Homeodomain proteins MEIS1 and PBXs regulate the lineage-specific transcription of the platelet factor 4 gene.
Platelet factor 4 (PF4) is expressed during megakaryocytic differentiation. We previously reported that GATA-1 and ETS-1 regulate the rat PF4 promoter and transactivate the PF4 gene. For the present study, we investigated the regulatory elements and their transcription factors responsible for the lineage-specific expression of the PF4 gene. The promoter activities of deletion constructs were evaluated, and a novel regulatory element termed TME (tandem repeat of MEIS1 binding element) (-219 to -182) was defined. Binding proteins to TME were strongly detected in HEL nuclear extracts by electrophoresis mobility shift assay (EMSA), and they were purified by DNA affinity chromatography. By performing Western blottings and supershift assays, the binding proteins were identified as homeodomain proteins, MEIS1, PBX1B, and PBX2. These factors are expressed in megakaryocytes differentiated from CD34+ cells in human cord blood. MEIS1 and PBXs bind to the TME as MEIS1/PBX complexes and activate the PF4 promoter. In nonmegakaryocytic HepG2 cells, GATA-1 and ETS-1 activate the PF4 promoter approximately 10-fold. Surprisingly, we found that additional expression of both MEIS1 and PBX2 multiplied this major activation another 2-fold. This activation was not observed when MEIS1 binding sites in the TME were disrupted. Furthermore, inhibition of the binding of endogenous MEIS1/PBX complexes to the TME decreased the promoter activity by almost one half, in megakaryocytic HEL cells. Thus, these studies demonstrate that the homeodomain proteins, MEIS1, PBX1B, and PBX2, play an important role in megakaryocytic gene expression.
Okada
Yoshiaki
Y
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Japan.
Nagai
Ryohei
R
Sato
Takahiro
T
Matsuura
Eri
E
Minami
Takashi
T
Morita
Ikuo
I
Doi
Takefumi
T
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004357:Drug Synergism; D024202:Electrophoretic Mobility Shift Assay; D005312:Fetal Blood / Q000166:cytology; D015870:Gene Expression; D005786:Gene Expression Regulation; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010978:Platelet Factor 4 / Q000235:genetics*; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D017385:Sequence Homology; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C000616391:MEIS1 protein, human; C000616392:Meis1 protein, mouse; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D009363:Neoplasm Proteins; C090465:PBX2 protein, human; C490382:Pbx2 protein, mouse; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; C083400:PBX1 protein, human; D010978:Platelet Factor 4