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Leukocyte mono-immunoglobulin (Ig)–like receptor 5 (LMIR5)/CD300b is a DAP12-coupled activating receptor predominantly expressed in myeloid cells. The ligands for LMIR have not been reported. We have identified T cell Ig mucin 1 (TIM1) as a possible ligand for LMIR5 by retrovirus-mediated expression cloning. TIM1 interacted only with LMIR5 among the LMIR family, whereas LMIR5 interacted with TIM4 as well as TIM1. The Ig-like domain of LMIR5 bound to TIM1 in the vicinity of the phosphatidylserine (PS)-binding site within the Ig-like domain of TIM1. Unlike its binding to TIM1 or TIM4, LMIR5 failed to bind to PS. LMIR5 binding did not affect TIM1- or TIM4-mediated phagocytosis of apoptotic cells, and stimulation with TIM1 or TIM4 induced LMIR5-mediated activation of mast cells. Notably, LMIR5 deficiency suppressed TIM1-Fc–induced recruitment of neutrophils in the dorsal air pouch, and LMIR5 deficiency attenuated neutrophil accumulation in a model of ischemia/reperfusion injury in the kidneys in which TIM1 expression is up-regulated. In that model, LMIR5 deficiency resulted in ameliorated tubular necrosis and cast formation in the acute phase. Collectively, our results indicate that TIM1 is an endogenous ligand for LMIR5 and that the TIM1–LMIR5 interaction plays a physiological role in immune regulation by myeloid cells.

Fusion protein AML1-ETO, resulting from t(8;21) translocation, is highly related to leukemia development. It has been reported that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here, we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a-activated genes, resulting in enrichment of H4 arginine 3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses the self-renewal capability of AE9a, suggesting a potential role of PRMT1 in regulating leukemia development. (Blood. 2012;119(21):4953-4962) IntroductionAcute myeloid leukemia (AML) is closely associated with chromosomal abnormalities. 1 The AML1 gene (also known as CBFA2, PEBP2␣B, and RUNX1) was initially identified as a target of chromosomal translocation in t(8;21), which is associated with 15% of de novo AML cases and Յ 40% in the AML subtype M2 of the French-American-British classification. 2,3 This specific translocation at t(8;21) involves the AML1 gene on chromosome 21 and the ETO (also known as MTG8) gene on chromosome 8 and generates an AML1-ETO fusion transcription factor. 4 AML1-ETO inherits the DNA binding RUNT domain from AML1 and is functionally characterized as a transcription factor for both gene repression and activation. 3,[5][6][7] It has been shown that AML1-ETO negatively regulates AML1 target genes, possibly through interaction with corepressor proteins such as mSin3A, N-CoR/SMRT (nuclear receptor corepressor/silencing mediator for retinoic acid receptor and thyroid hormone receptor), and HDACs (histone deacetylases). [8][9][10] AML1-ETO could also act as a transactivator on certain genes. One of the possible mechanisms is by recruiting histone modifiers to make chromatin structure more accessible to the transcription activation machinery, resulting in gene activation. A recent finding shows that p300 binds to NHR1 domain of AML1-ETO to facilitate transcription. 11 A variety of posttranslational modifications, including acetylation, methylation, and phosphorylation, on specific residues of histones and their corresponding enzymes has been discovered. 12 It is well documented that a specific histone modification on a promoter could determine the state of transcription. Specifically, methylation on histone H4 arginine 3 (Arg3) by PRMT1 (protein arginine methyltransferase 1) generally correlates with transcription activation. 13 PRMT1 is the most predominant arginine ...

The mechanisms by which AML1/ETO (A/E) fusion protein induces leukemogenesis in acute myeloid leukemia (AML) without mutagenic events remain elusive. Here we show that interactions between A/E and hypoxia-inducible factor 1α (HIF1α) are sufficient to prime leukemia cells for subsequent aggressive growth. In agreement with this, HIF1α is highly expressed in A/E-positive AML patients and strongly predicts inferior outcomes, regardless of gene mutations. Co-expression of A/E and HIF1α in leukemia cells causes a higher cell proliferation rate in vitro and more serious leukemic status in mice. Mechanistically, A/E and HIF1α form a positive regulatory circuit and cooperate to transactivate DNMT3a gene leading to DNA hypermethylation. Pharmacological or genetic interventions in the A/E-HIF1α loop results in DNA hypomethylation, a re-expression of hypermethylated tumor-suppressor p15(INK4b) and the blockage of leukemia growth. Thus high HIF1α expression serves as a reliable marker, which identifies patients with a poor prognosis in an otherwise prognostically favorable AML group and represents an innovative therapeutic target in high-risk A/E-driven leukemia.