Biological characteristics of the leukemia-associated transcriptional factor AML1 disclosed by hematopoietic rescue of AML1-deficient embryonic stem cells by using a knock-in strategy

Abstract

AML1 is one of the most frequently mutated genes associated with human acute leukemia and encodes the DNA-binding subunit of the heterodimering transcriptional factor complex, core-binding factor (CBF) (or polyoma enhancer binding protein 2 [PEBP2]). A null mutation in either AML1 or its dimerizing partner, CBFbeta, results in embryonic lethality secondary to a complete block in fetal liver hematopoiesis, indicating an essential role of this transcription complex in the development of definitive hematopoiesis. The hematopoietic phenotype that results from the loss of AML1 can be replicated in vitro with a two-step culture system of murine embryonic stem (ES) cells. Using this experimental system, we now demonstrate that this hematopoietic defect can be rescued by expressing the PEBP2alphaB1 (AML1b) isoform under the endogenous AML1-regulatory sequences through a knock-in (targeted insertion) approach. Moreover, we demonstrate that the rescued AML1(-/-) ES cell clones contribute to lymphohematopoiesis within the context of chimeric animals. Rescue requires the transcription activation domain of AML1 but does not require the C-terminal VWRPY motif, which is conserved in all AML1 family members and has been shown to interact with the transcriptional corepressor, Groucho/transducin-like Enhancer of split. Taken together, these data provide compelling evidence that the phenotype seen in AML1-deficient mice is due solely to the loss of transcriptionally active AML1.

FIG. 1

Rescue strategy of AML1-deficient ES cells by expressing PEBP2αB1 (AML1b) cDNA from a knock-in allele. (A) The AML1 loci of the ES cell lines were disrupted by targeting exon 4, which encodes the middle of the Runt domain; one allele was replaced by a hygromycin resistance cassette [KO(hygr)] and the other by a neomycin resistance cassette [KO(neo)] (37). To rescue the AML1-deficient ES cell phenotype, we designed a replacement-type vector (knock-in vector) which creates an artificial allele [KI(puro)] that expresses the PEBP2αB1 isoform of the AML1 protein in place of the disrupted exon 4 by means of homologous recombination. Clones which had undergone targeted insertion at either of the hygromycin or neomycin alleles were detected by Southern blot analyses with a 5′ outside probe (B) and 3′ outside probe (C), in which the knock-in alleles were detectable as XbaI-restricted fragments of ca. 11 and 7 kb, respectively. Abbreviations: hygr, hygromycin B resistance cassette; neo, neomycin resistance cassette; puro, puromycin resistance cassette; DT-A, diphtheria toxin-A suicide cassette. Arrows above selection cassettes indicate the orientation of the transcription. (D) Expression of the AML1 message in the knock-in ES clones was confirmed by RT-PCR analysis, which amplified the 292-bp fragment of the cDNA with a primer pair corresponding to exons 3 and 4 of the gene locus (top panel). Parallel amplification for the HPRT gene (bottom panel) demonstrates the integrity of the RNA samples obtained from the undifferentiated ES cell clones.

FIG. 2

Results of the EB differentiation experiments of knock-in clones. (A) Appearance of the representative day 14 EBs derived from the ES cell clones of AML1^+/− and AML1^−/− and knock-in with wild-type AML1b (AML1^−/KI) genotypes. AML1^+/− ES cell clones formed EBs, the majority of which was surrounded by hematopoietic cells (upper left panel) consisting primarily of mature macrophages and a few erythroid and granulocytic cells (lower left panel). EBs derived from AML1^−/− clones developed no such cells (center). In contrast, knock-in ES clones (AML1^−/KI) formed hematopoietic cells around the EBs (upper right panel), consisting of macrophages with occasional granulocytes and erythrocytes (lower right panel). The morphology of the cells derived from the knock-in clones was indistinguishable from that of cells developed from control AML1^+/− clones. Original magnifications: upper panels ×20, lower panels, ×132. (B) Incidence of hematopoietic differentiation of the day 14 EBs derived from ES cell clones of AML1^+/−, AML1^−/−, AML1^−/− with a knock-in AML1b at the hygromycin allele [AML1^−/KI(hygr)], and AML1^−/− with a knock-in AML1b for the neomycin allele [AML1^−/KI(neo)] genotypes in a representative experiment. Dark bars indicate the percentage of the EBs with visible hematopoietic cells whereas light bars represent those without a hematopoietic element. Actual numbers of the EBs grown per 300 ES cells are given above each bar as the mean ± standard deviation of triplicate cultures.

FIG. 3

Results of the two-step differentiation experiments of the knock-in clones. (A) Schematic representation of the method used in the present study to induce the hematopoietic differentiation of ES cells. When ES cells are cultured to form EBs in semisolid media without feeder cells or leukemia inhibitory factor, they develop hematopoietic cells of primitive and definitive origins, which are detectable by second-step cultures in the presence of appropriate CSF. In contrast, when AML1^−/− ES cells were subjected to this analysis, they failed to develop hematopoietic cells of definitive origin. (B) In vitro differentiation of ES cell clones of each of the AML1 genotypes into hematopoietic precursors in a representative experiment. Colonies of Ery-P, definitive erythroid (Ery-D), and granulocyte-macrophage and macrophage (Myeloid), as well as mixed lineages including definitive erythroid (E-Mix), were scored in a two-step replating assay. ES cells were cultured to form EBs and then disrupted on day 6 for Ery-P and on day 10 for definitive precursors, after which 10⁵ cells were subjected to second-step cultures with appropriate CSF (see Materials and Methods). Open bars, grey bars, and dark bars represent AML1^+/−, AML1^−/−, and AML1^−/− with knock-in clones, respectively. (C) Representative colonies of the primitive erythroid lineage observed in the two-step replating assay with EBs on day 6 of culture. The morphology of the colonies in situ (top panels) and of composing primitive erythroid cells stained with May-Gruenwald-Giemsa staining (lower panels) was almost identical among the clones. Magnifications: top panels, ×50; bottom panels, ×330. (D) Representative hematopoietic colonies of definitive origin observed in the second-step cultures of day 10 EBs derived from AML1^+/− and knock-in clones. As shown, the appearance of the colonies and the morphology of the composing cells of definitive erythroid (a), macrophage (b), granulocyte-macrophage (c), and mixed lineages including definitive erythroid (d) of the rescued knock-in clones (AML1^−/KI) were indistinguishable from those observed in the cultures of the control heterozygous clones (AML1^+/−). Magnifications: upper panels of a, ×50; upper panels of b to d, ×20; lower panels, ×198.

FIG. 4

Contribution of AML1^−/−, AML1^+/−, and AML1^−/KI ES cells to tissues in chimeric mice. Representative results for the GPI analysis of chimeric mice derived from AML1^+/− (A) and AML1^−/− (B) and knock-in clones for the hygromycin allele [AMLl−/KI(hygr)] (C) and neomycin allele [AML1−/KI(neo)] (D). ES cells contain the GPI-A isoform as indicated in lanes 8 and 16, whereas the host contains the GPI-B isoform. Results from the following tissues are shown: brain (Br), kidney (K), thymus (T), liver (L), spleen (Sp), bone marrow (BM), and peripheral blood (PB), and ES cells (ES) were controls. WT, wild type.

FIG. 5

Biological properties of the C-terminal deletion mutants of AML1b. (A) Schematic representation of the module structure according to Kanno et al. (14) of the wild-type PEBP2αB1 (AML1b) and each mutant analyzed. (B) The integrity of the mutant constructs was confirmed by Western blot analysis of transiently transfected COS-7 cells. (C) Expression of the exogenous genes in ES cells were confirmed by RT-PCR analysis with primers from exons 3 and 4 (see the legend for Fig. 1D). (D) Hematopoietic differentiation of AML1^−/− ES knock-in clones expressing the different AML1b mutants in a representative experiment. Dark bars indicate the percentage of the EBs with visible hematopoietic cells whereas light bars represent those without a hematopoietic element. Actual numbers of the EBs grown per 300 ES cells are given above each bar as the mean ± standard deviation of triplicate cultures.