Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

Abstract

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABP␣/␤ to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest "repressing repressors" as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle. INTRODUCTIONDuchenne's muscular dystrophy (DMD) is a fatal neuromuscular disease caused by gene mutations leading to qualitative or quantitative disturbances in dystrophin expression (Hoffman et al., 1987). Dystrophin-related protein or utrophin is considered the autosomal homologue of dystrophin, because it shares extensive sequence homology as well as its size and abundance in muscle (Love et al., 1989;Khurana et al., 1990;Tinsley et al., 1992). Utrophin and dystrophin also share functional properties such as the ability to associate with the dystroglycan complex and bind F-actin (Matsumura et al., 1992;Winder and Kendrick, 1995;Rybakova et al., 2002). Direct evidence for the ability of utrophin to functionally substitute comes from experiments demonstrating that utrophin overexpression driven either by transgenic means Rafael et al., 1998;Tinsley et al., 1998;Fisher et al., 2001) or viral vectors (Gilbert et al., 1999;Wakefield et al., 2000;Cerletti et al., 2003) can rescue dystrophin-deficient muscle. Despite these functional similarities, dystrophin and utrophin exhibit distinct localization in normal adult tissues.Perinatally, utrophin is expressed throughout the sarcolemma, however, its expression declines as that of dystrophin increases (Khurana et al., 1991;Clerk et al., 1993), leading to its spatially restricted expression at neuromuscular and myotendinous junctions (NMJs and MTJs, respectively) of adult myofibers. These features and relevance toward a therapy for DMD provide a powerful impetus for b...

Microutrophin Delivery Through rAAV6 Increases Lifespan and Improves Muscle Function in Dystrophic Dystrophin/Utrophin-deficient Mice

“…The uniform expression pattern of microutrophin likely reflects the use of the CMV promoter, which lacks N-boxes that can restrict transcription to nuclei adjacent to neuromuscular junctions, and the lack of most of the untranslated regions that also can influence mRNA trafficking and/or stability. [44–46] The extrasynaptic localization of microutrophin is vital in the consideration of a possible treatment for DMD.…”

The therapeutic potential of skeletal muscle plasticity in Duchenne muscular dystrophy: phenotypic modifiers as pharmacologic targets

“…In addition to the recent description of the corepressors RIP140 and NCoR1, transcriptional repressors have been identified, such as Ets‐2 repressor factor (ERF) and E2F1, that also regulate skeletal muscle plasticity (68, 69) (Fig. 1).…”

A Method of Utrophin Up-Regulation through RNAi-Mediated Knockdown of the Transcription Factor EN1

“…21 Many research groups are utilizing different strategies to design therapeutic options by attempting the functional recovery of dystrophic muscle. 22,23 Currently, the upregulation of utrophin has become a feasible approach for the treatment of DMD. The full-length product of the UTRN gene is driven by two promoters (A and B) and one downstream enhancer element.…”

“…Both the ε-subunit promoter of the nicotinic acetylcholine receptor (nAChR) and promoter A in the UTRN gene contain an Nbox motif. 22 The N-box motif region has been shown to be critical for transcriptional activation at the sub-synaptic nuclei by neurite-associated heregulin. 26 Utrophin is a protein with a similar structure to dystrophin that can be found in high concentrations within muscle cells during fetal development.…”