Supplementary Figure 8: Generation of Kif1bp^−/− mESCs and model of regulation of mitochondrial biogenesis in mESCs.

(a) Generation of Kif1bp^−/− mESCs by CRISPR Genome Editing. Schematic representation of Kif1bp genomic locus and gRNA target location. Exon 1 refers to mouse Kif1bp gene in NC_000076.6 Reference GRCm38.p4 C57BL/6J and in NM_028197.2. In the bottom panel, full-length wild-type genomic DNA template and truncated mutant sequences identified by TOPO-TA cloning of Kif1bp PCR from two independent mESCs knockout clones are depicted. Clone #1 sequence and chromatogram revealed an identical indel event at both alleles. Clone #2 sequence and chromatogram identified a 107bp deletion event at both alleles. (b) Protein extracts from wild type mESCs (WT) and Kif1bp-/- mESCs (two different clones) were immunoblotted for the indicated proteins. Unprocessed original scans of blots are shown Supplementary Fig. 9. This experiment was performed three times. (c) The TDH-dependent production of acetyl-CoA at mitochondria of naïve mESCs promotes the GCN5L1-mediated acetylation of KBP on Lysine 501. This event, in turn, allows KBP to be recognized by and degraded via Fbxo15, attenuating mitochondrial biogenesis during self-renewal. This molecular mechanism limits the accumulation of mitochondrial mass in self-renewing mESCs and preserves their optimal fitness. In human, TDH is an expressed pseudogene, a unique case among all metazoans (including chimpanzees) whose genomes have been sequenced to date (Pruitt et al. Nucleic Acids Res. 2007). However, the fact that the degron of KBP is conserved and acetylated in humans, together with the finding that its presence is necessary for KBP binding to Fbxo15, suggests that AcetylCoA is necessary for Fbxo15-mediated degradation of KBP in human cells as well, even if the source of AcetylCoA is different. Altogether, our data suggest that the molecular mechanism we have identified in mouse, with some minor differences, is conserved in humans. (d) Upon differentiation, levels of Fbxo15 and TDH decrease, acetylation of KBP on Lysine 501 is inhibited, and KBP levels accumulate. We hypothesize that this accumulation results in the increase in cellular mitochondrial mass, which contributes to the metabolic switch in differentiating cells.