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Gene Regulatory Network of Ikaros in T cell development and leukemogenesis Dr. Jiangwen Zhang Lab, School of Biological Sciences Fig. 3. Screening of the functional sites along the enhancer-H by in situ mutagenesis on endogenous enhancer locus. Introduction: Ikaros is a master regulator of lymphogenesis, especially critical for lymphoid differentiation and maturation. Recurrent genomic lesions of Ikaros have been found associated with high risks of relapse of leukemia and poor outcome of therapy. However, it remains unclear about the gene regulatory network associated with Ikaros. How exactly the transcription of Ikaros itself is regulated? Ikaros can positively or negatively regulate its target genes, and how Ikaros' activity is regulated by its partners? In additional to deletion and mutations, aberrant epigenetic regulation, e.g. DNA methylation of the promoter, can also disturb Ikaros gene transcription. In this project, we are seeking to address these questions, and reveal the gene regulatory network of Ikaros in T cell development and leukemogenesis. Fig. 4. Confirmation of the functional site 4 (also called site A) along the enhancer-H by luciferase reporter and EMSA. Objectives: 1. Characterize the enhancer-H critical for Ikaros expression in T cell locating at 4th intron of Ikaros locus. 2. Characterize how Ikaros activity is regulated by its partner, Heb transcriptional factor. Methods: 1.To characterize enhancer-H , we have created a powerful system allowing us to introduce point mutations to the endogenous chromosome on Ikaros enhancer locus. In addition to construction of enhancer-H mutated cell line through CRISPR/Cas9 system, we have employed conventional pGL3 luciferase reporter system to confirm the enhancer activity. 2. To interrogate the function interaction between Heb and Ikaros, we have employed CRISPR/Cas9 system to deplete Heb expression and test its effect on Ikaros target genes’ expression. Fig. 5. Heb interacts with Ikaros: 1) venn diagram of direct gene targets of Ikaros and Heb based on integrative analysis of ChIP-seq and microarray datasets; 2) Ikaros and Heb co-occupy some genomic regions by Ward’s hierarchical clustering of Ikaros, HEB and H3K4me3, H3K4me2 and H3K4me1 modifications at distal regions. Gancinovir selection + Results: Fig.1. Replacement of enhancer-H on endogenous chromosome by Bsd/Tk flanked by loxP was confirmed by sequencing. Fig. 6. Gene set enrichment analysis to determine the correlation of Ikaros DNA binding with gene expression change upon knockdown of Heb. And occupancy of transcription factors and H3 modifications at Ccnd3 locus. All Heb targets Fig. 2. Endogenous Ikaros expression was lost upon replacement of enhancer-H by Bsd/Tk, and recovered by back insertion of enhancer-H flanked by loxP. <- Ikaros binding Up-by-Heb Down-by-Heb Conclusions: 1. Construct cre/loxp system for the dissection of enhancer facilitated by CRISPR/Cas9 technique. 2. Find one functional site on Ikaros enhancer-H, responsible for more than half of the activity. 3. Functional interaction between Heb and Ikaros has direct effect on downstream gene expression of Ikaros.