In vivo analysis of retroviral enhancer mutations in hematopoietic cells: SP1/EGR1 and ETS/GATA motifs contribute to long terminal repeat specificity.
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.
Wahlers
Anke
A
Department of Cell and Virus Genetics, Heinrich Pette Institute, D-20251 Hamburg, Germany.
Zipfel
Peter F
PF
Schwieger
Maike
M
Ostertag
Wolfram
W
Baum
Christopher
C
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
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