R of Crtl1 expression during cardiac development. Providing further indication that Mef2c could be involved in the transcriptional regulation of Crtl1, we found that the Crtl1 promoter contains two Mef2 ML 240 site transcription factor binding sites that are conserved between human, mouse, and rat. Testing the hypothesis that these Mef2 binding sites indeed can bind Mef2c and activate Crtl1 transcription, we performed DNA affinity precipitation, ChIP analysis, and luciferase assays. Combined, these in vitro and in vivo studies provided evidence that Mef2c binds to the Crtl1 59UTR and activates Crtl1 transcription. To ascertain the importance of each Mef2 binding site, we mutated these two sites and evaluated the Crtl1 promoter response to exogenous Mef2c. Mutation of the Mef2c binding site at 2707/2698 resulted 
in reduction of Crtl1 promoter activity both in the presence and absence of exogenous Mef2c protein, while mutation of the Mef2c binding site at 2923/2913 only resulted in 18334597 a slight reduction of Crtl1 promoter activity. The Crtl1 promoter fragment that was used in these experiments also contained a previously described Sox9 binding site that has been demonMef2c Regulates Crtl1 TranscriptionFigure 5. Crtl1 promoter activity is regulated by Mef2c. Fold change in luciferase activity driven by approximately 1 kb of the Crtl1 promoter in the pGL3 luciferase reporter vector was assayed in fetal chicken VICs (A and B) and in NIH3T3 cells (C and D). In fetal chicken VICs (A) and NIH3T3 cells (C), Crtl1 promoter activity was MedChemExpress GSK -3203591 significantly increased with increasing concentrations of Mef2c. (B) Crtl1 promoter activity in the presence of 100 ng Mef2c with the addition of 200 ng of the Mef2-Engrailed dominant negative expression construct resulted in an approximately 30 reduction in Crtl1 reporter activity. (D) Mutations were introduced into the Crtl1 promoter construct at Mef2 Site 1 and Mef2 Site 2 (Crtl1-Mutant 1 and Crtl1Mutant 2 respectively). Crtl1-Mutant 1 results in an approximately 30 reduction in Crtl1 promoter activation in the presence of 100 ng of Mef2c and Crtl1-Mutant 2 results in an approximately 50 reduction of Crtl1 activity.(*p,0.05, #p,0.1). doi:10.1371/journal.pone.0057073.gstrated to be important in the regulation of Crtl1 in cartilage and bone formation [10,30]. In the developing bone, Sox9 and Mef2c have been shown to activate the Col10a1 promoter independently or co-activate Col10a1 in an additive fashion [13,15]. Deletion of either the Sox9 binding site or the Mef2c binding site in the Col10a1 promoter results in a reduction in Col10a1 activation. However deletion of both Sox9 and Mef2c binding sites are needed to result in complete abolishment of promoter activity [15]. It is therefore possible that Crtl1, an important ECM component in developing bone as well as valves, may be similarly regulated and that mutation of the Sox9 binding site within the Crtl1 promoter may be needed to achieve a complete loss of promoter activity. As described above, during the process of valve remodeling, Crtl1 expression becomes restricted as the mesenchyme within the valves becomes condensed. Tgfb2 has been demonstrated to be necessary for the repression of Crtl1 during late stages of valve development in order to prevent ectopic differentiation into a cartilage-lineage [35], while Sox9 has been shown to be critical for Crtl1 expression during the early stages of cushion and valve development [12]. Based on expression patterns of Mef2c, itsbind.R of Crtl1 expression during cardiac development. Providing further indication that Mef2c could be involved in the transcriptional regulation of Crtl1, we found that the Crtl1 promoter contains two Mef2 transcription factor binding sites that are conserved between human, mouse, and rat. Testing the hypothesis that these Mef2 binding sites indeed can bind Mef2c and activate Crtl1 transcription, we performed DNA affinity precipitation, ChIP analysis, and luciferase assays. Combined, these in vitro and in vivo studies provided evidence that Mef2c binds to the Crtl1 59UTR and activates Crtl1 transcription. To ascertain the importance of each Mef2 binding site, we mutated these two sites and evaluated the Crtl1 promoter response to exogenous Mef2c. Mutation of the Mef2c binding site at 2707/2698 resulted in reduction of Crtl1 promoter activity both in the presence and absence of exogenous Mef2c protein, while mutation of the Mef2c binding site at 2923/2913 only resulted in 18334597 a slight reduction of Crtl1 promoter activity. The Crtl1 promoter fragment that was used in these experiments also contained a previously described Sox9 binding site that has been demonMef2c Regulates Crtl1 TranscriptionFigure 5. Crtl1 promoter activity is regulated by Mef2c. Fold change in luciferase activity driven by approximately 1 kb of the Crtl1 promoter in the pGL3 luciferase reporter vector was assayed in fetal chicken VICs (A and B) and in NIH3T3 cells (C and D). In fetal chicken VICs (A) and NIH3T3 cells (C), Crtl1 promoter activity was significantly increased with increasing concentrations of Mef2c. (B) Crtl1 promoter activity in the presence of 100 ng Mef2c with the addition of 200 ng of the Mef2-Engrailed dominant negative expression construct resulted in an approximately 30 reduction in Crtl1 reporter activity. (D) Mutations were introduced into the Crtl1 promoter construct at Mef2 Site 1 and Mef2 Site 2 (Crtl1-Mutant 1 and Crtl1Mutant 2 respectively). Crtl1-Mutant 1 results in an approximately 30 reduction in Crtl1 promoter activation in the presence of 100 ng of Mef2c and Crtl1-Mutant 2 results in an approximately 50 reduction of Crtl1 activity.(*p,0.05, #p,0.1). doi:10.1371/journal.pone.0057073.gstrated to be important in the regulation of Crtl1 in cartilage and bone formation [10,30]. In the developing bone, Sox9 and Mef2c have been shown to activate the Col10a1 promoter independently or co-activate Col10a1 in an additive fashion [13,15]. Deletion of either the Sox9 binding site or the Mef2c binding site in the Col10a1 promoter results in a reduction in Col10a1 activation. However deletion of both Sox9 and Mef2c binding sites are needed to result in complete abolishment of promoter activity [15]. It is therefore possible that Crtl1, an important ECM component in developing bone as well as valves, may be similarly regulated and that mutation of the Sox9 binding site within the Crtl1 promoter may be needed to achieve a complete loss of promoter activity. As described above, during the process of valve remodeling, Crtl1 expression becomes restricted as the mesenchyme within the valves becomes condensed. Tgfb2 has been demonstrated to be 
necessary for the repression of Crtl1 during late stages of valve development in order to prevent ectopic differentiation into a cartilage-lineage [35], while Sox9 has been shown to be critical for Crtl1 expression during the early stages of cushion and valve development [12]. Based on expression patterns of Mef2c, itsbind.