E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation

Abstract

The E2A proteins are basic helix-loop-helix transcription factors that regulate proliferation and differentiation in many cell types. In muscle cells, the E2A proteins form heterodimers with muscle regulatory factors such as MyoD, which then bind to DNA and regulate the transcription of target genes essential for muscle differentiation. We now demonstrate that E2A proteins are primarily localized in the nucleus in both C2C12 myoblasts and myotubes, and are degraded by the ubiquitin proteasome system evidenced by stabilization following treatment with the proteasome inhibitor, MG132. During the differentiation from myoblast to myotube, the cellular abundance of E2A proteins is relatively unaltered, despite significant changes (each B5-fold) in the relative rates of protein synthesis and protein degradation via the ubiquitin-proteasome system. The rate of ubiquitin-proteasome-mediated E2A protein degradation depends on the myogenic differentiation state (t½B2 h in proliferating myoblasts versus t½>10 h in differentiated myotubes), and is also associated with cell cycle in non-muscle cells. Our findings reveal an important role for both translational and post-translational regulatory mechanisms in mediating the complex program of muscle differentiation determined by the E2A proteins.

Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts

“…These data are consistent with a different transcriptional activity pattern, which in turn may be related to a MyoD protein level regulation dictated by relative rates of differentiation stagedependent protein synthesis [Sun et al, 2005] and degradation, as already described for MyoD itself [Lingbeck et al, 2005] and other myogenic transcription factors, such as the E2A and Id proteins [Sun et al, 2007]. The detection of different degree of MyoD labeling can also be observed among the nuclei of the same myotube, where it is suggestive of the presence of myonuclear domains [Gundersen and Bruusgaard, 2008], characterized by a specific gene expression and behavior [Rosser et al, 2002;Gundersen and Bruusgaard, 2008].…”

“…In addition, myogenin protein half-life might be regulated by the relative rates of differentiation stage-dependent protein synthesis and degradation. This regulation mechanism could affect both myogenin itself, as already described for MyoD [Lingbeck et al, 2005;Sun et al, 2007], and/or other myogenic factors able to modulate myogenin half-life, such as MyoD or Id proteins [Viñ als and Ventura, 2004;Sun et al, 2005]. In particular, it should be considered that myogenin expression is enhanced by MyoD [Thayer et al, 1989], whereas it is down-regulated by MRF4 [Zhang et al, 1995].…”

Calcium Regulation of Myogenesis by Differential Calmodulin Inhibition of Basic Helix-Loop-Helix Transcription Factors

“…In developing skeletal muscle, E2A expression increases during the early stages of muscle differentiation and returns to low or undetectable levels in fully differentiated muscle fibers (Rutherford and LeBrun, 1998). E2A proteins are primarily localized in the nucleus in both C2C12 myoblasts and myotubes and the cellular abundance of E2A is relatively stable during this differentiation (Sun et al, 2007). HEB is also expressed in developing muscle (Conway et al, 2004).…”

Control of myogenesis by rodent SINE-containing lncRNAs

“…After using confocal microscopy to analyze DAPI-stained cells that had been transfected with control siRNA and finding that multinucleated MTs were apparent as early as day 3 (Supplemental Fig. 6B), the differentiation rates of all transfected cells were assessed using one or both of two methods: (1) transmitted light microscopy to visualize changes in cell morphology and (2) Western blotting to quantitate the level of three myogenic markers-namely, the induction of mMyogenin production and mMHC production (Sun et al 2006) and the inhibition of mb-actin production (Sun et al 2006)-relative to the level of mCalnexin.…”