å­¦ Cell Cycle-specific Function Of Ikaros In Human Leukemia

Ikaros encodes a zinc finger protein that is essential for hematopoiesis and that acts as a tumor suppressor in leukemia. Loss of Ikaros activity is associated with the development of B and T cell leukemia Ikaros function depends on its ability to localize to pericentromeric heterochromatin (PC-HC). Ikaros protein binds to the upstream regulatory elements of target genes, aids in their recruitment to PC-HC, and regulates their transcription via chromatin remodeling. Proteins generated from the Ikaros gene contain two separate zinc finger domains. Four zinc fingers in the amino half of the protein take part in sequence-specific DNA binding . The two zinc fingers at the C-terminus of the protein are responsible for protein-protein interaction. In humans, the loss of Ikaros function has been associated with childhood and adult ALL, and deletion of an IKAROS allele has been identified as a poor prognostic marker for childhood ALL. These data established Ikaros as a major tumor suppressor in human leukemia. The specific mechanisms by which Ikaros regulates cellular proliferation in humans remains largely unknown. Data on Ikaros tumor suppressor activity have come from studies of murine Ikaros–very few studies have examined the function of human Ikaros proteins in leukemia.Previous studies demonstrated that Ikaros has essential roles in tumor suppression and in normal hematopoiesis. However, Ikaros is abundantly expressed in all hematopoietic cells and throughout the cell cycle, suggesting that its function might be regulated by post-translational modifications. Cell cycle-specific phosphorylation has been shown to regulate Ikaros'function during mitosis. We present the first functional analysis of Ikaros isoforms during cell cycle in human leukemia.focus on DNA-binding ability, phosphorylation and subcellular localization of Ikaros isoforms in human leukemia cells.Gel shift retardation assays showed that the DNA-binding activity of Ikaros protein complexes changes as the cell cycle progresses. These data showed that the ability of human Ikaros to bind DNA is strongest during G1 phase of the cell cycle,Following the G1-to-S phase transition, the DNA-binding activity of Ikaros toward regulatory regions of its target genes is decreased, while its binding to pericentromeric heterochromatin is preserved,and that the ability of human Ikaros to bind DNA is strongest during G1 phase of the cell cycle,The S phase-specific changes in Ikaros DNA binding ability are controlled by its phosphorylation via the CK2 kinase pathway. As predicted, based on murine studies, the ability of human Ikaros to bind DNA is almost abolished during mitosis.Phosphopeptide mapping showed that the IK-VI and IK-H isoforms have unique phopshorylation patterns that change during G1, S, and M phase of the cell cycle. Confocal microscopy revealed that IK-VI and IK-H exhibit unique subcellular localization patterns during different phases of the cell cycle. These patterns are unique with respect to homologous murine isoforms. These experiments show that human Ikaros isoforms exhibit unique biochemical properties compared to those expressed in the mouse. These data provide a foundation for studies to determine the role of Ikaros in controlling cell cycle progression in human leukemia. For our analyses we used human T cell leukemia cells, thus allowing for more valid comparisons between our human data and that obtained in murine studies. We report that the largest human Ikaros isoforms, IK-VI and IK-H, exhibit cell cycle-specific DNA-binding affinity, subcellular localization, and phosphorylation patterns, suggesting that Ikaros proteins have distinct cellular functions at different stages of the cell cycle. This is important as IK-H is abundant in human, but not murine, hematopoietic tissues. Our results expose significant differences in the properties of human Ikaros proteins and those expressed in the mouse. Presented data suggest that Ikaros function in human cell cycle regulation and proliferation may occur through more complicated mechanisms than in the mouse.Results provide the first functional analysis of human Ikaros isoforms during each stage of the cell cycle. The cell cycle-specific functions of Ikaros in human leukemia are distinct from those in mouse, and are regulated by phosphorylation. Determining the function of Ikaros proteins will aid in our understanding of the role of Ikaros as a tumor suppressor and shed light on the mechanisms of malignant transformation and immune.