Characterization of human mesoderm induction-early response 1 (hMI-ER1) as a nuclear hormone receptor cofactor

Savicky, Marianne (2004) Characterization of human mesoderm induction-early response 1 (hMI-ER1) as a nuclear hormone receptor cofactor. Masters thesis, Memorial University of Newfoundland.

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Abstract

Fibroblast growth factors (FGFs) are a family of proteins whose signaling in cells play important roles in cell growth and development, cell differentiation, and angiogenesis [Powers, et al., 2000]. Investigations into the role of FGF in cell growth and differentiation in Xenopus laevis embryos led to the identification of a novel immediate early gene, or initial target, of FGF signal transduction, which was later named mesoderm induction early response 1 (mi-er1) [Paterno, et al., 1997]. A human orthologue of xmi-er1, hmi-er1, was also cloned in our laboratory [Paterno, et al., 1998; Paterno, et al., 2002]. hMI-ER1 was found to be consistently expressed at low levels in normal human tissues, however, breast carcinoma cell lines and tumour tissue samples displayed elevated levels, indicating that hMI-ER1 may have a role in the neoplastic state (Paterno, et al., 1998; Paterno, et al., 2002]. -- Examination of the protein structure of hMI-ER1 revealed the presence of a number of protein interacting motifs, namely an acidic activation domain, ELM2 and SANT domains. Each of these motifs are present in both hMI-ER1α and hMI-ER1β isoforms. Notably, the hMI-ER1α isoform also contains an LXXLL motif (L represents the amino acid leucine and X can be any amino acid), a domain not found in hMI-ER1β but commonly found in nuclear hormone receptor interacting proteins. Therefore, my hypothesis is that hMI-ER1α is involved in protein-protein interactions involving nuclear hormone receptors. The finding that hMI-ER1 recruits HDAC (histone deacetylase) activity through the ELM2 domain [Ding, et al., 2002], indicates that hMI-ER1 is involved in transcriptional regulation, and will therefore, also likely play a role in the regulation of transcription by nuclear hormone receptors. -- The studies described in this thesis revealed that both hMI-ER1α and hMI-ER1β interact with a number of nuclear hormone receptors (ERα, RARα, RARβ, RARγ, RXRα, and RXRγ) in vitro and with ERα (estrogen receptor alpha) in vivo. Further characterization of the in vitro interaction between hMI-ER1 and ERα demonstrated that the region important for interaction is located between amino acids 325-357 of hMI-ER1. -- In vivo studies confirmed the interaction between hMI-ER1α or hMI-ER1β and ERα in the absence of the ER ligand, estrogen (E₂). Interaction in the presence of E₂ appeared to depend on the antibody used for immunoprecipitation. -- Studies into the functional consequences of the interaction show that hMI-ER1β enhances estrogen response element (ERE)-driven transcription while hMI-ER1α has a negative, or negligible, effect on ERE-driven transcription. Overall, these results indicate that both hMI-ER1α and hMI-ER1β interact with the estrogen receptor alpha and do indeed play a role in the functioning of the estrogen receptor in cells.

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