| Nuclear receptors constitute an important superfamily of transcription regulators that are involved in widely diverse physiological functions, including control of embryonic development, cell differentiation and homeostasis. Most nuclear receptors have a similar structural organization which is divided into six portions, namely A to F. The A/B domain, which is also called activation function domain 1 (AF-1 domain) at amino-terminal, is involved in hormone-independent activating of transcription. The C domain is highly conserved and contains the DNA binding domain (DBD) that confers sequence-specific DNA binding activity. A hinge region, called the D domain, links the C domain with the carboxyl-terminal E/F domain that includes the ligand-binding domain (LBD). The E/F domain contains the other activation function domain, AF-2. Nuclear receptors regulate transcription by binding to specific DNA sequences in target genes known as hormone response elements or HREs after their binding with the ligands.It has been realized now that there are various bridge proteins or so-called nuclear receptor coregulators existing in cells. Coregulators are involved in the modulation of nuclear receptor's function . These nuclear receptor coregulators are divided into coactivators and corepressors according to their opposite functions hi regulating nuclear receptor's function on the transcription of the target genes. Generally, they regulate transactivation of nuclear receptors by protein-protein interaction, instead of binding to DNA directly.Estrogen receptor-related receptors, ERRs, share high nucleic acid and amino acid sequence homology with estrogen receptors. ERR a 1 is the major isoform of the human ERR a gene. The DNA-binding domain (DBD) and ligand-binding domain (HBD) of the human ERR a 1 share 70% amino acid homology and 35% amino acid identity with that of ER a , respectively. Based on their own studies, researchers in our lab and some other labs hypothesized that ERR a 1 may play a critical role in the etiology of some breast cancers, and therefore it may be a novel therapeutic target in breast cancer treatment. It is known that nuclear receptor coactivator SRC-1, GRIP1, PNRC and PNRC2 can interact with ERR a 1and enhance its transactivation function. However, the detailed mechanism revealed is very limited. To further our understanding of the molecular mechanism of transcription regulationcontrolled by ERR a 1 in breast cancer and to identify coactivators interacting with ERR a 1, we carried out this study, and the major results are summarized below:1. Ligand binding domain of ERR a 1 was used as the "bait" to screen human mammary gland cDNA expression library in a Gal4 based yeast two-hybrid system. Fast skeletal muscle troponin I, TNNI2, along with some known coactivators, was isolated.2. It was found that TNNI2 interacted with PR, TR and RXR in a ligand-dependent manner in yeast two-hybrid assay, and also interacted with some "orphan" receptors including mERR a , SF1 and hERR a 1 in a ligand-independent manner. The interaction between TNNI2 and ERR a 1 is the strongest. In addition, functional AF-2 domain is required for nuclear receptors to interact with TNNI2.3. In transient transfection experiments, TNNI2 was found to enhance the transcription mediated by ER, ERR a 1, SF1 and ARP1. However, TNNI2 alone did not change the transcription rate of the luciferase gene significantly. These results confirmed that TNNI2 functions as a nuclear receptor coactivator.4. TNNI2 was found to interact directly with ERR a 1 in GST pull-down assay.5. By a combination of mutagenesis and yeast two-hybrid assay, the ERR a 1-interacting domain on TNNI2 has been mapped to a region encompassing amino acids 1-128. This region consists of the N-terminal, inhibitory region (amino acids 96-116) and a nuclear receptor-binding site, LXXLL motif (also named NR box). The TNNI2 1-128 fragment can dominant negatively inhibit the coactivation function of TNNI2 by competing with wild type TNNI2 for nuclear receptor binding. T...
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