![Fig. 5. The NFY site positively regulates the human CD34 promoter and functionally interacts with c-myb binding site. (A) A comparison of oligonucleotide probes used in Fig 2 (+48/+80) and the +54/+89 sequence (Y+M+) shown previously to bind c-myb.89 NFY and c-myb sites are boxed. Arrows under the Y+M+ sequence indicate point mutations introduced into either the Y box or the c-myb site individually (to generate Y−M+ or Y+M− oligonucleotides) or to both sites simultaneously (to generate the Y−M− oligonucleotide). (B) EMSA with Y+M+ probe and no protein (lanes 1 and 7) or 20 μg of RPMI-8402 nuclear extract (N.E.; lanes 2 to 6 and 8 to 12). Reactions in lanes 3 and 4 contained antibodies raised to NFYA [NFYA(1) was from Rockland Immunochemicals and NFYA(2) was described29], and anti-NFYB antibody29 was added in lane 5. Competitors were added to the reactions as follows: CD10-derived NFY binding oligonucleotide41 to lane 6; +48/+80 region of human CD34 to lane 9; self-competitor (Y+M+) to lane 10; +54/+89 oligonucleotide with Y box mutated (Y−M+) to lane 11; +54/+89 oligonucleotide with both the Y box and c-myb site mutated (Y−M−) to lane 12. Reactions in lanes 13 and 14 contain Y−M+ probe incubated in the absence or presence of nuclear extract, respectively. The NFY binding complex is indicated by an arrow. Supershifted complexes (s.s.) and free probe are marked with brackets. Stars indicate nonspecific complexes. (C) A schematic drawing of constructs used in transient transfection assays shown in (D). All constructs contained the human CD34 promoter (base pairs −391/+175) with wild-type Y box and c-myb site (Y+M+), Y box mutated (Y−M+), c-myb site mutated (Y+M−), or both sites mutated (Y−M−). (D) CD34 promoter/luciferase constructs in (C) were transfected to KG1a cells together with an internal control plasmid, CMV-driven Renilla luciferase (pCVM-RL). The results are presented as mean firefly luciferase relative light units (FL RLU) and normalized to Renilla luciferase relative light units (RL RLU). Standard deviations are represented by error bars.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/94/11/10.1182_blood.v94.11.3772/4/blod42319005aw.jpeg?Expires=1736051149&Signature=RkzGY8D3dbA5KZgfe3MYDNVGCtXCp5KFl96yZZ5fnD4FKW2gg5OTkYqqWRFHDdNG3Kz20pKwHI8VzATiv25baqbZDtW2Lm7neBIiG7AgoCvrBYarAI0mPnVBla0nBzI1IPHZc2URUlRa18PE3QqagO2bHtrx3PEzlCiXVuqqy8teaeVp4zcOyb7xk-UzIjpX75oIUzDGZz5OS9YTI81thlQwPbmfXnqzAkBV3rI~HLZW8CKq1qjv~nTbJwwWL5~Jsl7cBzROcfShU5PUBTXq3fwB8zOQmiFkdVopblVGqnaicD4pzbRri62q-OJgVl0rKh65r0QVZZ0WG4t83ngliA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The NFY site positively regulates the human CD34 promoter and functionally interacts with c-myb binding site. (A) A comparison of oligonucleotide probes used in Fig 2 (+48/+80) and the +54/+89 sequence (Y+M+) shown previously to bind c-myb.8,9 NFY and c-myb sites are boxed. Arrows under the Y+M+ sequence indicate point mutations introduced into either the Y box or the c-myb site individually (to generate Y−M+ or Y+M− oligonucleotides) or to both sites simultaneously (to generate the Y−M− oligonucleotide). (B) EMSA with Y+M+ probe and no protein (lanes 1 and 7) or 20 μg of RPMI-8402 nuclear extract (N.E.; lanes 2 to 6 and 8 to 12). Reactions in lanes 3 and 4 contained antibodies raised to NFYA [NFYA(1) was from Rockland Immunochemicals and NFYA(2) was described29], and anti-NFYB antibody29 was added in lane 5. Competitors were added to the reactions as follows: CD10-derived NFY binding oligonucleotide41 to lane 6; +48/+80 region of human CD34 to lane 9; self-competitor (Y+M+) to lane 10; +54/+89 oligonucleotide with Y box mutated (Y−M+) to lane 11; +54/+89 oligonucleotide with both the Y box and c-myb site mutated (Y−M−) to lane 12. Reactions in lanes 13 and 14 contain Y−M+ probe incubated in the absence or presence of nuclear extract, respectively. The NFY binding complex is indicated by an arrow. Supershifted complexes (s.s.) and free probe are marked with brackets. Stars indicate nonspecific complexes. (C) A schematic drawing of constructs used in transient transfection assays shown in (D). All constructs contained the human CD34 promoter (base pairs −391/+175) with wild-type Y box and c-myb site (Y+M+), Y box mutated (Y−M+), c-myb site mutated (Y+M−), or both sites mutated (Y−M−). (D) CD34 promoter/luciferase constructs in (C) were transfected to KG1a cells together with an internal control plasmid, CMV-driven Renilla luciferase (pCVM-RL). The results are presented as mean firefly luciferase relative light units (FL RLU) and normalized to Renilla luciferase relative light units (RL RLU). Standard deviations are represented by error bars.
