PU.1 regulates the expression of the vav proto-oncogene.
Vav is a guanine nucleotide exchange factor for the rho/rac GTPases that is upregulated in the embryo during the transition from primitive to definitive hematopoiesis. It is one of several genetic markers that correlates with the differentiation of the intraembryonic definitive hematopoietic stem cell. Subsequently, in the adult, vav is expressed predominantly in cells of the hematopoietic system. A heat-resistant protein complex that bound to a 23-bp segment, which is essential for vav promoter activity, was found to be present in myeloid cells but not T-cells. The complex was absent in non-hematopoietic cells which normally do not express vav. Using a saturation mutagenesis method, Mutex, a "footprint" of the protein binding site (AGAGGAAGT) was obtained that was consistent with the consensus binding site for PU.1. A specific antibody to PU.1 supershifted the complex and identified the presence of PU.1 within the complex. A GST fusion protein of the human PU.1 bound to the same consensus sequence as the heat-resistant complex from myeloid lineages. Specific mutation of the GGAA PU.1 core binding site silenced vav promoter activity and a dominant negative PU.1 inhibited the transactivation of PU.1 at the vav promoter as measured by the expression of the EGFP reporter gene. In addition, PCR analysis of immunoprecipitated chromatin using specific antibodies for PU.1 detected the co-immunoprecipitation of DNA containing the vav promoter. These results suggest that PU.1 is essential for transcriptional activity of the vav promoter in myeloid cells.
Denkinger
Diane J
DJ
Department of Pharmacology, University of Nebraska Medical Center, 986260 Nebraska Medical Center, Omaha, Nebraska 68198-6260, USA.
Lambrecht
Taryn Q
TQ
Cushman-Vokoun
Allison M
AM
Kawahara
Rodney S
RS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018983:DNA Footprinting / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter; D006410:Hematopoiesis / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D022423:Myeloid Cells / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051567:Proto-Oncogene Proteins c-vav; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology; D014407:Tumor Cells, Cultured; D000099232:Proto-Oncogene Protein Spi-1
D015513:Oncogene Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C000715470:MAS1 protein, human; C496618:VAV1 protein, human; C496619:Vav1 protein, mouse