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The TDH–GCN5L1–Fbxo15–KBP axis limits mitochondrial biogenesis in mouse embryonic stem cells

Nature Cell Biology volume 19, pages 341–351 (2017)Cite this article

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Abstract

Self-renewing naive mouse embryonic stem cells (mESCs) contain few mitochondria, which increase in number and volume at the onset of differentiation. KBP (encoded by Kif1bp) is an interactor of the mitochondrial-associated kinesin Kif1Bα. We found that TDH, responsible for mitochondrial production of acetyl-CoA in mESCs, and the acetyltransferase GCN5L1 cooperate to acetylate Lys501 in KBP, allowing its recognition by and degradation via Fbxo15, an F-box protein transcriptionally controlled by the pluripotency core factors and repressed following differentiation. Defects in KBP degradation in mESCs result in an unscheduled increase in mitochondrial biogenesis, enhanced respiration and ROS production, and inhibition of cell proliferation. Silencing of Kif1Bα reverts the aberrant increase in mitochondria induced by KBP stabilization. Notably, following differentiation, Kif1bp−/− mESCs display impaired expansion of the mitochondrial mass and form smaller embryoid bodies. Thus, KBP proteolysis limits the accumulation of mitochondria in mESCs to preserve their optimal fitness, whereas KBP accumulation promotes mitochondrial biogenesis in differentiating cells.

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Figure 1: KBP is an Fbxo15 substrate that is recognized in an acetylation-dependent manner.
Figure 2: GCN5L1 and TDH are necessary for the Fbxo15-mediated degradation of KBP in mESCs.
Figure 3: Expression of a stable mutant of KBP in mESCs induces mitochondrial biogenesis.
Figure 4: Failure to degrade KBP in mESCs induces mitochondrial biogenesis.
Figure 5: The mitochondrial biogenesis induced by KBP stabilization depends on Kif1Bα.
Figure 6: Effects of KBP accumulation on mitochondrial respiration.
Figure 7: In vivo and in vitro effects of KBP stabilization and loss of Fbxo15 on mESC growth.
Figure 8: Ectopic expression of Fbxo15 and TDH reduces the mitochondrial content in differentiating mESCs, and Kif1bp knockout impairs mitochondrial biogenesis and differentiation.

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Acknowledgements

The authors thank C. Haynes, J. K. Pagan and T. Pozzan for critical reading of the manuscript; I. Aifantis (NYU School of Medicine, USA), R. Lammers (University of Tubingen, Germany), M. N. Sack (National Institutes of Health, USA) and F. Zhang (Massachusetts Institute of Technology, USA) for reagents. M.P. is grateful to T. M. Thor and T. B. Balduur for continuous support. V.D. is grateful to F. Catalanotti, G. Donato and A. Donato for continuous support. P.P. is grateful to C. d. Scrovegni for providing continuous support. This work was funded by grants from the National Institutes of Health (R37-CA076584, R01-GM057587 and R21-CA161108) and New York State Health Department (NYSTEM-N11G-255) to M.P.; the American-Italian Cancer Foundation and the National Institutes of Health (5T32CA009161-40) to V.D.; and The Italian Ministry of Health and the Italian Ministry of Education, University and Research (COFIN no. 20129JLHSY_002, FIRB no. RBAP11FXBC_002, and Futuro in Ricerca no. RBFR10EGVP_001), Telethon (GGP15219/B), the Italian Cystic Fibrosis Research Foundation (19/2014) and the Italian Association for Cancer Research (IG-18624) and local funds from the University of Ferrara and the Italian Ministry of Health to P.P. The authors also thank the NYU Genome Technology Center (partially funded by the Perlmutter Cancer Center Support Grant P30CA016087) for expert library preparation and sequencing for RNA-seq, and the NYU Applied Bioinformatics Laboratories for providing bioinformatics support and helping with the analysis and interpretation of the RNA-seq data using computing resources at the NYU High Performance Computing Facility. A.S., L.F. and M.P.W. are supported by the Stowers Institute for Medical Research. M.P. is an Investigator with the Howard Hughes Medical Institute.

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  1. Davide Sartini

    Present address: Present address: Department of Clinical Sciences and Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, Ancona 60131, Italy.,

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA

    Valerio Donato, Daniele Simoneschi, Davide Sartini, Yasusei Kudo & Michele Pagano

  2. NYU Perlmutter Cancer Center, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA

    Valerio Donato, Daniele Simoneschi, Davide Sartini, Yasusei Kudo & Michele Pagano

  3. Howard Hughes Medical Institute, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA

    Valerio Donato & Michele Pagano

  4. Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Via Fossato di Mortara 70, Ferrara 44121, Italy

    Massimo Bonora & Paolo Pinton

  5. Department of Oral Molecular Pathology, Tokushima University Graduate School 3-18-15 Kuramoto, Tokushima 770-8504, Japan

    Yasusei Kudo

  6. The Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA

    Anita Saraf, Laurence Florens & Michael P. Washburn

  7. Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA

    Michael P. Washburn

  8. Skirball Institute of Biomolecular Medicine and Helen L. and Martin S. Kimmel Center for Biology and Medicine, New York University School of Medicine, 522 First Avenue, New York, New York 10016, USA

    Matthias Stadtfeld

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Contributions

V.D. conceived the project, planned and performed most experiments, and co-wrote the manuscript. M.P. directed and coordinated the study, oversaw the results, and co-wrote the manuscript. M.B. and P.P. performed analyses of mitochondrial morphology and physiology. D.Simoneschi generated the CRISPR knockout mESC clones and helped with biochemical experiments and with RNA-seq data analysis. D.Sartini helped with some biochemical experiments. Y.K. performed the Fbxo15+/+ and Fbxo15−/− mouse breeding. M.S. provided reagents and advice. A.S., L.F. and M.P.W. performed the mass spectrometry analyses of the purifications performed by V.D. All authors discussed the results and commented on the manuscript.

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Correspondence to Michele Pagano.

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Integrated supplementary information

Supplementary Figure 1 Multidimensional Protein Identification Technology analysis of the Fbxo15 complex and its validation, and generation of Fbxo15−/− mESCs by CRISPR Genome Editing.

(a) HEK293T cells were transfected with either Strep-FLAG-tagged human Fbxo15 or Strep-FLAG-tagged human Fbxo21 (the latter used as control). The table shows the mass spectrometry analysis of the two immunopurifications, listing normalized spectral abundance factors (NSAFs) for the indicated proteins. The complete list of Fbxo15 interactors is reported in Supplementary Table 1. This experiment was performed twice. (b) HEK293T cells were transfected with an empty vector (EV) or constructs for the indicated human proteins. Whole cell extracts (WCE) were immunoprecipitated (IP) with anti-FLAG resin, and immunocomplexes were immunoblotted as indicated. (c) mESCs were transfected with either an siRNA to Fbxo15 (oligo #1) or a nontargeting (N/T) siRNA. Protein extracts were then immunoblotted for the indicated proteins. (d) mESCs were transfected with either a nontargeting (N/T) siRNA or siRNA to Fbxo15 and treated for 4 h with MG132, MLN4924, or DMSO. Cells were then collected and lysed for immunoblotting as indicated. The Nedd8-activating enzyme inhibitor MLN4924 blocks the neddylation of Cul1, which is required for the SCF activity. p27, an established substrate of the proteasome and the F-box protein Skp2, is used as positive control for MG132 and MLN4924, and as a negative control for Fbxo15 silencing. (e) mESCs were transfected with either an siRNA to Fbxo15 (oligo #1) or a nontargeting (N/T) siRNA. Cells were treated with cycloheximide (CHX) for the indicated times and protein extracts were then immunoblotted for the indicated proteins. Long (l. e.) and short (s. e.) exposures are shown for KBP. (f) Schematic representation of Fbxo15 genomic locus and gRNA target location. Exon 1 refers to mouse Fbxo15 gene in NC_000084.6 Reference GRCm38.p4 C57BL/6J and in NM_015798.3. In the bottom panel, full-length wild-type genomic DNA template and truncated mutant sequences identified by TOPO-TA cloning of Fbxo15 PCR from two independent mESCs knockout clones are depicted. Clone #1 sequence and chromatogram revealed two different deletion events; one allele contains a 31bp deletion, while the other allele contains a 16bp deletion. Clone #2 sequence and chromatogram identified a 14bp deletion event at both alleles. Unprocessed original scans of blots are shown Supplementary Fig. 9. Unless otherwise noted, experiments were performed at least three times.

Supplementary Figure 2 Identification of the KBP degron.

(a–d) HEK293T cells were transfected with either an empty vector (EV), wild type human KBP, or the indicated FLAG-tagged human KBP mutants (either deletion, truncation, or point mutation mutants, as indicated). Whole cell extracts (WCE) were immunoprecipitated (IP) with an anti-FLAG resin, and immunoprecipitates were probed with the indicated antibodies. (e) Alignment of the Fbxo15 binding motif in KBP orthologs. The critical lysine required for Fbxo15 binding is highlighted in red. (f) Schematic representation of KBP mutants. KBP mutants found to interact with Fbxo15 are indicated by the symbol (+). (±) denotes reduced binding, and (−) denotes a lack of binding. (g) HEK293T cells were transfected with either an empty vector (EV), wild type human KBP, or the indicated FLAG-tagged human KBP point mutation mutants. Whole cell extracts (WCE) were immunoprecipitated (IP) with an anti-FLAG resin, and immunoprecipitates were probed with the indicated antibodies. (h) Strep-FLAG-tagged human KBP was immunoprecipitated from HEK293T cells and subjected to PTM analysis. The table lists the number of spectra matching acetylated and phosphorylated peptides as a fraction of the total spectra detected for peptides bearing each modified residue and their corresponding modification percentage. This experiment was performed once. (i) HEK293T cells were transfected with either an empty vector (EV) or vectors expressing FLAG-tagged wild type human KBP or FLAG-tagged human KBP(KK/RR). Whole cell extracts were immunoprecipitated (IP) with anti-FLAG resin, and proteins were immunoblotted as indicated. Unprocessed original scans of blots are shown Supplementary Fig. 9. Unless otherwise noted, experiments were performed at least three times.

Supplementary Figure 3 GCN5L1 and TDH allow KBP degradation via Fbxo15.

(a) mESCs were transfected with either a nontargeting (N/T) siRNA or siRNAs to Fbxo15 (oligo #2), GCN5L1, or KBP. Cells were either maintained in LIF-containing medium or induced to differentiate for 24 h after LIF withdrawal and exposure to retinoic acid (RA). Cells were then collected and lysed for immunoblotting as indicated. (b) mESCs were treated for 12 h with either DMSO or the TDH inhibitor Qc-1. Protein extracts were then immunoblotted for the indicated proteins. (c)The experiment was performed as in b, except that cells were treated with cycloheximide (CHX) for the indicated times. (d) mESCs were transfected with either a nontargeting (N/T) siRNA or siRNAs to Fbxo15 (oligo #1), GCN5L1, KBP, or TDH. Cells were either maintained in LIF-containing medium or induced to differentiate for 24 h after LIF withdrawal and exposure to neurobasal (NB) medium on non-adherent petri dishes. Cells were then collected and lysed for immunoblotting as indicated. Unprocessed original scans of blots are shown Supplementary Fig. 9. Unless otherwise noted, experiments were performed at least three times.

Supplementary Figure 4 Accumulation of KBP does not induce any major transcriptional rearrangement.

mESCs were infected with lentiviruses expressing either HA-tagged wild type mouse KBP or HA-tagged mouse KBP(KK/RR) and maintained in self renewing conditions. RNA-seq analysis was performed and results are shown as a MA plot of log2 fold changes versus the mean of normalized counts. Genes were considered differentially expressed when the P adjusted value was <0.05. ∼290 genes displayed differential expression, with modest fold changes (the relative expression for each affected transcript is comprised between −0.2 and +0.3). Neither a variation in the expression of the pluripotency core factors nor changes in the major metabolic drivers were detected. The complete list of FPKM reads is reported in Supplementary Table 2. n = 3 biologically independent experiments.

Supplementary Figure 5 Inhibition of KBP degradation promotes mitochondria-to-microtubule interaction and mitochondrial network expansion in mESCs.

(a) mESCs were transfected with either a nontargeting (N/T) siRNA or an siRNA to Fbxo15 (oligo #1) and immunostained for TOM20 and β-tubulin. Microtubule-mitochondria co-localization was detected using confocal microscopy. Scale bar, 15 μm. In the bottom graph, the Manders’ Overlap Coefficient is shown. n = 61 and 40 cells (N/T and Fbxo15 siRNA, respectively). P value was calculated by unpaired t-test. Error bars indicate ± s.d. (b) mESCs were treated for 12 h with DMSO or Qc-1 and immunostained for TOM20 and β-tubulin. Microtubule-mitochondria co-localization was detected by confocal microscopy. Scale bar = 15 μm. In the bottom graph, the Manders’ Overlap Coefficient is shown. n = 32 and 47 cells (DMSO and Qc-1 treatment, respectively). P values were calculated by unpaired t-test. Error bars indicate ± s.d. (c) mESCs were either maintained in LIF-containing medium or induced to differentiate for 24 h after LIF withdrawal and exposure to retinoic acid (RA). Cells were stained with Mitotracker and DAPI and then analyzed by 3D deconvolution microscopy. Representative MIPs are displayed. Scale bar = 15 μm. (d–e) mESCs were transfected with either a nontargeting (N/T) siRNA or an siRNA to Fbxo15 (oligo #1) in the presence or absence of an siRNA to KBP, and maintained in LIF-containing medium. Cells were stained with Mitotracker and DAPI prior to fixation and then analyzed by confocal microscopy. Panel (d) shows the MIPs of representative cells (scale bar = 15 μm). Panel (e) shows the quantification of average mitochondrial content. n = 62, 62 and 33 cells (N/T, Fbxo15 and Fbxo15 + KBP siRNA, respectively). P value was calculated by one-way ANOVA. Error bars indicate ± s.d. (f) mESCs were transfected with either an siRNA to Fbxo15 (oligo #1) or a nontargeting (N/T) siRNA. Total cellular DNA was extracted and mtDNA copy number was evaluated by qPCR normalized using β-actin DNA copy number. mtDNA amount in mESCs expressing transfected with a nontargeting (N/T) siRNA was set as 100 and the graph shows the fold change. P value was calculated by t-test. Error bars indicate ± s.d. n = 4 biologically independent experiments (see Supplementary Table 6). (g) mESCs were transfected with either an siRNA to Fbxo15 (oligo #1) or a nontargeting (N/T) siRNA. Protein extracts were then immunoblotted for the indicated proteins. Unprocessed original scans of blots are shown Supplementary Fig. 9. Unless otherwise noted, experiments were performed at least three times.

Supplementary Figure 6 Inhibition of KBP degradation induces a gain in mitonchondrial mass similar to that observed during differentiation.

(a) mESCs stably infected with lentiviruses expressing either HA-tagged wild type mouse KBP or HA-tagged mouse KBP(KK/RR) were treated for 12 h with Qc-1. Cells were collected and lysed for immunoblotting as indicated. (b–c) mESCs infected with lentiviruses expressing either HA-tagged wild type mouse KBP or HA-tagged mouse KBP(KK/RR) were either maintained in LIF-containing medium or induced to differentiate for 24 h after LIF withdrawal and exposure to retinoic acid (RA). Where indicated, cells were treated for 12 h with the TDH inhibitor Qc-1. Panel (b) shows the MIPs of representative cells (scale bar, 15 μm). Panel (c) shows the quantification of average mitochondrial count. Error bars indicate ± s.d. n = 108, 17, 56, 129, 66 and 39 cells [KBP – DMSO, Qc-1 and differentiation- and KBP(KK/RR) – DMSO, Qc-1 and differentiation-, respectively]. P value was calculated by two-way ANOVA and multiple comparison. Unprocessed original scans of blots are shown Supplementary Fig. 9. Unless otherwise noted, experiments were performed at least three times.

Supplementary Figure 7 Defects in KBP degradation do not affect mitochondrial dynamics.

(a–f) mESCs were infected with lentiviruses expressing either HA-tagged wild type mouse KBP or HA-tagged mouse KBP(KK/RR) and then transiently transfected with a plasmid expressing mitochondrial-targeted GFP. Panels (a) and (d) show representative 3D reconstructions of the mitochondria. Panels (b) and (e) show the entity of mitochondrial size changes registered in a and d, respectively. Panels (c) and (f) show the quantification of the events of mitochondrial fission and fusion. n = 6 and 7 independent field acquisitions [KBP and KBP(KK/RR), respectively]. Statistics was calculated with unpaired t-test. This experiment was performed once.

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.

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Donato, V., Bonora, M., Simoneschi, D. et al. The TDH–GCN5L1–Fbxo15–KBP axis limits mitochondrial biogenesis in mouse embryonic stem cells. Nat Cell Biol 19, 341–351 (2017). https://doi.org/10.1038/ncb3491

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