2021 – TRANSCRIPTIONAL REGULATION OF MITOCHONDRIAL METABOLISM BY TIF1GAMMA DRIVES ERYTHROID PROGENITOR DIFFERENTIATION

Understanding in-vivo mechanisms of erythropoiesis is critical for directed differentiation approaches to treat blood disorders. Zebrafish moonshine (mon) mutant embryos defective for the conserved transcriptional intermediary factor 1 gamma (tif1γ) do not specify enough erythroid progenitors. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we performed a chemical suppressor screen using 3,100 compounds and identified inhibitors of the essential mitochondrial pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH). Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar rescue the formation of erythroid progenitors in 61% (38/62) and 68% (50/74) of mon embryos, respectively. Beyond changes in nucleotide metabolism, in-vivo metabolic analyses revealed low levels of TCA cycle metabolites which were functionally linked to a reduced oxygen consumption rate. In addition, an increased 2HG/αKG ratio was associated with higher histone methylation states at H3K27, H3K36 and H4K20 as assessed by quantitative targeted mass spectrometry, which may contribute to the erythroid differentiation block upon tif1γ loss. DHODH is the only pyrimidine de novo synthesis enzyme located in mitochondria and its activity is coupled to that of the electron transport chain (ETC) via coenzyme Q (CoQ). Rotenone, a potent ETC complex I inhibitor reversed the rescue by DHODH inhibition in mon embryos. Through parallel genome-wide transcriptome and chromatin immunoprecipitation analyses, we found that genes encoding CoQ metabolic enzymes are direct TIF1γ targets. Treatment with the CoQ analog decylubiquinone rescued erythroid progenitors in 26% (33/126) of mon embryos. Our work highlights the importance of transcription regulatory processes for tuning metabolism during lineage differentiation and could have therapeutic implications.