A Nuclear Factor Y (NFY) Site Positively Regulates the Human CD34 Stem Cell Gene by Hanna S. Radomska, Anne B. Satterthwaite, Natalie Taranenko, Sailaja Narravula, Diane S. Krause, and Daniel G. Tenen Blood Volume 94(11):3772-3780 December 1, 1999 ©1999 by American Society of Hematology

The 5′ untranslated region of human CD34 is required for maximum promoter activity. The 5′ untranslated region of human CD34 is required for maximum promoter activity. (A) A diagram of CD34/luciferase constructs used in transient transfections shown in (B) and (C). All constructs have the same 5′ flanking sequences of CD34 (391 bp), but the 3′ end points are at the base pairs +12, +75, +175, and +300. (B) CD34+ human KG1a and (C) murine 416B cells were transiently transfected with 20 μg of each plasmid depicted in (A) and luciferase activity determined. (D) CD34 promoter/luciferase constructs containing various 5′ end truncations and identical 3′ ends at the base pair +175 (diagrammed on the left) were analyzed in transient transfection assays in human CD34+ RPMI-8402 cells. For comparison, a promoterless pXP2 luciferase vector is included. The internal deletion of base pairs +40/+75 in the context of the −391/+175 promoter is shown on the bottom. (E) The cell type specific effect of the internal deletion of the +40/+75 region in the context of the −391/+175 promoter was tested in transiently transfected human CD34+, KG1a and RPMI-8402, murine CD34+ M1, and human CD34− BJA-B cells. The error bars indicate the standard deviations. The data are presented as mean relative light units (RLU) normalized to the expression of a cotransfected pCMV-human growth hormone (ng GH). Hanna S. Radomska et al. Blood 1999;94:3772-3780 ©1999 by American Society of Hematology

A nuclear factor binds to the critical element in the human CD34 5′ UTR. (A) Sequence of the oligonucleotide +48/+80 used as a probe in EMSA. Putative Sp1 and μE4 sites are overlined. A nuclear factor binds to the critical element in the human CD34 5′ UTR. (A) Sequence of the oligonucleotide +48/+80 used as a probe in EMSA. Putative Sp1 and μE4 sites are overlined. The potential Y box motif is boxed. (B) Nuclear extracts (10 μg) from CD34+ (KG1a and RPMI-8402; lanes 2 and 4, respectively) and CD34− (Jurkat and HeLa; lanes 3 and 5, respectively) were incubated with radiolabeled +48/+80 probe. The protein/DNA complexes (marked with an arrowhead) were resolved on native acrylamide gel. No protein was added to the reaction in lane 1. (C) EMSA with the +48/+80 probe and 10 μg of nuclear extract prepared from cell lines indicated above lanes. The gel contained 4% acrylamide:bis-acrylamide at the ratio 29:1 to allow the separation of 2 closely migrating complexes (marked with arrows). The electrophoresis proceeded for 4 hours at 4°C, thus the unbound probe migrated out of the gel. (D) Sp1 is not involved in the binding to the +48/+80 region. Probe shown in (A) was incubated with 10 μg of nuclear extracts of KG1a (lanes 1 to 6) or Jurkat (lanes 7 to 12) with electrophoresis conditions as in (C). Lanes 2 and 8 contained unlabeled self-competitor, lanes 3 and 9 contained the Sp1-binding oligonucleotide from the human CD34 promoter (base pairs −68/−44) as competitor, and lanes 4 and 10 contained the Sp1-binding oligonucleotide from the murine PU.1 promoter (base pairs −114/−90).36 An antiserum raised against Sp1 or nonimmune serum were added to the binding reactions shown in lanes 5 and 11, or 6 and 12, respectively. Hanna S. Radomska et al. Blood 1999;94:3772-3780 ©1999 by American Society of Hematology

Comparison of sequences from the 5′ UTRs of the human (top strand; hCD34) and murine (lower strand; mCD34) CD34 genes. Comparison of sequences from the 5′ UTRs of the human (top strand; hCD34) and murine (lower strand; mCD34) CD34 genes. Transcription start sites are marked with arrows. The regions critical for human (NFY) and murine (Sp1/3) gene activity are indicated as ovals. A potential CCAAT box in the murine sequence is double underlined. Hanna S. Radomska et al. Blood 1999;94:3772-3780 ©1999 by American Society of Hematology

Identification of the +48/+80 binding activity as NFY Identification of the +48/+80 binding activity as NFY. Binding reactions were performed in the absence (lane 1) or presence of 20 μg of nuclear extract from KG1a (lanes 2 to 12), RPMI-8402 (lanes 13 to 16), Jurkat (lanes 17 to 19), HeLa (lanes 20 to 22), an... Identification of the +48/+80 binding activity as NFY. Binding reactions were performed in the absence (lane 1) or presence of 20 μg of nuclear extract from KG1a (lanes 2 to 12), RPMI-8402 (lanes 13 to 16), Jurkat (lanes 17 to 19), HeLa (lanes 20 to 22), and CD34+ peripheral blood cells cultured with G-CSF for 22 days (75% CD15+; lanes 23 and 24). The unlabeled competitors were as follows: self-competitor in lane 3; oligonucleotide −78/−28 from the murine CD34 promoter in lane 4; 5′ untranslated region from the murine CD34 (base pairs +48/+119) in lane 5; NFY binding site from the human CD10 promoter41 in lanes 6, 15, 18, 21, and 24; NF1 binding site41 in lane 7; C/EBP consensus oligonucleotide61 in lane 14. The reactions in lanes 8, 16, 19, and 22 included anti-NFYA antibodies provided by Benoit de Crombrugghe.28 Lanes 10 and 11 contained, respectively, anti-NFYA and anti-NFYB antibodies supplied by Roberto Mantovani.29 Supershifted complexes (s.s.) as well as free probe are indicated by arrows. Hanna S. Radomska et al. Blood 1999;94:3772-3780 ©1999 by American Society of Hematology

Hanna S. Radomska et al. Blood 1999;94:3772-3780 ©1999 by American Society of Hematology

Hanna S. Radomska et al. Blood 1999;94:3772-3780 ©1999 by American Society of Hematology

Binding of NFY is stabilized by the integrity of the adjacent c-myb binding site. Binding of NFY is stabilized by the integrity of the adjacent c-myb binding site. EMSA was performed with the Y+M+ probe (lanes 1 to 3, 7, and 8), Y+M− probe (lanes 4 to 6), and +48/+80 probe (lanes 9 and 10). The reactions contained no protein (lanes 1, 4, 7, and 9) or 20 μg of RPMI-8402 nuclear extract (N.E.). In addition, reactions shown in lanes 3 and 6 contained anti-NFYA antibody (NFYA Ab; from Rockland Immunochemicals). The migration of NFY binding complex (NFY), supershifted complexes (s.s.), or free probe is shown by arrows. Nonspecific complexes are marked by stars. Hanna S. Radomska et al. Blood 1999;94:3772-3780 ©1999 by American Society of Hematology