GABP transcription factor (nuclear respiratory factor 2) is required for mitochondrial biogenesis

Abstract

Mitochondria are membrane-bound cytoplasmic organelles that serve as the major source of ATP production in eukaryotic cells. GABP (also known as nuclear respiratory factor 2) is a nuclear E26 transformation-specific transcription factor (ETS) that binds and activates mitochondrial genes that are required for electron transport and oxidative phosphorylation. We conditionally deleted Gabpa, the DNA-binding component of this transcription factor complex, from mouse embryonic fibroblasts (MEFs) to examine the role of Gabp in mitochondrial biogenesis, function, and gene expression. Gabpα loss modestly reduced mitochondrial mass, ATP production, oxygen consumption, and mitochondrial protein synthesis but did not alter mitochondrial morphology, membrane potential, apoptosis, or the expression of several genes that were previously reported to be GABP targets. However, the expression of Tfb1m, a methyltransferase that modifies ribosomal rRNA and is required for mitochondrial protein translation, was markedly reduced in Gabpα-null MEFs. We conclude that Gabp regulates Tfb1m expression and plays an essential, nonredundant role in mitochondrial biogenesis.

FIG 1

Disruption of Gabpa in MEFs. (A) Targeting strategy illustrating the wild-type, floxed, and Cre-mediated recombined Gabpa alleles. Exon 8, which encodes part of the Gabpa ETS DNA-binding domain, is indicated; triangles represent loxP sites; arrows indicate PCR primers used for genotyping. (B to D) Analysis of Gabpa^fl/fl MEFs infected with control (Ctrl) or Cre-expressing (Cre) retrovirus for the presence in genomic DNA of Gabpa exon 8 (with primers 1 and 2 in panel A) and deletion of exon 8 (with primers 1 and 3) by real-time PCR (B), quantitative PCR of reverse-transcribed Gabpa mRNA (C), and immunoblotting to detect Gabpα and β-actin protein expression (D).

FIG 2

Gabpa deletion reduces mitochondrial mass in MEFs. (A) Gabpa^fl/fl MEFs infected with control or Cre-expressing (KO) retrovirus were examined by electron microscopy (×14,000 magnification). (B) Summary of mitochondrial counts per microscopy view (n = 3). (C) Flow cytometry analysis of MEFs stained with NAO (left) and MitoTracker Green (MT-Green, right). Geometric means and P values of the differences are indicated. (D) Ratio of COX I (mitochondrial) to actin B (nuclear) DNA by quantitative real-time PCR. The P values of the differences are indicated. (E) Immunoblotting of retrovirus-infected MEFs with antibodies against Vdac and β-actin.

FIG 3

Mitochondrial function in Gabpa^−/− MEFs. Wild-type or Gabpa^fl/fl MEFs were infected with control or Cre-expressing (KO) retrovirus, stained with MitoTracker JC-1 (A) or MitoTracker Red (mt-Red) (B), and then subjected to flow cytometry to evaluate mitochondrial membrane potential. Valinomycin was used to dissipate the membrane potential and served as a negative control. Values are the percentages of cells in the indicated quadrants or gates. (C) ATP generation measured by luciferase relative light units (RLU) by wild-type MEFs treated with oligomycin or valinomycin (left) or Gabpa^fl/fl MEFs infected with the control or Cre retrovirus in the absence or presence of 10% fetal calf serum (right).

FIG 4

Oxygen consumption and glycolysis in Gabpa^−/− MEFs. Shown are the oxygen consumption rate (OCR) (A) and glucose metabolism (B) of Gabpa^fl/fl (fl/fl) MEFs infected with control or Cre (KO) retrovirus, as measured with a Seahorse XF24 analyzer. ECAR, extracellular acidification rate; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, the ion-uncoupling reagent; 2-DG, 2-deoxy-d-glucose, an unmetabolizable glucose analog.

FIG 5

Apoptosis following Gabpa deletion. (A) Caspase 3/7 activity of untreated or staurosporine (Stauro)-treated wild-type (+/+) MEFs or Gabpa^fl/fl (fl/fl) MEFs infected with the control (Ctrl) or Cre retrovirus. (B) Immunoblotting for PARP or β-actin protein from wild-type (+/+) MEFs treated with staurosporine for the indicated numbers of hours and Gabpa^fl/fl MEFs infected with the control or Cre retrovirus.

FIG 6

Expression of key mitochondrial genes and proteins in Gabpa^−/− MEFs. Gabpa^fl/fl MEFs infected with the control (Ctrl) or Cre retrovirus were analyzed by quantitative RT-PCR of key mitochondrial genes (A) and transcription factors and cofactors (B) that affect mitochondrial biogenesis. Cyto C, cytochrome c; *, P < 0.03; **, P < 0.01. (C) Analysis of protein expression from these MEFs by immunoblotting for Tfb1m and β-actin. (D) ChIP of F1 ATP synthase β and ATP synthase cofactor 6 with antibodies against IgG, GABPα (α), or GABPβ (β). neg, no DNA; input, genomic DNA. (E) Wild-type and Gabpa^fl/fl MEFs infected with the control or Cre retrovirus analyzed by immunoblotting for the expression of mitochondrial membrane proteins Tomm20 and Tomm70. (F) Analysis of mitochondrial protein synthesis in these MEFs by radioactive ³⁵S pulse-labeling in the presence of emetine. Immunoblotting for β-actin indicated equal loading of total cellular proteins. Mitochondrial (mt) translation products are identified on the left.