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. Author manuscript; available in PMC: 2016 May 9.
Published in final edited form as: Cancer Res. 2012 Sep 17;72(22):5988–6001. doi: 10.1158/0008-5472.CAN-12-0614

Figure 2. OTX2 functions as a transcriptional repressor of MyoD.

Figure 2

(A) OTX2 homeobox domain (HD) mediates transcriptional repression. OTX2, OTX2-3M, OTX2-HD (HD) or OTX2 deleted of HD (OTX2-ΔHD) were cloned in fusion with Gal4 DNA binding domain (BD, aa1-147) and co-transfected in D425 cells along with a luciferase reporter construct containing 4x Gal4 binding sites (Gal4-seq).

(B) Gal4-OTX2 represses the trans-activity of Gal4-MyoD in D425 cells. Gal4-MyoD was transfected along with empty vector or with indicated Gal4-OTX2 constructs. On the right panel, anti-Gal4 western blot showed the expression levels of transfected constructs.

(C) Fusion protein of MyoD and OTX2-HD (HD) showed repressed transcription activity. OTX2-HD (HD), OTX2-HD-3M (HD-3M) or OTX2-ΔHD were cloned in fusion with Gal4-MyoD and tested for their trans-activities in D425 cells.

(D) Fusion of OTX2-HD decreases the myogenic potential of MyoD protein in D283 medulloblastoma cells. 3xFLAG-tagged MyoD, MyoD-OTX2-HD (MyoD-HD), MyoD-OTX2-HD-3M (MyoD-HD-3M) or MyoD-OTX2-ΔHD (MyoD-ΔHD) was transiently transfected in D283 cells for 2 days. Cell lysates were blotted for myogenic marker MYH.

(E) A summary of the OTX2 mutants used in the study.