Chromatin structure of the regulatory regions of pS2 and cathepsin D genes in hormone-dependent and -independent breast cancer cell lines

Abstract

We have compared the DNase I hypersensitivity of the regulatory region of two estrogen-regulated genes, pS2 and cathepsin D in hormone-dependent and -independent breast carcinoma cell lines. This strategy allowed the identi®cation of two important control regions, one in pS2 and the other in cathepsin D genes. In the hormonedependent MCF7 cell line, within the pS2 gene 5'-anking region, we detected two major DNase I hypersensitive sites, induced by estrogens and/or IGFI: pS2-HS1, located in the proximal promoter and pS2-HS4, located 710.5 Kb from the CAP site, within a region that has not been cloned. The presence of these two DNase I hypersensitive sites correlates with pS2 expression. Interestingly in MCF7 cells, estrogens and IGFI induced indistinguishable chromatin structural changes over the pS2 regulatory region, suggesting that the two transduction-pathways converge to a unique chromatin target. In two cell lines that do not express pS2, MDA MB 231, a hormone-independent cell line that lacks the estrogen receptor a, and HE5, a cell line derived from MDA MB 231 by transfection that expresses estrogen receptor a, there was only one hormoneindependent DNase I hypersensitive site. This site, pS2-HS2, was located immediately upstream of pS2-HS1. In MCF7 cells, two major DNase I hypersensitive sites were present in the 5'-¯anking sequences of the cathepsin D gene, which is regulated by estrogens in these cells. These sites, catD-HS2 and catD-HS3, located at positions 72.3 Kb and 73.45 Kb, respectively, were both hormone-independent. A much weaker site, catD-HS1, covered the proximal promoter. In MDA MB 231 cells, that express cathepsin D constitutively, we detected an additional strong hormoneindependent DNase I hypersensitive site, catD-HS4, located at position 74.3 Kb. This region might control the constitutive over-expression of cathepsin D in hormone-independent breast cancer cells. All together, these data demonstrate that a local reorganization of the chromatin structure over pS2 and cathepsin D promoters accompanies the establishment of the hormone-independent phenotype of the cells.

Eliminating epigenetic barriers induces transient hormone-regulated gene expression in estrogen receptor negative breast cancer cells

“…We confirmed that the presence of ERa in the cell is sufficient to confer hormone-regulated Cat-D expression, since both HE-5 and MDA-66 exhibit a 1.8-fold increase in Cat-D mRNA levels similar to the twofold stimulation observed in MCF-7 cells (Figure 1c). These results taken together with previous observations that the chromatin structure of the PR and pS2/TFF1 promoters remains insensitive to DNAseI cleavage in the HE-5 cell line (Giamarchi et al, 1999) led us to postulate that promoter accessibility may hamper ERa binding to PR and, to some extent, to pS2/TFF1 in MDA-MB231-derived cell lines.…”

“…Some reports claim that genes such as PR or pS2/ TFF1 could be expressed simply by ectopically introducing ERa into these cell lines (Lazennec et al, 1999;Metivier et al, 2003), while other reports indicate that expression of ERa is not sufficient to transcribe these silenced genes (Touitou et al, 1991;Giamarchi et al, 1999). However, it was previously shown that treatment of ERa-negative breast cancer cell lines such as MDA-MB231 or MDA-MB435 with DNA methylation inhibitors can lead to expression of ERa protein (Yang et al, 2001).…”

FOXP1, an Estrogen-Inducible Transcription Factor, Modulates Cell Proliferation in Breast Cancer Cells and 5-Year Recurrence-Free Survival of Patients with Tamoxifen-Treated Breast Cancer

“…Salmon sperm DNA/protein A-agarose (Upstate Biotechnology, Lake Placid, NY) was added and incubated for 2 h. Precipitated DNA was used as a template for qPCR using an Applied Biosystems 7000 sequence detector (Foster City, CA) based on SYBR Green I fluorescence. A genomic fragment containing ERE in the enhancer region of TFF1 (405/393 bp from the transcription start site) was used as a positive control for ER binding [19]. Sequences of PCR primers are as follows: TFF1_ERE, 5′-TGAGATTCAGAAAG TCCCTCTTTC-3′ and 5′-TGGGCTTCATGAGCTCCTT-3′; FOXP1_ER_790, 5′ -AACATCTGACAAATTATT GGGTGGTT-3′ and 5′-TGGCTTACCAGTTTAATGTCC CATA-3′; FOXP1_ER_791, 5′-AGGGTGAACCACA GCCTGTT-3′ and 5′-AAAGTGACAGTTTCCCAAGTA CATGT-3′; FOXP1_ER_792, 5′-TGCAAGGTCTGTTTAA CAGACACA-3′ and 5′-CCCCTTCATCCAAGCAAAAG-3′.…”

“…2d). ERα was enriched about sixfold on the ERBS within the TFF1 gene region, used as a positive control [19].…”

Estrogen Receptor α and the Activating Protein-1 Complex Cooperate during Insulin-like Growth Factor-I-induced Transcriptional Activation of the pS2/TFF1 Gene

“…The pS2/TFF1 gene has a composite promoter with an ERE (located between Ϫ405 and Ϫ393) and a TRE (AP1 response element, located between Ϫ338 and Ϫ332), and it has been reported that its activation can be achieved by estradiol or IGF-I (27). Our laboratory has shown that both estradiol and IGF-I treatments induce a rapid chromatin remodeling of the pS2/TFF1 promoter in a region encompassing the ERE and the TRE (28). The role of the AP1 element in the response of pS2/TFF1 to estradiol has been analyzed in detail by others in a cell line derived from the HepG2 hepatocarcinoma cell line, re-expressing ER␣.…”