MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption

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Abstract

Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A1.1 contains a macrodomain capable of binding NAD+-derived metabolites. Here we report that macroH2A1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing, and that myotubes that lack macroH2A1.1 have a defect in mitochondrial respiratory capacity. We found that the metabolite-binding macrodomain was essential for sustained optimal mitochondrial function but dispensable for gene regulation. Through direct binding, macroH2A1.1 inhibits basal poly-ADP ribose polymerase 1 (PARP-1) activity and thus reduces nuclear NAD+ consumption. The resultant accumulation of the NAD+ precursor NMN allows for maintenance of mitochondrial NAD+ pools that are critical for respiration. Our data indicate that macroH2A1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD+ consumption and establishing a buffer of NAD+ precursors in differentiated cells.

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Figure 1: A splicing switch upregulates macroH2A1.1 during myogenic differentiation.
Figure 2: MacroH2A1.1 knockdown results in metabolic changes without affecting myogenic differentiation.
Figure 3: Knockdown of macroH2A1.1 causes defective mitochondrial respiration.
Figure 4: Gene regulation by macroH2A1.1 is independent of ADP ribose binding.
Figure 5: The binding pocket of macroH2A1.1 is required for mitochondrial activity and for binding and inhibition of PARP-1.
Figure 6: Inhibition of PARP-1 rescues the mitochondrial phenotype.
Figure 7: The influence of macroH2A1.1 on mitochondrial function is mediated by the NAD+ precursor NMN.

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Acknowledgements

We thank P. Muñoz Canoves for tools, training and advice; E. Gallardo (Institut de Recerca Hospital de la Santa Creu i Sant Pau, Barcelona, Spain) for primary human myoblasts; S. Samino for help with metabolomics analyses; S.-V. Forcales, A. Mikoč and the Ahel lab for helpful discussions; and the departments of pathology and biochemistry of the Hospital Universitari Germans Trias I Pujol (HGTP) for excellent support. This project was supported by MINECO (grants SAF2012-39749 and BFU2015-66559-P to M.B.; grant SAF2012-37427 to M.S.), AFM-Téléthon (grant 18738 to M.B.), the Marie Skłodowska Curie training network 'ChroMe' (grant H2020-MSCA-ITN-2015-675610 to M.B., A.G.L., O.Y. and J.A.P.), the Minerva Foundation (ARCHES award to T.P.), DZD (T.P.), the ERC (grants 281641 and 682679 to J.A.P.), DFG (grants SFB 646 and SFB 1064 to A.G.L.), the Wellcome Trust (grant 101794 to I.A.), Cancer Research UK (grant C35050/A22284 to I.A.), the Unity through Knowledge Fund (grant UKF 1B 2/13 to I.A.), ISCIII (grant PI15/00701 to P.M.G.-R.), MECD (FPU14/06542 to D.C.), AGAUR (FI fellowship to M.P.M.), and a Juan de la Cierva fellowship (JCI-2011-10831 to J.D.). Work in the Buschbeck lab is further supported by the Deutsche José Carreras Leukaemie Stiftung (DJCLS R 14/16), MINECO–ISCIII (PIE16/00011) and AGAUR (2014-SGR-35). Research at the IJC is supported by the 'La Caixa' Foundation, the Fundació Internacional Josep Carreras, Celgene Spain and the CERCA Programme/Generalitat de Catalunya.

Author information

M.P.M., R.T. and M.B. conceived the project; M.P.M., S.H.-B., M.S., P.B., I.A., A.G.L., P.M.G.-R., O.Y., J.A.P., R.T. and M.B. designed experiments and interpreted data; O.Y. contributed methods; M.P.M., S.H.-B., M.L., V.V., H.D., M.N., D.C., I.G., J.D. and P.G.-P. performed experiments; R.M. analyzed high-content data; and M.P.M., J.A.P., R.T. and M.B. wrote the manuscript.

Correspondence to Raffaele Teperino or Marcus Buschbeck.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Analysis of macroH2A1 splice variants during differentiation of primary myoblasts.

(a) Scheme of the mouse macroH2A1 transcript containing the region of the two alternative exons that give rise to macroH2A1.1 (exon 7) and macroH2A1.2 (exon 6). The macro domain of macroH2A1.1 variant was shown to bind ADPR, o-acetyl-ADPR and poly-ADPR in vitro (REF 12,13).(b) Schematic representation of the RFLP analysis of mouse and human spliced transcripts encoding macroH2A1 isoforms. Exon numbers and the length of digested fragments (in bp) are indicated.(c) mRNA levels of macroH2A variants were measured by RT-qPCR during mouse primary myoblasts differentiation and normalized to equimolar reference samples to allow direct comparison. Data is represented as mean + s.e.m. of three independent cell culture experiments. Mouse Myogenin (Myog) and muscle-type creatine kinase (Ckm) genes are used as markers of differentiation. Time point 0 refers to subconfluent cells grown in GM medium (same for d). Asterisks indicate significantly higher expression compared to the other isoform at the same time point (p<0.05).(d) Same as (c) analyzing human primary myoblasts. Cells from two different donors were used. Data is the mean + s.e.m. of two independent cell cultures.(e) Immunoblot analysis of mouse primary myoblasts using indicated antibodies.(f) Comparative immunoblot of differentiated C2C12 cells and FLAG-tagged reference samples. All three macroH2A proteins, histone H3 and FLAG were analyzed. A sample of C2C12 cells treated with macroH2A1.1-specific siRNA was also included. Uncropped blot images of (e) and (f) are shown in Supplementary Data Set 1.

Supplementary Figure 2 Nuclear localization and chromatin association of macroH2A1.1.

(a) Cross-sections of snap-frozen human muscle were fixed in cold 100% aceton and immunostained with anti-macroH2A1.1 antibody. Arrowheads indicate the positive signal of laterally located myonuclei in the muscle fibers. Scale bar is 50 μm.(b) C2C12 cells were fixed at day 1.5 of differentiation and analyzed by immunofluorescence with anti-macroH2A1.1 antibody. DNA was stained with DAPI. Scale bar is 20 μm.(c) Immunoblot analysis after cellular fractionation of differentiated C2C12 cells. Uncropped blot images are shown in Supplementary Data Set 1.(d) Overview table of peaks called by SICER in proliferating myoblasts and after 5 days of differentiation.(e) UCSC genome browser window of a representative 3 Mb window of chromosome 8 (8:121.500.000-124.500.000). Peaks called by SICER are indicated below the profiles.(f) Overlap of SICER peaks at bp resolution.

Supplementary Figure 3 MacroH2A1.1 depletion has little influence on key metabolic genes.

(a) Overview of up- and downregulated probes comparing si macroH2A1.1 (mH2A1.1) to si ctrl myotubes after 4 days of differentiation. The numbers above and below brackets indicate the number of genes corresponding to these probes. For full detail please see Supplementary Data File 1.(b) The expression of known regulatory genes of glucose uptake (Glut1, Glut4), glycolysis (Hk2, Pdk4, Pdh1Ea) and fatty acid uptake and metabolism were analyzed by semi-quantitative in control and si macroH2A1.1 knockdown cells at day 4 of differentiation. Data is represented as mean + s.d. of three independent experiments (*p<0.05).

Supplementary Figure 4 MacroH2A1.1-sensitive genes are not rescued by NMN or by PARP inhibition.

(a) The purity of isolated mitochondria was checked by western blot. (b) The mRNA levels of Nmnat3 in siRNA-treated C2C12 cells was analyzed by RT-qPCR. Data is the mean + s.d. of three independent experiments (*p<0.05).(c) No rescue of gene expression. The mRNA levels of si macroH2A1.1-sensitive genes were analyzed at day 4 of differentiation under control and rescue conditions with PARP inhibitor (100 nM PARP inhibitor ABT-888 for 16 hours as in Figure 6g) and NMN (500 nM for 24 hours as in Figure 7e) by RT-qPCR. Data is represented as mean + s.d. of three independent experiments. Differences between si macroH2A1.1 alone and co-treatments were not significant.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Table 1. (PDF 814 kb)

Life Sciences Reporting Summary (PDF 141 kb)

Supplementary Data Set 1

Uncropped images and blots. (PDF 4648 kb)

Supplementary Data Set 2

Expression array data. (XLS 296 kb)

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Posavec Marjanović, M., Hurtado-Bagès, S., Lassi, M. et al. MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption. Nat Struct Mol Biol 24, 902–910 (2017) doi:10.1038/nsmb.3481

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