The mouse Runx1 +23 hematopoietic stem cell enhancer confers hematopoietic specificity to both Runx1 promoters

Abstract

The transcription factor Runx1 plays a pivotal role in hematopoietic stem cell (HSC) emergence, and studies into its transcriptional regulation should give insight into the critical steps of HSC specification. Recently, we identified the Runx1 ؉23 enhancer that targets reporter gene expression to the first emerging HSCs of the mouse embryo when linked to the heterologous hsp68 promoter. Endogenous Runx1 is transcribed from 2 alternative promoters, P1 and P2. Here, we examined the in vivo cis-regulatory potential of these alternative promoters and asked whether they act with and contribute to the spatiotemporal specific expression of the Runx1 ؉23 enhancer. Our results firmly establish that, in contrast to zebrafish runx1, mouse Runx1 promoter sequences do not confer any hematopoietic specificity in transgenic embryos. Yet, both mouse promoters act with the ؉23 enhancer to drive reporter gene expression to sites of HSC emergence and colonization, in a ؉23-specific pattern. (Blood. 2009;113:5121-5124) IntroductionThe transcription factor RUNX1 is a critical regulator of definitive hematopoiesis, and genomic aberrations of the gene encoding RUNX1 are frequently found in human acute leukemia. 1 In the mouse, Runx1 null mutations result in the absence of functional hematopoietic stem cells (HSCs) and definitive progenitors, leading to embryonic lethality. [2][3][4][5][6] During development, Runx1 is first expressed in the emerging hematopoietic system, including definitive HSCs. 7,8 Its highly regulated spatiotemporal expression pattern and pivotal role in HSC emergence prompted us to study its transcriptional regulation, to obtain insight into the molecular mechanisms underlying de novo HSC generation. We recently identified the Runx1 ϩ23 hematopoietic enhancer, located 23.5 kb downstream of the ATG in exon 1. 9 We showed that this ϩ23 enhancer targets reporter gene expression, from a heterologous hsp68 core promoter, to the emerging HSCs and putative HSCfated cells in the mouse embryo, and acts directly downstream of Gata2, SCL, and Ets transcription factors. Whether the ϩ23 enhancer is equally active with the endogenous Runx1 promoters has not been assessed.Runx1 is transcribed from 2 alternative promoters ( Figure 1A), a distal P1 and proximal P2, with the P1 being specific to vertebrates. [10][11][12][13] Both the P1 and P2 promoters were reported to be transcriptionally active in the emerging hematopoietic system of the mouse embryo, at the stages of yolk sac (YS), aortagonad-mesonephros (AGM), and fetal liver (FL) hematopoiesis, with P1-derived transcripts particularly prevalent among enriched FL HSCs. 11,14,15 The P2 promoter was shown to be active in HSC-fated cells 16 and to be critically required for FL hematopoiesis. 17 In vitro transfection assays suggested that neither P1 nor P2 RUNX1 promoter elements harbored tissue-specific cis-regulatory elements. 10 However, in vivo mouse promoter assays have not been reported, and it is therefore not clear to what extent cis-elements elsewhere in the locus are req...

A Runx1-Smad6 Rheostat Controls Runx1 Activity during Embryonic Hematopoiesis

“…It is significant in this regard that the signaling Smads, Smad1 and Smad5, no longer are required for HSC maintenance after these cells have been specified (34). Runx1 levels in circulating HSCs likely are maintained by the activity of Fli1-Gata2-Scl on Runx1 regulatory elements such as the Runx1ϩ23 enhancer and promoter (3). The role of the Runx1-Smad6 rheostat would be to stabilize Runx1 levels within a set range during hematopoiesis.…”

“…At a transcriptional level, the Runx1ϩ23 enhancer mediates expression in the developing hematopoietic system in conjunction with the distal and/or proximal Runx1 promoter (3). Feed-forward loops involving upstream TFs, such as Gata2, Fli1, and Scl, upregulate Runx1 expression in cells during hematopoietic commitment (16,23).…”

Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo

“…In our study, the hematopoietic defect of gata2b morphants is partially rescued by provision of runx1 mRNA, suggesting that loss of runx1 accounts for the hematopoietic phenotype observed with gata2b knockdown. Hemogenic endothelial expression of Runx1 is governed by an intronic enhancer element that contains consensus sites for Runx, Cmyb, Gata, ETS and E-box transcription factors (Bee et al, 2009;Nottingham et al, 2007), but not for the Notch partner RBPjκ, suggesting that Notch signaling might be required through the induction of intermediate transcription factors to promote Runx1 expression in hemogenic endothelium. The GATA binding site is crucial for the activity of this enhancer (Nottingham et al, 2007).…”

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

“…P1 is bound and repressed by RUNX1 itself [44,[105][106][107], while in T-cells P2 is activated by NFAT binding [108]. To date, an enhancer has been characterized at +23 kb in the Runx1 locus, conferring hematopoietic specificity to both isoforms [109]. Runx1 expression is controlled by TAL1/SCL via binding at the +23 kb enhancer [96,110].…”