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Defining the sequence specificity of Ikaros DNA binding zinc fingers and their role in T cell activation and tumor suppression


Ikaros (Ikzf1) is a transcription factor necessary for the development of lymphoid and other hematopoietic lineages. Several studies have identified Ikaros as an important lymphoid tumor suppressor both in murine models and human cases of acute lymphocytic leukemia (ALL). Few gene targets of Ikaros regulation have been identified, and the mechanistic link to tumor suppression is unknown. Ikaros binds DNA through a region of four tandem C2H2 DNA-binding zinc fingers, and early characterization suggested that the first and fourth fingers have modulatory binding that allows for targeting of different consensus sequences. However, the binding sequence specificity of each of these fingers remained ambiguous. The Smale lab has developed a mutant mouse strain with a deletion of the exon encoding the fourth zinc finger, Ikzf1δF4/>δF4, which among other phenotypes, develops spontaneous aggressive T cell lymphomas with 100% penetrance. These results suggest that the binding specificity of zinc finger 4 is necessary for regulating target genes that maintain Ikaros tumor suppressor function. This thesis examines the DNA binding specificity of Ikaros zinc fingers 1 and 4, and identifies a putative consensus sequence for each. The kinetics and stability of binding for purified proteins without zinc fingers 1 or 4 is also characterized using surface plasmon resonance. Mice with a heterozygous deletion of the Ikaros DNA binding domain, Ikzf1DN/>+, also develop thymic lymphomas and have been observed to show a decreased threshold of stimulation for T cell activation. This phenotype is thought to have a connection with the malignant transformation of these cells. Ikzf1δF4/>δF4 T cells are tested by in vitro assays and found to also have a significantly decreased threshold for activation. These assays are used to further investigate gene deregulation in activated Ikzf1δF4/>δF4 T cells using RNA high throughput sequencing. Many deregulated genes with potential contribution to decreased activation threshold and loss of tumor suppressor function were identified by RNA-seq analysis. In combination with RNA-seq, Ikaros ChIP-seq data could be used to validate functional targets and associate loss of Ikaros occupancy with deregulation of gene expression. Ikaros target genes have remained largely elusive in part due to difficulty with traditional chromatin immunoprecipitation (ChIP) methods. A new technique is tested and optimized for future application to Ikaros ChIP-seq.

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