Genome-wide analysis of aberrant methylation in human breast cancer cells using methyl-DNA immunoprecipitation combined with high-throughput sequencing

<p>Abstract</p> <p>Background</p> <p>Cancer cells undergo massive alterations to their DNA methylation patterns that result in aberrant gene expression and malignant phenotypes. However, the mechanisms that underlie methylome changes are not well understood nor is the genomic distribution of DNA methylation changes well characterized.</p> <p>Results</p> <p>Here, we performed methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) to obtain whole-genome DNA methylation profiles for eight human breast cancer cell (BCC) lines and for normal human mammary epithelial cells (HMEC). The MeDIP-seq analysis generated non-biased DNA methylation maps by covering almost the entire genome with sufficient depth and resolution. The most prominent feature of the BCC lines compared to HMEC was a massively reduced methylation level particularly in CpG-poor regions. While hypomethylation did not appear to be associated with particular genomic features, hypermethylation preferentially occurred at CpG-rich gene-related regions independently of the distance from transcription start sites. We also investigated methylome alterations during epithelial-to-mesenchymal transition (EMT) in MCF7 cells. EMT induction was associated with specific alterations to the methylation patterns of gene-related CpG-rich regions, although overall methylation levels were not significantly altered. Moreover, approximately 40% of the epithelial cell-specific methylation patterns in gene-related regions were altered to those typical of mesenchymal cells, suggesting a cell-type specific regulation of DNA methylation.</p> <p>Conclusions</p> <p>This study provides the most comprehensive analysis to date of the methylome of human mammary cell lines and has produced novel insights into the mechanisms of methylome alteration during tumorigenesis and the interdependence between DNA methylome alterations and morphological changes.</p

MicroRNA-338-3p and microRNA-451 contribute to the formation of basolateral polarity in epithelial cells

MicroRNAs are small noncoding RNA species, some of which are playing important roles in cell differentiation. However, the level of participations of microRNAs in epithelial cell differentiation is largely unknown. Here, utilizing an epithelial differentiation model with T84 cells, we demonstrate that miR-338-3p and miR-451 contribute to the formation of epithelial basolateral polarity by facilitating translocalization of β1 integrin to the basolateral membrane. Among 250 microRNAs screened in this study, the expression levels of four microRNAs (miR-33a, 210, 338-3p and 451) were significantly elevated in the differentiated stage of T84 cells, when epithelial cell polarity was established. To investigate the involvement of these microRNAs in terms of epithelial cell polarity, we executed loss-of- and gain-of-function analyses of these microRNAs. The blockade of endogenous miR-338-3p or miR-451 via each microRNA-specific antisense oligonucleotides inhibited the translocalization of β1 integrin to the basolateral membrane, whereas inhibition of miR-210 or miR-33a had no effect on it. On the other hand, simultaneous transfection of synthetic miR-338-3p and miR-451 accelerated the translocalization of β1 integrin to the basolateral membrane, although the introduction of individual synthetic microRNAs exhibited no effect. Therefore, we concluded that both miR-338-3p and miR-451 are necessary for the development of epithelial cell polarity

Sip1, an AP-1 Accessory Protein in Fission Yeast, Is Required for Localization of Rho3 GTPase

<div><p>Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of <i>rho3</i>⁺ suppressed defective membrane trafficking associated with <i>sip1-i4</i> mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in <i>sip1-i4</i> mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because <i>Sip1-i4</i>-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the <i>sip1-i4</i> mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the <i>sip1-i4</i> mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in <i>sip1-i4</i> mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi/endosomal trafficking in fission yeast.</p> </div

Rho3 suppresses various phenotypes associated with <i>sip1-i4</i> mutant cells.

<p>(A) Rho3 suppresses the defective secretion of acid phosphatase in <i>sip1-i4</i> mutant cells. Wild-type (wt) and <i>sip1-i4</i> cells, which were transformed with either the pDB248 vector or <i>rho3</i>⁺-containing vector, were assayed for acid phosphatase activity. Data are representative of 3 independent experiments. (B) Rho3 suppresses the defects in vacuole fusion in <i>sip1-i4</i> cells. The wt and <i>sip1-i4</i> cells transformed with pDB248 or the vector containing <i>rho3</i>⁺ were cultured in YPD medium at 27°C. Cells were harvested, labeled with FM4-64 fluorescent dye for 60 min, resuspended in water, and examined by fluorescence microscopy. Bar, 10 µm. The number in the image indicates the percentage of cells with fragmented vacuoles. Data from at least 3 independent experiments are expressed as means ± standard deviations. (C) Rho3 suppresses GFP-Syb1 mislocalization in <i>sip1-i4</i> mutant cells. The wt and <i>sip1-i4</i> cells expressing GFP-Syb1 transformed with pDB248 or the vector containing <i>rho3</i>⁺ were cultured in YPD medium at 27°C. GFP-Syb1 localization was examined under a fluorescence microscope. Arrowheads indicate the dot-like structures of GFP-Syb1 and the Golgi/endosomes stained with FM4-64, double arrowheads indicate cytoplasmic accumulation, and arrows indicate the concentrated fluorescence at the medial region and cell surface. Bar, 10 µm. (D) Quantitative analysis of the number of Syb1 dots that co-localized with FM4-64/cell. (E) Percentage of cells in which Syb1 was localized at the cell surface. Cells in D and E were the same as those indicated in C.</p

The Sip1 C-terminus is dispensable for the Sip1 association with the AP-1 complex or Rho3.

<p>(A) Schematic representation of Sip1 protein, <i>Sip1-i4</i> mutant protein and Sip1ΔN protein. The Sip1 protein is 1919 amino acids long and contains HEAT repeats (black). Star represents termination codon at the amino acid position 1434 found in the <i>sip1-i4</i> allele. The <i>Sip1-i4</i> mutant protein lacks the C-terminal 485 amino acids. The Sip1ΔN protein lacks the N-terminal 1414 amino acids. (B) Binding assay involving <i>Sip1-i4</i> and the 4 subunits of the AP-1 complex. GST pull-down experiments were performed using <i>Sip1-i4</i>-GST, <i>Sip1-i4</i>-GST expressed under the control of the <i>nmt1</i> promoter. Cells that expressed GFP alone or GFP-tagged to the 4 subunits of the AP-1 complex were harvested, and their lysates were incubated with the purified <i>Sip1-i4</i> fused GST protein. GST-tagged proteins were analyzed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068488#pone-0068488-g003" target="_blank">Figure 3B</a>. (C) Binding assay involving Sip1ΔN and the 4 subunits of the AP-1 complex. The binding assay was performed as described in B. (D) Binding assay involving <i>Sip1-i4</i> and various mutant forms of Rho3. GST pull-down experiments were performed using <i>Sip1-i4</i>-GST expressed under the control of the <i>nmt1</i> promoter. Cells that expressed GFP alone or various GFP-tagged mutant forms of Rho3 were harvested, and their lysates were incubated with the purified <i>Sip1-i4-</i>GST protein. GST-tagged <i>Sip1-i4</i> was precipitated with glutathione beads, washed extensively, subjected to SDS-PAGE, and immunoblotted using anti-GFP or anti-GST antibodies. (E) Binding assay involving Sip1ΔN and various mutant forms of Rho3. The binding assay was performed as described in (D). Lower panel: Quantitation of GFP-tagged various mutant forms of Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels as shown in D and E. Data from at least three independent experiments are expressed as means ± standard deviations.</p

Sip1 links Rho3 to AP-1 complex.

<p>(A) Binding assay involving Apm1 and Rho3 in wild-type, or <i>sip1-i4</i> cells harboring the control vector or Sip1ΔN. GST pull-down experiments were performed using Apm1-GST expressed in wild-type (wt) and <i>sip1-i4</i> mutant (<i>sip1-i4</i>) cells, which were transformed with the pDB248 multi-copy vector or the vector containing <i>sip1</i>ΔN expressed under the control of the <i>nmt1</i> promoter. Cells that expressed GFP alone or GFP-Rho3 were harvested, and their lysates were incubated with purified full-length Apm1 fused to GST. Proteins bound to glutathione beads were analyzed by SDS-PAGE and visualized by autoradiography. Right panel: Quantitation of GFP-Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels in wild-type cells, <i>sip1-i4</i> cells or <i>sip1-i4</i> cells with Sip1ΔN expression as shown in A. Data from at least three independent experiments are expressed as means ± standard deviations. (B) Subcellular localization of GFP-Rho3 in wild-type cells, <i>sip1-i4</i> cells or <i>sip1-i4</i> cells with Sip1ΔN expression. GFP-Rho3 expressed in wild-type (wt) and <i>sip1-i4</i> mutant (<i>sip1-i4</i>) cells, which were transformed with the pDB248 multi-copy vector or the vector containing <i>sip1</i>ΔN expressed under the control of the <i>nmt1</i> promoter. Cells were cultured in YPD medium at 27°C, following which they were incubated with FM4-64 dye for 5 min at 27°C to visualize the Golgi/endosomes. FM4-64 fluorescence was examined using a fluorescence microscope. Arrowheads indicate the dot-like structures of GFP-Rho3 and the Golgi/endosomes stained with FM4-64, double arrowheads indicate cytoplasmic accumulation of GFP-Rho3, and arrows indicate the concentrated fluorescence at the cell division site. Bar, 10 µm. (C) Percentage of cells in which Rho3 were localized at the cell division site in wild-type (wt) and <i>sip1-i4</i> cells, which were transformed with the pDB248 multi-copy vector or the vector containing <i>sip1</i>ΔN expressed under the control of the <i>nmt1</i> promoter. (D) Quantitative analysis for the number of Rho3 dots co-localizing with FM4-64/cells in wt and <i>sip1-i4</i> cells, which were transformed with the pDB248 multi-copy vector or the vector containing <i>sip1</i>ΔN expressed under the control of the <i>nmt1</i> promoter. Data are the means ± standard deviations of 3 independent experiments with 150 cells in B.</p

Functional and physical interactions between Rho3 and Sip1.

<p>(A) Rho3 suppresses <i>sip1-i4</i> mutant cells (<i>sip1-i4</i>) in a GTP- and effector domain-dependent manner. The <i>sip1-i4</i> cells were transformed with the pDB248 multi-copy vector or the vector containing <i>rho3</i>^<i>+</i>, <i>rho3</i>GV, <i>rho3</i>TN, and <i>rho3</i>EV expressed from its endogenous promoter. These cells were streaked onto YES plates and then incubated at 27°C for 4 d or at 36°C for 3 d, respectively. (B) Binding assay for Sip1 and Rho3. GST pull-down experiments were performed using chromosome-borne GST-Sip1 expressed under the control of the <i>nmt1</i> promoter. Cells expressing GFP alone, or GFP-Rho3, GFP-Rho3GV, GFP-Rho3TN, or GFP-Rho3EV were harvested and their lysates were incubated with the purified full-length Sip1 fused GST protein. GST-tagged Sip1 was precipitated with glutathione beads, washed extensively, subjected to SDS-PAGE, immunoblotted using anti-GFP or anti-GST antibodies and visualized by autoradiography. Lower panel: Quantitation of GFP-tagged various mutant forms of Rho3 beads protein levels by densitometry of the expressed bands against that of the lysate protein levels as shown in B. Data from at least three independent experiments are expressed as means ± standard deviations.</p

The C-terminus of Sip1 is important for its Golgi/endosomal localization.

<p>(A) Co-localization of Sip1-GFP, <i>Sip1-i4</i>-GFP or Sip1ΔN-GFP with FM4-64 in wild-type cells. The wild-type (wt) cells that expressed chromosome-borne Sip1-GFP or wt cells transformed with pREP1-<i>Sip1-i4</i>-GFP or pREP1-Sip1ΔN-GFP were examined by fluorescence microscopy under repressed conditions. The cells were incubated with FM4-64 fluorescent dye for 5 min at 27°C to visualize the Golgi/endosomes. FM4-64 fluorescence was examined using a fluorescence microscope. Arrowheads indicate the dot-like structures and the Golgi/endosomes. Bar, 10 µm. (B) Sip1ΔN suppresses <i>sip1-i4</i> cells similar to Sip1. The wt and <i>sip1-i4</i> cells were transformed with the pDB248 multi-copy vector or the vector containing <i>sip1</i>^<i>+</i>, <i>sip1-i4</i>, and <i>sip1</i>ΔN expressed under the control of the <i>nmt1</i> promoter. Cells were streaked onto plates containing 0.5 µg/mL FK506 and then incubated at 27°C for 4 d or at 36°C for 3 d, respectively.</p