Precise control of GATA3 expression in CD4+ T cell differentiation via selective activation of primed enhancers

GATA3 is a master regulator with expression that must be precisely controlled during differentiation of naïve CD4+ T cells into diverse subtypes to ensure correct immune responses. The regulatory landscape of GATA3 is complex with dozens of potential enhancers in different states. Here, we use thousands of tiling deletions to systematically map regulatory sequences that affect GATA3 expression. We identify and characterize 34 regulatory sequences in naïve CD4+ T cells, including 4 active enhancers and, unexpectedly, 10 primed enhancers. Focusing on the differentiation of naïve CD4+ T cells into T helper 2 cells where GATA3 expression is highest, we find that upregulation of GATA3 is achieved by retaining active enhancers and activating poised and primed enhancers, including one which harbors candidate causal genetic variants for allergic diseases. We focused on the 2.02Mbp GATA3 region (hg19 chr10: 7,096,667-9,117,164) to identify regulatory elements for GATA3 in human T cells. We designed 14,769 pairs of single guide RNAs (sgRNAs) to tile ~1kb deletions across the 2 Mb genome region centered on GATA3. The targeted deletions have a median size of 1043 bp and median step size of 96 bp, such that each base in the screened region is covered by a median of 8 programmed deletions. We cloned oligos encoding the sgRNAs and generated a lentiviral library to deliver and constitutively express both the sgRNA pairs and spCas9. After transducing the lentiviral library into Jurkat T cells, which we use as a model for naïve CD4+ T cells, we selected for cells with viral genome integration and flow sorted them into pools based on GATA3 protein expression. Finally, we performed deep sequencing of the sgRNA pairs in each expression pool. We performed four biological replicates of the screen, sorting into three expression level pools in the first two replicates and seven expression level pools in the other two replicates.