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

Jaafar Alomairi,Salvatore Spicuglia,David Santiago-Algarra,Guillaume Charbonnier,Nori Sadouni,Saadat Hussain,Tom Sexton,Anne M Molitor,Jean Christophe Andrau,Denis Puthier,Magali Torres,Wiam Saadi,Lan T M Dao

doi:10.1371/journal.pone.0233191

Abstract

The Ikzf1 locus encodes the lymphoid specific transcription factor Ikaros, which plays 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 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.

Highlights

Cell-type specific regulation of gene expression requires the activation of promoters by distal genomic elements defined as enhancers
J1 mouse embryonic stem (ES) cells were grown on gamma-irradiated mouse embryonic fibroblast cells under standard conditions (4.5 g/L glucose-DMEN, 15% FCS, 0.1 mM non-essential amino acids, 0.1 mM beta-mercaptoethanol, 1 mM glutamine, 500 U/mL LIF, gentamicin), passaged onto feeder-free 0.2% gelatincoated plates for at least two passages to remove feeder cells before 4C
We identified thirteen DNAse I hypersensitive sites (DHSs) within the two superenhancers in DP thymocytes (Fig 1A; S4 Table)

Summary

Introduction

Cell-type specific regulation of gene expression requires the activation of promoters by distal genomic elements defined as enhancers. The classical view of enhancer function is that they contribute to increasing the overall level of gene expression by inducing transcription from associated promoters [1]. Complex gene regulation is mediated by the association of clusters of enhancers, called super-enhancers [2]. Whether the individual components (i.e. single enhancers) synergistically contribute to transcription regulation of their target genes or have distinct specialized functions has been a matter of debate [2,3,4,5].

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Results

Conclusion