MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERα) in regulation of the MUC1 gene expression

BACKGROUND: The MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter. RESULTS: Using TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERα, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed. CONCLUSION: Analysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERα in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy

<i>Islet1</i> and Its Co-Factor <i>Ldb1</i> Are Expressed in Quiescent Cells of Mouse Intestinal Epithelium

<div><p><i>Islet1</i> belongs to Lim homeobox (<i>Lhx</i>) gene family which encodes transcription factors that have been conserved in evolution. They form complexes with other transcriptional regulators, among them obligatory co-factors encoded by <i>Ldb</i> genes. <i>Isl1 (Islet1)</i>, <i>Lhx</i> and <i>Ldb1</i> genes play a crucial role in organ patterning, cell fate determination and cell differentiation in both embryonic and adult tissues. In this study we analyzed expression pattern of <i>Isl1</i> and its co-factor <i>Ldb1</i> in small intestine. We also studied the biological role of <i>Ldb1</i> in gut endoderm. Quantitative PCR analysis revealed a relatively high level of expression of <i>Lhx1, Isl1, Isl2, Lmx1a, Ldb1</i> and <i>Ldb2</i> mRNAs in the gut tissue as compared to the level of less abundant detectable <i>Lmx1b</i> mRNA. Immunohistochemical studies demonstrated a unique pattern of Ldb1 and Islet1 proteins in the crypt compartment. Ldb1 is produced at a low level in majority of crypt cells; but, its abundant expression was demonstrated for some single cells. Islet1 is also expressed in single cells of the crypt. Double staining experiments with Ldb1 and Isl1 antibodies showed that both genes are co-expressed in certain cells of the crypt. Further analysis revealed the Ldb1-expressing cells in the gut are both of endodermal and mesodermal origin. Proliferation studies using antibodies to phospho-histone H3 and Ki-67 antigens, as well as long-term BrdU labeling, showed that cells prominently expressing Ldb1/Islet1 are quiescent but do not belong to any known terminally differentiated cell lineages. They may represent a group of stem-like cells in the crypt. Further experiments by cell lineage tracing should be performed to better characterize this cell population. Functional studies of mice with <i>Ldb1</i> gene ablated in gut endoderm revealed no specific role of <i>Ldb1</i> in that tissue.</p></div

Quantitative PCR analysis of mRNA expression of the LIM homeobox genes and their cofactors.

<p>The <i>Lhx1</i>, <i>Isl1</i>, <i>Isl2</i>, <i>Lmx1a</i>, <i>Lmx1b</i>, <i>Ldb1</i> and <i>Ldb2</i> mRNAs have been detected in mouse small intestine. <i>Lhx1</i>, <i>Islet1</i>, <i>Ldb1</i> and <i>Ldb2</i> mRNAs were expressed at high level. <i>Lhx2</i>, <i>Lhx3</i>, <i>Lhx4</i>, <i>Lhx5</i>, <i>Lhx6</i> and <i>Lhx8</i> mRNAs were not detected. All values are in log2 scale and were normalized to the level of <i>Lmx1b</i> mRNA which is considered 1. At least three independent experiments were performed for each gene. The error bars show standard deviations.</p

Cell proliferation in the crypt.

<p>Cell proliferation is evaluated with antibodies for Ki-67 antigen. Cells expressing Ldb1 (a–c) and Isl1 (d–f) are negatively stained by Ki-67 proliferation marker. <b>a</b>. Ldb1-expressing cells (green); <b>b</b>. Ki-67-expressing proliferative cells (red); <b>c</b>. Merge of <b>a</b> and <b>b</b> (x1000). <b>d</b>. Isl1-expressing cells (red); <b>e</b>. Ki-67-expressing cells (green); <b>f</b>. Merge of <b>d</b> and <b>e</b> (x1000). Arrows point on cells expressing either Ldb1 (a, b, c) or Isl1 (d, e, f). Nuclei are counterstained with DAPI (blue).</p

Localization of Ldb1- and Isl1-expressing cells in small intestine.

<p>Low level of Ldb1 protein is detected in most cells of the crypt (b and c). In a few single cells Ldb1 is expressed abundantly (arrows in b and c). Isl1 expression is observed in single cells of the proliferative zone of the crypt (arrowheads in e and f). No cells producing Ldb1 and Isl1 are found in the villous epithelium (data not shown). <b>a</b> and <b>d</b>. Negative control with pre-immune serum (x400). <b>b</b>. Expression of Ldb1 protein in the crypt (x400). <b>c</b>. High magnification of the selected area from <b>b</b> (x1000). Arrows point on cells which are highly expressing Ldb1. <b>e</b>. Expression of Isl1 protein in the crypt (x400). <b>f</b>. High magnification of the selected area from <b>e</b> (x1000). Arrowheads show cells which are prominently expressing Isl1. <b>g-i</b>. Co-localization of Ldb1 and Isl1 (x1000). Ldb1 (green) and Isl1 (red) are co-expressed (yellow) in single cells of the crypt. <b>g</b>. Ldb1 expression, <b>h</b>. Isl1 expression. <b>i</b>. Merge of <b>g</b> and <b>h</b>. Arrow pointed on cell which is co-expressing Ldb1 and Isl1. <b>j</b>. Location of Ldb1^high cells in the crypt. Ldb1^high cells occupied predominately position +4–+7.</p

Gene targeting of <i>Ldb1</i> in the <i>villin-cre Ldb1^Δ/Δ</i> mutant intestine.

<p>No defects in cell morphology of either villous or cryptal epithelium are observed in <i>villin-cre Ldb1^Δ/Δ</i> mutant intestines (b, d) as compared to the <i>Ldb1^fl/fl</i> control littermates (a, c). Most cells of the wild type crypt are expressed Ldb1 at low level. Strong positive signal is detected in a few cells of the crypt (arrows) as well as in the epithelial stroma (arrowheads) (x400). No Ldb1 protein is found in the crypts of the <i>villin-cre Ldb1^Δ/Δ</i> mutant intestines, while stromal cells of the mutant are still expressed Ldb1 (arrowheads).</p