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Characterization of Tetratricopeptide 39c (Ttc39c) in Skeletal Muscle
Author(s) -
Hayes Caleb,
Waddell David
Publication year - 2017
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.31.1_supplement.1058.4
Subject(s) - myogenesis , myod , reporter gene , myogenin , biology , microbiology and biotechnology , ectopic expression , myocyte , c2c12 , transfection , gene , transcriptional regulation , gene expression , regulation of gene expression , myogenic regulatory factors , genetics
Ttc39c has been identified as a novel gene in skeletal muscle that is differentially regulated under conditions of neurogenic atrophy in wild‐type mice compared to Muscle RING Finger 1 (MuRF1) knockout mice. MuRF1 is a ubiquitin E3 ligase that is known to be an important mediator of muscle wasting; however, evidence presented here suggests that MuRF1 may also function as a regulator of atrophy‐induced gene expression. Western blot analysis comparing undifferentiated and differentiated C 2 C 12 cell lysates demonstrates that Ttc39c protein is expressed in proliferating myoblasts, but is reduced in differentiated myotubes. The transcriptional regulation of two distinct transcripts (termed 001 and 004) of Ttc39c have been examined by cloning the alternative promoter regions of each transcript into to a reporter plasmid. These reporter constructs were then transfected into muscle cells along with expression plasmids for MyoD and myogenin, two myogenic regulatory factors (MRFs) that regulate the expression of muscle‐specific genes by binding canonical E‐box elements (5′‐CANNTG‐3′). The reporter gene constructs for both Ttc39c transcripts show significant transcriptional activity in cultured muscle cells and are transcriptionally responsive to MRF ectopic expression. Furthermore, a conserved E‐box element in the Ttc39c‐004 proximal promoter was also identified, mutated, and analyzed for its role in the regulation of Ttc39c‐004 expression. The mutation of the conserved E‐box rendered the Ttc39c‐004 reporter gene inactive, demonstrating that the element is necessary for Ttc39c‐004 expression. Finally, the 004 transcript reporter construct was also transfected into muscle cells along with an expression plasmid for MuRF1. MuRF1 overexpression repressed Ttc39c‐004 reporter activity, while overexpression of a MuRF1 RING domain mutant failed to repress the Ttc39c‐004 reporter. The identification and characterization of novel genes that are activated during neurogenic atrophy helps improve our understanding of the molecular genetic events that lead to muscle wasting and could eventually lead to new therapeutic targets in the future. Support or Funding Information The work was support by University of North Florida Transformational Learning Opportunity grants to D.W.