b-globin enhancers regulate bistable gene expression states

Multiple enhancers can coordinately regulate a target gene to ensure robust expression. Under the failure rate model, each redundant enhancer contributes a probability of target gene expression in the cell, and genetic deletion will cause single-cell expression phenotypes. However, this model has been challenging to test in human cells. To examine the role of the human ß-globin enhancers in controlling single-cell expression phenotypes, we engineered a haploid locus in K562 cells that fluorescently reports the expression of HBG1. We isolated combinatorial enhancer deletion clones having single-cell expression phenotypes. Genetic and epigenetic analysis shows that deletion of HS3 or HS4 yields clonal cells having bimodal expression of HBG1. Time course analysis indicates that clonal cells can transition between bimodal expression states, and that GFP+ cells have more open chromatin at the HBG1 promoter than GFP- cells. Finally, we derive failure rate contributions for several enhancers. Thus, ß-globin enhancers regulate bistable gene expression states, supporting the failure rate model of enhancer redundancy in human cells. Overall design: Multiple cell lines were sequenced with the goal of finding beta globin (bgl) enhancer deletions for downstream analysis. Prospective knockouts were prepared in duplicate for ATAC-seq, with several samples sequenced multiple times for increased coverage.