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 nuclear 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 enzymes 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. Using acute perturbation of the complex we further could show that inhibition of the enzymatic activity of the complex results in rapid changes in chromatin accessibility at at the timescale of minutes. These findings highlight the tight interplay between cellular metabolism, chromatin structure, and gene expression.
In contrast to the classical chromatin protein of the remodeller families, 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. We further could show that MTHFD1 links cellular metabolism to purine responses, thereby integrating metabolic signals from the environment, cellular metabolism, and chromatin organization.
In summary, our research has yielded novel insights into basic cell biology, novel therapeutic targets, as well as novel technology for monitoring and controlling subcellular protein localization.
