Periodic Reporting for period 3 - CHROMABOLISM (Chromatin-localized central metabolism regulating gene expression and cell identity) Reporting period: 2021-05-01 to 2022-10-31 Summary of the context and overall objectives of the project Epigenetics research has revealed that in the nucleus of mammalian cells the localization of almost all biomolecules – DNA, RNAs, proteins, protein posttranslational modifications – is tightly regulated to ensure they occupy distinct nuclear territories and specific sites in the genome. In contrast, small molecules and cellular metabolites are generally considered to passively enter the nucleus and lack locus-specific roles due to their fast diffusion. The CHROMABOLISM proposal tests the hypothesis that phase separation, proximity to the nuclear pore and recruitment of metabolic enzymes to chromatin can result in localized roles for at least some metabolites. A better understanding of the interplay between cellular metabolism, chromatin structure and gene regulation can have important societal implications, particularly for the characterization, prevention and treatment of human diseases. Through the close link between chromatin structure, gene expression and cell identify, our research aims to uncover novel opportunities to establish desirable cell identify for regenerative medicine and to interfere with aberrant identity of cancer cells. Our overall objectives are generating comprehensive maps of chromatin-bound metabolic enzymes and nuclear metabolomes in representative leukemia cell lines, developing technology to perturb these nuclear metabolite patterns, and modeling the effects of cellular metabolites on cancer cell identity to identify novel therapeutic targets in leukemia. Successful completion of this project has the potential to transform our understanding of nuclear metabolism in control of gene expression and cellular identity. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Since the start of the CHROMABOLISM grant, we conducted the groundwork and proof of concept experiments towards achieving the overall project goals. For mapping nuclear metabolic enzymes and metabolites we have developed and optimized protocols for the rapid isolation of nuclei and of chromatin. In selected leukemia cell lines, we then performed mass-spectrometry based proteomics and metabolomics analyses to determine the cell-type variation of nucelar metabolic enzymes and metabolites in steady state. In parallel, we developed a novel complementary method for microscopy-based live-cell imaging of hundreds of metabolic enyzmes in parallel. In short, insertion of a fluorescent tag as a synthetic exon into introns of metabolic enzymes allows the generation of a cell pool, for which in each cell a different enzyme is tagged. The responses of these cells to drugs and other perturbations can then be monitored by live-cell microscopy. Finally, we use in situ sequencing to identify which enzyme is tagged in which cell. In addition to these untargeted approaches, we conducted detailed characterization of two metabolic enzymes in the nucleus. We systematically studied the BAF complex, a chromatin remodelling ATPase, and identified subunit specific roles and cancer vulnerabilities. In contrast to this classical chromatin protein, the nuclear role of the second metabolic enzyme we studied, MTHFD1, was unexpected. We had identified MTHFD1 from a genome-scale genetic screen for factors phenocopying inhibition of the histone acetyl-binder BRD4 and could show that the proteins physically interact in the nucleus. The results for the first project period have already validated some of the hypothesis underlying our proposal and they provide extensive starting points for further experiments in the next 2.5 years according to our experimental plan. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In the first period of the CHROMABOLISM project, we have applied proteomics and metabolomics methods to uncover novel roles of metabolic enzymes and metabolites in the cell’s nucleus. To complement these methods, we have established an entirely novel method for pooled tagging of hundreds of metabolic enzymes. We have shown that this method can be applied to identify expected and novel effects of small molecule perturbations on the localizations and levels of selected metabolic enzymes. In addition, this method together with a detailed proteomics study, has identified dozens of metabolic enzymes in the nucleus for which such a localization has not been previously studied. We expect our method of pooled intron tagging to have broad application in cell and chemical biology, and plan to systematically apply this method to assess dynamic responses of nuclear metabolic enzymes to various stimuli until the end of the project. In addition to method development, we have uncovered novel roles of the human BAF complex, a chromatin remodelling ATPase in the nucleus. Using systematic knock-out of subunits of this protein complex, we have been able to assign subunit-specific effects on chromatin accessibility and transcription. In addition, we have also uncovered three novel intra-complex synthetic lethalities, that may in the future be exploited as therapeutic targets for the development of therapies for BAF mutant cancers. Until the end of the project period, we plan to further deepen our insights into this complex, and in particular study chromatin remodelling with tight temporal resolution following BAF perturbations and investigate the interplay between nuclear ATP levels, complex activity and mutations. Finally, we have uncovered a novel role for MTHFD1 in the nucleus. We identified this folate metabolic enzyme from a genetic screen for genes that when knocked-out phenocopy inhibition of the important chromatin protein BRD4. Current work is ongoing to dissect the role of the folate pathway in transcriptional control in more detail and to develop chemical probes against selected enzymes of the pathway. In summary, the first period CHROMABOLISM project enabled us to significantly advance the state of the art in three subprojects. We anticipate that the preliminary results we generated will enable us to further transform our knowledge on central metabolic activities in the cell’s nucleus.