Elucidating a Putative Enhancer Element for the Human LAT Gene

The LAT gene encodes a transmembrane adaptor protein required for the function of several immune system cell types, including T cells, mast cells, and natural killer cells. In order to better understand transcriptional regulation of the LAT gene, the laboratory sought to identify and characterize distal regulatory regions, specifically enhancer elements. Data available through the University of California Santa Cruz Genome Browser was assessed to identify genomic regions displaying features consistent with enhancer elements such as DNA sequence conservation, DNase hypersensitivity, and specific post‐translational histone modifications. Identified regions were then PCR amplified and tested for enhancer activity using transient transfection assays in Jurkat T cells and HMC‐1 mast cells. Deletion analysis narrowed enhancer activity to a highly conserved 300 bp region that contains a number of putative transcription factor binding sites, including two Ets and two GATA sites. Point mutations within these Ets and GATA sites significantly inhibited enhancer activity. CRISPR/Cas9‐mediated deletion was then employed in Jurkat T cells to confirm the functional relevance of this region in the regulation of LAT gene expression. Surprisingly, only heterozygous deletion clones were obtained. Ongoing steps focus on attempting once again to obtain homozygous knockout clones and performing RT‐qPCR to analyze LAT gene expression in both heterozygous and homozygous deletion clones. Collectively, these results identified a putative 300 bp enhancer for the LAT gene which appears to function in part through Ets and GATA transcription factor binding sites. Support or Funding Information National Institutes of Health grant # 1R15GM107734‐01