Identification of an upstream enhancer region that increases the transcription of the miR-200b~200a~429 promoter in epithelial breast cancer cells.

<p>(A) A series of 5’ deletions of the human miR-200b~200a~429 locus comprising the promoter and potential enhancer were cloned into a firefly luciferase reporter plasmid. (B) The reporter plasmids were transiently transfected along with the Renilla pTK vector into epithelial HMLE cells (white bars) or mesenchymal HMLE cells (black bars). Luciferase activity was assayed approximately 48 hours later using the Dual-Luciferase Reporter Assay System (Promega). Data are expressed as normalized luciferase activity and represent means ± SD of at least four independent experiments. (C) The enhancer region (-5771/-4607 ENH) was cloned in both directions immediately upstream of the minimal miR-200b~200a~429 promoter (-321/+19 PRO) or the Luciferase coding region creating PRO&ENH and ENH as well as PRO&ENH (-) and ENH (-) with ENH oriented in the sense and antisense orientation, respectively. Luciferase activity was assay as described in (B).</p

Gene expression analysis of <i>miR-200b eRNA</i>, miR-200b~200a~429 and EMT-affiliated genes.

<p>(A) Real-time PCR of <i>miR-200b </i><i>eRNA</i>, EMT markers and miR-200 genes in a panel of breast epithelial and mesenchymal cell lines. mRNA (top panel) is normalized to GAPDH while miRNA (bottom panel) is normalized to U6 snRNA. Comparative quantitation was used to determine expression profiles of the genes in the cell line panel. Expression of E-cadherin and <i>miR-200b </i><i>eRNA</i> in HMLE cells is set to a value of 1, whereas Zeb-1 is expressed relative to mesHMLE cells having a value of 1. Error bars represent mean ± SD of three independent experiments. (B) Relative expression levels of <i>miR-200b </i><i>eRNA</i> during EMT time course of HMLE cells treated with TGF-β1 for up to 18 days. The realtime PCR data is normalized to GAPDH. Expression of <i>miR-200b </i><i>eRNA</i> is set to a value of 1 in HMLE cells (C) Relative concentration of <i>miR-200b </i><i>eRNA</i>, U6, HOTAIR, GAPDH and βActin RNA transcripts in the nucleus and cytoplasm of HMLE cells. Absolute quantitation was used to determine the expression level of each gene in the cytoplasmic and nuclear fractions. Error bars represent mean ± SD of three independent experiments.</p

Over-expression of <i>miR-200b eRNA</i> has no effect on miR-200b~200a~429 promoter activity in epithelial and mesenchymal HMLE cells.

<p>Schematic representation of the miR-200b~200a~429 luciferase reporter constructs, PRO (-321/+19), LOCUS (-5771/+19), PRO&ENH (-321/+19 to -5711/4607) and ENH (-5711/-4607). The miR-200b~200a~429 reporters, pcDNA3.1, pcDNA3.1 Zeb1, pcDNA3.1 Zeb2 or the pcDNA3.1 <i>miR-200b </i><i>eRNA</i> plasmids, and the Renilla vector were co-transfected into epithelial HMLE (white bars) or mesenchymal HMLE (black bars) cells. Transiently transfected cells were incubated for approximately 24-48 hours. Data are normalized by Renilla luciferase activity and represent means ± SD of at least three independent experiments.</p

An active chromatin domain is located upstream of the miR-200b~200a~429 locus.

<p>Normalized ChIP-seq signal profiles were generated for H3K4me1, H3K4me3, H3K9/14ac, H3K27ac and H3K27me3 at the miR-200b~200a~429 locus (chr1:1,090,000-1,105,000) in (A) epithelial HMLE cells and (B) mesenchymal HMLE that have undergone EMT. The x-axis shows the distance in kilobases (kb) relative to the TSS (designated 0), the chromosomal coordinate marking the TSS is indicated and a schematic diagram of the primary miR-200b~200a~429 transcript is positioned boxes indicate the mature miRNA hairpin transcripts. The y-axis shows the sequencing coverage per million reads for each histone modification normalized to the Input control sample. (C) CpG methylation analysis of the miR-200b~200a~429 locus in epithelial HMLE cells (left panel) and in mesenchymal HMLE cells following 46 days of TGF-β1 (right panel) treatment using Illumina HM450K methylation array [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075517#B36" target="_blank">36</a>]. An arrow marks the TSS. The x-axis indicates the distance in kb from the TSS designated 0. The y-axis shows the % CpG methylation occurring at each genomic region.</p

<i>miR-200b eRNA</i> is transcribed from the upstream intergenic enhancer region of miR-200b~200a~429.

<p>(A) Schematic representation of the miR-200b~200a~429 locus. Black box indicates the position of the potential enhancer. A black arrow marks the TSS direction of the primary miR-200b~200a~429 transcript. Grey boxes indicated the mature miR-200b, miR-200a and miR-429 genes. Black bars indicate the positions (in kilobases, kb) of the PCR primers used for qRT-PCR. (B) Expression levels of HOTAIR, <i>miR-200b </i><i>eRNA</i> and the primary miR-200b~200a~429 transcript as determined by qRT-PCR in epithelial and mesenchymal HMLE cells using random hexamer primed cDNA synthesized from total RNA. The x-axis shows the distance from the miR-200b~200a~429 TSS in kb. Data represents mean ± SD of three independent experiments. (C) Schematic representation of the enhancer region located relative to the miR-200b~200a~429 TSS. Boxes indicate the locations of PCR amplicons used to detect the <i>miR-200b </i><i>eRNA</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075517#pone-0075517-g003" target="_blank">Figure 3B</a>. RACE PCR primers and their start locations relative to the miR-200b~200a~429 are indicated. 5’ and 3’ RACE-seq analysis of the <i>miR-200b </i><i>eRNA</i> with cDNA prepared from total RNA of HMLE, mesHMLE, MDA-MB-468 and MDA-MB-231 cells as described in the Materials and Methods. 5’ and 3’ ends of the <i>miR-200b </i><i>eRNA</i> transcript are mapped as % total reads for each cell line with extreme 5’ and 3’ ends indicated by colored arrows below. A consensus <i>miR-200b </i><i>eRNA</i> transcript is indicated.</p