1. by Toshibumi Shimokawa, and Chisei Ra
C/EBPα functionally and physically interacts with GABP to activate the human myeloid IgA Fc receptor (FcαR, CD89) gene promoter
by Toshibumi Shimokawa, and Chisei Ra
Blood
Volume 106(7):
October 1, 2005
©2005 by American Society of Hematology

2. Transcription factor-binding motifs in the FCAR promoter and oligonucleotides used for EMSA. Binding sequences for Ets and C/EBP family members (CE1, CE2, and CE3) in the regulatory region of the FCAR gene are indicated by arrows.
Transcription factor-binding motifs in the FCAR promoter and oligonucleotides used for EMSA. Binding sequences for Ets and C/EBP family members (CE1, CE2, and CE3) in the regulatory region of the FCAR gene are indicated by arrows. The numbers indicate nucleotide positions relative to the translation initiation ATG at +1. Wild-type oligonucleotides between -103 and -74 (wtETS) and between -98 and -79 (wtETS-s) used in EMSA contain an Ets-binding site. Specific base pair substitutions used to mutate the Ets-binding site in mutant oligonucleotides (mutETS and mutETS-s) are indicated in bold.
Toshibumi Shimokawa, and Chisei Ra Blood 2005;106:
©2005 by American Society of Hematology

3. Effect of artificial spacing between the adjacent Ets- and C/EBP-binding sites or mutation in the Ets-binding site on transactivation of the FCAR promoter by C/EBPα and C/EBPβ.
Effect of artificial spacing between the adjacent Ets- and C/EBP-binding sites or mutation in the Ets-binding site on transactivation of the FCAR promoter by C/EBPα and C/EBPβ. (A) A schematic representation for the structure of the wild-type FCAR upstream-luciferase (Luc) construct (pGL-259) and the mutated FCAR upstream-luciferase constructs. The numbers indicate nucleotide positions relative to the translation initiation ATG at +1. The major transcription start site (-197) identified by de Wit et al48 is indicated as bent arrow. The 3 C/EBP-binding sites at -139, -127, and -74 and an Ets-binding site at -92 are indicated. X indicates mutated site. (B-C) Jurkat cells were transfected with 0.7 μg luciferase reporter constructs containing the indicated FCAR promoter mutant in the absence or presence of 0.1 μg expression vector for C/EBPα (hCMV-C/EBPα) (B) or C/EBPβ (pD3NF-IL6) (C), along with 0.03 μg pRL-SV40; each sample was transfected with the additional corresponding empty vector pCMV-Empt (B) or pcDNA3.1(+) (C) to bring the amount of transfected DNA in each sample to 1 μg. Firefly and Renilla luciferase activities were determined 24 hours after transfection. Values are corrected for transfection efficiency and presented as the fold stimulation of each construct by C/EBPα (B) or C/EBPβ (C), compared with the promoter activity seen without the expression vector. Thin bars represent the SE from 3 to 4 transfections. *Significant decrease (P < .01) compared with pGLmCE by 2-tailed t test.
Toshibumi Shimokawa, and Chisei Ra Blood 2005;106:
©2005 by American Society of Hematology

4. DNA binding of renatured nuclear proteins fractionated by SDS-PAGE.
DNA binding of renatured nuclear proteins fractionated by SDS-PAGE. U937 nuclear proteins were separated by SDS-PAGE. The gel was cut crosswise in fractions on the basis of molecular weight, and the nuclear proteins were eluted and renatured. (A) Distribution of DNA-binding activity in gel size-fractionated nuclear proteins. Renatured proteins (10 μL) from different molecular weight fractions were analyzed by EMSA using the labeled FCAR Ets oligonucleotide (wtETS). The molecular weights indicated correspond to molecular weight size markers (Prestained SDS-PAGE Standard; Bio-Rad, Hercules, CA) run alongside the nuclear extract. U937, one third of the binding reaction with 6 μg U937 nuclear extract was applied to the electrophoresis. ns indicates nonspecific bands identified previously.27 (B) DNA-binding specificity and supershift EMSA of size-fractionated nuclear factors that bind to the FCAR Ets site. Renatured proteins (6 μL) from fraction 12 were analyzed by EMSA using the labeled FCAR Ets oligonucleotide (wtETS) in the absence (lane 1) or presence of 100-fold molar excess of unlabeled wild-type (lane 2) and mutant FCAR Ets oligonucleotide competitors (mutETS) (lane 3), or 2 μg antibody against Elf-1 (lane 4). Arrow indicates the binding complex comigrating with HEL-NF1. The supershifted band is indicated by open circle. (C) DNA binding of gel size-fractionated nuclear proteins in the presence of the GABPβ subunit. The indicated SDS-PAGE fractions (10 μL) were incubated with the labeled wtETS-s oligonucleotide in the absence (-) or presence of 1 μL in vitro-translated GABPβ (+). U937, one third of the binding reaction with 6 μg U937 nuclear extract was applied to the electrophoresis.
Toshibumi Shimokawa, and Chisei Ra Blood 2005;106:
©2005 by American Society of Hematology

5. GABP binds to the FCAR promoter Ets-binding site.
GABP binds to the FCAR promoter Ets-binding site. (A) EMSA of the FCAR promoter Ets-binding site using nuclear extracts. The labeled double-stranded oligonucleotide corresponding to the FCAR promoter sequence from -98 to -79 (wtETS-s) was incubated without (lane 1) and with 6 μg nuclear extracts from U937 (lanes 2-4), Jurkat (lanes 5-7), and HeLa cells (lanes 8-10) in the absence (lanes 1, 2, 5, and 8) or presence of 100-fold molar excess of unlabeled wild-type (wtETS-s; W) (lanes 3, 6, and 9) and mutant FCAR Ets oligonucleotide competitors (mutETS-s; M) (lanes 4, 7, and 10). Arrow indicates the location of HEL-NF1. Newly detected binding species (complexes I, II, III, and IV) are also indicated on left. Right panel shows long exposure of lanes 8 to 10 to show complex I. (B) EMSA of the FCAR promoter Ets-binding site using in vitro-translated GABP. The labeled double-stranded oligonucleotide corresponding to the sequence from -98 to -79 (wtETS-s) was incubated without (lane 1) and with 1 μL unprogrammed reticulocyte lysate (lane 2), 1 μL in vitro-translated GABPα (lane 3), 1 μL each of in vitro-translated GABPα and GABPβ (lanes 4-6), or 6 μg nuclear extracts from U937 (lane 7), Jurkat (lane 8), and HeLa cells (lane 9) in the absence (lanes 1-4, 7-9) or presence of 100-fold molar excess of unlabeled wild-type (wtETS-s; W) (lane 5) and mutant FCAR Ets oligonucleotide competitors (mutETS-s; M) (lane 6). Arrow indicates the location of HEL-NF1. Complexes I and III detected in nuclear extracts are also indicated on right. (C) Supershift EMSA of U937 nuclear extracts using the FCAR promoter Ets-binding site. The labeled double-stranded oligonucleotide corresponding to the sequence from -98 to -79 (wtETS-s) was incubated with 6 μg nuclear extracts from U937 cells in the presence of rabbit IgG (lane 1) or 1 μL antiserum against GABPβ (lane 2). Arrow indicates the location of HEL-NF1. (D) ChIP assay of HA-tagged GABPα binding in vivo. Crosslinked protein-DNA complexes from U937/HA-GAα cells were immunoprecipitated without antibody (lanes 2 and 7), with control rabbit IgG (lanes 3 and 8), and with antibodies against HA (lanes 4 and 9) and C/EBPα (lanes 5 and 10), and they were analyzed by PCR using primers corresponding to the FCAR promoter region from -143 to +8 (lanes 1-5) and a region within the FCAR EC1 exon (lanes 6-10). Positive control is chromatin prior to immunoprecipitation (lanes 1 and 6).
Toshibumi Shimokawa, and Chisei Ra Blood 2005;106:
©2005 by American Society of Hematology

6. The FCAR promoter is activated by GABP
The FCAR promoter is activated by GABP. (A) A schematic representation for the structure of the wild-type and mutant FCAR upstream-luciferase (Luc) constructs.
The FCAR promoter is activated by GABP. (A) A schematic representation for the structure of the wild-type and mutant FCAR upstream-luciferase (Luc) constructs. X indicates Ets site mutation. (B) HeLa cells were transfected with 0.7 μg luciferase reporter constructs containing the indicated FCAR promoter in the absence or presence of 0.1 μg each of expression vector(s) for GABPα and/or GABPβ, along with 5 ng pRL-CMV; each sample was transfected with the additional corresponding empty vector pCR3.1E to bring the amount of transfected DNA in each sample to 1 μg. Firefly and Renilla luciferase activities were determined 24 hours after transfection. Values are corrected for transfection efficiency and presented as the fold stimulation of each construct by GABPα and/or GABPβ, compared with the promoter activity seen without the expression vector(s). Thin bars represent the SE from 3 transfections.
Toshibumi Shimokawa, and Chisei Ra Blood 2005;106:
©2005 by American Society of Hematology

7. C/EBP proteins physically interact with GABPα.
C/EBP proteins physically interact with GABPα. (A) In vitro-translated 35S-C/EBPα protein was mixed with GST alone (lane 2), GST-fused GABPα protein (lane 3), and GST-fused GABPβ protein (lane 4). The proteins bound to each GST-fusion protein beads were analyzed by SDS-PAGE and autoradiography (top); lane 1 shows 5% of the input 35S-C/EBPα; the molecular weight markers (Prestained SDS-PAGE Standard; Bio-Rad) are shown (left). The input GST-fused proteins were analyzed by SDS-PAGE and Coomassie blue staining (bottom); the molecular weight markers are shown in lane 1. (B) In vitro-translated 35S-C/EBPβ protein was mixed with GST alone (lane 2) and GST-fused GABPα protein (lane 3). The proteins bound to each GST-fusion protein beads were analyzed by SDS-PAGE and autoradiography (top): lane 1 shows 5% of the input 35S-C/EBPβ; the molecular weight markers (Prestained SDS-PAGE Standard; Bio-Rad) are shown (left). The input GST-fused proteins were analyzed by SDS-PAGE and Coomassie blue staining (bottom); the molecular weight markers are shown in lane 1. (C) Schematic structure of GST-fused GABPα deletion mutants. ETS domain indicates E26 transformation-specific domain. (D) In vitro-translated 35S-C/EBPα protein was mixed with GST-fused GABPα protein (lane 2), the indicated GST-fused GABPα deletion mutants (lanes 3-5), and GST alone (lane 6). The proteins bound to each GST-fusion protein beads were analyzed by SDS-PAGE and autoradiography (top); lane 1 shows 5% of the input 35S-C/EBPα. The input GST-fused proteins were analyzed by SDS-PAGE and Coomassie blue staining (bottom); the molecular weight markers are shown in lane 1.
Toshibumi Shimokawa, and Chisei Ra Blood 2005;106:
©2005 by American Society of Hematology

8. GABP cooperates with C/EBPα to strongly activate the FCAR promoter.
GABP cooperates with C/EBPα to strongly activate the FCAR promoter. (A,D) A schematic representation for the structure of the FCAR upstream-luciferase (Luc) constructs. The numbers indicate nucleotide positions relative to the translation initiation ATG at +1. The major transcription start site is indicated as bent arrow. X indicates mutated site. (B-C,E) HeLa cells were transfected with 0.7 μg indicated FCAR upstream-luciferase constructs in the absence or presence of 0.1 μg each of expression plasmids for GABPβ (pC3E4TF1-53S) and GABPα (pC3E4TF1-60S) or its deletion mutant, GABPαΔETS (pC3GAαΔETS) or GABPα (pC3GAα ), along with 3 ng C/EBPα expression plasmid (hCMV-C/EBPα) or the empty vector (pCMV-Empt) (B,E), or with 3 ng C/EBPβ expression plasmid (pD3NF-IL6) or the empty vector [pcDNA3.1(+)] (C); each sample was transfected with 5 ng pRL-CMV and the additional empty vector pCR3.1E to bring the amount of transfected DNA in each sample to 1 μg. Firefly and Renilla luciferase activities were determined 24 hours after transfection. Values are corrected for transfection efficiency and presented as the fold stimulation of each construct by the indicated transcription factor(s), compared with the promoter activity seen without the expression vector(s). Thin bars represent the standard errors (SE) from 3 to 6 transfections. *Significant decrease (P < .05) compared with pGLmCE by one-tailed t test.
Toshibumi Shimokawa, and Chisei Ra Blood 2005;106:
©2005 by American Society of Hematology