Dennis, Jonathan Hancock; (2001) Transcriptional regulation by Brn-3 POU domain-containing transcription factors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Brn-3a and Brn-3b are closely related members of the POU domain-containing transcription factors. While Brn-3a is associated with neuronal differentiation and Brn-3b with neuronal proliferation, their expression is not strictly limited to the nervous system. It has been shown that these Brn-3 proteins are expressed in non-neuronal tissues including cervical epithelium, testis and breast. Moreover, these Brn-3 proteins functionally interact with the estrogen receptor and in association with the estrogen receptor have differing transactivation potentials. The p160 steroid receptor coactivators (Srcs) are also able to interact with the estrogen receptor in a ligand dependent manner and are able to enhance estrogen dependent transcription. This work describes a functional interaction between Brn-3 proteins and members of the steroid receptor coactivator family. In affinity chromatography and coimminoprecipitation experiments, Src-1 proteins were shown to physically interact with Brn-3a and Brn-3b. In addition, the transactivation potential of the Brn-3/Src complexes was tested on two promoters in three different cell lines. The steroid receptor coactivators potentiated the transcriptional effects of Brn-3 a and Brn-3b. The Src proteins, however, differed in their ability to potentiate the transcriptional activity of Brn-3 proteins on different promoters in transiently transfected cells. In addition, maximal activation of the Brn-3/Src complex differed between cell lines, indicating that additional cell-type specific proteins are important for maximal activation by the complex. It has recently been shown that Brn-3b may play a role in regulating BRCA-1 in mammary tumors as its expression is enhanced in primary breast tumors with reduced BRCA-1 expression. Moreover, this elevated Brn-3b expression is not seen in normal mammary cells, benign tumors, or malignant tumors which do not have reduced levels of BRCA-1. This work also describes the effects of stable Brn-3b overexpression and reduction in the estrogen receptor positive breast adenocarcinoma cell line MCF7. Overexpression of Brn-3b resulted in increased growth rate, saturation density, proliferation, and ability to form anchorage independent colonies when compared to mock transfected cells. MCF7 cells with reduced Brn-3b levels exhibited a significant decrease in growth rate, saturation density, proliferation, and ability to form anchorage independent colonies when compared to mock transfected controls. Differential gene expression in these cell lines was examined using both specific western immunodetection as well as comprehensive DNA array technology. Immunodetection revealed increases in ER, HSP-27, and Brn-3a in Brn-3b overexpressing cells as well as a decrease in these proteins in the Brn-3b reduced cell lines. The DNA array including 1200 cancer related genes revealed several genes differentially expressed when the Brn-3b overexpressing and reduced cell line mRNAs were compared. Finally, the results of three of the genes shown to be differentially regulated in the cDNA array differential display were corroborated by semi-quantitative RT-PCR.

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