Neddylation Regulates Class IIa and III Histone Deacetylases to Mediate Myoblast Differentiation

Abstract

As the largest tissue in the body, skeletal muscle has multiple functions in movement and energy metabolism. Skeletal myogenesis is controlled by a transcriptional cascade including a set of muscle regulatory factors (MRFs) that includes Myogenic Differentiation 1 (MYOD1), Myocyte Enhancer Factor 2 (MEF2), and Myogenin (MYOG), which direct the fusion of myogenic myoblasts into multinucleated myotubes. Neddylation is a posttranslational modification that covalently conjugates ubiquitin-like NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to protein targets. Inhibition of neddylation impairs muscle differentiation; however, the underlying molecular mechanisms remain less explored. Here, we report that neddylation is temporally regulated during myoblast differentiation. Inhibition of neddylation through pharmacological blockade using MLN4924 (Pevonedistat) or genetic deletion of NEDD8 Activating Enzyme E1 Subunit 1 (NAE1), a subunit of the E1 neddylation-activating enzyme, blocks terminal myoblast differentiation partially through repressing MYOG expression. Mechanistically, we found that neddylation deficiency enhances the mRNA and protein expressions of class IIa histone deacetylases 4 and 5 (HDAC4 and 5) and prevents the downregulation and nuclear export of class III HDAC (NAD-Dependent Protein Deacetylase Sirtuin-1, SIRT1), all of which have been shown to repress MYOD1-mediated MYOG transcriptional activation. Together, our findings for the first time identify the crucial role of neddylation in mediating class IIa and III HDAC co-repressors to control myogenic program and provide new insights into the mechanisms of muscle disease and regeneration.

Keywords: histone deacetylases; myoblast differentiation; neddylation.

Figure 1

Neddylation is differentially regulated during C2C12 differentiation. (A–C) Western blot of the indicated proteins (A), and quantifications of NAE1, UBC12, and neddylated Cullins (N8-CULs) (B); quantifications of MYOD1, MYOGENIN, and MYOSIN (skeletal, fast) (MyHC) (C) in proliferative C2C12 cells cultured in growth media (i.e., day 0, D0) and for the indicated days after differentiation induction (D1 to D5). Proteins were quantified and normalized to ACTB. Data were presented as fold changes with D0 cells set at 1. N8-Conj.: NEDD8-conjugated. Arrows indicate neddylated CULs and CUL1. (D) Representative immunofluorescence images of C2C12 cells stained with NEDD8 (green) in growth media (D0) and for 3 and 6 days in differentiation media (D3, D6). Nuclei were counterstained with Hoechst 33342. Scale bars: 50 μm. ** p < 0.005 versus D0. One-way ANOVA. Three independent experiments.

Figure 2

Neddylation inhibition mediated by a specific NAE1 inhibitor MLN4924 blocks C2C12 myotube formation. (A,B) Western blot analysis of neddylated Cullins (N8-CULs), CUL1, and muscle differentiation markers in vehicle (Veh)-treated and MLN4924 (MLN, 0.5 μM)-treated C2C12 for 6 days (D6) in differentiation media. Proteins were quantified and normalized to TUBA1A. Arrow indicates neddylated CUL1. Data were presented as fold changes with Veh-treated cells set at 1. * p < 0.05; ** p < 0.005 vs. Veh. Unpaired t tests. (C) Representative bright-field and immunofluorescence images of Veh-treated and MLN-treated C2C12 at D6 of differentiation. Cells were stained with MyHC (red), and nuclei were counterstained with DAPI (blue). Scale bar: 200 μm. (D) Representative bright-field images of Veh- and MLN-treated C2C12 cells at D6 in differentiation media with insulin. MLN at indicated doses was only treated for the first 2 days of differentiation. Scale bar: 200 μm. Three independent experiments.

Figure 3

Neddylation inhibition mediated by knockout of NAE1 inhibits C2C12 differentiation. C2C12 cells were infected with lentiviruses expressing gRNAs (gRNA1 and gRNA2) against murine Nae1, respectively, to generate bulk cultures of cells with NAE1 knockout (KO1 and KO2). Cells infected with lentiviruses expressing no gRNA were used as controls (Ctrl). (A) Western blot analysis of NAE1, neddylated Cullins (N8-CULs), CUL1, myogenic transcription factors, and muscle differentiation markers during the differentiation. Arrow indicates neddylated CUL1. (B) Quantifications of protein expressions as normalized to TUBA1A at D6. Data were presented as fold changes with Ctrl set at 1. (C) qRT-PCR analysis of Myog gene expression at D0, D3, and D6 of myoblast differentiation. (D) Representative bright-field and immunofluorescence images at 6 days in differentiation medium. Cells were stained with MyHC (red), and nuclei were counterstained with DAPI (blue). Scale bar: 200 μm. * p < 0.05; ** p < 0.01 vs. Ctrl. One-way ANOVA. Three independent experiments.

Figure 4

Neddylation controls the expression of class IIa and class III HDACs during myoblast differentiation. (A) Western blots of HDAC4, 5 and SIRT1 in NAE1-KO C2C12 cells before (D0) and after 3 and 6 days (D3 and D6) of myoblast differentiation as compared with vector controls (Ctrl). (B) Protein expressions at D0 and D3 were quantified by normalizing to ACTB. Data were presented as fold changes with D0 Ctrl cells set at 1. (C) qRT-PCR gene expression analysis of Hdac4/5 and Sirt1 at D0 and D3 of myoblast differentiation. D0 Ctrl cells were set at 1. * p < 0.05; ** p < 0.005 vs. Ctrl at the same day of differentiation. # p < 0.05 vs. D0 Ctrl cells. Two independent experiments in triplicates. (D) Western blots of neddylated Cullins (N8-CULs), CUL1, HDAC4, 5, and SIRT1, and myogenic transcription factors in vehicle (Veh) and 0.5 μM MLN4924 (MLN) treated C2C12 cells before and during the first three days of myogenic differentiation. Arrow indicates neddylated CUL1. Three independent experiments.

Figure 5

Neddylation inhibition dysregulates SIRT1 intracellular localization. (A) Representative immunofluorescence images of Ctrl and NAE1-KO (KO1, KO2) C2C12 cells at 3 days in differentiation media. Cells were stained with SIRT1 (red), and nuclei were labeled with Hoechst 33342 (blue). Scale bar: 20 μm. (B) Western blots of whole cell lysates (WCL), cytosol, and nucleus fractions of Ctrl and NAE1-KO (KO1 and KO2) C2C12 cells at 3 days in differentiation media. GAPDH and LMNB1 were used as cytosol and nucleus marker proteins, respectively. (C) Quantifications of nuclear fraction of SIRT1 as normalized to LMNB1 with Ctrl set as 1. * p < 0.05 vs. Ctrl. One-way ANOVA. Three independent experiments.