Abstract
Sumoylation, the conjugation of a small ubiquitin-like modifier (SUMO) protein to a target, has diverse cellular effects. However, the functional roles of the SUMO modification during myogenesis have not been fully elucidated. Here, we report that basal sumoylation of histone deacetylase 1 (HDAC1) enhances the deacetylation of MyoD in undifferentiated myoblasts, whereas further sumoylation of HDAC1 contributes to switching its binding partners from MyoD to Rb to induce myocyte differentiation. Differentiation in C2C12 skeletal myoblasts induced new immunoblot bands above HDAC1 that were gradually enhanced during differentiation. Using SUMO inhibitors and sumoylation assays, we showed that the upper band was caused by sumoylation of HDAC1 during differentiation. Basal deacetylase activity was not altered in the SUMO modification-resistant mutant HDAC1 K444/476R (HDAC1 2R). Either differentiation or transfection of SUMO1 increased HDAC1 activity that was attenuated in HDAC1 2R. Furthermore, HDAC1 2R failed to deacetylate MyoD. Binding of HDAC1 to MyoD was attenuated by K444/476R. Binding of HDAC1 to MyoD was gradually reduced after 2 days of differentiation. Transfection of SUMO1 induced dissociation of HDAC1 from MyoD but potentiated its binding to Rb. SUMO1 transfection further attenuated HDAC1-induced inhibition of muscle creatine kinase luciferase activity that was reversed in HDAC1 2R. HDAC1 2R failed to inhibit myogenesis and muscle gene expression. In conclusion, HDAC1 sumoylation plays a dual role in MyoD signaling: enhancement of HDAC1 deacetylation of MyoD in the basally sumoylated state of undifferentiated myoblasts and dissociation of HDAC1 from MyoD during myogenesis.
Highlights
Similar to ubiquitination, sumoylation is a covalent binding of small ubiquitin-like modifiers (SUMOs) to specific lysine residues of target proteins
Additional immunoreactivity with histone deacetylase 1 (HDAC1) during myoblast differentiation Because HDAC1 interacts with MyoD,[17] a key myogenic transcription factor, we examined whether either the protein level or the posttranslational modification of HDAC1 is altered during myogenesis
We assessed whether differentiation media (DM) induces the additional band of HDAC2 and HDAC1 in the nuclear fractions of 2-day DM-treated C2C12 cells; the HDAC1-associated upper bands appeared, no clear upper band was observed with HDAC2 (Figure 1c)
Summary
Sumoylation is a covalent binding of small ubiquitin-like modifiers (SUMOs) to specific lysine residues of target proteins. Since initially being discovered in 1997,1 sumoylation has been implicated in many biological functions and in disease status because it regulates diverse cellular processes. By identifying novel sumoylated proteins and evaluating the altered function of the sumoylated targets, we can extend the current understanding of the roles of sumoylation in cellular function.[2] SUMOs comprise 92–97 amino acids, and sumoylation is different from classically characterized posttranslational modifications with relatively small chemical groups, such as acetylation, phosphorylation and methylation. Several hundred sumoylation target proteins are involved in various processes, such as chromatin organization, transcriptional regulation, DNA repair, macromolecular assembly, protein turnover, intracellular localization and signal transduction.[3] Inversely, SUMO/sentrin-specific proteases remove SUMOs from target proteins, resulting in the tight balance of the sumoylation status.[4]
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