VWRPY motif–dependent and –independent roles of AML1/Runx1 transcription factor in murine hematopoietic development

Abstract

AML1/Runx1 is a frequent target of leukemia-associated gene aberration, and it encodes a transcription factor essential for definitive hematopoiesis. We previously reported that the AML1 molecules with trans-activation subdomains retained can rescue in vitro hematopoietic defects of AML1-deficient mouse embryonic stem (ES) cells when expressed by using a knock-in approach. Extending this notion to in vivo conditions, we found that the knock-in ES cell clones with AML1 mutants, which retain trans-activation subdomains but lack C-terminal repression subdomains including the conserved VWRPY motif, contribute to hematopoietic tissues in chimera mice. We also found that germline mice homozygous for the mutated AML1 allele, which lacks the VWRPY motif, exhibit a minimal effect on hematopoietic development, as was observed in control knock-in mice with full-length AML1. On the other hand, reduced cell numbers and deviant CD4 expression were observed during early T-lymphoid ontogeny in the VWRPY-deficient mice, whereas the contribution to the thymus by the corresponding ES cell clones was inadequate. These findings demonstrate that AML1 with its trans-activating subdomains is essential and sufficient for hematopoietic development in the context of the entire mouse. In addition, its transrepression activity, depending on the Cterminal VWRPY motif, plays a role in early thymocyte development. IntroductionVertebrate hematopoietic development is characterized by the sequential appearance of two cell populations, further defined as primitive and definitive hematopoiesis. 1 In mice, for example, primitive hematopoiesis is first seen in the form of blood islands in the yolk sac of the 7.5-day-old (E7.5) mouse embryo. It is thought that this cell population is directly differentiated from hemangioblasts, a bipotent precursor. Primitive hematopoiesis consists predominantly of a large and nucleated erythroid population containing embryonic-type hemoglobin. In contrast to the restricted and temporal development of this first wave, which diminishes at midgestation, definitive hematopoiesis originates from the aorta-gonad-mesonephros (AGM) region, where stem cells with long-term repopulating ability of multilineage hematopoiesis emerge at approximately E9.5, as a result of budding, from the ventral endothelial cells of the great vessels. 2,3 The stem cells then migrate into the fetal liver and proliferate to rapidly establish the definitive hematopoiesis of all lineages, including progenitors for T-and B-lymphoid populations. Active sites for definitive hematopoiesis are transferred to bone marrow and spleen before birth and function throughout life within these organs.These stem cells are equipped with a number of critical transcription factors that play pivotal roles in determining the fate of the cells at discrete developmental stages. Most of these molecules have been identified by isolating DNA-binding proteins to known cis-regulatory elements of lineage-specific genes or by cloning the DNA targets of leukemia-associated chrom...

Functional Domains of Runx1 Are Differentially Required for CD4 Repression, TCRβ Expression, and CD4/8 Double-Negative to CD4/8 Double-Positive Transition in Thymocyte Development

“…Because Runx1 mutants that lack the VWRPY motif could fully restore hematopoiesis in Runx1-deficient cells in these two studies, the VWRPY motif does not seem to be necessary for hematopoiesis. On the contrary, because mice in which cDNA for the VWRPY-deficient Runx1 mutant had been homozygously knocked-in to the Runx1 alleles exhibited a reduced number of thymocytes and deviant CD4 expression during thymocyte ontogeny (27), the VWRPY motif seems to play a role in thymocyte development, although the precise molecular mechanism is unclear. In the present study, although the VWRPY-deficient Runx1 mutant (⌬447) could restore not only maturation to the DN4 subset but also TCR␤ expression in cko FL-derived thymocytes as efficiently as wild-type Runx1 (Fig.…”

“…Okuda et al (26) examined the ability of fulllength and mutant Runx1 genes to rescue the hemopoietic defect in Runx1-deficient embryonic stem cells through a knock-in approach and demonstrated that the activation domain, but not the VWRPY motif, is indispensable for definitive hematopoiesis. No alterations in thymocyte subpopulations were detected in mice in which the VWRPY motif of Runx1 is genetically disrupted, although they have a significantly small thymus (27). In their study, the roles of the activation domain during thymocyte development were not assessed, due to a profound defect in hematopoiesis in the absence of the activation domain of Runx1.…”

Mechanisms of Compartmentalized Expression of Mrg Class G-Protein-Coupled Sensory Receptors

“…The generation of Runx1 conditional mutant mice, Wnt1-Cre transgenic mice, ⌬446 mice, Smad4 conditional null mice, MrgD-GFP mice and SNS-Cre transgenic mice has been described previously (Jiang et al, 2000;Yang et al, 2002;Agarwal et al, 2004;Nishimura et al, 2004;Growney et al, 2005;Zylka et al, 2005). The morning that vaginal plugs were observed was considered as E0.5.…”

“…Runx1 therefore can act as either a transcriptional repressor or activator (Durst and Hiebert, 2004). To determine whether Runx1 repressor activity contributes to nociceptor phenotype specification, including compartmentalized expression of Mrg genes, we analyzed ⌬446 mice (Nishimura et al, 2004). ⌬446 encodes a truncated Runx1 protein that lacks the C-terminal repression motif, the VWRPY peptide, but retains the activation domain (Fig.…”

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription

“…RUNX1 recruits the mSin3A co-repressor through a domain adjacent to the DNA-binding domain (repression domain 1) (Lutterbach et al, 2000). In addition, RUNX1 contacts Groucho family members to repress transcription in a promoter-dependent manner through a C-terminal motif termed repression domain 3 (Aronson et al, 1997;Levanon et al, 1998;Javed et al, 2000;Lutterbach et al, 2000;Nishimura et al, 2004). Repression domain 2 was simply defined as an approximately 100 amino-acid sequence that was required for repression (Lutterbach et al, 2000).…”