Characterization of the novel transcriptional regulator human mesoderm induction early response gene 1 (hMI-ER1): its promoters, interacting proteins and transcriptional regulatory functions

Ding, Zhihu (2004) Characterization of the novel transcriptional regulator human mesoderm induction early response gene 1 (hMI-ER1): its promoters, interacting proteins and transcriptional regulatory functions. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Mesoderm induction early response 1 (mi-er1), previously called er1, was first isolated as a novel fibroblast growth factor-regulated early-response gene from Xenopus embryonic cells induced to differentiate into mesoderm. The human orthologue of xmi-er1, hmi-er1, was found to have ubiquitous, but low level, expression in normal human tissues (Paterno et al., 1998; Paterno et al., 2002). Breast carcinoma cell lines and tumours, on the other hand, showed elevated levels (Paterno et al., 1998), suggesting that hmi-er1 expression is associated with the neoplastic state in human breast carcinoma. Structurally, hMI-ER1 has conserved domains found in a number of transcriptional regulators, including an acid activation domain (Paterno et al., 1997), an ELM2 domain (Solari et al., 1999) and a signature SANT domain (Aasland et al., 1996). -- My hypothesis is that hMI-ER1 may be a potent transcriptional regulator, and deregulation of its expression and/or functions may contribute to tumorigenesis. The purpose of this project was to: (1) isolate and characterize the hmi-er1 promoters; (2) investigate the role of hMI-ER1 in transcriptional regulation; and (3) identify and characterize hMI-ER1-interacting proteins. -- Cloning and sequence analysis of one of the hmi-er1 promoters, P2, showed the absence of a TATA box, but presence of a CpG island, multiple Sp1 binding sites, and other factor binding sites which may be important in the regulation of gene expression. Functional characterization of the promoter revealed that Sp1 binds to the promoter and plays a positive role in the regulation of the P2 promoter activity of hmi-er1. hMI-ER1 was found to function as transcriptional repressor on the G5TKCAT promoter by recruitment of HDAC1 via the ELM2 domain. hMI-ER1α and β also function as transcriptional repressors on their own P2 promoter, through interaction with Sp1 and interference with the Sp1-DNA binding via the SANT domain. In order to better understand the function of hMI-ER1, an attempt was made to identify more hMI-ER1 interacting proteins by yeast two-hybrid cDNA library screening. HSP40 and TRABID were identified as proteins that interact specifically with hMI-ER1α and β. Furthermore, hMI-EMR1α and β were found to interact with the tumour suppressor RB and may participate in cell growth regulation. These results suggest that hMI-ER1 may function as a transcriptional regulator, through distinct mechanisms, to regulate cellular functions.

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