The precise control of gene transcription is of fundamental importance to prevent pathological disorders and carcinogenesis. Dysregulation of the transcription factor ER81 is involved in breast cancer, where ER81 is a downstream effector of HER2/Neu, and in those Ewing's sarcomas, where the ER81 gene is affected by a chromosomal translocation. They hypothesize that ER81 is activated by HER2/Neu via the mitogen-activated protein kinase signaling pathways. Activated ER81, in conjunction with the coactivators CBP and p300, leads to stimulation of target genes, similarly as in Ewing's sarcoma due to the presence of a constitutively active EWS-ER81 fusion protein. This dysregulation of ER81 target genes leads to pathological levels of certain proteins contributing to cell transformation.
Two specific aims are proposed to test these hypothesis. (1) They will study how mitogen-activated protein kinases stimulate ER81 upon HER2/Neu overexpression and how CBP/p300 support ER81-dependent transcription. Utilizing in vitro and in vivo phoyphorylation studies, they will determine the phosphoylation sites within ER81 and identify the protein kinases involved. Mutation of the phosphorylation sites will demonstrate how they affect ER81 and identify the protein kinases involved. Mutation of the phosphorylation sites will demonstrate how they affect ER81 function. Furthermore, it will be analyzed whether acetylation of ER81 by CBP/p300 regulates its transactivation function. Additionally, the impact of HER2/Neu on the interaction between ER81 and CBP/p300 will be investigated. (2) In order to identify ER81 target genes, human cells will be transfected with the constitutively active EWS-ER81 molecule. RNA from these cells and control cells will be employed to identify target genes utilizing DNA microarrays. Identified target genes will then be analyzed for their capacity to transform cells and contribute to metastasis. The analysis how the ER81-CBP/p300 complex is stimulated by HER2/Neu may point out novel strategies of interference with breast cancer development. The identification of ER81 target genes will shed light on how the pathological disorders in breast cancer and Ewing's sarcoma are elicited and how they could be cured.
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