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.

Mammalian Transcription Factor Networks: Recent Advances in Interrogating Biological Complexity

“…Synthetic TFs such as Zinc Fingers, Transcription Activator-Like Effectors (TALEs), and Cas9, can also be used to perturb endogenous enhancer activity and study TF networks. This is achieved by fusing genome-specific synthetic DNA binding domains to transcriptional effector domains such as the VP64 (activator) and KRAB (repressor) domains (Gao et al, 2013, Wilkinson et al, 2014). These approaches provide an opportunity to inducibly activate or silence enhancers, or synthetically engineer more complex transcriptional circuitry.…”

“…PetriNets, a mathematical modeling approach to graphically model networks, have also been successfully used to computationally encode TF networks (Bonzanni et al, 2013). Alternative methods have been used to “reverse engineer” networks from gene expression data, such as by using mutual information or partial correlation analysis including the ARACNE algorithm (Margolin et al, 2006), and have been recently applied to single cell gene expression data (Wilkinson et al, 2014, Moignard et al, 2015). We refer readers to recent reviews of TF network modeling for further information (Woodhouse et al, 2016, Le Novère, 2015).…”

Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors

“…4g). The transcription factors involving hematopoietic development, such as Lyl1 47 , Etv6 48 , Prdm5 9 , Myb 49 , Sfpi1 50–52 , and Meis1 53 , were widely expressed among day-14, 17, and 21 iHPC populations. (Fig.…”

The RUNX1–PU.1 axis in the control of hematopoiesis

“…PU.1 has been shown to play a role also in early committing blood precursor stages. TALE-effectormediated PU.1 repression in a murine ESC differentiation confirmed the presence of PU.1 at sites of mouse definitive hematopoiesis [35]. However, using the same experimental model, Lancrin et al [36] investigated the capability of the major downstream targets of RUNX1, i.e., GFI1, GFI1B and PU.1, to rescue the defective phenotype of Runx1…”