Integration of high-throughput reporter assays identify a critical enhancer of the Ikzf1 gene

The Ikzf1 locus encodes the lymphoid specific transcription factors Ikaros, which play an essential role in both T and B cell differentiation, while deregulation or mutation of IKZF1/Ikzf1 is involved in leukemia. Tissue-specific and cell identity genes are usually associated with clusters of enhancers, also called super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. Several potential regulatory regions have been identified in close proximity of Ikzf1, however, the full extent of the regulatory landscape of the Ikzf1 locus is not yet established. In this study, we combined epigenomics and transcription factor binding along with high-throughput enhancer assay and 4C-seq to prioritize an enhancer element located 120 kb upstream of the Ikzf1 gene. We found that the deletion of the E120 enhancer resulted in a significant reduction of Ikzf1 mRNA. However, the epigenetic landscape and 3D topology of the locus were only slightly affected, highlighting the complexity of the regulatory landscape regulating the Ikzf1 locus Overall design: We used CRISPR/Cas9 technology to delete the IkE120 enhancer in the P5424 cell line. ChIP-seq was used to assess H3K27ac enrichment in wild-type and knock-out cells; 4C was used to interrogate chromatin interactions with the Ikzf1 promoter.