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.
Highlights
Transcriptions factors (TFs) are key regulators of cell identity and fate
Design and validation of Transcription activator-like effectors (TALEs) targeting conserved regions within haematopoietic enhancers We identified regions within the Scl+40kb and PU.1-14kb elements that were perfectly conserved between human and mouse
Transgenic mouse studies confirmed that the PU.1-14kb enhancer is active at mid-gestation in the dorsal aorta (DA), where the endothelial-to-haematopoietic transition (EHT) occurs in vivo, and that PU.1 expression can be detected from the endothelium stage of this process
Summary
Cell type-specific transcriptional regulation is thought to largely occur by TF binding to distal cis-regulatory elements (Heinz et al, 2010). Two well-studied examples of such haematopoietic cisregulatory elements are the PU.1-14kb (Rosenbauer et al, 2004; Okuno et al, 2005; Huang et al, 2008; Staber et al, 2013) and Scl +40kb (Delabesse et al, 2005; Ogilvy et al, 2007; Ferreira et al, 2013). The Scl+40kb element is thought to regulate expression of the 3′ flanking gene, Map (Pdzk1ip1) (Tijssen et al, 2011; Ferreira et al, 2013)
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