Dev115709 1..13

Transcription factors (TFs) act withinwider 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 novelclassofgenetic toolbasedon themodularDNA-bindingdomainsof 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 bothPU.1 activation and repressiononwider TFnetworks 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 andPU.1 is detectable in haemogenicendotheliumandearly 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.