Abstract
Abstract 1309
Acute Myeloid Leukemia 1 (AML1; also called as Runx1: Runt-related transcription factor 1) belongs to the Class II group of leukemia-associated mutation-target genes, and it encodes the DNA-binding subunit of the hetero-dimeric transcription factor complex, Core-Binding Factor (CBF). CBF plays pivotal roles in initial hematopoietic development during embryogenesis and in cellular differentiation of thrombotic and lymphatic lineages throughout adult life. Recent researches revealed that cellular AML1 polypeptide is processed with post-translational modifications, including phosphorylation, acethylation, ubiquitination, and methylation. Biological significance of these modifications on the AML1's function as the hematopoietic regulator, however, largely remains to be elucidated. In this study, we focused on the arginine-methylation as an initial step towards the comprehensive understanding for the AML1-regulating mechanism through these modifications. Arginine residues just downstream to the Runt-domain, which is located at N-terminal region of the molecule and functions as the binding site to DNA and CBF beta: the hetero-dimerization partner, are recently reported to be methylated, resulting in the inhibition of the corepressor-binding thus enhancing its trans- activating activity. In order to elucidate biological significance of these methylations, we performed a series of genetic experiments: First, we generated the non-methylatable double arginine-to-lysine (RRKK) mutant of AML1 at these residues, which should keep AML1-corepressor-binding. When this mutant was subjected to the luciferase reporter-assay using a target-gene construct, it showed lower trans- activating activity in comparison to that for wild-type molecule, as expected. However, this loss-of-function mutation appeared to be dispensable at least for in vitro function for hematopoietic regulation in that this RRKK mutant did rescue hematopoietic differentiation of the AML1-deficient murine ES cells in culture when expressed from a knock-in allele as was the case for the wild-type cDNA of mouse AML1. To further evaluate the biological activity of this mutant in the context of an entire animal, we introduced this mutant cDNA into AML1/Runx1 locus of mouse ES cells by means of a targeted-insertion (knock-in) strategy. Germline mutant mouse lines were successfully established, following blastocyst-injection of these ES cell clones. Heterozygous mice were healthy and fertile, and genotyping for the live pups generated from heterozygotes-crossing revealed that this arginine-mutant allele segregated according to the Mendelian ratio. Homozygous AML1RRKK/RRKK mice were born alive and grew up adult, circumventing the mid-embryonic death due to hematopoietic block that was originally described for the AML1-deficient mice, thus the in vitro notion that these arginine-methylations were not essential for the early hematopoietic development described above was further underscored. There were no significant differences so far observed in peripheral blood cell counts among mice of the AML1RRKK/RRKK or AML1WT/WT genotypes, in comparison to their wild-type littermates. Preliminary studies revealed that AML1RRKK/RRKK mice showed imbalance of the peripheral T cell populations, implying that these methylations may have roles in these cellular lineages. We are currently focusing on further examination of these mutant mice, paying special attention to the cellular lineages where genetic manifestations were observed for AML1 haploinsufficient mice and/or conditional AML1-deficient mice. We hope that these efforts will unveil the biological significance of the AML1 methylation in hematopoietic regulation.
Disclosures:
No relevant conflicts of interest to declare.
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