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. Author manuscript; available in PMC: 2016 Feb 12.
Published in final edited form as: Cell. 2015 Jan 29;160(4):595–606. doi: 10.1016/j.cell.2015.01.009

Figure 5. Regulation of MLN Transcription by MyoD and MEF2.

Figure 5

(A) A fragment of the MLN promoter (−506 to +86, relative to the transcriptional start site) containing a highly conserved MyoD E-box (CACCTG) and MEF2 site (CTAATAACAG) was cloned in front of the luciferase reporter gene (MLN-Luc). The MLN-Luc reporter was robustly transactivated by the skeletal muscle transcription factors MyoD and MEF2 in COS7 cells (gray and red bars respectively). Mutation of the E-box (acCCgt) or Mef2 site (CTgggAACAG) (indicated by an X) abrogated transactivation by MyoD:E12 heterodimer or Mef2c, respectively. All luciferase values were normalized to the transactivation of a basal luciferase reporter (TATA-Luc) with MyoD or Mef2, respectively.

(B) Transfection of the MLN-Luc reporter or mutant luciferase vectors into C2C12 myoblasts or myotubes showed they are essential for the transactivation of the MLN promoter.

(C) X-gal and H&E staining of E10.5 mouse embryos harboring either the MLN promoter-lacZ transgene (WT) or mutations in the MLN promoter (ΔMef2 or ΔE-box). The MLN-promoter showed expression in the myotomal compartment of the somites (orange arrow) and premyogenic cells in the mandibular arch (red arrow). Mutation of the MEF2 or E-box sequences in the MLN promoter-lacZ transgene abrogated or abolished muscle specific expression.

See also Figure S4.