Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: regulated use of a facultative intron determines subcellular localization

Paterno, Gary D.; Ding, Zhihu; Lew, Yuan-Y.; Nash, Gord W.; Mercer, FV; Gillespie, Laura L.

doi:10.1016/s0378-1119(02)00823-5

Cited by 15 publications

(45 citation statements)

References 33 publications

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“…The MI-ER1a antibody is a rabbit polyclonal antibody directed against a synthetic peptide representing alpha-specific sequence (amino acids 413 -426); its production has been described and its specificity has been determined previously (Paterno et al, 2002;Thorne et al, 2008). Purified IgG was prepared from pre-immune or immune serum using the Melon Gel IgG purification kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.…”

Section: Antibodiesmentioning

confidence: 99%

“…Control cell lines were generated by transfection with the pTRE-tight empty vector. Stable clones were induced to express MI-ER1a using 2 mg ml À1 doxycycline (dox) and expression was verified by western blot analysis of whole cell extracts, using our anti-MI-ER1a-specific antibody (Paterno et al, 2002).…”

Section: Establishment Of Stably Transfected T47d Cell Clones and Doxmentioning

confidence: 99%

“…The MI-ER1a antibody used in this study has been well characterised and shown to be specific for the MI-ER1a protein (Paterno et al, 2002;Thorne et al, 2008). For this study, we used purified anti-MI-ER1a IgG and performed initial tests to confirm the specificity of antibody staining on paraffin sections of human tissue.…”

Section: Immunohistochemical Analysis Of Mi-er1a In Normal Breast Andmentioning

confidence: 99%

“…Characterisation of the human mi-er1 gene revealed that there are two major protein isoforms, MI-ER1a and MI-ER1b, which differ in their C-terminal sequence (Paterno et al, 2002). Of particular interest is MI-ER1a, as it contains in its C terminus a consensus LXXLL interaction domain characteristic of nuclear hormone receptor (NR) coregulators.…”

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confidence: 99%

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Changes in subcellular localisation of MI-ER1α, a novel oestrogen receptor-α interacting protein, is associated with breast cancer progression

et al. 2008

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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.

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Section: Antibodiesmentioning

confidence: 99%

Section: Establishment Of Stably Transfected T47d Cell Clones and Doxmentioning

confidence: 99%

Section: Immunohistochemical Analysis Of Mi-er1a In Normal Breast Andmentioning

confidence: 99%

mentioning

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Changes in subcellular localisation of MI-ER1α, a novel oestrogen receptor-α interacting protein, is associated with breast cancer progression

et al. 2008

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“…hmi-er1 is transcribed from two distinct promoters P1 and P2 (4), and transcripts undergo alternative splicing to produce six protein isoforms differing in their N and C termini (4). Two of the N-terminal variants differ in the sequence of their 5Ј-UTR, 1 whereas the third contains an additional exon that encodes 25 amino acids (aa).…”

mentioning

confidence: 99%

The SANT Domain of Human MI-ER1 Interacts with Sp1 to Interfere with GC Box Recognition and Repress Transcription from Its Own Promoter

Ding

Gillespie

Mercer

et al. 2004

Journal of Biological Chemistry

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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.

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Protein expression of the transcriptional regulator MI-ER1 alpha in adult mouse tissues

et al. 2007

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MI-ER1 is a novel transcriptional regulator that plays a critical role in embryonic development and is differentially expressed in breast carcinoma. The MI-ER1 protein sequence is highly conserved among species, with 95% identity between mouse and humans and 72% between Xenopus and mouse. There are two major protein isoforms, MI-ER1alpha and MI-ER1beta, which differ in the sequence of their C-terminus. MI-ER1alpha is of particular interest because it contains a consensus LXXLL nuclear receptor interaction motif and the current study was undertaken to determine the expression pattern of MI-ER1alpha protein in adult mouse tissues. Immunohistochemical analysis of paraffin-embedded tissue using an MI-ER1alpha-specific antibody revealed that the majority of mouse adult tissues examined showed very weak or no immunoreactivity; these included tissues of the lung, liver, intestine, uterus, spleen, lymph node, bladder as well as skeletal muscle. Interestingly, a subset of endocrine tissues displayed intense staining for MI-ER1alpha. Specifically, the islets of Langerhans, the zona glomerulosa and medulla of the adrenal gland, the ovary and the hypothalamus were intensely stained. In addition, both anterior and posterior pituitary showed moderate immunoreactivity, as did the parafollicular cells of the thyroid gland and Leydig cells and spermatids in the testes. Negative endocrine tissues included follicular cells of the thyroid gland and the X zone of the adrenal cortex. A few non-endocrine tissues displayed moderate immunoreactivity; these included all tubules and collecting ducts in the kidney, myocardial and endocardial layers of the heart, the hippocampal formation, pyramidal neurons in the cortex and the ductal epithelium of the mammary gland. In all cases, MI-ER1alpha immunoreactivity was cytoplasmic. This study represents the first immunohistochemical analysis of MI-ER1alpha expression in mammals and our data suggest that this transcriptional regulator plays a role in specific endocrine pathways.

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Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: regulated use of a facultative intron determines subcellular localization

Cited by 15 publications

References 33 publications

Changes in subcellular localisation of MI-ER1α, a novel oestrogen receptor-α interacting protein, is associated with breast cancer progression

Changes in subcellular localisation of MI-ER1α, a novel oestrogen receptor-α interacting protein, is associated with breast cancer progression

The SANT Domain of Human MI-ER1 Interacts with Sp1 to Interfere with GC Box Recognition and Repress Transcription from Its Own Promoter

Protein expression of the transcriptional regulator MI-ER1 alpha in adult mouse tissues

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