MCB Accepts, published online ahead of print on 23 June 2014 Mol. Cell. Biol. doi:10.1128/MCB.00492-12 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

GABP Transcription Factor (Nuclear Respiratory Factor 2) is Required for Mitochondrial Biogenesis

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Zhong-Fa Yang,1* Karen Drumea,2* Stephanie Mott,3 Junling Wang,1 and Alan G Rosmarin1 #

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1

University of Massachusetts Medical School, Worcester, MA 2

Rambam Medical Center, Haifa, Israel

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Brown University, Rhode Island Hospital, Providence, RI

*

These authors contribute equally to the work

#

Corresponding Author University Hospital H8-533 55 Lake Avenue North Worcester, MA 01655 [email protected]

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Running title: GABP IN MITOCHONDRIAL BIOGENESIS

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Mitochondria are membrane-bound cytoplasmic organelles that serve as the

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major source for ATP production in eukaryotic cells. GABP (also known as

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Nuclear Respiratory Factor 2) is a nuclear-encoded ets transcription factor that

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binds and activates mitochondrial genes which are required for electron transport

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and oxidative phosphorylation. We conditionally deleted Gabpa, the DNA-binding

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component of this transcription factor complex, in mouse embryonic fibroblasts

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(MEFs) to examine the role of Gabp in mitochondrial biogenesis, function, and gene

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expression. Gabpα loss modestly reduced mitochondrial mass, ATP production,

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and oxygen consumption,and mitochondrial protein synthesis, but did not alter

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mitochondrial morphology, membrane potential, apoptosis, or expression of several

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genes that were previously reported to be GABP targets. However, expression of

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Tfb1m, a methyltransferase that modifies ribosomal rRNA and is required for

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mitochondrial protein translation, was markedly reduced in Gabpα null MEFs. We

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conclude that Gabp regulates Tfb1m expression and plays an essential, non-

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redundant role in mitochondrial biogenesis.

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Mitochondria are semi-autonomous membrane-bound cytoplasmic organelles that

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are the major source for ATP production in the eukaryotic cell. In addition to their roles

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in generating cellular energy through electron transfer and oxidative phosphorylation,

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mitochondria are also required for lipid biosynthesis, cell signaling, apoptosis, and other

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essential cellular functions. Because the 16 kilobase (kb) mitochondrial genome does not

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encode any transcription factors, it depends on nuclear transcription factors for

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expression of mitochondrial DNA (mtDNA)-encoded genes. Mitochondrial Transcription

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Factor A (TFAM) and Transcription Factor B (TFBM) are nuclear-encoded transcription

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factors that exclusively regulate mtDNA genes. Other transcription factors, including

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NRF1 (Nuclear Respiratory Factor 1) and NRF2, control both mtDNA genes and nuclear

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genes (3, 30, 35, 36).

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There are two distinct TFBM proteins: TFB1M and TFB2M, both of which have

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rRNA methyltransferase activity (23, 26). Shadel and colleagues reported that TFB1M

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and TFB2M play crucial, but distinct roles, in controlling mitochondrial biogenesis in

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both human and Drosophila cells (5, 6, 25). TFB2M mainly regulates mitochondrial

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DNA replication and mitochondrial gene transcription. Ectopic expression of TFB1M did

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not significantly affect mitochondrial function, but reduced expression of TFB1M

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decreased mitochondrial protein translation through impaired methylation of the 12S

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rRNA, impaired mitochondrial ribosome assembly, and abolished mitochondrial

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translation. Genetic disruption of Tfb1m caused early (E8.5) embryonic lethality, but

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neither activated nor repressed mitochondrial gene transcription (27). Thus, adequate

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levels of TFB1M are required for normal mitochondrial protein synthesis and biogenesis.

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Scarpulla and colleagues recognized that the multiprotein NRF2 complex is the

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human homologue of GABP, or GA-Binding Protein (40). GABP is the only obligate

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multimer among the more than two dozen mammalian ets factors (13). The tetrameric

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GABP complex includes two distinct proteins: GABPα binds to DNA through its ets

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domain, and it recruits GABPβ, which activates transcription through a glutamine-rich

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region in its carboxy terminus (34). GABPA is a unique gene in the human and murine

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genomes (24), and GABPα is the only protein that can recruit GABPβ to DNA to form

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the transcriptionally active complex (4). GABP regulates lineage-restricted myeloid and

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lymphoid genes that are required for innate immunity (34), is required for cell cycle

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control in fibroblasts (42), and has been implicated in the regulation of more than one

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dozen mitochondrial genes (30). Genetic disruption (33, 42) and knock-down (41) of

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mouse Gabpa caused early embryonic lethality, and mitochondrial dysfunction was

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proposed as a possible explanation for embryonic loss (33). However, as a member of the

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large ets family of transcription factors that bind to similar DNA motifs, it has been

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unclear if GABP is an essential, non-redundant regulator of mitochondrial gene

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expression.

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To examine the role of GABP in the regulation of mitochondrial biogenesis,

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function, and gene expression, we conditionally deleted Gabpa in cultured primary

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mouse embryonic fibroblasts (MEFs). Mitochondrial mass in Gabpα null MEFs was

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reduced by approximately one-third, and ATP production, oxygen consumption, and

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mitochondrial protein were decreased proportionally; however, mitochondrial structure,

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membrane potential and apoptosis were not significantly altered by loss of Gabpα.

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Expression of several mitochondrial genes that were previously implicated as GABP

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targets was not significantly affected, but we observed a marked reduction in expression

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of the mitochondrial methyltransferase, Tfb1m, which is essential for mitochondrial

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protein translation. We conclude that Gabpα is required for mitochondrial biogenesis and

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energy production, at least in part, through its essential and non-redundant control of

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Tfb1m expression.

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MATERIALS AND METHODS

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Cell culture and Microscopy

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Mice with loxP recombination sites which flank exons that encode the Gabpa ets

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domain (floxed Gabpa, Gabpafl/fl) were previously described (42). MEFs were prepared

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from Gabpafl/fl or Gabpa+/+ embryos at embryonic day (E) 12.5 - 14.5 and maintained in

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DMEM (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum

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(FBS; Invitrogen), except where serum-starvation is explicitly described. Retroviruses

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were packaged in helper-free Phoenix packaging cell line (ATCC, Manassas, VA). For

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retroviral infection, MEFs at passage 2 were infected with either pBabe-Puro (empty

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virus) or pBabe-Cre for 48 hours, and selected for 3-5 days in medium containing

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2.5μg/ml puromycin (Sigma-Aldrich, St. Louis, MO).

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fluorescent microscopy were supported by Rhode Island Hospital Core Services.

Electron microscopy and

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Semi-quantitative reverse transcription (RT)-PCR and real-time PCR

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Total RNA was prepared with Qiagen (Valencia, CA) RNeasy, reverse

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transcribed with the Invitrogen Superscript II RT system and poly (dT), and serial two-

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fold dilutions of cDNA were subjected to PCR within the linear range of the assay (25 to

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35 cycles, depending on the transcript). PCR products were resolved on 1.5% agarose

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gel and visualized by ethidium bromide and scanned by Amersham Typhone Image

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Scanner (Amersham, Piscataway, NJ). Real-time PCR was performed with Qiagen real-

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time PCR master kit on real-time thermal-cycler from Stratagene. Primer sequences for

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RT-PCR, real-time PCR, or genomic PCR will be provided upon request.

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Analysis of Apoptosis

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Apo-ONE Homogeneous Caspase-3/7 (Promega Corporation, Madison, WI)

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assay was performed according to manufacturer’s protocols. Staurosporine (Sigma-

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Aldrich) was applied to cells at 1 μM for 24-48 hours to induce apoptosis. Intracellular

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ATP detection was performed according to manufacturer’s protocol (Roche Applied

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Science).

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Mitochondrial quantification and Membrane Potential

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Mitochondria in wild type and Gabpα null MEFs were counted in three separate

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fields. Mitochondrial mass was evaluated by staining cells with 50 nM NAO (Nonyl

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Acridine Orange - a membrane potential-insensitive mitochondrial dye; Molecular

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Probes, Grand Island, NY), or MitoTracker Green FM (Invitrogen) for 30-60 minutes at

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37°C (30). Mitochondrial membrane potential was measured by incubating MEFs with

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mito-tracker probe 5 µg/mL 5,5’,6,6’-Tetrachloro-1,19,3,39-tetraethybenzimidazol

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carbocyanine iodide (JC-1; Molecular Probes), or MitoTracker Red (Invitrogen) at 37°C

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for 30-60 minutes (9), followed by flow cytometry or fluorescence microscopy. As a

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control, wild-type MEFs were incubated with DMSO or 100 nM valinomycin (a

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potassium ionophore that dissipates the membrane potential), for 24 hours before JC-1

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staining.

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Western Blotting

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Cells were washed in phosphate-buffered saline, and total cellular proteins were

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prepared by addition of Laemmli sample buffer, followed by incubation at 100o C for 5

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minutes. Extracts that corresponded to 5 X 105 cells were electrophoresed in 8-10%

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polyacylamide gel, transferred to Amersham Hybond-P membrane and probed with the

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following antibodies: GABPα, β-Actin, PARP, Tomm20, Tomm70 (Santa Cruz

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Biotechnology), Vdac1 (Cell Signalling), and TFBIM (a gift of Gerald Shadel, Yale

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University, New Haven, CT).

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Oxygen consumption and glycolysis

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Log-phase cells were grown in 24-well XF24 assay plates (Seahorse Bioscience)

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at 37°C with 5% CO2 for 24 hours before analysis. Cells were then incubated with assay

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media and transferred to non-CO2 incubator for 1 hour. Oxygen consumption and

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glycolysis were measured by Seahorse XF24 Analyzer with cell mito stress test kit and

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glycolysis stress test kit, according to manufacturer’s protocols.

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Chromatin Immunoprecipitation (ChIP)

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Chromatin was immunoprecipitated with antiserum against IgG and GABPα;

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ChIP was performed as previously described (42). Pulse labeling of mitochondrial protein translation

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Labeling of mitochondrial translation in MEFs was performed as previously

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described (5). Briefly, MEFs were pre-incubated with methionine-free DMEM media for

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15 minutes, followed by the methionine-free DMEM labeling medium containing 100

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μCi/ml

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St. Louis, MO) and chased for 10 minutes in regular DMEM. Total cellular protein was

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run on 4-20% polyacrylamide gradient gels, gels were dried for 1 hour, and

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autoradiographed.

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S (MP Biomedicals, Santa Ana, CA) and 100 μg/ml emetine (Sigma-Aldrich,

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RESULTS

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Disruption of Gabpα in mice and MEFs We used homologous recombination to generate mice in which loxP

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recombination sites flank exons that encode the Gabpa ets DNA binding domain (Fig.

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1A). Heterozygous floxed mice (Gabpafl/+) were bred with mice that bear the CMV-Cre

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transgene, which express Cre recombinase in all tissues. The resultant Gabpa+/- mice

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were phenotypically normal, but intercrossing of these hemizygous mice generated no

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nullizygous mice among 35 pups. This finding is consistent with previous reports that

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Gabpα nullizygosity causes an embryonic lethal defect (33, 42).

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We prepared MEFs from wild type (Gabpa+/+) or homozygous floxed (Gabpafl/fl)

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embryos and infected them with either pBabe-Cre, which expresses Cre recombinase, or

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the empty control retrovirus, pBabe-Puro. Cre efficiently deleted Gabpa (Fig. 1B), the

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Gabpa transcript was absent (Fig. 1C), and Gabpα protein was not detected (Fig. 1D) in

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Gabpafl/fl MEFs (hereinafter referred to as Gabpa-/- or Gabpα null cells).

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Gabpα-/- MEFs have reduced mitochondrial cell mass

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We sought to determine if deletion of Gabpa affected the morphology or the total

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cellular content of mitochondria.

Electron microscopy demonstrated no discernable

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abnormalities in mitochondrial morphology (Fig. 2A), but there was a statistically

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significant reduction of mitochondrial content in Gabpα null MEFs (Gabpafl/fl MEFs

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infected with pBabe-Cre) compared with control cells (Gabpafl/fl MEFs infected with

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pBabe-Puro) (Fig. 2B; p