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Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Resource Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks Graphical Abstract Authors Davide Pellacani, Misha Bilenky, Nagarajan Kannan, ..., Samuel Aparicio, Martin Hirst, Connie J. Eaves Correspondence [email protected] (M.H.), [email protected] (C.J.E.) In Brief Pellacani et al. present comprehensive histone and DNA modification profiles for four cell types in normal human breast tissue and three immortalized human mammary epithelial cell lines. Analysis of activated enhancers place luminal progenitors in between bipotent progenitor-containing basal cells and nonproliferative luminal cells. Explore consortium data at the Cell Press IHEC webportal at http://www.cell.com/ consortium/IHEC. Highlights d Epigenomes of four normal human breast cell fractions and three cell lines are reported d Luminal progenitor and mature luminal cell epigenomes differ greatly d Enhancers define luminal progenitors as intermediate between basal and luminal cells d Transcription factor binding sites in active enhancers point to distinct regulators Pellacani et al., 2016, Cell Reports 17, 2060–2074 November 15, 2016 ª 2016 The Author(s). http://dx.doi.org/10.1016/j.celrep.2016.10.058 Cell Reports Resource Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks Davide Pellacani,1 Misha Bilenky,2 Nagarajan Kannan,1 Alireza Heravi-Moussavi,2 David J.H.F. Knapp,1 Sitanshu Gakkhar,2 Michelle Moksa,3 Annaick Carles,3 Richard Moore,2 Andrew J. Mungall,2 Marco A. Marra,2 Steven J.M. Jones,2 Samuel Aparicio,4,5 Martin Hirst,2,3,* and Connie J. Eaves1,6,* 1Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC V5Z 1L3, Canada 2Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC V5Z 1L3, Canada 3Michael Smith Laboratories, Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada 4Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC V5Z 1L3, Canada 5Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada 6Lead Contact *Correspondence: [email protected] (M.H.), [email protected] (C.J.E.) http://dx.doi.org/10.1016/j.celrep.2016.10.058 SUMMARY 3D cultures (Eirew et al., 2008; Kannan et al., 2013; Raouf et al., 2008). Most colonies derived from the BCs contain a The normal adult human mammary gland is a contin- mixture of cells with either basal or luminal features, although uous bilayered epithelial system. Bipotent and sometimes only basal progeny are produced. Human LPs myoepithelial progenitors are prominent and unique contain all of the in vitro progenitor activity of the luminal components of the outer (basal) layer. The inner compartment and produce colonies of cells expressing luminal (luminal) layer includes both luminal-restricted pro- features exclusively. Because LCs lack substantial proliferative genitors and a phenotypically separable fraction potential in vitro, they are assumed to represent a mature subset of luminal cells (Fridriksdottir et al., 2015; Kannan et al., 2013). that lacks progenitor activity. We now report an epi- When transplanted into immunodeficient mice, a small propor- genomic comparison of these three subsets with one tion (<1%) of human BCs, but not LPs or LCs, can also individu- another, with their associated stromal cells, and with ally regenerate normal-appearing bilayered mammary structures three immortalized, non-tumorigenic human mam- that contain the same spectrum of cell types and progenitors mary cell lines. Each genome-wide analysis contains found in the normal human gland. This property suggests the profiles for six histone marks, methylated DNA, and cells of origin of these structures represent a human mammary RNA transcripts. Analysis of these datasets shows stem cell population (Eirew et al., 2008; Kuperwasser et al., that each cell type has unique features, primarily 2004; Lim et al., 2009; Nguyen et al., 2014). within genomic regulatory regions, and that the cell Despite these advances, the molecular regulators that specify lines group together. Analyses of the promoter and the unique features of each of these normal human mammary enhancer profiles place the luminal progenitors in subsets have only recently become amenable to interrogation. Transcriptome profiling (Choudhury et al., 2013; Gascard et al., between the basal cells and the non-progenitor 2015; Huh et al., 2015; Kannan et al., 2013; Lim et al., 2009, luminal subset. Integrative analysis reveals networks 2010; Makarem et al., 2013; Maruyama et al., 2011; Raouf of subset-specific transcription factors. et al., 2008; Dos Santos et al., 2015), together with functional studies, has enabled several key transcription factors (TFs) to INTRODUCTION be identified in the subpopulations of normal human as well as analogous mouse mammary cell types (Fu et al., 2014; Visvader The normal adult human female mammary gland is a continuous and Stingl, 2014). Thus implicated in BCs are SLUG, TP63 and bilayered, branching epithelial structure containing three pheno- TP53, SOX9, STAT3, MYC, and TAZ. In cells with luminal fea- typically distinct subsets of cells. These can now be isolated and tures, CEBPB and NOTCH3 (Raouf et al., 2008), together with biologically distinguished (Fu et al., 2014). They are referred to as GATA3, ELF5 (Chakrabarti et al., 2012), and FOXA1, which reg- basal cells (BCs), luminal progenitors (LPs), and luminal cells ulates estrogen receptor activity (Theodorou et al., 2013), have (LCs) because their surface marker expression profiles identify been identified as important. their location within the outer (basal) or inner (luminal) layer of Previous human mammary epigenome profiles produced as the gland. A prominent fourth cell population in the breast is part of the Roadmap Epigenomics Consortium (Roadmap Epi- comprised of mesodermally derived stromal cells (SCs). Approx- genomics Consortium et al., 2015) have provided an initial global imately 20%–30% of human mammary BCs and LPs have annotation of active enhancer regions (Gascard et al., 2015). We progenitor (clonogenic) activity demonstrable in both 2D and now report a more extensive set of reference epigenome profiles 2060 Cell Reports 17, 2060–2074, November 15, 2016 ª 2016 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). A Normal FACS 6 samples pooled Molecular analyses Integrative analyses breast ( 5x107 cells per fraction) samples RNA Datasets n = 6 RNA-seq CD45- CD31- LC DNA Expression Chromatin states single cells WGBS LC LP Chromatin LP H3K4me1 H3K4me3 Enhancers BC BC H3K9me3 EpCAM-PE Promoters H3K27ac SC SC H3K27me3 Transcription H3K36me3 factor CD49f-APC networks Colony assays B C Type Study LP vs BC LP vs LC Up: 806 BC CEMT Do Do BC Nguyen BC 1 Nguyen 1 BC Nguyen (RPKM) (RPKM) 10 myo REMC 10 myo REMC myo REMC LC log LP CEMT BC log LP Nguyen LP Nguyen LP Nguyen 1 1 lum REMC LP log10(RPKM) LP log10(RPKM) LC CEMT lum REMC BC vs LC DE genes lum REMC Do MCF10A CEMT LP vs BC 184−L9 CEMT 184−L2 CEMT 1 vHMEC REMC (RPKM) vHMEC REMC 10 LP vs LC SC CEMT log fibr REMC LC fibr REMC BC vs LC 0.20 0.15 0.10 0.05 0.00 1 246810 BC log10(RPKM) Dist. = 1−Spearman | Link. = complete absolute log2( D E Type Study myo REMC BC CEMT myo REMC SC CEMT LP CEMT LC CEMT lum REMC lum REMC fibr REMC fibr REMC vHMEC REMC MCF10A CEMT 184−L9 CEMT 184−L2 CEMT 0.4 0.3 0.2 0.1 0.0 Dist. = 1−Spearman | Link. = complete Figure 1. Isolation of Purified Cell Subsets and Epigenomic Profiling (A) Experimental design. (B) Unsupervised clustering of RNA-sequencing experiments comparing the samples analyzed in this study (CEMT) with other studies (Nguyen et al., 2015; Roadmap Epigenomics Consortium et al., 2015; Gascard et al., 2015). (legend continued on next page) Cell Reports 17, 2060–2074, November 15, 2016 2061 that include a separate analysis of human LPs and LCs and pro- These findings are consistent with and extend previously re- files for SCs isolated from the same normal breast tissue, as well ported findings obtained both by RNA-seq (Gascard et al., as profiles generated from three widely used immortalized, but 2015; Nguyen et al., 2015) and by 30 tag-based and microarray non-tumorigenic, human mammary epithelial cell lines: i.e., analysis (Kannan et al., 2013; Lim et al., 2009; Raouf et al., MCF-10A cells (Debnath et al., 2003) and 184-hTERT-L2 (184- 2008), including an identification of many of the same genes as L2) and 184-hTERT-L9 (184-L9) cells (Burleigh et al., 2015). ‘‘BC specific’’ (e.g., TP63 (deltaN), ACTA2, MME, KRT14, The results demonstrate that the cell lines are not representative THY1, and KRT5) or ‘‘LP specific’’ (e.g., MUC1, PROM1, KIT, of any freshly isolated normal human mammary subset. They KRT18, EPCAM, and KRT8)(Figure S2A). Similar clustering have also allowed a hierarchy of enhancer states in normal and differences between the same four cell types were human mammary tissue and their corresponding cell-specific observed when either microRNA (miRNA) or large intergenic TF networks to be identified. noncoding RNA (lincRNA) profiles were analyzed (Figures 1D, S2D, and S2E). RESULTS AND DISCUSSION Notably, the transcriptomes for all three immortalized human mammary cell lines clustered independently from both the pri- Generation and Validation of Comprehensive Reference mary epithelial subsets and the SCs isolated directly from the Epigenomes same freshly dissociated normal human breast tissue (Figure 1B). Reference epigenomic profiles were generated following the rec- Moreover, 66 of the 100 genes whose differential expression ommendations of the International Human Epigenome Con- contributed most to the separate clustering of the mammary sortium (IHEC; http://ihec-epigenomes.org).
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