Genetic Selection for Modulators of a Retinoic-Acid-Responsive Reporter in Human Cells

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Genetic Selection for Modulators of a Retinoic-Acid-Responsive Reporter in Human Cells

Burt Richards^a, Jon Karpilow^a, Christine Dunn^a, Isaac Peterson^a, Andrew Maxfield^a, Ludmilla Zharkikh^a, Majid Abedi^a, Anthony Hurlburt^a, Joshua Hardman^a, Forrest Hsu^a, Wenhua Li^a, Matthew Rebentisch^a, Robert Sandrock^a, Tanya Sandrock^a, Alexander Kamb^a, and David H.-F. Teng^a
^a Deltagen Proteomics, Salt Lake City, Utah 84108

Corresponding author: David H.-F. Teng, Salt Lake City, UT 84108.

Communicating editor: S. HENIKOFF

We used a genetic screening methodology, a human cell line bearinga retinoic-acid-responsive enhanced GFP reporter, and a flowsorter to recover dominant modulators of reporter expression.Four inducers and three suppressors that were fused to the Cterminus of a protein scaffold for stability were isolated andtheir mechanisms of action studied. Mutagenesis experimentsindicated that six of these dominant agents exerted their effectsat the protein level. The single cDNA coding fragment that wasisolated comprised the central 64-amino-acid section of humancyclophilin B, which contained its peptidyl-prolyl isomerasedomain; this cyclophilin fragment repressed expression of theretinoic-acid-responsive reporter. The remaining clones encodedpeptides shorter than 30 amino acids unrelated to known geneopen reading frames. Genetic epistasis studies between the strongestinducer, R3, and a dominant-negative mutant of RAR ${alpha}$ suggest thatthe two factors function in the same pathway. Transcript microarrayanalyses suggest that R3 induced a subset of the retinoid-responsivegenes in melanoma cells. Finally, yeast two-hybrid assays andco-immunoprecipitation studies of human cell extracts identifiedPAT1 as a protein that interacts with R3.