title: Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification.
abstract: Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs, including Scl (Tal1) and PU.1 (Spi1), are known regulators of developmental and adult haematopoiesis, but how they act within wider TF networks is still poorly understood. Transcription activator-like effectors (TALEs) are a novel class of genetic tool based on the modular DNA-binding domains of Xanthomonas TAL proteins, which enable DNA sequence-specific targeting and the manipulation of endogenous gene expression. Here, we report TALEs engineered to target the PU.1-14kb and Scl+40kb transcriptional enhancers as efficient new tools to perturb the expression of these key haematopoietic TFs. We confirmed the efficiency of these TALEs at the single-cell level using high-throughput RT-qPCR, which also allowed us to assess the consequences of both PU.1 activation and repression on wider TF networks during developmental haematopoiesis. Combined with comprehensive cellular assays, these experiments uncovered novel roles for PU.1 during early haematopoietic specification. Finally, transgenic mouse studies confirmed that the PU.1-14kb element is active at sites of definitive haematopoiesis in vivo and PU.1 is detectable in haemogenic endothelium and early committing blood cells. We therefore establish TALEs as powerful new tools to study the functionality of transcriptional networks that control developmental processes such as early haematopoiesis.
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Adam C
authors.initials: AC
authors.lastname: Wilkinson
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
authors.forename: Viviane K S
authors.initials: VK
authors.lastname: Kawata
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Judith
authors.initials: J
authors.lastname: Schütte
authors.affiliation: Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK.
authors.forename: Xuefei
authors.initials: X
authors.lastname: Gao
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Stella
authors.initials: S
authors.lastname: Antoniou
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Claudia
authors.initials: C
authors.lastname: Baumann
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Steven
authors.initials: S
authors.lastname: Woodhouse
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Hannah
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Gemma
authors.initials: G
authors.lastname: Swiers
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Victoria
authors.initials: V
authors.lastname: Moignard
authors.affiliation: Microsoft Research Cambridge, 21 Station Road, Cambridge CB1 2FB, UK Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Fisher
authors.affiliation: Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575, Japan.
authors.forename: Shimauchi
authors.initials: S
authors.lastname: Hidetoshi
authors.affiliation: Department of Haematology, University of Cambridge and National Health Service Blood and Transplant, Cambridge CB2 0PT, UK.
authors.forename: Marloes R
authors.initials: MR
authors.lastname: Tijssen
authors.affiliation: MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, UK.
authors.forename: Marella F T R
authors.initials: MF
authors.lastname: de Bruijn
authors.affiliation: Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK.
authors.forename: Pentao
authors.initials: P
authors.lastname: Liu
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK bg200@cam.ac.uk.
authors.forename: Berthold
authors.initials: B
authors.lastname: Göttgens
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006410:Hematopoiesis / Q000502:physiology*; D006412:Hematopoietic Stem Cells; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008822:Mice, Transgenic; D010641:Phenotype; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D059010:Single-Cell Analysis; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D019076:Transgenes; D014974:Xanthomonas / Q000378:metabolism; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1
keywords: Haematopoiesis; PU.1; Regulatory networks; Transcription activator-like effectors