Nonredundant roles for Runx1 alternative promoters reflect their activity at discrete stages of developmental hematopoiesis

Abstract

The transcription factor Runx1 is a pivotal regulator of definitive hematopoiesis in mouse ontogeny. Vertebrate Runx1 is transcribed from 2 promoters, the distal P1 and proximal P2, which provide a paradigm of the complex transcriptional and translational control of Runx1 function. However, very little is known about the biologic relevance of alternative Runx1 promoter usage in definitive hematopoietic cell emergence. Here we report that both promoters are active at the very onset of definitive hematopoiesis, with a skewing toward the P2. Moreover, functional and morphologic analysis of a novel P1-null and an attenuated P2 mouse model revealed that although both promoters play important nonredundant roles in the emergence of definitive hematopoietic cells, the proximal P2 was most critically required for this. The nature of the observed phenotypes is indicative of a differential contribution of the P1 and P2 promoters to the control of overall Runx1 levels, where and when this is most critically required. In addition, the dynamic expression of P1-Runx1 and P2-Runx1 points at a requirement for Runx1 early in development, when the P2 is still the prevalent promoter in the emerging hemogenic endothelium and/or first committed hematopoietic cells. (Blood. 2010;115(15): 3042-3050) IntroductionThe generation of the definitive hematopoietic system during embryogenesis critically depends on the transcription factor Runx1. In mice, homozygous loss of Runx1 function results in embryonic lethality attributable to a complete lack of functional definitive hematopoietic stem cells (HSCs) and progenitor cells and hemorrhages in the central nervous system. 1-3 Runx1 belongs to the family of runt-domain transcription factors. The 3 mammalian members of this family, Runx1, 2, and 3, all are important developmental regulators and bind to the same DNA motif. 4 Although both Runx2 and Runx3 have been implicated in hematopoiesis, only Runx1 has a role in the emergence of definitive hematopoietic cells, 5 reflecting its specific expression at hemogenic sites. 6,7 Recently, it was shown that Runx1 is required in VE-cadherin ϩ cells of the embryo, within the developmental window that starts with the initiation of Runx1 expression in these cells and ends when/before definitive HSCs reach the embryonic day (E) 11 fetal liver (FL). 8 Although the precise developmental stage(s) at which Runx1 is required within this window remains to be determined, it is generally believed to be at the transition of hemogenic endothelium to definitive hematopoietic cells. 6,[8][9][10] In the adult, Runx1 is no longer critically required in HSCs, although it still plays important roles in maintaining hematopoietic homeostasis and in the generation of specific hematopoietic cells/lineages. [11][12][13] Not only the expression pattern of Runx1 but also its levels need to be tightly controlled for the normal emergence of HSCs in the embryo. 3 To gain insight into how this is achieved, we have initiated studies into the transcriptional regulation of Runx1. 14,15...

A short history of hemogenic endothelium

“…Combination of VECadh or CD31 with the hematopoietic markers CD41 [40][41][42][43], CD45 [44] and Ter119 [45] has been used to separate endothelial from hematopoietic cells (e.g. [38,[46][47][48]), and other identification strategies have also been employed e.g. [49] [50].…”

“…This enhancer mediates reporter gene expression specifically to sites of HSPC emergence and colonization, but not to other areas of Runx1 expression later in development. In enhancerreporter transgenic mice, hematopoietic cells are marked throughout development [48,58]. In addition, we recently showed that this +23 enhancer-reporter transgene marks all hemogenic endothelium in the embryo, thus providing us with a unique marker to isolate hemogenic endothelium for detailed functional and molecular studies.…”

“…However, the expression of smooth muscle actin (SMA) in these cells argues that the majority of the peri-aortic Runx1 + cells are smooth muscle ([75]; Figure 2b), though a very small number may be the recently identified pre-HSCs [59]. In the absence of Runx1 there is a compaction of the cell layers ventral to the dorsal aorta [36,48], suggesting that Runx1 plays a role in the migration/organization of the smooth muscle cell layer. Interestingly, Runx1 affects the expression of a negative regulator of hematopoiesis, Dlk1 in the AGM smooth muscle cells [75].…”

Runx1/Cbfβ2 Complexes Are Required for Lymphoid Tissue Inducer Cell Differentiation at Two Developmental Stages

“…r gene (the Runx1 P1N allele) (26). Although NKT cell development was almost abolished in the Runx1 P1N/P1N mice, as reflected in the conditional inactivation of the Runx1 gene in CD4 + CD8 + double-positive thymocytes (21), other phenotypes, such as thymic hypocellularity, CD4 derepression in doublenegative thymocytes, and the reduction of CD4 + T cell numbers, were milder than those caused by inactivation of Runx1 gene (Supplemental Fig.…”

“…In , and Runx1 PIN/P1N mice have been described previously (15,18,26). The generation of Cbfb 2m/2m mice by homologous recombination in embryonic stem cells will be described elsewhere.…”