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Engineering small molecule‐dependent molecular switches to control gene expression
Author(s) -
Rohatgi Priyanka,
Doyle Donald Francis
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a75-b
Subject(s) - retinoid x receptor , nuclear receptor , transcription factor , biology , reporter gene , chemistry , ligand (biochemistry) , microbiology and biotechnology , retinoid x receptor alpha , fusion protein , receptor , biochemistry , gene , gene expression , recombinant dna
Small molecule dependent molecular switches that control gene expression are important tools in understanding and controlling biological processes, and for applications in gene therapy, drug discovery and biosensors. To develop these molecular switches, ligand activated transcription factors called nuclear receptors, specifically retinoid X receptors (RXR) was used. Structurally, nuclear receptors contain a DNA binding domain (DBD) and a ligand‐binding domain (LBD). RXR LBD was modified (Q275C, I310M, F313I) such that it preferentially binds a synthetic ligand LG335 and not its natural ligand, 9cRA. This variant showed reverse ligand specificity and has an EC 50 for LG335 of 1μm. This variant was then fused to Gal4 yeast DBD to recognize Gal4 response element. This modified chimeric fusion protein does not heterodimerize with RXR wild type or with other nuclear receptor such as retinoic acid receptor. To improve the affinity of the molecular switch with LG335, we applied a structure‐based approach to generate a library of RXR variants and screened in yeast using chemical complementation. The library produced switches with LG335 that have a variety of EC 50 s (40 nM to > 2 mM) and activation levels (10% to 80% of wild‐type RXR with 9cRA) in yeast. The variant RXR (I268V;A272V;I310L;F313M) has an EC 50 for LG335 of 1nM, and an EC 50 for 9cRA of greater than 10 mM in mammalian cells. A retroviral vector was constructed that contained the variant and the reporter for in vivo studies in mice. This molecular switch will be used to regulate differentiation of hematopoietic stem cells into blood cells.