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. 2004 May;24(9):3757–3768. doi: 10.1128/MCB.24.9.3757-3768.2004

FIG. 5.

FIG. 5.

The conserved region of the HRC enhancer contains a high-affinity, functional MEF2 site. (A) MEF2 binds specifically to the HRC MEF2 site in vitro. MEF2A was transcribed and translated in vitro and used in EMSA analyses with radiolabeled double-stranded oligonucleotides representing the HRC MEF2 site (lanes 1 to 6) or a mutant version of the HRC MEF2 site (lanes 7 and 8). MEF2 efficiently bound to the HRC MEF2 site (lane 2) but failed to bind to the mutant MEF2 site (lane 8). Binding of MEF2 to the HRC MEF2 site was specific, since a 100-fold excess of unlabeled HRC MEF2 site efficiently competed for binding (lane 3) but a mutant version of the HRC MEF2 site (mHRC) failed to compete for binding even at a 100-fold excess (lane 4). Likewise, an unlabeled control MEF2 site from the myogenin gene (My) efficiently competed for binding (lane 5), but a 100-fold excess of a mutant myogenin MEF2 site (mMy) did not compete for binding (lane 6). In samples where in vitro-translated proteins were absent (lanes 1 and 7), an equal amount of unprogrammed reticulocyte lysate was included. Lysate-derived, nonspecific mobility shifts are noted. (B) The HRC enhancer is activated directly by MEF2 factors through the MEF2 site in the enhancer. MEF2A expression plasmid (lanes 3 and 4), MEF2C expression plasmid (lanes 5 and 6), or parental expression vector (lanes 1 and 2) was cotransfected with a full-length HRC-lacZ reporter plasmid (lanes 1, 3, and 5) or a mutant version of that reporter containing a disrupted MEF2 site (lanes 2, 4, and 6) into 10T1/2 fibroblasts. The parental expression vector failed to significantly activate the HRC enhancer (lane 1). MEF2A and MEF2C were each able to significantly trans-activate the HRC-dependent reporter (lanes 3 and 5, respectively). Neither MEF2A nor MEF2C activated the MEF2 mutant enhancer (lanes 4 and 6, respectively). The data shown represent the mean values obtained in three independent transfections and analyses. Error bars represent the standard errors of the means. (C) The HRC MEF2 site is not bound by SRF. Either MEF2A (lanes 2 to 4) or SRF (lanes 5 to 7 and 9 to 11) was transcribed and translated in vitro and used in EMSA analyses with radiolabeled double-stranded oligonucleotides representing the HRC MEF2 site (lanes 1 to 7) or the SMaa intronic CArG box (lanes 8 to 11). MEF2 efficiently bound to the HRC MEF2 site (lane 2), whereas SRF was completely unable to bind to the HRC MEF2 site (lane 5) under conditions in which it efficiently bound to the SMaa CArG box (lane 9). In samples where in vitro-translated proteins were absent (lanes 1 and 8), an equal amount of unprogrammedreticulocyte lysate was included. Lysate-derived, nonspecific mobility shifts are noted. Wild-type (HRC and CArG) and mutant (mHRC and mCArG) competitors were used at a 100-fold excess where indicated. (D) The HRC enhancer is not trans activated by SRF. Expression plasmids for MEF2C (lane 2), SRF (lanes 3 and 5), or the parental expression vector (lanes 1 and 4) were cotransfected with a full-length HRC-lacZ reporter plasmid (lanes 1 to 3) or a SMaa-lacZ reporter plasmid (lanes 4 and 5) into 10T1/2 fibroblasts. SRF failed to activate the HRC reporter (lane 3) under conditions in which MEF2C activated the HRC reporter more than 10-fold (lane 2) over the background level of activation indicated by parental expression vector cotransfection (lane 1). By contrast, SRF activated the SMaa reporter in the same experiment more than 16-fold (lane 5) over the background level of activation indicated by parental expression vector cotransfection (lane 4). The data shown represent the mean values obtained in three independent transfections and analyses. Error bars represent the standard errors of the means.