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

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.

Regulation of proliferation and differentiation of mouse tooth germ epithelial cells by distinct isoforms of p51/p63.

OBJECTIVES: p51/p63 gene, one of the p53 families, is specifically expressed in tooth germ epithelial cells and is essential for tooth development. This study aims to elucidate roles of p51/p63 in ameloblastic cell differentiation. MATERIALS AND METHODS: We determined expression pattern of each of p51/p63 isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting using emtg (epithelium of molar tooth germ)-1, -2, -3, -4, and -5 cell lines established from a mandibular molar tooth germ of p53-deficient mice and SF2 cells which differentiates into ameloblasts upon exposure to NT4. Furthermore, we investigated the function of p51/p63 in these cells by Tet system, which enables inducible expression and knock down of the target genes of interest by exposing cells to doxycycline. RESULTS: The expression of ΔNp51B/ΔNp63α, an isoform without transactivation domain, was detected at high level in immature cells, while the expression of TAp51/TAp63 isoforms, isoforms of with the transactivation domain, was detected at high level in mature cells. Moreover, induction of TAp51A/TAp63γ expression led to down-regulation of ΔNp51B/ΔNp63α expression and cell proliferation. Interestingly, this also led to up-regulation of ameloblastin expression, a differentiation marker of amelogenesis. CONCLUSIONS: The results suggested that p51/p63 might regulate the cell proliferation and differentiation of tooth germ epithelial cells.