mi-er1 (previously called er1) was first isolated from Xenopus laevis embryonic cells as a novel fibroblast growth factor-regulated immediate-early gene. Xmi-er1 was shown to encode a nuclear protein with an Nterminal acidic transcription activation domain. The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level and was shown to be upregulated in breast carcinoma cell lines and tumors. Alternative splicing at the 3 end of hmi-er1 produces two major isoforms, hMI-ER1␣ and hMI-ER1␤, which contain distinct C-terminal domains. In this study, we investigated the role of hMI-ER1␣ and hMI-ER1␤ in the regulation of transcription. Using fusion proteins of hMI-ER1␣ or hMI-ER1␤ tethered to the GAL4 DNA binding domain, we show that both isoforms, when recruited to the G5tkCAT minimal promoter, function to repress transcription. We demonstrate that this repressor activity is due to interaction and recruitment of a trichostatin A-sensitive histone deacetylase 1 (HDAC1). Furthermore, deletion analysis revealed that recruitment of HDAC1 to hMI-ER1␣ and hMI-ER1␤ occurs through their common ELM2 domain. The ELM2 domain was first described in the Caenorhabditis elegans Egl-27 protein and is present in a number of SANT domain-containing transcription factors. This is the first report of a function for the ELM2 domain, highlighting its role in the regulation of transcription.mi-er1 (mesoderm induction early response 1), previously called er1, was first isolated as a novel fibroblast growth factorregulated immediate-early gene from Xenopus embryonic cells induced to differentiate into mesoderm (22). Xmi-er1 encodes a nuclear protein that contains an N-terminal acidic domain with potent transcriptional activation activity (22).The human orthologue of mi-er1 (hmi-er1) displays 91% similarity to the Xenopus sequence at the amino acid level (23) and has been shown to undergo tissue-specific alternative splicing to produce six protein isoforms that differ in their amino and carboxyl termini (24). The hmi-er1 isoform expression pattern is complex, but overall, expression of hmi-er1 isoforms is very low or undetectable in healthy adult tissues (23,24). Human breast carcinoma cell lines and breast tumors, on the other hand, displayed elevated levels of hmi-er1 (23).Alternate use of a facultative intron at the 3Ј end of hmi-er1 produces two major isoforms, hMI-ER1␣ and hMI-ER1␤, which contain distinct C-terminal domains (24). The ␣ C terminus consists of 23 amino acids (aa) and includes the sequence LXXLL, a motif important for interaction with nuclear hormone receptors (15). This motif also bears some similarity to the Sin3A interaction domain of the MAD family of transcriptional repressors (4). The ␤ C terminus contains 102 aa and includes the functional nuclear localization signal (NLS) (24,25). The divergent amino acid sequences of the ␣ and ␤ C termini suggest that the two serve distinct functions.

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We have utilized the polymerase chain reaction (PCR)-based differential display methodology (Liang, P., and Pardee, A. B. (1992) Science 257, 967-969) to identify a novel transcript whose expression levels increased in Xenopus embryo explants during mesoderm induction by fibroblast growth factor. The PCR product was used to clone a 2.3-kilobase pair cDNA representing this transcript, which we have named er1 (early response 1). The er1 cDNA contained a single open reading frame predicted to encode a protein of 493 amino acid residues. A data base homology search revealed that the predicted ER1 amino acid sequence contains three regions of similarity to the rat and human proteins encoded by the metastasis-associated gene, mta1, and two regions of similarity to the Caenorhabditis elegans sequence that is similar to mta1. The fibroblast growth factor-induced increase in er1 steady-state levels was not dependent on de novo protein synthesis, demonstrating that er1 is an immediate-early gene. Northern blot analysis revealed a single 2.8-kilobase pair mRNA that was observed predominantly during the initial cleavage and blastula stages of Xenopus development, with little or no detectable mRNA during subsequent development. Quantitative PCR analysis of early developmental stages showed that er1 peaked during late blastula. Computer-assisted analysis of the predicted ER1 amino acid sequence revealed two putative nuclear localization signals, four highly acidic regions clustered at the N terminus and a proline-rich region located near the C terminus. Subcellular localization by immunocytochemistry revealed that the ER1 protein was targeted exclusively to the nucleus. Transactivation assays using various regions of ER1 fused to the DNA binding domain of GAL4 demonstrated that the N-terminal acidic region is a potent transactivator. These data suggest that ER1 may function as a transcription factor.

ld;&.2qTo gain insight into the regulation of hmi-er1 expression, we cloned a human genomic DNA fragment containing one of the two hmi-er1 promoters and consisting of 1460 bp upstream of the translation initiation codon of hMI-ER1. Computer-assisted sequence analysis revealed that the hmi-er1 promoter region contains a CpG island but lacks an identifiable TATA element, initiator sequence and downstream promoter element. This genomic DNA was able to direct transcription of a luciferase reporter gene in a variety of human cell lines, and the minimal promoter was shown to be located within ؊68/؉144 bp. Several putative Sp1 binding sites were identified, and we show that Sp1 can bind to the hmi-er1 minimal promoter and increase transcription, suggesting that the level of hmi-er1 expression may depend on the availability of Sp1 protein. Functional analysis revealed that hMI-ER1 represses Sp1-activated transcription from the minimal promoter by a histone deacetylase-independent mechanism. Chromatin immunoprecipitation analysis demonstrated that both Sp1 and hMI-ER1 are associated with the chromatin of the hmi-er1 promoter and that overexpression of hMI-ER1 in cell lines that allow Tet-Oninducible expression resulted in loss of detectable Sp1 from the endogenous hmi-er1 promoter. The mechanism by which this occurs does not involve binding of hMI-ER1 to cis-acting elements. Instead, we show that hMI-ER1 physically associates with Sp1 and that endogenous complexes containing the two proteins could be detected in vivo. Furthermore, hMI-ER1 specifically interferes with binding of Sp1 to the hmi-er1 minimal promoter as well as to an Sp1 consensus oligonucleotide. Deletion analysis revealed that this interaction occurs through a region containing the SANT domain of hMI-ER1. Together, these data reveal a functional role for the SANT domain in the action of co-repressor regulatory factors and suggest that the association of hMI-ER1 with Sp1 represents a novel mechanism for the negative regulation of Sp1 target promoters.

The oestrogen receptor-a (ERa) plays a key role in breast development and tumorigenesis and inhibiting its activity remains a prime strategy in the treatment of ERa-positive breast cancers. Thus, elucidation of the molecular mechanisms responsible for regulating ERa activity may facilitate the design of new, more effective breast cancer therapies. The MI-ER1a is a novel transcriptional repressor that contains an LXXLL motif for interaction with nuclear hormone receptors. We investigated the ability of MI-ER1a to bind to ERa in HEK293 and MCF-7 breast carcinoma cells, using co-immunoprecipitation assays. In both cell lines, MI-ER1a interacted with ERa in the presence and absence of oestrogen, but the interaction was stronger in the absence of ligand. Functional analysis revealed that overexpression of MI-ER1a in T47D breast carcinoma cells results in inhibition of oestrogen-stimulated anchorage-independent growth, suggesting that MI-ER1a may play a role in regulating breast carcinoma cell proliferation in vivo. To explore this further, we performed an immunohistochemical analysis of normal breast tissue and breast carcinoma; a total of 110 cases were examined in whole tissue sections and 771 cases were analysed in tissue microarrays. No consistent difference in the MI-ER1a expression level between normal breast tissue and breast carcinoma was discernible. However, there was a dramatic shift in the subcellular localisation: nuclear MI-ER1a was detectable in 75% of normal breast samples and in 77% of hyperplasia, but in breast carcinoma, only 51% of DCIS, 25% of ILC and 4% of IDC contained nuclear staining. This shift from nuclear to cytoplasmic localisation of MI-ER1a during breast cancer progression suggests that loss of nuclear MI-ER1a might contribute to the development of invasive breast carcinoma.