Nuclear localization of MTHFD2 is required for correct mitosis progression

Article https://doi.org/10.1038/s41467-024-51847-z Nuclear localization of MTHFD2 is required for correct mitosis progression Received: 6 June 2023 Accepted: 20 August 2024 Natalia Pardo-Lorente1, Anestis Gkanogiannis 1,3, Luca Cozzuto1,3, Antoni Gañez Zapater 1, Lorena Espinar 1, Ritobrata Ghose 1, Jacqueline Severino 1, Laura García-López1, Rabia Gül Aydin 1, Laura Martin Maria Victoria Neguembor 1, Evangelia Darai1, Maria Pia Cosma 1,2, Laura Batlle-Morera1,2, Julia Ponomarenko1,2 & Sara Sdelci 1,2 1 , Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we show that the nuclear localization of the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) ensures mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability, including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces severe methylation defects and impedes correct mitosis completion. MTHFD2 deficient cells display chromosome congression and segregation defects and accumulate chromosomal aberrations. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells, whereas restricting MTHFD2 to the nucleus is sufficient to ensure correct mitotic progression. Our discovery uncovers a nuclear role for MTHFD2, supporting the notion that translocation of metabolic enzymes to the nucleus is required to meet precise chromatin needs. 1234567890():,; 1234567890():,; Check for updates Enzymes of central metabolism can translocate to the nucleus and influence chromatin remodeling, epigenetics, and transcription regulation1–5. A clear example of this in loco regulatory activity of metabolic enzymes is the synthesis of acetylation and methylation cofactors required for histone modification directly on chromatin6–9. One-carbon folate metabolism is a pivotal pathway, being indispensable for the de novo synthesis of nucleotides, amino acid homeostasis, DNA and histone methylation, and the maintenance of the cellular redox state10. Folate metabolism is compartmentalized between the cytosol and the mitochondria, and the mitochondrial onecarbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) emerged as the most consistently overexpressed metabolic gene in cancer11. While expressed during embryonic development and cancer, MTHFD2 is barely expressed in adult tissues11, making it a very attractive anticancer target12–14. High levels of MTHFD2 are associated with a worse outcome in several cancer types, including breast15, colon16, and lung17 cancer. MTHFD2 has been shown to support cancer cell proliferation and survival in vitro and tumor growth in vivo16–20, and to promote metastatic features such as cell migration and invasion18,21. MTHFD2 can localize in the nucleus22,23. However, its nuclear function remains poorly characterized and elusive. During cell division, cells duplicate their genetic material, which is divided between two daughter cells during mitosis. Mitotic defects lead to the accumulation of genetic aberrations that can result in cellular transformation or lead to cell death24. Several mechanisms ensure faithful chromosome segregation, and centromeres are important pillars in this process. Centromeres orchestrate the chromosomal attachment to spindle fibers through the assembly of the kinetochore complex25–27. Centromeric and peri-centromeric DNA is compacted into 1 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain. 2Universitat Pompeu e-mail: Fabra (UPF), Barcelona, Spain. 3These authors contributed equally: Anestis Gkanogiannis, Luca Cozzuto. Nature Communications | (2024)15:9529 1 Article heterochromatin28 and heterochromatin marks H3K9me3, H3K27me3, and H4K20me1 decorate the centromere, contributing to the formation of the kinetochore29–32. Moreover, DNA methylation at centromeres is abundant and maintains chromatin structure, preventing errors in chromosome segregation and genomic instability33–37. The expression of the centromeric region regulates the loading of the centromeric histone variant CENP-A and contributes to the recruitment of inner kinetochore proteins to the centromeric region38. Here, we discovered that MTHFD2 localizes in the nucleus to regulate DNA and centromeric histone methylation, centromeric expression, and proper mitotic progression. MTHFD2 nuclear partners are mostly cell cycle regulators and methyltransferases responsible for depositing methylation marks at centromeres, such as KMT5A29,39, DNMT3B40, and PRMT141. The absence of MTHFD2 leads to a drastic reduction of DNA and centromeric histone methylation, increased centromeric alpha-satellite expression, and accumulation of genomic aberrations. Consequently, cell cycle progression is also impaired when cells lack MTHFD2, with a significant reduction in mitotic events. The absence of total MTHFD2 or its nuclear inhibition alone induces chromosome congression and segregation defects, as well as micronuclei accumulation, indicating that the nuclear localization of MTHFD2 plays an active enzymatic role in controlling centromeric heterochromatin maintenance and correct mitotic cell division. Results MTHFD2 localizes within the nucleus in proliferating cells A meta-analysis comprising microarray expression data covering 19 types of tumors highlighted the mitochondrial folate enzyme MTHFD2 as the top-scoring upregulated metabolic enzyme in cancer11. To corroborate this finding, we retrieved RNA-sequencing data from The Cancer Genome Atlas (TCGA) database42. We filtered for solid tumor types where paired normal tissue data were available (Supplementary Fig. 1a) and confirmed that MTHFD2 was significantly upregulated in 13 out of 15 evaluated tumor types. Among these, breast carcinoma, colon adenocarcinoma, lung adenocarcinoma and lung squamous cell carcinoma showed the most significantly increased MTHFD2 levels (Fig. 1a). Then, we asked whether MTHFD2 expression alone could be used to predict the status of a sample (tumor versus healthy) in breast, lung and colon cancer. Training a tree-based algorithm with a subset of the TCGA expression data yielded a prediction accuracy (true positive rate) over 0.84 and AUC (area under the curve) values between 0.77 and 0.88 on unseen data, confirming MTHFD2 expression’s predictive usefulness (Supplementary Fig. 1b–d). Although MTHFD2 primarily localizes within the mitochondria, its nuclear23 and chromatin43 localizations have been previously reported in some cancer cell lines. Therefore, we asked whether MTHFD2 chromatin localization is a generalizable event. We selected a panel of breast, colon and lung cancer cell lines in which MTHFD2 was highl (...truncated)