. However, endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related coactivators, ATAC and SAGA, assemble by using co-translational pathways, but their subcellular localization, cytoplasmic abundance and functions are distinct.

molecular biology↗

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↗

bioRxiv2023

Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1 - the largest protein in the complex - as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

molecular biology↗

bioRxiv2023

An EOMES induced epigenetic deflection initiates lineage commitment at mammalian gastrulation

Summary paragraph Different cell types are determined by cell lineage-specific transcriptional programmes and by epigenetic regulation of chromatin1, 2. Yet, the functional relationships between dynamically expressed transcription factors (TFs) and chromatin changes guiding lineage specification often remain elusive3. First mammalian embryonic lineages segregate when pluripotent cells become committed to either Mesoderm and Endoderm (ME) or Neuroectoderm (NE). NE forms by default in the absence of signalling-induced ME specification4, 5, resulting from global asymmetries in chromatin state favouring NE gene programme activation as recently demonstrated6-8. In this study, we unravel the initiation of ME lineage specification by the genome-wide, de novo formation of chromatin accessibility at ME enhancers that epigenetically deflects pluripotent cells from default NE differentiation. The Tbx TF Eomes, previously considered a transcriptional regulator, acts as global chromatin organizer that establishes ME lineage competence. EOMES recruits the canonical ATP-dependent chromatin remodelling complex SWI/SNF to broadly generate the chromatin- accessible ME enhancer landscape. This lineage competence is generated independently of ME gene transcription that fully depends on ME-inducing signalling pathways including Wnts and TGF{beta}/NODAL9. This study thus resolves the successive steps of ME lineage differentiation by globally establishing chromatin accessibility for lineage competence, followed by signal-encoded transcriptional regulation of different ME lineage-defining gene programmes.

developmental biology↗

bioRxiv2022

Epigenetic-focused CRISPR/Cas9 screen identifies ASH2L as a regulator of glioblastoma cell survival

Glioblastoma is the most common and aggressive primary brain tumor with poor prognosis, highlighting an urgent need for novel treatment strategies. In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma.

cancer biology↗

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↗