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. 2015 Jan;4(1):125-35.
doi: 10.1002/cam4.347. Epub 2014 Oct 15.

δEF1 associates with DNMT1 and maintains DNA methylation of the E-cadherin promoter in breast cancer cells

Akihiko Fukagawa  1 , Hiroki Ishii, Keiji Miyazawa, Masao Saitoh
Affiliations

δEF1 associates with DNMT1 and maintains DNA methylation of the E-cadherin promoter in breast cancer cells

Akihiko Fukagawa et al. Cancer Med. 2015 Jan.

Abstract

Abnormal DNA methylation at the C-5 position of cytosine (5mC) of CpG dinucleotides is a well-known epigenetic feature of cancer. Levels of E-cadherin, which is regularly expressed in epithelial tissues, are frequently reduced in epithelial tumors due to transcriptional repression, sometimes accompanied by hypermethylation of the promoter region. δEF1 family proteins (δEF1/ZEB1 and SIP1/ZEB2), key regulators of the epithelial-mesenchymal transition (EMT), suppress E-cadherin expression at the transcriptional level. We recently showed that levels of mRNAs encoding δEF1 proteins are regulated reciprocally with E-cadherin level in breast cancer cells. Here, we examined the mechanism underlying downregulation of E-cadherin expression in three basal-type breast cancer cells in which the E-cadherin promoter region is hypermethylated (Hs578T) or moderately methylated (BT549 and MDA-MB-231). Regardless of methylation status, treatment with 5-aza-2'-deoxycytidine (5-aza), which inhibits DNA methyltransferases, had no effect on E-cadherin expression. Knockdown of δEF1 and SIP1 resulted in recovery of E-cadherin expression in cells lacking hypermethylation, whereas combined treatment with 5-aza synergistically restored E-cadherin expression, especially when the E-cadherin promoter was hypermethylated. Moreover, δEF1 interacted with DNA methyltransferase 1 (DNMT1) through the Smad-binding domain. Sustained knockdown of δEF1 family proteins reduced the number of 5mC sites in the E-cadherin promoter region, suggesting that these proteins maintain 5mC through interaction with DNMT1 in breast cancer cells. Thus, δEF1 family proteins appear to repress expression of E-cadherin during cancer progression, both directly at the transcriptional level and indirectly at the epigenetic level.

Keywords: Cancer cells; DNA methylation; E-cadherin; EMT; δEF1.

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Figures

Figure 1
Figure 1
Expression profiles of E-cadherin, δEF1, and DNMT1 in human breast cancer cells. (A) Protein levels of E-cadherin, δEF1, and DNMT1 were determined by immunoblot analysis of whole-cell extracts. α-tubulin levels were monitored as a loading control. Molecular subtypes are as reported by Neve et al. . and Charafe-Jauffret et al. . (B, C, and D) BT549, MDA-MB-231, and Hs578T cells were transfected with siRNAs against both δEF1 and SIP1, and then treated with 5 μmol/L of 5-aza-2′-deoxycytidine (5-aza) for 48 h (for BT549 and MDA-MB-231 cells) or 1 μmol/L of 5-aza for 72 h (for Hs587T cells). Cells were then harvested and examined for expression of δEF1, SIP1, and E-cadherin by quantitative RT-PCR (B), immunoblotting (C), or immunocytochemistry (D). NC, negative control.
Figure 2
Figure 2
DNA methylation at the C-5 position (5mC) of cytosine in CpG dinucleotides in human breast cancer cells. (A) Schematic illustration of the promoter region of human E-cadherin (−162 to +37). Numbers in parentheses represent individual CpG sites in the region. E-boxes 1 and 2 have been already reported as binding sites for δEF1 and SIP1–. (B) Bisulfite sequencing was performed using bisulfite-treated templates from MCF7 and T47D cells. White and black circles represent unmethylated and methylated CpG (5mC) sites, respectively. (C) MCF7 and T47D cells were infected with lentiviral vectors encoding FLAG-δEF1. After 48 h, immunoblots were performed on whole-cell extracts. α-tubulin levels were monitored as a loading control. (D) Bisulfite sequencing was performed using bisulfite-treated templates from BT549, MDA-MB-231, and Hs578T cells. White and black circles represent unmethylated and methylated CpG (5mC), respectively. (E) The number of 5mC sites was compared among BT549, MDA-MB-231, and Hs578T cells. The Mann–Whitney U-test was used for assessing distributional differences of variance across different test samples. *Mann–Whitney U-test, P < 0.01. (F) Hs578T cells transfected with siRNAs against both δEF1 and SIP1 were treated with 1 μmol/L 5-aza-2′-deoxycytidine (5-aza) for 72 h. After bisulfite sequencing was performed on 11 clones of Hs578T treated with the indicated combinations, the number of methylated CpG (5mC) sites was counted. Median values are represented by horizontal bars (E and F). NC, negative control.
Figure 3
Figure 3
Interaction of δEF1 with DNMT1. (A–C) HEK293 cells were transiently transfected with the indicated expression plasmids. Twenty-four hours after transfection, cells were harvested, lysed, and subjected to immunoprecipitation (IP) with anti-FLAG antibody, followed by immunoblotting (IB) with anti-Myc antibody. Schematic illustrations depict wild-type (WT), N-terminally truncated mutants (ΔA–ΔD), and C-terminally truncated mutants (ΔE–ΔF) of δEF1 (left panels in B and C). (D) MCF7 and Hs578T cells were harvested and subjected to immunoprecipitation (IP) with anti-DNMT1 antibody or IgG, followed by immunoblotting (IB) with anti-DNMT1 or anti-δEF1 antibodies. α-tubulin levels were monitored as a loading control. NZF, N-terminal zinc finger; SBD, Smad-binding domain; HD, homeodomain; CtBP, CtBP-binding domain; CZF, C-terminal zinc finger.
Figure 4
Figure 4
Evaluation of 5mC sites after sustained knockdown of both δEF1 and SIP1 in Hs578T. (A, B, and C) Lentiviral vectors encoding δEF1 and SIP1 shRNAs were used to infect Hs578T cells. Twenty days after infection, the cells were harvested and examined for expression of δEF1/SIP1 and E-cadherin by quantitative RT-PCR (A), immunoblotting (B), or immunofluorescence (C). (D) Schematic illustration of the promoter region of human E-cadherin is shown (top). After bisulfite sequencing was performed, the number of methylated CpG (5mC) sites ay (11)–(17) was counted (right). White and black circles represent unmethylated and methylated CpG (5mC) sites, respectively (left). Median values are represented as horizontal bars (right). NC, negative control.
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