Estrogen Receptor Regulating Factor Ga9 The Functional Studies

Estrogen receptor(ER) is a member of a large superfamily of nuclear receptors that regulate the transcription of estrogen-responsive genes. ER is an effective treatment target as well as a prognostic factor of breast cancer. There are two forms of ER, ERa and ERβ,which exhibit similar, yet distinct, structural and functional features. Both ERs have two important transcription activation function regions, AF1 and AF2. The effective transactivation of AF1 and AF2 depends on their interaction with other proteins. Recently, endocrine therapy of breast cancer is acquired mainly by decreasing ER transcriptional activity. Therefore, research of identifying and characterizing the proteins regulating ER transcriptional activity has great significance in developing drugs for breast cancer.Epigenetic modifications such as DNA and histone modifications, play a very important role in the development and progression of many important human diseases including cancers. Most epigenetic changes are reversible, which provides optimistic outlook for the treatment of diseases. Histone modifications are diverse and they are important for the regulation of gene expression. These modifications can affect the affinity of histones and DNA, thereby changing the state of chromatin, or affect the combination of transcrition factors and DNA sequences, then leading to activate or inhibit the transcription of downstream genes. It has become widely accepted that histone modifications are associated with the initiation and development of tumor. Therefore, further study of histone modifications will have important significance for the diagnosis, prevention and prognosis of cancer. And it also represents a new target for the therapy and for developing dugs of cancer.GA9 is a novel protein. This article is the preliminary study of GA9 based on yeast two-hybrid system. The aim is to confirm the expression of GA9 in breast cancer tissues and cells, to indentify the interactions between GA9 and ER, to investigate the effects of GA9 on the transcriptional activity of ER, and to explore the biological functions of GA9. Western blot analysis showed that GA9 is expressed in breast cancer cells. Immunofluorescence and Immunohistochemisty analysis showed that GA9 was expressed in the nuclei of breast cancer cells. GST pull-down demonstrated that GA9 could interact with ER in vitro. Co-immunoprecipitation showed that GA9 also bound to ER in vivo in a ligand-independent manner. GA9 bound specifically to the estrogen-independent AF1 and the AF2 domain of ER. Luciferase reporter assays showed that GA9 repressed estrogen-responsive gene transcription in a dose-dependent manner through interaction with ER in breast cancer cells. Meanwhile, knockdown of endogenous GA9 protein expression with GA9 small interfering RNA increased ER transcriptional activity. Further evidence showed that GA9 affected recruitment of ER to an estrogen-responsive promoter. Overexpression of GA9 in breast cancer cells decreased expression of estrogen-responsive proteins, whereas knockdown of endogenous GA9 with GA9 siRNA increased the expression of these proteins. These results indicated that GA9 is a novel ER co-repressor.Meanwhile, we also successfully amplified nucleosmal histone family members H2A, H2B, H3 and H4 from mammary gland cDNA library by PCR, constructed it in procaryotic or eukaryotic expression vectors, and expressed it in the corresponding cells. GST pull-down demonstrated that GA9 could interact with H2A, H2B, H3 and H4. Further study of methylase assay showed that GA9 may be related to histone methylation.Taken together, the results suggest that GA9 might be a new regulating factor of ER signaling pathway through interaction with ER. Further study will help us understand its specific regulating mechanism in ER signaling pathway and clinical therapeutic effects of GA9 on breast cancer, and perhaps it represents a new target for therapeutic intervention.