Mammalian Vestigial-like 2, a Cofactor of TEF-1 and MEF2 Transcription Factors That Promotes Skeletal Muscle Differentiation

Abstract

Expression of many skeletal muscle-specific genes depends on TEF-1 (transcription enhancer factor-1) and MEF2 transcription factors. In Drosophila, the TEF-1 homolog Scalloped interacts with the cofactor Vestigial to drive differentiation of the wing and indirect flight muscles. Here, we identify three mammalian vestigiallike genes, Vgl-1, Vgl-2, and Vgl-3, that share homology in a TEF-1 interaction domain. Vgl-1 and Vgl-3 transcripts are enriched in the placenta, whereas Vgl-2 is expressed in the differentiating somites and branchial arches during embryogenesis and is skeletal musclespecific in the adult. During muscle differentiation, Vgl-2 mRNA levels increase and Vgl-2 protein translocates from the cytoplasm to the nucleus. In situ hybridization revealed co-expression of Vgl-2 with myogenin in the differentiating muscle of embryonic myotomes but not in newly formed somites prior to muscle differentiation. Like Vgl-1, Vgl-2 interacts with TEF-1. In addition, we show that Vgl-2 interacts with MEF2 in a mammalian two-hybrid assay and that Vgl-2 selectively binds to MEF2 in vitro. Co-expression of Vgl-2 with MEF2 markedly co-activates an MEF2-dependent promoter through its MEF2 element. Overexpression of Vgl-2 in MyoDtransfected 10T 1 ⁄2 cells markedly increased myosin heavy chain expression, a marker of terminal muscle differentiation. These results identify Vgl-2 as an important new component of the myogenic program.Commitment of pluripotent cells to the skeletal muscle lineage involves multiple inductive signals and the hierarchical activation of several transcription factors (1). Differentiation of skeletal muscle cells is coupled to withdrawal from the cell cycle and is accompanied by transcriptional activation of muscle-specific genes. The MyoD family of basic-helix-loop-helix transcription factors, including MyoD, Myf5, myogenin, and MRF4, plays a central role in activating the muscle differentiation program (2). Activation of muscle gene expression by the MyoD family of factors is dependent on their association with members of the MEF2 family of MADS box transcription factors (3). Together, MyoD and MEF2 account for a large part of muscle-specific gene activation. In addition, many muscle genes contain muscle-specific cytidine-adenosine-thymidine cis-elements (5Ј-CATDSH-3Ј) that are also required for musclespecific expression (4, 5). The proteins that bind to these elements belong to the transcription enhancer factor-1 (TEF-1)

The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members

“…YAP65 and TAZ did not interact with GAL4. Both GAL4-TEF-1-(79-426) and GAL4-RTEF-1-(79-426) were relatively weak transcriptional activators, with RTEF-1 being slightly stronger: 1-2-fold compared with 3-8-fold [26]. At all concentrations of input DNA, TAZ interacted with both GAL4-TEF-1-(79-426) and GAL4-RTEF-1-(79-426) and strongly activated transcription.…”

“…Expression vectors for human ETF-1 and DTEF-1 were kindly provided by Dr Irwin Davidson (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France). Expression vectors for human (h) RTEF-1-and hTEF-1-GAL4 fusion proteins {pM, pM-hTEF-1(1-426), equivalent to rat TEF-1ζ (see Figure 2 and [40]), pM-hTEF-1(79-426), and pM-hRTEF-1(79-426)} and a GAL4-UAS (upstream activator sequence)-dependent luciferase reporter (pG5-luc) were kindly provided by Dr Alexandre Stewart (University of Ottawa, Heart Institute, Ottawa, Ontario, Canada) [11,26]. pCMV-FL-TEF-1ε: TEF-1ε [40] was released from XJ40-TEF-1ε-HSV with XhoI and cloned into pCMV-FLAG-5a (Sigma) digested with SalI to create pCMV-FL-TEF-1ε.…”

“…To test this, modified mammalian two-hybrid experiments were performed as above. Amino acids 79-426 (C-terminus) of human TEF-1 and human RTEF-1 were fused to the GAL4 DNA-binding domain to yield GAL4-TEF-1-(79-426) and GAL4-RTEF-1-(79-426) (kindly provided by Dr Alexandre F. R. Stewart, University of Ottawa Heart Institute, Ottawa, Ontario, Canada) [11,26]. Increasing amounts (5-100 ng) of expression vectors for GAL4-TEF-1-(79-426) and GAL4-RTEF-1-(79-426) were cotransfected into H1299 cells, with a constant amount of expression vector for TAZ and a GAL-UAS-dependent luciferase reporter, and extracts were prepared and analysed for luciferase activity ( Figure 7B).…”

“…It is proposed that activity of TEF-1 proteins is controlled by interactions with specific cofactors with limited expression patterns. Known vertebrate TEF-1 co-factors include PARP [27], the Vgl family of proteins [25,26,43], YAP65 [24] and p160 [22]. These co-factors are broadly expressed (PARP and p160), expressed in the same tissues as TEF-1 proteins (YAP65) or restricted in their expression to one or two tissues (Vgl proteins).…”

“…Class 2: interacting proteins without co-activation function [TONDU (Vgl-1, Vestigial-like protein-1), Vgl-2 and Vgl-3]. TONDU, Vgl-2 and Vgl-3, vertebrate proteins related to the Drosophila co-activator Vg [20,25,26], interact with TEF-1 proteins, but do not contain a transactivation domain. Expression of these is tissue restricted (placenta, skeletal muscle and heart), supporting the hypothesis that TEF-1 activity is regulated by tissue-specific co-factors.…”