Abstract
Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.
Highlights
As an effort to understand the role of Hop in muscle-specific gene regulation, we sought a Hop-binding candidate by the yeast two-hybrid screening technique and found that Enhancer of Polycomb1 (Epc1), a novel Hop-interacting partner, initiates skeletal muscle differentiation and that its interaction with Hop is required for the full activity
To delineate the functional role of Hop in the muscle differentiation by seeking its interacting protein, we utilized yeast two-hybrid assay with the human adult heart cDNA library and maturation is impaired in Hop knock-out mice
Our this, the expressions of myogenin and MHCemb were investi- results highlight Epc1 as a regulator in skeletal muscle differentiagated in postnatal day 1 mice with either wild type or Hop tion, in association with Hop, by inducing the expression of key knock-out
Summary
Animals and Plasmid Constructs—Pregnant CD1 mice were purchased from Daehan Biolink (Daejeon, Korea). PcDNA3.1-mouse Hop-myc was described previously [6]. Complementary sequences of the 1–516-nucleotide region of rat Epc was amplified and subcloned into pcDNA6/myc-HisA (Invitrogen) containing the blasticidine resistance gene. Transfection of pcDNA3.1-HOP-V5/His and/or pCMV-myc-Epc to H9c2 cells was repeated every other day; the cells were lifted at the 5th days, and the numbers were counted as described previously [10]. 293T cells were transfected with pCMV-FLAG-Epc full or truncated mutants together with pcDNA3.1-HOP-V5/His by FuGENE 6 (Roche Diagnostics). Total RNA from embryonic mouse heart and differentiated H9c2 cells was extracted using TRIzol reagent (Invitrogen) and subjected to reverse transcription reaction followed by semi-quantitative PCR amplification. In Vivo Wound Healing Assay—Seven- or 8-week-old wild type or Hop knock-out mice were utilized for in vivo wound healing assay as described previously [14]. The transverse sections at suture sites were obtained and utilized for hematoxylin and eosin staining or desmin immunohistochemistry
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