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. 2002 Mar 1;30(5):1213–1223. doi: 10.1093/nar/30.5.1213

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Copyright © 2002 Oxford University Press

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YY1 transactivated Msx2 promoter by protein–DNA binding to three YY1 binding sites on the Msx2 proximal promoter. Three candidate YY1 binding sites encompassing the core binding motif 5′-CAT-3′ were identified on the proximal Msx2 promoter and designated as YY1 sites 1, 2 and 3 (A). Using site directed mutagenesis these sites were mutated in the core sequence from 5′-CAT-3′ to 5′-GGT-3′. The three sites were mutated individually and resulted in constructs designated M1, M2 and M3, or in combinations to yield M123. Additionally, the initiator on the Msx2 promoter was also mutated from 5′-CAC-3′ to 5′-GGC-3′ to generate M123/Inr construct. These Msx2 promoter constructs (x-axis) were co-transfected with LacZ as control or YY1 expression vector into P19 cells cultured on 10-cm plates. Luciferase activities were assayed 24 h post-transfection (B). Basal wild-type Msx2 promoter activity in the presence of LacZ control was designated as 100%. YY1 induced a significant increase in this Msx2 promoter activity. However, all mutant Msx2 promoters, except M2, exhibited reduced level of activities in response to YY1 when compared with wild-type and their respective LacZ controls. *P < 0.01. M2 showed slightly decreased activity. Three sets of oligonucleotides, corresponding to the three YY1 binding sites on the Msx2 promoter, were radiolabeled and tested in EMSA for their binding to P19 nuclear extract or to a GST–YY1 fusion protein. All three probes bound, albeit at different intensities (sites 1–3, lanes 1–3, respectively). The results for all three sets of oligonucleotides were similar for additional assays and those for site 3 are shown in lanes 4–11. The site 3 probe bound to the P19 nuclear extract (lane 4), and this binding was competed off by 100-fold molar excess of cold site 3 probe (lane 5), but not by excess mutant site 3 probe (lane 6) or mutant YY1 consensus oligonucleotides (lane 7). Similarly, the site 3 probe bound to the GST–YY1 fusion protein (lane 8), and this binding was competed off by 100-fold molar excess of cold site 3 probe (lane 9) or YY1 consensus oligonucleotides (lane 10). The band was super-shifted when YY1 antibody was also added to the site 3 probe and GST–YY1 complex (lane 11).

YY1 transactivated Msx2 promoter by protein–DNA binding to three YY1 binding sites on the Msx2 proximal promoter. Three candidate YY1 binding sites encompassing the core binding motif 5′-CAT-3′ were identified on the proximal Msx2 promoter and designated as YY1 sites 1, 2 and 3 (A). Using site directed mutagenesis these sites were mutated in the core sequence from 5′-CAT-3′ to 5′-GGT-3′. The three sites were mutated individually and resulted in constructs designated M1, M2 and M3, or in combinations to yield M123. Additionally, the initiator on the Msx2 promoter was also mutated from 5′-CAC-3′ to 5′-GGC-3′ to generate M123/Inr construct. These Msx2 promoter constructs (x-axis) were co-transfected with LacZ as control or YY1 expression vector into P19 cells cultured on 10-cm plates. Luciferase activities were assayed 24 h post-transfection (B). Basal wild-type Msx2 promoter activity in the presence of LacZ control was designated as 100%. YY1 induced a significant increase in this Msx2 promoter activity. However, all mutant Msx2 promoters, except M2, exhibited reduced level of activities in response to YY1 when compared with wild-type and their respective LacZ controls. *P < 0.01. M2 showed slightly decreased activity. Three sets of oligonucleotides, corresponding to the three YY1 binding sites on the Msx2 promoter, were radiolabeled and tested in EMSA for their binding to P19 nuclear extract or to a GST–YY1 fusion protein. All three probes bound, albeit at different intensities (sites 1–3, lanes 1–3, respectively). The results for all three sets of oligonucleotides were similar for additional assays and those for site 3 are shown in lanes 4–11. The site 3 probe bound to the P19 nuclear extract (lane 4), and this binding was competed off by 100-fold molar excess of cold site 3 probe (lane 5), but not by excess mutant site 3 probe (lane 6) or mutant YY1 consensus oligonucleotides (lane 7). Similarly, the site 3 probe bound to the GST–YY1 fusion protein (lane 8), and this binding was competed off by 100-fold molar excess of cold site 3 probe (lane 9) or YY1 consensus oligonucleotides (lane 10). The band was super-shifted when YY1 antibody was also added to the site 3 probe and GST–YY1 complex (lane 11).