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Expression and function of a human thyroid hormone receptor-derived DNA-binding domain protein
R M Hu1, A Sakurai, T Miyamoto
1Department of Medicine, University of Chicago, IL 60637.
Molecular and Cellular Endocrinology
|April 1, 1992
Summary
DNA binding domain proteins (DBDPs) from human thyroid hormone receptors (hTR) bind to DNA response elements, requiring zinc. These proteins, however, do not activate gene expression in cells, suggesting a lack of nuclear translocation or a missing transactivation domain.
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- The DNA binding domain (DBDs) of thyroid hormone receptors (TRs) are crucial for mediating the effects of thyroid hormone.
- Understanding the function of isolated DBDs can elucidate their role in DNA binding and transcriptional regulation.
Purpose of the Study:
- To characterize the DNA binding properties of isolated human thyroid hormone receptor alpha and beta DNA binding domain proteins (hTR DBDPs).
- To investigate the role of zinc and the functional activity of hTR DBDPs in gene transactivation.
Main Methods:
- Expression of hTR alpha and beta DBDPs in E. coli using pET constructs.
- Gel shift assays to assess DNA binding to thyroid hormone response elements (TREs).
- Expression in COS-7 cells to evaluate transactivation of a TRE-CAT fusion gene.
Main Results:
- Both hTR DBDPs bound to a TRE-containing oligonucleotide, with binding dependent on zinc ions.
- The DBDPs competed with full-length hTR alpha 1 for DNA binding.
- Expressed hTR DBDPs failed to induce transactivation of a TRE-CAT fusion gene in COS-7 cells.
Conclusions:
- hTR DBDPs possess DNA binding capacity, likely as monomers, and require zinc for this function.
- The lack of transactivation suggests that isolated DBDPs may not be efficiently translocated to the nucleus or lack essential transactivation domains.