Study On The New Function Of RBP4 As A Nuclear Coactivator

The control of eukaryote gene transcription is a very complex procedure. It involves the interactions between cis-acting elements and trans-acting factors as well as the interactions between different trans-acting factors. The nuclear receptor superfamily is composed of a large number of ligand-dependent transcriptional factors that mediate expression of genes associated with eukaryotic growth, development, and homeostasis. The mechanism by which the nuclear receptors regulate gene expression is currently under extensive investigation. Many studies show that nuclear receptors interact with a series of coregulators during their regulation procedure of target genes. Those coregulators, including coactivators and corepressors, bridge nuclear receptors with basal transcriptional machine, regulate promoter-, ligand-, and cell-specific gene expression controlled by nuclear receptors. Until now, many nuclear receptor coactivators were cloned including RIP 140, CBP/P300, SRC/pl60, CARM-1, TRAP/DRIP, and the newly discovered SRA (a small RNA molecular serving as a coactivator). Most nuclear receptor coactivators have molecular masses of approximately 160kDa and share a common motif containing a core consensus sequence LXXLL (also known as nuclear receptor box, NR box), which is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors.The human estrogen receptor-related receptor 1 (hERRl) is one of the firstly cloned orphan nuclear receptors. It is a member of nuclear receptor superfamily. It shares a very high sequence homology with the human estrogen receptor (hER). hERRl widely expressed in many tissues and has important functions in bone development and formation, and fat metabolism. The hERRl response element (ERRE) shares a very high homology with that of ER and SF1, therefore, the target genes of hERRl overlap with that of hER and SF1 widely. Many evidences show that hERRl affects the regulation functions of hER by competing with hER for the binding to target sequences or by direct protein-protein interaction with hER. hERRl could also interact with TFIIB and many coactivators such as ACTR, GRIP1, SRC-1, and PNRC during its regulation of gene expression. Our studies showed that hERRl is expressed in normal breast tissue and also expressed at higher levels in breast cancer tissue, indicating the close relationship between hERRl and breast cancer. Based on thisinformation, my project aims to study the molecular mechanism of hERRl in gene expression. Basically, the specific aims and results of my research are as follows:1. To identify new proteins which interacts with hERRl in breast tissue by yeast two-hybrid screening of breast cDNA expression library using hERRl/HBD as a bait, and to characterize these proteins functionally.In order to explore the molecular mechanism of hERRl in breast cancer development, the hormone-binding domain of hERRl (hERRl/HBD) was used as a bait protein to screen the breast tissue cDNA library by yeast two-hybrid approach for potential proteins that could interact with hERRl in normal breast tissue. The true interactions between hERRl and the positive clones identified from the screening were further confirmed by yeast two-hybrid and p-galactosidase activity assays. The true positive clones were sequenced, and the gene identities of the positive clones were made by BLAST search against GenBank database. The results showed that: 1) Total 12 genes were cloned from this library screening through their interactions with hERRl/HBD, including several known nuclear receptor coactivators such as SRC-1, RIP 140 and TRIP-1; 2) The interactions between hERRl and these proteins including RBP4 were further confirmed by yeast two-hybrid assays as well as P-galactosidase assays.2. To detect the interactions between RBP4 and multiple nuclear receptors, and to demonstrate the interactions are functional AF-2 (activation function 2) domain-dependent.Yeast two-hybrid assay was applied to detect the interactions between RBP4 and hERRl and the other nuclear...