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Global increases in O‐GlcNAc levels lead to differentiation of myoblasts
Author(s) -
Vaidyanathan Krithika,
Wells Lance
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.607.4
Subject(s) - c2c12 , myogenesis , microbiology and biotechnology , chemistry , transcription factor , activator (genetics) , blot , enzyme , repressor , myocyte , biochemistry , biology , gene
O‐GlcNAc is a posttranslational modification occurring on hundreds of nuclear and cytoplasmic proteins, which is cycled by the joint action of OGT, for its addition, and OGA, for its removal. O‐GlcNAc modification has been demonstrated to regulate transcription, differentiation, disease pathology and cell cycle regulation in various settings. In order to further understand the regulation of OGA, our laboratory performed a yeast two‐hybrid screen with the enzyme as bait and identified IFRD1, a transcriptional co‐activator/co‐repressor, as a candidate interacting protein. We have successfully demonstrated the OGA/IFRD1 interaction through reciprocal immunoprecipation, western blotting experiments. This led to our interest in examining the role of this interaction in skeletal muscle cells where IFRD1 is known to play a role in myoblast differentation. When OGA and IFRD1 are co‐expressed, there is a global increase in O‐GlcNAc levels suggesting that OGA could either be sequestered by IFRD1 thus allowing for increased global O‐GlcNAc levels, or IFRD1 inhibits OGA activity. When C2C12 cells were treated with GlcNAcstatin, an OGA inhibitor for 18 hours at 60% confluency, they spontaneously differentiate into myotubes in a dose‐dependent manner. Current work is recapitulating these results via genetic manipulation of the O‐GlcNAc cycling enzymes and more carefully defining the differentiation process using a variety of markers. We are currently localizing the two proteins as well as determining the activity of IFRD1‐associated OGA using immunofluorescence, biochemical fractionation, and enzyme activity assays. Current work is aimed at exploring the mechanism by which elevated O‐GlcNAc levels lead to myoblast differentiation with the working hypothesis that the IFRD1/OGA interaction positively regulates myogenesis.