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

Adam C. Wilkinson, Viviane K. S. Kawata, Judith Schütte, Xuefei Gao, Stella Antoniou, Claudia Baumann, Steven Woodhouse, Rebecca Hannah, Yosuke Tanaka, Gemma Swiers, Victoria Moignard, Jasmin Fisher, Shimauchi Hidetoshi, Marloes R. Tijssen, Marella F. T. R. de Bruijn, Pentao Liu, Berthold Göttgens
Source: Development
Publication Date: (2014)
Issue: 141(20): 4018-30
Research Area:
Basic Research
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Ainv18 ESCs (Kyba et al., 2002) were cultured as described previously (Ismailoglu et al., 2008) and transfected by nucleofection (Lonza).
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.