Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells
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{"title"=>"Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells", "type"=>"journal", "authors"=>[{"first_name"=>"Joanne L.", "last_name"=>"Attema", "scopus_author_id"=>"6602561159"}, {"first_name"=>"Andrew G.", "last_name"=>"Bert", "scopus_author_id"=>"7005146371"}, {"first_name"=>"Yat Yuen", "last_name"=>"Lim", "scopus_author_id"=>"52663775100"}, {"first_name"=>"Natasha", "last_name"=>"Kolesnikoff", "scopus_author_id"=>"24833643800"}, {"first_name"=>"David M.", "last_name"=>"Lawrence", "scopus_author_id"=>"55431813700"}, {"first_name"=>"Katherine A.", "last_name"=>"Pillman", "scopus_author_id"=>"55625629800"}, {"first_name"=>"Eric", "last_name"=>"Smith", "scopus_author_id"=>"7408616287"}, {"first_name"=>"Paul A.", "last_name"=>"Drew", "scopus_author_id"=>"7101736891"}, {"first_name"=>"Yeesim", "last_name"=>"Khew-Goodall", "scopus_author_id"=>"6603769494"}, {"first_name"=>"Frances", "last_name"=>"Shannon", "scopus_author_id"=>"7202548949"}, {"first_name"=>"Gregory J.", "last_name"=>"Goodall", "scopus_author_id"=>"7007064596"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0075517", "sgr"=>"84884575728", "issn"=>"19326203", "scopus"=>"2-s2.0-84884575728", "pui"=>"369883016", "pmid"=>"24086551"}, "id"=>"12173227-c529-3b25-9700-c4138d6cb277", "abstract"=>"The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of miR-200b eRNA function will be necessary, it is possible that miR-200b eRNA may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells.", "link"=>"http://www.mendeley.com/research/identification-enhancer-increases-mir200b200a429-gene-expression-breast-cancer-cells", "reader_count"=>24, "reader_count_by_academic_status"=>{"Researcher"=>6, "Student > Ph. D. Student"=>9, "Student > Postgraduate"=>3, "Other"=>1, "Student > Master"=>1, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Researcher"=>6, "Student > Ph. D. Student"=>9, "Student > Postgraduate"=>3, "Other"=>1, "Student > Master"=>1, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>5, "Agricultural and Biological Sciences"=>15, "Medicine and Dentistry"=>3, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>15}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}}, "reader_count_by_country"=>{"Austria"=>1, "United States"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1215374"], "description"=>"<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>", "links"=>[], "tags"=>["chromatin", "upstream"], "article_id"=>807842, "categories"=>["Biological Sciences"], "users"=>["Joanne L. Attema", "Andrew G. Bert", "Yat-Yuen Lim", "Natasha Kolesnikoff", "David M. Lawrence", "Katherine A. Pillman", "Eric Smith", "Paul A. Drew", "Yeesim Khew-Goodall", "Frances Shannon", "Gregory J. Goodall"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075517.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_An_active_chromatin_domain_is_located_upstream_of_the_miR_200b_200a_429_locus_/807842", "title"=>"An active chromatin domain is located upstream of the miR-200b~200a~429 locus.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-25 01:40:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1215376"], "description"=>"<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>", "links"=>[], "tags"=>["upstream", "enhancer", "transcription", "promoter", "epithelial", "cancer"], "article_id"=>807844, "categories"=>["Biological Sciences"], "users"=>["Joanne L. Attema", "Andrew G. Bert", "Yat-Yuen Lim", "Natasha Kolesnikoff", "David M. Lawrence", "Katherine A. Pillman", "Eric Smith", "Paul A. Drew", "Yeesim Khew-Goodall", "Frances Shannon", "Gregory J. Goodall"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075517.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Identification_of_an_upstream_enhancer_region_that_increases_the_transcription_of_the_miR_200b_200a_429_promoter_in_epithelial_breast_cancer_cells_/807844", "title"=>"Identification of an upstream enhancer region that increases the transcription of the miR-200b~200a~429 promoter in epithelial breast cancer cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-25 01:40:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1215378"], "description"=>"<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>", "links"=>[], "tags"=>["transcribed", "upstream", "intergenic", "enhancer"], "article_id"=>807846, "categories"=>["Biological Sciences"], "users"=>["Joanne L. Attema", "Andrew G. Bert", "Yat-Yuen Lim", "Natasha Kolesnikoff", "David M. Lawrence", "Katherine A. Pillman", "Eric Smith", "Paul A. Drew", "Yeesim Khew-Goodall", "Frances Shannon", "Gregory J. Goodall"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075517.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_miR_200b_eRNA_is_transcribed_from_the_upstream_intergenic_enhancer_region_of_miR_200b_200a_429_/807846", "title"=>"<i>miR-200b eRNA</i> is transcribed from the upstream intergenic enhancer region of miR-200b~200a~429.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-25 01:40:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1215379"], "description"=>"<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>", "links"=>[], "tags"=>["emt-affiliated"], "article_id"=>807847, "categories"=>["Biological Sciences"], "users"=>["Joanne L. Attema", "Andrew G. Bert", "Yat-Yuen Lim", "Natasha Kolesnikoff", "David M. Lawrence", "Katherine A. Pillman", "Eric Smith", "Paul A. Drew", "Yeesim Khew-Goodall", "Frances Shannon", "Gregory J. Goodall"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075517.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gene_expression_analysis_of_miR_200b_eRNA_miR_200b_200a_429_and_EMT_affiliated_genes_/807847", "title"=>"Gene expression analysis of <i>miR-200b eRNA</i>, miR-200b~200a~429 and EMT-affiliated genes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-25 01:40:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1215381"], "description"=>"<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>", "links"=>[], "tags"=>["promoter", "epithelial", "mesenchymal", "hmle"], "article_id"=>807849, "categories"=>["Biological Sciences"], "users"=>["Joanne L. Attema", "Andrew G. Bert", "Yat-Yuen Lim", "Natasha Kolesnikoff", "David M. Lawrence", "Katherine A. Pillman", "Eric Smith", "Paul A. Drew", "Yeesim Khew-Goodall", "Frances Shannon", "Gregory J. Goodall"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075517.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Over_expression_of_miR_200b_eRNA_has_no_effect_on_miR_200b_200a_429_promoter_activity_in_epithelial_and_mesenchymal_HMLE_cells_/807849", "title"=>"Over-expression of <i>miR-200b eRNA</i> has no effect on miR-200b~200a~429 promoter activity in epithelial and mesenchymal HMLE cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-09-25 01:40:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1215402", "https://ndownloader.figshare.com/files/1215403", "https://ndownloader.figshare.com/files/1215404", "https://ndownloader.figshare.com/files/1215405", "https://ndownloader.figshare.com/files/1215406", "https://ndownloader.figshare.com/files/1215407", "https://ndownloader.figshare.com/files/1215408", "https://ndownloader.figshare.com/files/1215409", "https://ndownloader.figshare.com/files/1215410", "https://ndownloader.figshare.com/files/1215411", "https://ndownloader.figshare.com/files/1215412", "https://ndownloader.figshare.com/files/1215413", "https://ndownloader.figshare.com/files/1215414", "https://ndownloader.figshare.com/files/1215415", "https://ndownloader.figshare.com/files/1215416"], "description"=>"<div><p>The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated <i>miR-200b eRNA</i>. Over-expression of <i>miR-200b eRNA</i> had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of <i>miR-200b eRNA</i> function will be necessary, it is possible that <i>miR-200b eRNA</i> may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells.</p> </div>", "links"=>[], "tags"=>["enhancer", "cancer"], "article_id"=>807861, "categories"=>["Biological Sciences"], "users"=>["Joanne L. Attema", "Andrew G. Bert", "Yat-Yuen Lim", "Natasha Kolesnikoff", "David M. Lawrence", "Katherine A. Pillman", "Eric Smith", "Paul A. Drew", "Yeesim Khew-Goodall", "Frances Shannon", "Gregory J. Goodall"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0075517.s001", "https://dx.doi.org/10.1371/journal.pone.0075517.s002", "https://dx.doi.org/10.1371/journal.pone.0075517.s003", "https://dx.doi.org/10.1371/journal.pone.0075517.s004", "https://dx.doi.org/10.1371/journal.pone.0075517.s005", "https://dx.doi.org/10.1371/journal.pone.0075517.s006", "https://dx.doi.org/10.1371/journal.pone.0075517.s007", "https://dx.doi.org/10.1371/journal.pone.0075517.s008", "https://dx.doi.org/10.1371/journal.pone.0075517.s009", "https://dx.doi.org/10.1371/journal.pone.0075517.s010", "https://dx.doi.org/10.1371/journal.pone.0075517.s011", "https://dx.doi.org/10.1371/journal.pone.0075517.s012", "https://dx.doi.org/10.1371/journal.pone.0075517.s013", "https://dx.doi.org/10.1371/journal.pone.0075517.s014", "https://dx.doi.org/10.1371/journal.pone.0075517.s015"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Identification_of_an_Enhancer_That_Increases_miR_200b_200a_429_Gene_Expression_in_Breast_Cancer_Cells_/807861", "title"=>"Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-09-25 01:40:09"}

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Relative Metric

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