Binding-site resolution structure and dynamics of Cebpa regulatory regions during macrophage-neutrophil differentiation

During hematopoiesis, multipotential progenitors differentiate into all the types of cells found in blood by the action of complex gene regulatory networks (GRNs) comprising transcription factor (TF) genes that regulate each other's expression. The gene regulatory logic—by which we mean the identities of the TF regulators, where they bind, whether they activate or repress, and how they interact with each other—remains unknown for most hematopoietic genes. Here, we utilize high coverage ATAC-Seq and reporters integrated in a site-specific manner to investigate binding-site resolution chromatin accessibility dynamics of Cebpa enhancers during macrophage-neutrophil differentiation. Reporter genes were integrated into the ROSA26 locus using CRISPR/Cas9 in PUER cells, which can be differentiated into neutrophils or macrophages in vitro. Time series reporter data showed an upregulation of two enhancers during the first 48 hours of neutrophil differentiation, which mirrored the temporal expression pattern of the endogenous Cebpa gene. Chromatin accessibility was also profiled at several time points by high coverage (300 million reads per sample) ATAC-Seq. The accessibility patterns were highly correlated between time points, allowing us to pool the data and achieve approximately 50x coverage after filtering. These data revealed that the enhancers had many protected regions of low accessibility interspersed with short high-accessibility islands. All previously characterized TF binding sites and centers of ChIP-Seq peaks overlapped protected regions, indicating that they are TF footprints. Surprisingly, while the total accessibility of the enhancers increased during differentiation, this increase occurred after gene expression had peaked, implying that the total accessibility of the enhancers was not driving gene expression. However, Tn5 counts at the high-accessibility islands adjacent to TF footprints, especially for Cebp sites, increased at earlier time points, suggesting that TF occupancy may drive the upregulation in gene expression as well as the later increase in the total accessibility of the enhancers. Our high coverage accessibility maps reveal a complex regulatory architecture, with 5-7 TFs binding up to 20 sites in a single Cebpa enhancer. Furthermore, the temporal dynamics of accessibility and gene expression do not support a causal role for total accessibility in gene expression. More generally, these results show that profiling gene expression and accessibility at high temporal- and genomic-resolution have the potential to reveal the causal drivers of gene expression changes during development. Overall design: Time series of Assay for Transposase Accessible Chromatin using sequencing (ATAC-Seq) during the differentiation of a mouse cell-line, PUER, into macrophages and neutrophils. ATAC-Seq was performed in duplicate in undifferentiated PUER cells, at 5 timepoints in GCSF conditions (neutrophil) and 2 timepoints in IL3 conditions (macrophage).