Ns done by the Polyphen-2 software used by the Exome Variant Server (EVS) database to predict the effect of amino acid substitution on protein function (http://evs.gs.washington.edu/EVS/).The NFATC1 double mutant protein is partially retained in the cytoplasmIn order to assess the impact of the P66L and I701L mutations on NFATC1 structural and functional properties, site directed mutagenesis was done on a human NFATC1 cDNA (Isoform A, NP_765978.1) cloned in an expression vector. Three vectors were generated harboring P66L alone (P66L), I701L alone (I701L) and both mutations together (P66L/I701L). The generated plasmids were Epigenetics transfected into HeLa cells to study the cellular localization of the mutated protein. Immunostaining revealed that Wt NFATC1 and NFATC1 mutants are located in the cytoplasm in absence of PPP3CA(Figure 4A). Wt NFATC1, P66l, and I701L translocated to the nucleus when cotransfected with the activated form of PPP3CA (Figure 4B). However, NFATC1 double mutant P66L/ I701L failed to translocate to the nucleus in more than 80 of cotransfected cells (Figure 4B).transfection with PPP3CA, the activation of DEGS1 promoter increased to reach 6.2 without attaining a synergistic threshold. This synergy is however observed when the amount of Wt NFATC1 was increased (Figure 6 A). In comparison, the different 25033180 mutant NFATC1 proteins have a decreased transcriptional activity alone or in combination with PPP3CA. The same approach was 
adopted to assess NFATC1 regulation of CCND1 promoter, a recently described bona fide target of NFATC1 [33]. The Wt protein showed a dose dependent activation of the promoter that was increased in presence of PPP3CA. NFATC1 mutants (P66l, I701L, and P66L/I701L) showed decreased activation of the promoter that was more Epigenetics significant in the case of the double mutant P66L/I701L (Figure 6 B).The NFATC1 double mutant is unable to functionally interact with both GATA5 and HANDInteraction of GATA5 and NFATC1 on DEGS1 promoter was studied based on previous data implicating both proteins in having physical and functional interaction over the endothelin promoter [19]. Hela cells were transfected with GATA5 alone, PPP3CA alone, Wt NFATC1 alone or NFATC1 mutants, a combination of each two, or a combination of the three together. Wt NFATC1 alone, PPP3CA alone, and GATA5 alone resulted 23727046 in 1.8, 11.4 and 21.5 times fold activation respectively. Wt NFATC1 cotransfected with GATA5 caused a synergistic activation of 35 fold, while transfection of Wt NFATC1 with PPP3CA and GATA5 caused even a stronger synergy reaching 68 fold (Figure 7A). The combination of GATA5 with either the P66L or I701L NFATC1 mutants still yield a synergistic activation of the DEGS1 though at a much reduced magnitude as compared to the Wt. Only the double NFATC1 mutant failed to synergistically interact with GATA5. On the contrary, the interaction of NFATC1 and HAND2, a recently identified pathway implicated in chronic hypoxia, was totally disrupted over the DEGS1 promoter when any of the NFATC1 mutation was introduced (Figure 7B).Attenuated DNA binding affinity of the mutant NFATC1 mutant proteinsGel shift assays were carried out to assess the binding affinity of the mutated NFATC1 proteins to an NFAT consensus binding sites. Equal amounts of overexpressed proteins were verified by western blots (Figure 5A), and used for DNA-binding activity. Multiple assays with different amounts of proteins showed a consistent decrease in DNA binding affinity of aroun.Ns done by the Polyphen-2 software used by the Exome Variant Server (EVS) database to predict the effect of amino acid substitution on protein function (http://evs.gs.washington.edu/EVS/).The NFATC1 double mutant protein is partially retained in the cytoplasmIn order to assess the impact of the P66L and I701L mutations on NFATC1 structural and functional properties, site directed mutagenesis was done on a human NFATC1 cDNA (Isoform A, NP_765978.1) cloned in an expression vector. Three vectors were generated harboring P66L alone (P66L), I701L alone (I701L) and both mutations together (P66L/I701L). The generated plasmids were transfected into HeLa cells to study the cellular localization of the mutated protein. Immunostaining revealed that Wt NFATC1 and NFATC1 mutants are located in the cytoplasm in absence of PPP3CA(Figure 4A). Wt NFATC1, P66l, and I701L translocated to the nucleus when cotransfected with the activated form of PPP3CA (Figure 4B). However, NFATC1 double mutant P66L/ I701L failed to translocate to the nucleus in more than 80 of cotransfected cells (Figure 4B).transfection with PPP3CA, 
the activation of DEGS1 promoter increased to reach 6.2 without attaining a synergistic threshold. This synergy is however observed when the amount of Wt NFATC1 was increased (Figure 6 A). In comparison, the different 25033180 mutant NFATC1 proteins have a decreased transcriptional activity alone or in combination with PPP3CA. The same approach was adopted to assess NFATC1 regulation of CCND1 promoter, a recently described bona fide target of NFATC1 [33]. The Wt protein showed a dose dependent activation of the promoter that was increased in presence of PPP3CA. NFATC1 mutants (P66l, I701L, and P66L/I701L) showed decreased activation of the promoter that was more significant in the case of the double mutant P66L/I701L (Figure 6 B).The NFATC1 double mutant is unable to functionally interact with both GATA5 and HANDInteraction of GATA5 and NFATC1 on DEGS1 promoter was studied based on previous data implicating both proteins in having physical and functional interaction over the endothelin promoter [19]. Hela cells were transfected with GATA5 alone, PPP3CA alone, Wt NFATC1 alone or NFATC1 mutants, a combination of each two, or a combination of the three together. Wt NFATC1 alone, PPP3CA alone, and GATA5 alone resulted 23727046 in 1.8, 11.4 and 21.5 times fold activation respectively. Wt NFATC1 cotransfected with GATA5 caused a synergistic activation of 35 fold, while transfection of Wt NFATC1 with PPP3CA and GATA5 caused even a stronger synergy reaching 68 fold (Figure 7A). The combination of GATA5 with either the P66L or I701L NFATC1 mutants still yield a synergistic activation of the DEGS1 though at a much reduced magnitude as compared to the Wt. Only the double NFATC1 mutant failed to synergistically interact with GATA5. On the contrary, the interaction of NFATC1 and HAND2, a recently identified pathway implicated in chronic hypoxia, was totally disrupted over the DEGS1 promoter when any of the NFATC1 mutation was introduced (Figure 7B).Attenuated DNA binding affinity of the mutant NFATC1 mutant proteinsGel shift assays were carried out to assess the binding affinity of the mutated NFATC1 proteins to an NFAT consensus binding sites. Equal amounts of overexpressed proteins were verified by western blots (Figure 5A), and used for DNA-binding activity. Multiple assays with different amounts of proteins showed a consistent decrease in DNA binding affinity of aroun.