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 TR⅐DNA interaction in the absence of T 3 , which is repressed by T 3 through a transient TR⅐DNA 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 TR⅐DNA interaction was further confirmed by electrophoretic gel mobility shift assay, chromatin immunoprecipitation, and transfection assays using a non-DNA-binding TR␣1 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 TR⅐DNA 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.Thyroid hormone affects important biological functions such as growth, development, and metabolism. The biologically active thyroid hormone triiodothyronine (T 3 ) 1 regulates gene expression by interacting with two isoforms of the thyroid hormone receptor (TR), TR␣ and TR, each of which consists of a DNA binding domain (DBD), a ligand-binding domain, co-activator/co-repressor binding domains, and a transcription activation domain. The P-box of the zinc fingers within the DBD of TR recognizes the AGGTCA sequence of the thyroid hormoneresponsive element (TRE), which is diversely arranged in direct repeat (DRϩ4), palindrome, and inverted palindrome forms (1, 2). When unliganded TR binds to TRE, it recruits co-repressors (NCoR/SMRT) and histone deacetylases for repression. On the other hand, T 3 binding to TR induces conformational changes in TR to release co-repressors and recruit co-activators such as CBP/p300, pCAF, and SRC-1 for transactivation (3-8). Thus, although the mechanism of transcriptional regulation for the positive TRE (pTRE) is relatively well studied, T 3 -dependent negative gene regulation is still poorly understood and is limited to a few genes. It has not been possible to identify a consensus sequence of negative TRE (nTRE). Recently, Wondisford and coll...
We have identified a cell type-specific, negative thyroid hormone-responsive element in the human type 1 iodothyronine deiodinase (hdio1) gene. This fragment, termed a JEG response element, bound tightly to a JEG-cell nuclear protein [JEG cell-specific transcription factor (JTF)] also present in placenta but not in COS-7, HeLa, or human embryonic kidney-293 cells. In JEG-3 cells, three copies of the JEG response element conferred a more than 40-fold transcriptional stimulation to the heterologous rat GH promoter which was further increased 2-fold by apo-thyroid hormone receptor (TR) and reduced 3-fold by T(3). Dimethyl sulfide footprinting showed overlapping contact sites for the high-affinity interaction of JTF and low-affinity binding of TR-retinoid X receptor. Expression of the same construct was unaffected by TR or T(3) in COS cells, indicating JTF was required for negative regulation by T(3)-TR. Mutations of the critical thyroid hormone responsive element binding P box amino acids EG to GS in TRalpha1 or TRbeta2 eliminated the apo-TR and T(3)-TR effects. These studies identify a novel mechanism for cell type-specific, promoter-independent negative regulation by T(3).
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