Identification of CCR4 and other essential thyroid hormone receptor co-activators by modified yeast synthetic genetic array analysis
- Manjapra Govindana,
- Xianwang Mengb,
- Clyde L. Denisc,
- Paul Webbd,
- John D. Baxterd,1 and
- Paul G. Walfishb,1
- aDepartment of Molecular Biology, Biochemistry and Pathology, Centre de Recherche L'Hotel-Dieu de Quebec, Universite Laval, QC, Canada G1R 2J6;
- bEndocrine Division, Mount Sinai Hospital, University of Toronto Medical School, Toronto, ON, Canada M5G 1X5;
- cDepartment of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824; and
- dCenter for Diabetes Research, Methodist Hospital Research Institute, Houston, TX 77030
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Presented in part at the 77th Annual Meeting of the American Thyroid Association, Phoenix, October 11–15, 2006.
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Contributed by John D. Baxter, September 24, 2009 (received for review November 8, 2007)
Abstract
Identification of thyroid hormone receptor (TR) co-regulators has enhanced our understanding of thyroid hormone (TH) action. However, it is likely that many other co-regulators remained unidentified, and unbiased methods are required to discover these proteins. We have previously demonstrated that the yeast Saccharomyces cerevisiae is an excellent system in which to study TR action, and that defined TR signaling complexes in a eukaryotic background devoid of complicating influences of mammalian cell co-regulators can be constructed and analyzed for endogenous yeast genes, many of which are conserved in mammals. Here, a modified synthetic genetic array analysis was performed by crossing a yeast strain that expressed TRβ1 and the co-activator GRIP1/SRC2 with 384 yeast strains bearing deletions of known genes. Eight genes essential for TH action were isolated, of which 4 are conserved in mammals. Examination of one, the yeast CCR4 and its human homolog CCR4/NOT6 (hCCR4), confirmed that (i) transfected CCR4 potentiates a TH response in cultured cells more efficiently than established TR co-activators and (ii) knockdown of CCR4 expression strongly inhibited a TH response (>80%). TH treatment promoted rapid and sustained hCCR4 recruitment to the TH-responsive deiodinase 1 promoter and TR co-localizes with hCCR4 in the nucleus and interacts with hCCR4 in 2-hybrid and pull-down assays. These findings indicate that a modified yeast synthetic genetic array strategy is a feasible method for unbiased identification of conserved genes essential for TR and other nuclear receptor hormone functions in mammals.
Footnotes
- 1To whom correspondence may be addressed. E-mail: jbaxter918{at}aol.com, walfish{at}mshri.on.ca or pwalfish{at}mtsinai.on.ca
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Author contributions: M.G. and P.G.W. designed research; M.G., X.M., and P.G.W. performed research; M.G. and C.L.D. contributed new reagents/analytic tools; M.G., X.M., P.W., J.D.B., and P.G.W. analyzed data; and M.G., X.M., P.W., J.D.B., and P.G.W. wrote the paper.
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Conflict of interest statement: J.D.B. is a consultant to and shareholder of Karo Bio AB, which has commercial interests in thyroid hormone mimetics.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0910134106/DCSupplemental.
