An intronic Ikaros-binding element mediates retinoic acid suppression of the kappa opioid receptor gene, accompanied by histone deacetylation on the promoters

The mouse kappa opioid receptor (KOR) gene is constitutively expressed in mouse embryonal carcinoma P19 stem cells and suppressed by retinoic acid (RA) in cells undergoing neuronal differentiation. A negative regulatory element is located within intron 1 of the KOR gene, which contains an Ikaros (Ik)-binding site (GGGAAgGGGAT). This sequence is an Ik-1 respondive, functionally negative element as demonstrated in the context of both natural KOR and heterologous promoters. The two underlined G residues of the second half-site are critical for Ik-1 binding and Ik-mediated repression of the KOR gene. RA induces Ik-1 expression within 1 day of treatment and suppresses KOR expression between 2 and 3 days. Overexpression of Ik-1 in P19 suppresses endogenous KOR gene expression, accompanied by increased binding of Ik-1 to the Ik-binding site and chromatin histone deacetylation on KOR promoters. It is proposed that in an RA-induced P19 differentiation model, RA elevates Ik-1 expression, which recruits histone deacetylase to intron 1 of the KOR gene and silences KOR gene promoters.     

Zinc finger structure-function in Ikaros

The zinc finger motif was used as a vehicle for the initial discovery of Ikaros in the context of T-cell differentiation and has been central to all subsequent analyses of Ikaros function. The Ikaros gene is alternately spliced to produce several isoforms that confer diversity of function and consequently have complicated analysis of the function of Ikaros in vivo. Key features of Ikaros in vivo function are associated with six C2H2 zinc fingers; four of which are alternately incorporated in the production of the various Ikaros isoforms. Although no complete structures are available for the Ikaros protein or any of its family members, considerable evidence has accumulated about the structure of zinc fingers and the role that this structure plays in the functions of the Ikaros family of proteins. This review summarizes the structural aspects of Ikaros zinc fingers, individually, and in tandem to provide a structural context for Ikaros function and to provide a structural basis to inform the design of future experiments with Ikaros and its family members.     

The lymphoid transcription factor LyF-1 is encoded by specific, alternatively spliced mRNAs derived from the Ikaros gene.

The lymphocyte-specific DNA-binding protein LyF-1 interacts with a critical control element in the terminal deoxynucleotidyltransferase (TdT) promoter as well as with the promoters for other genes expressed during early stages of B- and T-cell development. We have purified LyF-1 and have obtained a partial amino acid sequence from proteolytic peptides. The amino acid sequence suggests that LyF-1 is a zinc finger protein encoded by the Ikaros gene, which previously was implicated in T-cell development. Recombinant Ikaros expressed in Escherichia coli bound to the TdT promoter, and antisera directed against the recombinant protein specifically blocked the DNA-binding activity of LyF-1 in crude extracts. Further analysis revealed that at least six distinct mRNAs are derived from the Ikaros/LyF-1 gene by alternative splicing. Only two of the isoforms possess the N-terminal zinc finger domain that is necessary and sufficient for TdT promoter binding. Although both of these isoforms bound to similar sequences in the TdT, lambda 5, VpreB, and lck promoters, one isoform contains an additional zinc finger that resulted in altered recognition of some binding sites. At least four of the Ikaros/LyF-1 isoforms were detectable in extracts from B- and T-cell lines, with the relative amounts of the isoforms varying considerably. These data reveal that the LyF-1 protein is encoded by specific mRNAs derived from the alternatively-spliced Ikaros gene, suggesting that this gene may be important for the early stages of both B- and T-lymphocyte development.     

Human Ikaros function in activated T cells is regulated by coordinated expression of its largest isoforms

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.     

尼罗罗非鱼Ikaros基因的克隆及表达分析

为揭示尼罗罗非鱼Ikaros基因结构特征及其在抗病原感染中的免疫调控机制, 实验采用RT-PCR和RACE方法克隆了尼罗罗非鱼Ikaros的cDNA序列以及利用PCR和染色体步移技术克隆了Ikaros的基因组DNA序列, 通过荧光定量PCR分析了Ikaros mRNA的组织分布及其对无乳链球菌感染的响应。结果表明, 克隆的尼罗罗非鱼Ikaros基因组DNA为20454 bp, 包括7个内含子和8个外显子, 经可变剪接可形成6种不同的mRNA剪接异构体, 其编码的氨基酸序列均具有Ikaros家族典型的锌指结构域且与硬骨鱼类Ikaros氨基酸序列同源性较高(70.6%—93.7%)。Ikaros基因在尼罗罗非鱼各组织中均有表达, 在血液中的表达量最高, 其次为胸腺、脾脏和头肾。人工感染无乳链球菌后, 血液、胸腺、脾脏、头肾中Ikaros基因的相对表达量均显著上调, 并在48h达到峰值, 这表明Ikaros基因参与调控尼罗罗非鱼抵御无乳链球菌的免疫应答反应。研究可为进一步探索Ikaros基因在罗非鱼抗病原感染中的作用机制奠定理论基础。     

Ikaros isoforms: The saga continues

Through alternate splicing, the Ikaros gene produces multiple proteins. Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity. Ikaros isoforms interact to form dimers and potentially multimeric complexes. Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions. Small dominant-negative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros. Here, we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H. Although IK-1 is described as full-length Ikaros, IK-H is the longest Ikaros isoform. IK-H, which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4), is abundant in human but not murine hematopoietic cells. Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1. Moreover, IK-H can potentiate or inhibit the ability of IK-1 to bind DNA. IK-H binds to the regulatory regions of genes that are upregulated by Ikaros, but not genes that are repressed by Ikaros. Although IK-1 localizes to pericentromeric heterochromatin, IK-H can be found in both pericentromeric and non-pericentromeric locations. Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H, but not other Ikaros isoforms. The unique features of IK-H, its influence on Ikaros activity, and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.     

hunchback and Ikaros-like zinc finger genes control reproductive system development in Caenorhabditis elegans

Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein-protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.