Human Ikaros Function in Activated T Cells Is Regulated by Coordinated Expression of Its Largest Isoforms

Tapani Ronni,Kimberly J Payne,Sam Ho,Michelle N Bradley,Glenn Dorsam,Sinisa Dovat

doi:10.1074/jbc.m605627200

Abstract

The Ikaros gene is alternately spliced to generate multiple zinc finger proteins involved in gene regulation and chromatin remodeling. Whereas murine studies have provided important information regarding the role of Ikaros in the mouse, little is known of Ikaros function in human. We report functional analyses of the two largest human Ikaros (hIK) isoforms, hIK-VI and hIK-H, in T cells. Abundant expression of hIK-H, the largest described isoform, is restricted to human hematopoietic cells. We find that the DNA binding affinity of hIK-H differs from that of hIK-VI. Co-expression of hIk-H with hIk-VI alters the ability of Ikaros complexes to bind DNA motifs found in pericentromeric heterochromatin (PC-HC). In the nucleus, hIK-VI is localized solely in PC-HC, whereas the hIK-H protein exhibits dual centromeric and non-centromeric localization. Mutational analysis defined the amino acids responsible for the distinct DNA binding ability of hIK-H, as well as the sequence required for the specific subcellular localization of this isoform. In proliferating cells, the binding of hIK-H to the upstream regulatory region of known Ikaros target genes correlates with their positive regulation by Ikaros. Results suggest that expression of hIK-H protein restricts affinity of Ikaros protein complexes toward specific PC-HC repeats. We propose a model, whereby the binding of hIK-H-deficient Ikaros complexes to the regulatory sequence of target genes would recruit these genes to the restrictive pericentromeric compartment, resulting in their repression. The presence of hIK-H in the Ikaros complex would alter its affinity for PC-HC, leading to chromatin remodeling and activation of target genes.

Highlights

Ikaros expression is essential for proper stem cell function [1] and for normal hematopoiesis in the lymphoid, myeloid, and
We report that the largest human Ikaros isoforms, hIK-VI and hIK-H, exhibit distinct DNA binding abilities and subcellular localization patterns
Using MOLT-4 nuclear extract, we show that the IK-H antibody detects only the hIK-H isoform, whereas the IKCTS antibody detected all of the three large human Ikaros isoforms

Summary

EXPERIMENTAL PROCEDURES

Cells—Human CCRF-CEM (CEM) and MOLT-4 human leukemia T-cell lines, the Ramos B cell line, the human 293T endothelial kidney cell line, and the murine the NIH/3T3 (3T3) fibroblast cell line were obtained from American Type Culture Collection (ATCC). T-cell activation with PMA and anti-CD3 has been described previously [23]. Antibodies used to detect the C terminus (IK-CTS) and the N terminus (IK-NTS) of murine and human Ikaros have been described previously [24]. ChIP (Chromatin Immunoprecipitation) Assay—In vivo DNA binding of Ikaros isoforms was tested using ChIP assays in 293T cells and in activated T cells as previously described [31] using 10 ␮g of IK-CTS or Ik-H antibodies. All samples within a particular set (anti-Ikaros antibodies, IgG, and total chromatin control) were analyzed at the same time and electrophoresed on the same agarose gel. The expression of Ikaros proteins in activated, primary, peripheral human T cells Overexpressed isoforms co-migrated with endogenous proteins in the MOLT-4 human T-cell line, confirming that the two top bands were hIK-H and hIK-VI. Using MOLT-4 nuclear extract, we show that the IK-H antibody detects only the hIK-H isoform, whereas the IKCTS antibody detected all of the three large human Ikaros isoforms

RESULTS

Ikaros has identical phosphorylation status and DNA binding

DISCUSSION