Control of transcription factor activity and osteoblast differentiation in mammalian cells using an evolved small-molecule-dependent intein.
Inteins are naturally occurring protein elements that catalyze their own excision from within a larger protein together with the ligation of the flanking "extein" sequences. Previously we reported the directed evolution of an intein-based molecular switch in which intein splicing in yeast cells was made dependent on the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). Here we show that these evolved inteins are effective means of rendering protein function and biological signaling pathway activation dependent on 4-HT in mammalian cells. We have characterized the generality, speed, and dose dependence of ligand-induced protein splicing in murine NIH3T3 cells and in human HEK293 cells. Evolved inteins were used to control in mammalian cells the function of Gli1 and a truncated form of Gli3, two transcriptional mediators of the Hedgehog signaling pathway. Finally, we show that a complex biological process such as osteoblast differentiation can be made dependent on 4-HT using the evolved intein system. Our findings suggest that evolved small-molecule-dependent inteins may serve as a general means of achieving gene-specific, dose-dependent, post-translational, and small-molecule-induced control over protein activity in mammalian systems.
Yuen
Courtney M
CM
Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
Rodda
Stephen J
SJ
Vokes
Steven A
SA
McMahon
Andrew P
AP
Liu
David R
DR
D000818:Animals; D002384:Catalysis; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D019020:Directed Molecular Evolution; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D006801:Humans; D047668:Inteins / Q000235:genetics; D051741:Kruppel-Like Transcription Factors / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008970:Molecular Weight; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism; D015513:Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D010006:Osteoblasts / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D015534:Trans-Activators / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D000071676:Zinc Finger Protein GLI1; D000074290:Zinc Finger Protein Gli3
C065683:GLI3 protein, human; C465571:Gli3 protein, mouse; D051741:Kruppel-Like Transcription Factors; D008024:Ligands; D009419:Nerve Tissue Proteins; D015513:Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; D000074290:Zinc Finger Protein Gli3; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; C016601:afimoxifene