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Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification.



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Wilkinson, Adam C 
Kawata, Viviane KS 
Schütte, Judith 
Gao, Xuefei 
Antoniou, Stella 


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.



Haematopoiesis, PU.1, Regulatory networks, Transcription activator-like effectors, Animals, Cell Differentiation, Coculture Techniques, Endothelial Cells, Gene Expression Profiling, Gene Expression Regulation, Developmental, Hematopoiesis, Hematopoietic Stem Cells, Humans, K562 Cells, Mice, Mice, Transgenic, Phenotype, Proto-Oncogene Proteins, Single-Cell Analysis, Trans-Activators, Transcription Factors, Transgenes, Xanthomonas

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The Company of Biologists
Wellcome Trust (100140/Z/12/Z)
Biotechnology and Biological Sciences Research Council (BB/I00050X/1)
Leukaemia & Lymphoma Research (12029)
BBSRC (via University of Birmingham) (12-0401; 12-0418)
Leukaemia & Lymphoma Research (7060)
Cancer Research Uk (None)
Medical Research Council (G0900951)
Wellcome Trust (097922/Z/11/Z)
Medical Research Council (MC_PC_12009)
Leukemia & Lymphoma Society (7001-12)
British Heart Foundation (None)
Medical Research Council (G0900951/1)
Research in the authors’ laboratories was supported by Leukaemia and Lymphoma Research, The Wellcome Trust, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, the National Institute of Health Research, the Medical Research Council and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute. V.K.S.K. was supported by a Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists.