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, CBF␤, 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 PEBP2␣B1 (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.Hematopoietic development in mammals is supported by two discrete cellular populations which are believed to be derived from distinct cellular origins (5, 22). In the mouse, for example, the first wave of primitive hematopoiesis emerges around day 7.5 postcoitus (E 7.5) in the yolk sac and consists primarily of large nucleated primitive erythrocytes, which diminish midgestation. By contrast, around E 9.5, the second wave of hematopoiesis emerges in the fetal liver and consists of so-called definitive hematopoietic cells, including enucleated erythrocytes containing adult-type globin molecules, myeloid cells, and lymphoid progenitors. The site of this second wave of hematopoiesis is subsequently shifted to the bone marrow and spleen prior to birth.Recent studies have revealed that a number of transcriptional factors play critical roles in regulating the fate of the hematopoietic stem cell populations. These factors are divided into two groups: one contains factors which regulate both primitive and definitive hematopoiesis, and the other contains factors whose activities are required for the development of some or all of the definitive hematopoietic lineages (reviewed in reference 44). The acute myeloid leukemia (AML) 1 gene, AML1, belongs to the latter group and is unique in that loss of this gene in mice results in the complete absence of all definitive hematopoietic lineages (37, 51).AML1 was originally cloned from the breakpoint of chromosome 21 in t(8;21)(q22;q22), which is associated with 40% of the AML cases of the French-British-American classification M2 subt...

Hematopoietic cytokines, transcription factors and lineage commitment

“…The rescue of AML-1 de®cient ES cells by knocking-in of a wild type AML-1b restored their ability to contribute to the formation of every lineage of hematopoietic cells in chimeric mice (Okuda et al, 2000). In vitro rescue experiment of the de®cient ES cells by the retroviral vectors carrying a series of AML1b mutants indicated that a 61aa of C-terminal region containing a VWRPY motif, required for interacting with transcriptional co-repressors, was not required for the de®nitive hematopoiesis (Okuda et al, 2000). Taken together, these results suggest that the transcriptional activation, rather than repression of target genes by AML-1 is required for de®nitive hematopoiesis.…”

Cooperating gene mutations in acute myeloid leukemia: a review of the literature

“…[114][115][116][117] Mice lacking AML1 or CBFb have no fetal liver hematopoiesis, showing that the heterodimeric complex CBF is essential for definitive hematopoiesis of all lineages. [118][119][120][121] AML1 is one of the genes most frequently deregulated in leukemia mainly through chromosomal translocations, point mutations and amplifications. Chromosomal translocations involving AML1 gene occur in different leukemia subtypes, leading to the formation of fusion genes encoding for chimeric proteins, including AML1-ETO (t(8;21)) in AML, AML1-ETV6 (t(12;21)) in childhood acute lymphoblastic leukemia and less often AML1-MDS1 (t(3;21)) in MDS and blastic phase of chronic myeloid leukemia (CML), or other rare translocations.…”

New mechanisms of AML1 gene alteration in hematological malignancies

“…For example, by AML1-ETO knock in strategy it was shown that mice heterozygous for AML1-ETO had a similar phenotype to mice lacking AML1 or CBF␤ indicating that AML1-ETO blocked normal AML1 functions. 16 It has also been shown that the coiled-coil region of ETO could oligomerize with AML1-ETO protein, thus recruiting the nuclear corepressor N-CoR responsible for transcriptional repression of AML1 target genes and resulting in impaired differentiation of primary hematopoietic precursors. 17,18 Recently, we and others have reported other mechanisms of inactivation of AML1 in hematological malignancies, through point mutations of the gene in AML and in MDS [19][20][21][22][23][24][25] or through gene amplification, [26][27][28][29][30][31][32] a rare event mainly observed in acute lymphoblastic leukemia (ALL).…”