SIRT1-HIF-1α Regulates Mitochondria by Modulating c-Myc’s Ability to Activate TFAM
These results raised the question of how HIF-1α, a nuclear protein, inhibits mitochondrial OXPHOS genes. Analysis of gene expression in SIRT1 iKO mice identified the nuclear-encoded mitochondrial factor TFAM as a candidate (Figures 5A and S5A). Consistent with this, TFAM promoter activity in SIRT1 iKO myoblasts was greatly reduced (Figure 5B), the reintroduction of TFAM into SIRT1 iKO cells restored levels of mitochondrially encoded mRNAs and ATP (Figure 5C-E), and in time course studies, TFAM levels declined 6 hr after VHL and HIF-1α (Figure 5F).
Knockdown of ARNT, a HIF-1α transcriptional binding partner (Wang et al., 1995), had no appreciable effect on mitochondrially encoded OXPHOS genes and ATP levels (Figures S5B–S5D), indicating that HIF-1α acts via a different mechanism. In cancer cells, metabolic reprogramming is mediated by crosstalk between HIF-1α and c-Myc (Gordan et al., 2007), raising the possibility that c-Myc was the missing factor. In fact, c-Myc DNA-binding sites are found at mitochondrial biogenesis genes (Kim et al., 2008, Li et al., 2005). Deletion of SIRT1 in primary myoblasts increased the binding between HIF-1α and c-Myc and reduced c-Myc reporter activity (Figures 5G and S5E). Similarly, knockdown of c-Myc completely blocked the ability of SIRT1 to induce mitochondrially encoded mRNAs and mtDNA (Figures S5F–S5H). Conversely, overexpression of c-Myc in myoblasts treated with a SIRT1 inhibitor, EX-527, prevented loss of mtDNA, mitochondrially encoded mRNA, and cellular ATP levels (Figures S5I–S5L).
We tested whether c-Myc directly controls TFAM promoter in myoblasts and is modulated by SIRT1-HIF-1α. TFAM is known to be regulated by PGC-1α, which interacts with NRF 1/2 bound at positions −311 and −154 in the TFAM promoter (Figure 5H). Knockdown of c-Myc reduced TFAM promoter activity (Figure 5I), consistent with a study in cancer cells (Li et al., 2005). We identified a putative c-Myc consensus sequence, CACGTG, 1,028 bp upstream of the ATG site—the mutation of which decreased promoter activity by about half without affecting PGC-1α-mediated induction (Figures 5J and 5K). Overexpression of SIRT1 also induced the TFAM promoter reporter, and mutation of the c-Myc binding site blocks this effect (Figure 5L). Chromatin IP experiments detected an interaction between c-Myc and the TFAM promoter, which was markedly reduced when SIRT1 was deleted, but not when HIF-1α was also knocked down (Figures 5M–O). We did not detect direct HIF-1α binding to TFAM (Figures 5M and 5N), with LDHA as a positive control (Figures S5M and S5N). Together, these data provide the first direct link between HIF-1α and the regulation of mitochondrially encoded genes in skeletal muscle and identify a mechanism of PGC-1α/β-independent regulation of mitochondrial function.