Interference of Myb transactivation activity by a conditional dominant negative protein: functional interference in a cytotoxic T-cell line results in G1 arrest.
The ability to ablate the activity of specific transcription factors in vivo is a potentially important tool to study their roles in cellular processes such as the cell cycle. Previously, production of a dominant interfering c-Myb protein (comprising a fusion of the c-Myb DNA binding domain with the Drosophila Engrailed transrepressor) was found to inhibit the proliferation of immature thymocytes in the developing thymus of transgenic mice. We report here the further development of this stratagem by rendering the c-Myb/Engrailed protein conditionally active by fusion to a modified estrogen receptor hormone binding domain, ER. Co-transfection experiments in NIH 3T3 fibroblasts showed that the resulting chimeric protein, Myb/En/ER, repressed transactivation of a c-Myb-responsive reporter only in the presence of the synthetic steroid, 4-hydroxytamoxifen (OHT). Additionally, we found that Myb/En/ER could counteract transactivation by C/EBP-beta of the mim-1 promoter, which contains juxtaposed Myb and C/EBP binding sites. Cytotoxic T-cells stably producing the inactive Myb/En/ER protein were readily obtained by gene transfection. The addition of OHT to these cells resulted in inhibition of proliferation and arrest in G1. The utility of this experimental system to study Myb and other transcription factors is discussed.
Lyon
J J
JJ
Ludwig Institute for Cancer Research, Imperial College School of Medicine at St.Mary's, London, W2 1PG, UK.
Watson
R J
RJ
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D007399:Interphase / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000494:pharmacology; D020598:Proto-Oncogene Proteins c-myb; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D012097:Repressor Proteins / Q000235:genetics*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D013950:Thymus Gland / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics
D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D013629:Tamoxifen; C016601:afimoxifene