Cloning and characterization of a novel MyoD enhancer-binding factor

Abstract

Glucocorticoid-induced gene-1 (Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 enhancer factor and papillomavirus binding factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF-E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF-E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 enhancer factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to study Gig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1(nlacZ) heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1(nlacZ) embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors.

OTX2 Represses Myogenic and Neuronal Differentiation in Medulloblastoma Cells

“…While OTX2 displayed no intrinsic trans-activity when fused with Gal4-binding domain (Gal4-BD) in D425 cells (Figure 1H), we fused OTX2 with the VP16 trans-activation domain to test its trans-activation ability with the pMyoD-CER construct, since this can serve as an indirect readout of the association of OTX2 with the MyoD CER (38). VP16-OTX2 was capable of activating pMyoD-CER significantly above VP16 vector alone, while the VP16-OTX2-3M construct containing triple DNA-binding mutations in the homeobox domain (HD) lost this trans-activity (Figure 1H).…”

MyoD-expressing progenitors are essential for skeletal myogenesis and satellite cell development

“…Whole mount in situ hybridization with digoxigenin-labeled probes was performed as previously described (Henrique et al, 1995; Yamamoto et al, 2007), with minor modifications related to color development and storage. Briefly, for color development, embryos were incubated in BM Purple (Roche Applied Science) overnight at room temperature.…”

“…Embryos were subsequently transferred to 70% ethanol for 30 min at room temperature, rinsed in MABT and stored at 4°C in MABT. The MyoD probe has been previously described (Yamamoto et al, 2007). The Myf-5 probe represents a 613 nucleotide fragment of the Myf-5 cDNA that was amplified by PCR using the following primers: Forward: 5′-CATATGGACATGACGGACGGC -3′, Reverse: 5′-AGCTGGACACGGAGCTTTTA -3′.…”

Identification of Progenitor Cells That Contribute to Heterotopic Skeletogenesis

“…X-gal Staining X-gal staining was performed as previously described 22 , with minor modifications. Mounted tissue sections were fixed in 0.2% glutaraldehyde for ten minutes on ice, rinsed briefly in phosphate-buffered saline solution, and rinsed in detergent solution (0.05% NP-40 and 0.01% sodium deoxycholate in phosphate-buffered saline solution) for ten minutes at 4°C.…”