The regulatory control of Cebpa enhancers and silencers in the myeloid and red-blood cell lineages

Abstract

During development, cell identity is determined by lineage-specific transcriptional programs en-coded in the cis-regulatory DNA sequence of developmental genes. The sequence-level regulatory logic--identities of bound transcription factors (TFs), TF binding sites, and TF regulatory roles--of most developmental cis-regulatory modules (CRMs) is yet to be determined. We had previously developed an approach for inferring regulatory logic de novo by training sequence-based thermo-dynamic models on comprehensive reporter activity and gene expression datasets and applied it to Cebpa, an important hematopoietic gene. Here, we experimentally test thermodynamic models to decode the cis-regulatory logic of 4 enhancers and 3 silencers neighboring Cebpa at the resolution of individual binding sites. Cebpa is expressed at high and intermediate levels in neutrophils and macrophages respectively and downregulated in non-myeloid lineages. We tested the binding sites and functional roles of inferred TFs by designing and constructing mutated CRMs and comparing theoretical predictions of their activity against empirical measurements. Reporter activity was measured in PUER cells, which can be induced to differentiate into macrophages or neutrophils. All four enhancers were found to be simultaneously active in undifferentiated PUER cells and early-stage macrophages and neutrophils, and activated by combinations of PU.1, C/EBP family TFs, Egr1, and Gfi1. We show that silencers repress the activity of the proximal promoter in a dominant manner in G1ME cells, which are derived from the red-blood cell lineage. Dominant repression in G1ME cells can be traced to binding sites for GATA and Myb, a motif shared by all of the silencers. Finally, we demonstrate that GATA and Myb act redundantly to silence the proximal promoter. The result that silencers quench the promoter selectively in non-myeloid cells indicates that dominant repression is a novel mechanism for resolving hematopoietic lineages. Furthermore, Cebpa has a fail-safe cis-regulatory architecture, featuring several functionally similar CRMs, each of which contains redundant binding sites for multiple TFs. Lastly, by experimentally demonstrating the predictive ability of our sequence-based thermodynamic models, this work highlights the utility of this computational approach for decoding the logic of mammalian gene regulation.