Molecular cloning of human er1 cDNA and its differential expression in breast tumours and tumour-derived cell lines

doi:10.1016/S0378-1119(98)00473-9

Gene

Volume 222, Issue 1, 5 November 1998, Pages 77-82

https://doi.org/10.1016/S0378-1119(98)00473-9 Get rights and content

Abstract

We recently cloned and characterized a novel immediate–early gene, called er1, from Xenopus embryos whose expression levels were increased during mesoderm induction by fibroblast growth factor (FGF). We describe here the isolation and expression pattern of the human er1 sequence. Human ER1 and Xenopus ER1 proteins display 91% similarity; the amino acid sequence motifs, including the putative DNA-binding SANT domain, the predicted nuclear localization signals (NLS) and the putative SH3 binding domain share 100% identity. er1 mRNA expression was negligible in all 50 normal human tissues analyzed. Examination of nine breast carcinoma-derived cell lines and eight breast tumour tissue samples by reverse transcription-polymerase chain reaction (RT-PCR) revealed that human er1 was consistently expressed in all tumour cell lines and tumour tissue while remaining undetectable in normal breast cell lines and breast tissue. These data suggest that er1 expression is associated with the neoplastic state in human breast carcinoma.

Introduction

Mesoderm differentiation in Xenopus can be induced by a number of factors, including members of the fibroblast growth factor (FGF) family. Many of the early events in mesoderm induction have been investigated and shown to involve a number of well-characterized intracellular signal transduction pathways, including PLC_γ and Ras [reviewed in Friesel and Maciag (1995)]. Little is known, however, about the genes that are the ultimate targets of these signal transduction pathways: the immediate–early genes. In a recent report, we described the cloning and characterization of one such gene, er1, from Xenopus embryos (Paterno et al., 1997). er1 is an immediate–early gene whose transcription is activated by FGF during mesoderm induction in Xenopus. ER1 protein was shown to be localized exclusively to the nucleus and to contain domains with potent transcriptional transactivation activity. The observation that er1 is an immediate–early transcription factor activated by FGF suggests that it may play a role in FGF-regulated cellular activities, including those associated with the pathology of human cancers.

In this report, we have isolated and characterized the cDNA for the human homologue of er1 and have investigated its expression pattern in normal and neoplastic tissue.

Section snippets

cDNA cloning and sequencing of human er1

Using forward (F) and reverse (R) primers 5′-TCCGTTACACCAGGATGTAG-3′ (F) and 5′-GGCTGAAATTCCAGTTGGTA-3′ (R), designed according to the Xenopus er1 sequence, a 440-bp product was amplified from a human testis cDNA library (Clontech, Palo Alto, CA), as described previously (Paterno et al., 1997). The cDNA was cloned into pCR2.1 (Invitrogen) (Paterno et al., 1997), and the sequence for both strands of this er1 cDNA and all subsequent cDNA inserts was determined as in Gillespie et al. (1995). A

cDNA cloning of human er1

We recently described the cloning and characterization of er1, a novel immediate–early gene from Xenopus laevis whose expression levels were increased by FGF (Paterno et al., 1997). Using primers based on the Xenopus sequence, we amplified a similar sequence from a human testis cDNA library. The full-length human cDNA consisted of a single open reading frame (ORF) of 1296 bp bracketed by a 68-bp 5′ UTR containing an in-frame stop codon and a 210-bp 3′ UTR containing an 18-bp poly-A tail (Fig. 1

Conclusions

This report presents the cloning and expression analysis of the human homologue of er1. Comparison of the Xenopus and human ER1 proteins reveals a high degree of conservation between lower and higher vertebrates.

Human er1 mRNA expression was negligible in all normal tissues and breast cell lines examined, but was upregulated in breast carcinoma cell lines and in breast tumours, suggesting that expression of er1 is associated with the neoplastic state in human breast carcinomas.

Acknowledgements

The authors thank Paula Ryan and Wendy Winsor for technical assistance. This work was supported by a grant from the Cancer Research Society to L.L.G. and G.D.P.

References (9)

R Aasland et al.
The SANT domain: a putative DNA-binding domain in the SWI-SNF and ADA complexes, the transcriptional co-repressor N-CoR and TFIIIB
Trends Biochem. Sci.
(1996)
L.L Gillespie et al.
Clonging of FGF receptor 1 splice variant from Xenopus embryos that lacks a protein kinase C site important for the regulation of receptor activity
J. Biol. Chem.
(1995)
A.E Krafft et al.
Optimization of the isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissue
Mol. Diag.
(1997)
G.D Paterno et al.
cDNA cloning of a novel, developmentally regulated immediate early gene activated by fibroblast growth factor and encoding a nuclear protein
J. Biol. Chem.
(1997)

There are more references available in the full text version of this article.

Cited by (20)

MIER3 induces epithelial-mesenchymal transition and promotes breast cancer cell aggressiveness via forming a co-repressor complex with HDAC1/HDAC2/Snail
2021, Experimental Cell Research
Citation Excerpt :
The MIER family consists of three related genes: MIER1, MIER2, and MIER3, which encoding ELM2-SANT containing proteins. Mesoderm induction early response 1 (MIER1) is a fibroblast growth factor that was first found in the embryonic cells of the African claw toad, which induce mesoderm differentiation in embryonic tissue and function as a transcription factor [6–8]. The N-terminus of MIER1 is an acidic domain with transcriptional activation activity, and MIER1 has a transcriptional inhibitory activity of ELM2 and SANT domain at the C-terminus [9,10].
Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer death in women. MIER3 (Mesoderm induction early response 1, family member3) is considered as a potential oncogene for breast cancer. However, the role of MIER3 in breast cancer remain largely unknown. The expression of MIER3 was detected and the relationship between its expression and clinicopathological characteristics was also analyzed. The effect of MIER3 on proliferation and migration of breast cancer cells was detected in vitro and in vivo. Western blot, IF, and Co-IP were employed to detect the relationship between MIER3, HDAC1, HDAC2, and Snail. ChIP assay was performed to determine the binding of MIER3/HDAC1/HDAC2/Snail complex to the promoter of E-cadherin. In this study, we found that MIER3 was upregulated in breast cancer tissue and closely associated with poor prognosis of patients. MIER3 could promote the proliferation, migration, and epithelial-mesenchymal transition (EMT) of breast cancer cells. Further studies showed that MIER3 interacted with HDAC1/HDAC2 and Snail to form a repressive complex which could bind to E-cadherin promoter and was related to its deacetylation. Our study concluded that MIER3 was involved in forming a co-repressor complex with HDAC1/HDAC2/Snail to promote EMT by silencing E-cadherin.
Proteomic analysis of a preneoplastic phenotype in ovarian surface epithelial cells derived from prophylactic oophorectomies
2005, Gynecologic Oncology
To study the pattern of protein expression associated with a predisposition to develop ovarian cancer.
Prophylactic oophorectomy is used to prevent ovarian carcinoma in high-risk populations who have a strong family history of breast/ovarian cancer. In ovarian specimens of these women, the ovarian surface epithelium (OSE), which is tissue of origin of epithelial ovarian cancer, often shows altered morphology, growth patterns and differentiation features that are believed to be preneoplastic. This study has used a proteomic approach, based on two-dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of OSE from women with a history of familial ovarian cancer (FH-OSE), i.e., at least two first-degree relatives with such cancer and/or testing positive for BRCA1 mutations, to those without such history (NFH-OSE).
Of >1500 protein spots, there were 8 proteins whose levels were significantly altered in FH-OSE. Three were known ovarian tumor associated proteins, others were novel changes. A number of the alterations seen were accompanied with protein modifications and have not been previously reported. There was a predominance of sequences related to the stress response pathway. Differential expression of selected genes was confirmed by Western blotting and real-time reverse transcription polymerase chain reaction.
Our findings define the OSE phenotype of women at a high risk of developing ovarian cancer. Protein alterations seen in these tissues may represent an early, irreversible, non-mutational step in ovarian epithelial neoplastic progression and may be potential early and sensitive markers for the evaluation of cancer risk.
Discordant expression of HLA class II-associated co-chaperones and HLA-DRB alleles in cultured fibroblast-like synoviocytes
2004, Human Immunology
Human leukocyte antigen (HLA)-DR-positive synovial fibroblasts are frequently observed in rheumatoid arthritis (RA) and may be implicated in the autoimmune reaction because RA is associated with certain HLA-DRB1* alleles. The question of whether components of the class II antigen presentation pathway and specific DRB alleles are efficiently expressed by synovial fibroblasts is germane to this hypothesis. To address this, cultured fibroblast-like synoviocytes (cFLS) were analyzed for constitutive and interferon (IFN)-γ-induced expression of specific DRB alleles and class II-associated cochaperones. IFN-γ induction of invariant chain, DM, and DR molecules was observed in all cFLS, but expression of specific DR allotypes was variable. Interestingly, DM-modulated epitopes on RA-associated DR molecules were either absent or delayed, despite strong DM expression and a paucity of major histocompatibility complex/class II-associated invariant chain peptide complexes. Altered expression of specific peptide-dependent epitopes on RA-associated HLA-DR molecules suggests differences in antigen presentation by cFLS, which may have implications for the immunopathogenesis of RA.
The SANT domain of human MI-ER1 interacts with Sp1 to interfere with GC box recognition and repress transcription from its own promoter
2004, Journal of Biological Chemistry
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-On-inducible 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.
Proline<sup>365</sup> is a critical residue for the activity of XMI-ER1 in Xenopus embryonic development
2003, Biochemical and Biophysical Research Communications
Xmi-er1 is an immediate-early gene encoding a transcriptional regulator whose expression is activated by fibroblast growth factor (FGF) during mesoderm induction in Xenopus. In this study, we examined the role of xmi-er1 in embryonic development and mesoderm induction and investigated the importance of various functional domains in the protein sequence. Overexpression of xmi-er1 in embryos resulted in truncations of the anteroposterior axis, with most of the abnormal embryos exhibiting deficiencies in both anterior and posterior structures. Whole mount in situ hybridization for the early mesodermal marker brachyury (Xbra) revealed a dramatic reduction of Xbra expression in xmi-er1-injected embryos, while mesoderm induction assays showed that overexpression of xmi-er1 significantly reduced the percentage of explants induced by FGF-2. Site-directed mutagenesis of several functional domains, including the ELM2 domain, the SANT domain, a putative MEK phosphorylation site, and a proline-rich region showed that only proline 365 in the proline-rich region is required for the effect on embryonic development and mesoderm induction. These data demonstrate that XMI-ER1 is a negative regulator of FGF, perhaps serving to limit the extent of mesoderm formation in vivo, and that this activity is mediated by proline 365.
Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: Regulated use of a facultative intron determines subcellular localization
2002, Gene
mi-er1 (previously called er1) is a fibroblast growth factor-inducible early response gene activated during mesoderm induction in Xenopus embryos and encoding a nuclear protein that functions as a transcriptional activator. The human orthologue of mi-er1 was shown to be upregulated in breast carcinoma cell lines and breast tumours when compared to normal breast cells. In this report, we investigate the structure of the human mi-er1 (hmi-er1) gene and characterize the alternatively spliced transcripts and protein isoforms. hmi-er1 is a single copy gene located at 1p31.2 and spanning 63 kb. It contains 17 exons and includes one skipped exon, a facultative intron and three polyadenylation signals to produce 12 transcripts encoding six distinct proteins. hmi-er1 transcripts were expressed at very low levels in most human adult tissues and the mRNA isoform pattern varied with the tissue. The 12 transcripts encode proteins containing a common internal sequence with variable N- and C-termini. Three distinct N- and two distinct C-termini were identified, giving rise to six protein isoforms. The two C-termini differ significantly in size and sequence and arise from alternate use of a facultative intron to produce hMI-ER1α and hMI-ER1β. In all tissues except testis, transcripts encoding the β isoform were predominant. hMI-ER1α lacks the predicted nuclear localization signal and transfection assays revealed that, unlike hMI-ER1β, it is not a nuclear protein, but remains in the cytoplasm. Our results demonstrate that alternate use of a facultative intron regulates the subcellular localization of hMI-ER1 proteins and this may have important implications for hMI-ER1 function.

View all citing articles on Scopus

View full text

Article preview

Gene

Abstract

Introduction

Section snippets

cDNA cloning and sequencing of human er1

cDNA cloning of human er1

Conclusions

Acknowledgements

References (9)

The SANT domain: a putative DNA-binding domain in the SWI-SNF and ADA complexes, the transcriptional co-repressor N-CoR and TFIIIB

Trends Biochem. Sci.

Clonging of FGF receptor 1 splice variant from Xenopus embryos that lacks a protein kinase C site important for the regulation of receptor activity

J. Biol. Chem.

Optimization of the isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissue

Mol. Diag.

cDNA cloning of a novel, developmentally regulated immediate early gene activated by fibroblast growth factor and encoding a nuclear protein

J. Biol. Chem.

Cited by (20)

MIER3 induces epithelial-mesenchymal transition and promotes breast cancer cell aggressiveness via forming a co-repressor complex with HDAC1/HDAC2/Snail

Proteomic analysis of a preneoplastic phenotype in ovarian surface epithelial cells derived from prophylactic oophorectomies

Discordant expression of HLA class II-associated co-chaperones and HLA-DRB alleles in cultured fibroblast-like synoviocytes

The SANT domain of human MI-ER1 interacts with Sp1 to interfere with GC box recognition and repress transcription from its own promoter

Proline<sup>365</sup> is a critical residue for the activity of XMI-ER1 in Xenopus embryonic development

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