Transcriptionally regulated energy metabolism drives early erythropoiesis (tif1g RNA-seq)

Transcription and metabolism both influence cell function yet dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. Through a chemical suppressor screen, we discovered that inhibition of the pyrimidine biosynthesis enzyme DHODH rescues erythroid differentiation in bloodless moonshine mutant embryos defective for the transcription elongation factor tif1?. This rescue depends on the functional link of DHODH to mitochondrial respiration. Low a-ketoglutarate levels caused by tif1? loss lead to histone hypermethylation. TIF? directly controls coenzyme Q synthesis gene expression and coenzyme Q levels are reduced in moonshine mutants. A coenzyme Q analogue rescues moonshine's bloodless phenotype. These results demonstrate mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage. Overall design: tif1? (ENSDART00000020116.9/NM_001002871.2) was knocked down (tif1? KD) by injecting 1 ng of an ATG-blocking morpholino (Gene Tools, 5'?3' morpholino sequence ATCTTGGCCTTTGTTGTCCGCCATC) into zebrafish wild-type (TU) embryos at the 1-2 cell stage. As a control (Ctrl KD), 1 ng of a Standard Control oligo (designed by Gene Tools, 5'?3' morpholino sequence CCTCTTACCTCAGTTACAATTTATA) was injected into zebrafish embryos at the 1-2 cell stage. tif1? KD and Ctrl KD embryos from each clutch were split in half and treated with either 7 µM leflunomide or the equivalent amount of DMSO from 5.3 hpf until 10 hpf, when embryos were harvested for RNA-seq by snap-freezing them.