An integrated multi-omics and network analysis of neutrophil differentiation from initial- to late-stage

Neutrophil differentiation is a well-orchestrated process that involves coordinated changes in the chromatin accessibility, transcription factor (TF) binding, 3D genome-structure and transcription. However, despite significant advances in understanding the later stages of neutrophil maturation, the initial molecular events that trigger and drive the commitment to neutrophil lineage remain poorly characterized, especially the functional roles of master TFs in orchestrating the earliest stages of lineage specification.

Here, we examine changes in the genome topology, transcriptome, and chromatin accessibility during the neutrophil differentiation process. We demonstrate striking changes in 3D genome structure and chromatin accessibility as early as 4 h after all-trans-retinoic acid treatment, which accommodate and regulate gene expression to guide neutrophil lineage differentiation. Analysis of early transcriptional changes confirmed CEBPA as a key TF. To further elucidate the relationships among CEBPA binding, chromatin accessibility, 3D genomic organization, and gene expression, we perform CEBPA HiCut, a technology that simultaneously profiles TF- DNA binding sites and TF-mediated 3D genome interactions. Our first TF-mediated HiCut experiment in neutrophils revealed the synergistic relationship and sequential regulation cascade between this core TF and chromatin accessibility, 3D genomic organization, and gene expression.

Our work systematically investigates coordinated chromatin accessibility, TF binding, 3D genome-structure and transcriptional changes during neutrophil differentiation, especially at initialization. Our study highlights the sequential interplay between the initial changes of chromatin state, 3D genome organization and pioneer TF s such as CEBPA.