A Novel Mechanism of Thyroid Hormone-dependent Negative Regulation by Thyroid Hormone Receptor, Nuclear Receptor Corepressor (NCoR), and GAGA-binding Factor on the Rat CD44 Promoter

Sung-Woo Kim,Sung-Chul Ho,Seong-June Hong,Kyung Min Kim,Edward C So,Marcelo Christoffolete,John W Harney

doi:10.1074/jbc.m411517200

Sung-Woo Kim, Sung-Chul Ho + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m411517200

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Abstract

CD44 is an adhesion molecule in the extracellular matrix that shows various functions, including tumor genesis and metastasis. A recent study showed that CD44 expression level was strongly correlated with the generation of papillary thyroid carcinomas, the most prevalent malignancy of the thyroid gland. We report here that CD44 is negatively regulated by thyroid hormone (T(3)) through a novel mechanism. We demonstrate that nuclear receptor corepressor (NCoR) enhances thyroid hormone receptor (TR)-mediated basal transactivation by a weak TR.DNA interaction in the absence of T(3), which is repressed by T(3) through a transient TR .DNA interaction. Initially, we identified that CD44 was negatively directly transcriptionally T(3) -responsive. Deletion and mutation analysis indicated that both a weak TR and a GAGA-binding factor (GAF) binding sites on the CD44 promoter were required for negative regulation by T(3). The weak TR.DNA interaction was further confirmed by electrophoretic gel mobility shift assay, chromatin immunoprecipitation, and transfection assays using a non-DNA-binding TRalpha1 mutant. More interestingly, NCoR acted as a co-activator to enhance TR-mediated basal transactivation in the absence of T(3). This effect was eliminated by removal of TR or NCoR binding. Most strikingly, T(3) induced a remarkable increase in TR.DNA binding at 40-60 min after T(3) exposure that rapidly returned to basal levels, suggesting a T(3)-induced remodeling of chromatin structure at the early stage of T(3) stimulation resulting in repression. Therefore, we propose a mechanism by which NCoR, GAF, and TR interact with the CD44 negative T(3)-responsive element to enhance basal transactivation, whereas T(3) induces the remodeling of chromatin structure for repression.

Highlights

We demonstrate that nuclear receptor corepressor (NCoR) enhances thyroid hormone receptor (TR)-mediated basal transactivation by a weak TR1⁄7DNA interaction in the absence of T3, which is repressed by T3 through a transient TR1⁄7DNA interaction
GAGA-binding factor (GAF) Binding Is Required for T3-dependent Negative Regulation of the CD44 Gene—We further examined whether TR binding to weak thyroid hormoneresponsive element (TRE) at Ϫ82ϳϪ87 bp was sufficient to bring about negative regulation by T3
Nuclear Receptor Corepressor (NCoR)-mediated Basal Transactivation—Different from the “squelching” model, which proposed that NCoR enhanced unliganded TR-mediated basal transactivation without TR1⁄7DNA interaction, our results indicate that T3-dependent negative regulation requires TR1⁄7nTRE interaction, even though its interaction was weak in the absence of T3

Summary

Methods

Cell Culture—Cells were cultured as described previously [23, 37]. For stimulation with T3, culture medium was removed, the cells were rinsed once with phosphatebuffered saline, and Ham’s F-10 medium containing charcoal-stripped 10% fetal bovine serum was added. T3 (50 nM) was diluted in Ham’s F-10 medium, and charcoal-stripped 10% fetal bovine serum was added to cells for the times indicated in figure legends. To generate a non-TRE binding GS71 sequence as already described in JEG-3 cells [23], mTR␣1 cDNA was inserted in a CDM expression vector in which two bases from TSS of the mTRa1 cDNA were mutated: 614 (A to G) and at 616 (G to A).

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