title: In vivo analysis of retroviral enhancer mutations in hematopoietic cells: SP1/EGR1 and ETS/GATA motifs contribute to long terminal repeat specificity.
abstract: The objective of this work was to identify, in the context of chromosomally integrated DNA, the contribution of defined transcription factor binding motifs to the function of a complex retrovirus enhancer in hematopoietic cells in vivo. Repopulating murine hematopoietic cells were transduced with equal gene dosages of replication-incompetent retrovirus vectors encoding enhanced green fluorescent protein. Enhancer sequences were derived from mouse spleen focus-forming virus. Destruction of GC-rich sites representing overlapping targets for SP1 or EGR1 uniformly attenuated gene expression (approximately 25 to 70% of wild-type levels) in all hematopoietic lineages, as shown by multicolor flow cytometry of peripheral blood and bone marrow cells at various time points posttransplantation. In contrast, a point mutation within a dual ETS/GATA motif that abolished transactivation by ETS factors but not by GATA-1 slightly increased activity in erythroid cells and significantly attenuated enhancer function in T lymphocytes. This study shows that controlled gene transfer in transplantable hematopoietic cells allows a functional analysis of distinct cis elements within a complex retrovirus enhancer, as required for the characterization and engineering of various cellular and viral regulatory sequences in basic research and gene therapy.
authors.affiliation: Department of Cell and Virus Genetics, Heinrich Pette Institute, D-20251 Hamburg, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Wahlers
authors.forename: Peter F
authors.initials: PF
authors.lastname: Zipfel
authors.forename: Maike
authors.initials: M
authors.lastname: Schwieger
authors.forename: Wolfram
authors.initials: W
authors.lastname: Ostertag
authors.forename: Christopher
authors.initials: C
authors.lastname: Baum
mesh_terms: D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000502:physiology; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D006412:Hematopoietic Stem Cells / Q000821:virology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D012190:Retroviridae / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000502:physiology; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D020079:Terminal Repeat Sequences; D014157:Transcription Factors / Q000502:physiology
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; D016329:Sp1 Transcription Factor; D014157:Transcription Factors