Maps of open chromatin highlight cell type-restricted patterns of regulatory sequence variation at hematological trait loci Supplemental Material Corresponding authors: Dirk S. Paul UCL Cancer Institute 72 Huntley Street, London, WC1E 6DD, UK Email: [email protected] Panos Deloukas Wellcome Trust Sanger Institute Hinxton, Cambridge, CB10 1SA, UK Email: [email protected] Table of contents: Note Members of the HaemGen Consortium ............................................................................................ 3 Supplemental Figures: Fig. 1. Characterization of primary human monocytes (MOs), megakaryocytes (MKs) and erythroblasts (EBs) .................................................................................................................................................. 4 Fig. 2. Overlap of NDRs across all samples assayed ................................................................................... 6 Fig. 3. Dendrogram of the hierarchical clustering of the overlap of NDRs identified in megakaryocytes (MKs), erythroblasts (EBs), monocytes (MOs) and pancreatic islets (ISLs) .................................... 8 Fig. 4. Distance of NDRs to the closest transcription start site (TSS) ......................................................... 9 Fig. 5. Enrichment of SNPs associated with hematological traits at NDRs compared to the median of 100,000 random sets of SNPs .......................................................................................................... 10 Fig. 6. Electrophoretic mobility shift assays (EMSAs) for platelet candidate functional SNPs ................ 11 Fig. 7. Functional follow-up of the ABCC4 platelet count locus ............................................................... 13 Fig. 8. Bootstrapped quantile-quantile plots of SNPs located at cell type-restricted NDRs ...................... 14 Supplemental Tables: Table 1. Overview of sequencing statistics ................................................................................................... 15 Table 2. Overview of peak statistics ............................................................................................................. 16 Table 3. Overview of peak statistics stratified for peak class ....................................................................... 17 Table 4. Fraction of overlap of FAIRE peaks across replicates/individuals, transcription factor binding sites and histone modifications ................................................................................................................ 18 Table 5. Trends of up- or down-regulation of genes close to FAIRE peaks during hematopoietic differentiation .................................................................................................................................. 19 Table 6. Ontology analysis of genes in proximity to NDRs using the Genomic Regions Enrichment of Annotations Tool (GREAT) ............................................................................................................ 20 Table 7. SNPs associated with platelet and erythrocyte phenotypes located at open chromatin in primary human MKs, EBs and MOs ............................................................................................................. 25 Table 8. Cell type distribution of NDRs containing candidate functional variants at platelet and erythrocyte QTLs ................................................................................................................................................ 35 Table 9. Oligonucleotide probes for EMSA studies ...................................................................................... 36 Table 10. Pearson’s correlation coefficients between erythrocyte traits ......................................................... 37 References ........................................................................................................................................................ 38 -2- Supplemental Note. Members of the HaemGen Consortium. Jan-Willem N Akkerman, Cornelis A Albers, Ale Algra, Abtehale Al-Hussani, Hooman Allayee, Franco Anni, Folkert W Asselbergs, Antony Attwood, Beverley Balkau, Stefania Bandinelli, François Bastardot, Saonli Basu, Sebastian E Baumeister, Jacques Beckmann, Beben Benyamin, Ginevra Biino, Joshua C Bis, Lorenzo Bomba, Amélie Bonnefond, Dorret I Boomsma, John R Bradley, François Cambien, John C Chambers, Marina Ciullo, William O Cookson, Francesco Cucca, Ana Cvejic, Adamo Pio D’Adamo, John Danesh, Fabrice Danjou, Debashish Das, Gail Davies, Paul IW de Bakker, Rudolf A de Boer, Eco JC de Geus, Ian J Deary, George V Dedoussis, Panos Deloukas, Maria Dimitriou, Christian Dina, Angela Döring, Ulrich Elling, David Ellinghaus, Paul Elliott, Gunnar Engström, Jeanette Erdmann, Tõnu Esko, David M Evans, Gudmundur I Eyjolfsson, Mario Falchi, Wei Feng, Manuel A Ferreira, Luigi Ferrucci, Krista Fischer, Aaron R Folsom, Paolo Fortina, Andre Franke, Lude Franke, Ian H Frazer, Philippe Froguel, Renzo Galanello, Santhi K Ganesh, Stephen F Garner, Paolo Gasparini, Bernd Genser, Quince D Gibson, Christian Gieger, Giorgia Girotto, Nicole L Glazer, Martin Gögele, Alison H Goodall, Andreas Greinacher, Daniel F Gudbjartsson, Chris Hammond, Sarah E Harris, Jaana Hartiala, Anna-Liisa Hartikainen, Stanley L Hazen, Susan R Heckbert, Bo Hedblad, Christian Hengstenberg, Micha Hersch, Andrew A Hicks, Hilma Holm, Jouke-Jan Hottenga, Thomas Illig, Marjo-Riitta Jarvelin, Jennifer Jolley, Steve Jupe, Mika Kähönen, Naoyuki Kamatani, Stavroula Kanoni, Ido P Kema, John P Kemp, Jyoti Khadake, Kay Tee Khaw, Marcus E Kleber, Jaspal S Kooner, Peter Kovacs, Brigitte Kühnel, Marie-Christine Kyrtsonis, Yann Labrune, Vasiliki Lagou, Claudia Langenberg, Terho Lehtimäki, Xinzhong Li, Liming Liang, Lifelines Cohort Study, Heather Lloyd-Jones, Ruth JF Loos, Lorna M Lopez, Thomas Lumley, Leo-Pekka Lyytikäinen, Winfried Maerz, Reedik Mägi, Massimo Mangino, Nicholas G Martin, Andrea Maschio, Irene Mateo Leach, Barbara McKnight, Stuart Meacham, Sarah E Medland, Christa Meisinger, Olle Melander, Yasin Memari, Andres Metspalu, Kathy Miller, Braxton D Mitchell, Miriam F Moffatt, Grant W Montgomery, Carmel Moore, Federico Murgia, Yusuke Nakamura, Matthias Nauck, Gerjan Navis, Ilja M Nolte, Ute Nöthlings, Teresa Nutile, Yukinori Okada, Isleifur Olafsson, Pall T Onundarson, Paul F O’Reilly, Willem H Ouwehand, Debora Parracciani, Afshin Parsa, Dirk S Paul, Josef M Penninger, Brenda W Penninx, Mario Pirastu, Nicola Pirastu, Giorgio Pistis, Eleonora Porcu, Laura Portas, David Porteous, Anneli Pouta, Peter P Pramstaller, Inga Prokopenko, Bruce M Psaty, Janne Pullat, Aparna Radhakrishnan, Olli Raitakari, Ramiro Ramirez- Solis, Augusto Rendon, Janina S Ried, Susan M Ring, Antonietta Robino, Jerome I Rotter, Daniela Ruggiero, Aimo Ruokonen, Cinzia Sala, Andres Saluments, Nilesh J Samani, Jennifer Sambrook, Serena Sanna, David Schlessinger, Carsten O Schmidt, Stefan Schreiber, Heribert Schunkert, James Scott, Joban Sehmi, Jovana Serbanovic-Canic, So-Youn Shin, Alan R Shuldiner, Rob Sladek, Johannes H Smit, George Davey Smith, J Gustav Smith, Nicholas L Smith, Harold Snieder, Nicole Soranzo, Rossella Sorice, Timothy D Spector, John M Starr, Kari Stefansson, Derek Stemple, Jonathan Stephens, Michael Stumvoll, Patrick Sulem, Atsushi Takahashi, Sian-Tsung Tan, Toshiko Tanaka, Clara Tang, Weihong Tang, WH Wilson Tang, Kent Taylor, Albert Tenesa, Alexander Teumer, Swee Lay Thein, Unnur Thorsteinsdottir, Daniela Toniolo, Anke Tönjes, Michela Traglia, Manuela Uda, Sheila Ulivi, Pim van der Harst, C Ellen van der Schoot, Wiek H van Gilst, L Joost van Pelt, Dirk J van Veldhuisen, Niek Verweij, Peter M Visscher, Uwe Völker, Peter Vollenweider, Katrin Voss, Nicholas J Wareham, Lorenz Wernisch, Harm-Jan Westra, John B Whitfield, H- Erich Wichmann, Kerri L Wiggins, Gonneke Willemsen, Bernhard R Winkelmann, Gerald Wirnsberger, Bruce HR Wolffenbuttel, Jian Yang, Tsun-Po Yang, Weihua Zhang, Jing Hua Zhao, Paavo Zitting, Jaap-Jan Zwaginga. -3- Supplemental Figure 1. Characterization of primary human monocytes (MOs), megakaryocytes (MKs) and erythroblasts (EBs). Left panel: Representative images of stained cytospins (magnification, x40). Right panel: Gated cell populations with cell type-specific markers are shown in green, with corresponding IgG control populations in red. Indicated ranges represent average expression of gated cell populations of independent biological triplicates. For all cell cultures, more than 90% of cells tested negative for CD34 expression and corresponding isotype controls, as determined by flow cytometric analysis. Markers of other lineages were not detected. (A) We isolated mature MOs based on morphological evaluation through microscopial analysis of stained cytospins (Goasguen et al. 2009). Isolated cells showed morphological characteristics of mature MOs, i.e. lobulated nucleus, condensed chromatin, occasional -4- granules but no visible nucleolus. Histograms of flow cytometric analysis determined that 98% (range, 98– 99%) of isolated cells expressed CD14 (and CD45, data not shown). (B) After megakaryocyte culture, analysis of stained cytospins and modified Wright’s stain showed that the majority of cells were progenitor cells (megakaryoblasts, MB). MK cultures also contained pro-megakaryocytes (Pro-MK, horseshoe-shaped nucleus) and mature MKs (multi-nucleated) (Zeuner et al. 2007). Flow cytometric characterization revealed that 77% (range, 71–83%) of cells expressed CD41a and 44% (range, 28–59%) also