SIRT1 Regulates Mitochondrial Homeostasis by Modulation of the TFAM Promoter through HIF-1α/c-Myc (A) TFAM mRNA analyzed by qPCR in gastrocnemius of WT and SIRT1 iKO animals. Values were normalized to WT mice (n = 5, ∗p < 0.05 versus WT). (B) TFAM promoter activity in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision for 24 hr (SIRT1 iKO). Relative luciferase values were normalized to vehicle cells (n = 6, ∗p < 0.05 versus vehicle). (C) Immunoblot for SIRT1, TFAM, and tubulin in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision (SIRT1 iKO) for 24 or 48 hr, after which cells were infected with control or TFAM adenovirus. (D) Expression of nuclear- versus mitochondrially encoded genes in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with OHT to induce SIRT1 excision (SIRT1 iKO) for 24 or 48 hr, after which cells were infected with TFAM adenovirus. Values were normalized to vehicle cells (n = 4, ∗p < 0.05 versus vehicle; #p < 0.05 versus SIRT1 iKO 24 hr; &p < 0.05 versus SIRT1 iKO 48 hr). (E) ATP content in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT for 24 or 48 hr as in (D) (n = 4, ∗p < 0.05 versus vehicle; #p < 0.05 versus SIRT1 iKO 24 hr; &p < 0.05 versus SIRT1 iKO 48 hr). (F) Immunoblot of SIRT1, VHL, HIF-1α, TFAM, and tubulin in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision and in cells treated with OHT for 24 hr, after which SIRT1 was added back by adenoviral infection. (G) Interaction of HIF-1α and c-Myc determined by immunoprecipitation of HIF-1α in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with OHT to excise SIRT1. (H) The c-Myc-binding site on the TFAM promoter. (I) TFAM promoter activity in primary myoblasts transduced with c-Myc or nontargeting shRNA (n = 4, ∗p < 0.05 versus shNT). (J–L) TFAM promoter full-length activity or with mutation of c-Myc-binding site (Δ c-Myc) in primary myoblasts overexpressing c-Myc (J), PGC-1α (K), SIRT1 (L), or empty vector (n = 4, ∗p < 0.05 versus empty; #p < 0.05 versus Δ c-Myc empty). (M and N) Chromatin immunoprecipitation (ChIP) (M) and respective quantification by qPCR (N) of c-Myc and HIF-1α to the TFAM promoter in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision (n = 3, ∗p < 0.05 versus vehicle). (O) ChIP of c-Myc to the TFAM promoter in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA treated with vehicle or OHT to induce SIRT1 excision for 24 hr (SIRT1 iKO). Mitochondrially encoded genes were ND1, Cytb, COX1, ATP6. Values are expressed as mean ± SEM. See also Figure S5.