Date Published: June 10, 2019
Publisher: Public Library of Science
Author(s): Andrea Repele, Shawn Krueger, Tapas Bhattacharyya, Michelle Y. Tuineau, Jörn Lausen.
Cebpa encodes a transcription factor (TF) that plays an instructive role in the development of multiple myeloid lineages. The expression of Cebpa itself is finely modulated, as Cebpa is expressed at high and intermediate levels in neutrophils and macrophages respectively and downregulated in non-myeloid lineages. The cis-regulatory logic underlying the lineage-specific modulation of Cebpa’s expression level is yet to be fully characterized. Previously, we had identified 6 new cis-regulatory modules (CRMs) in a 78kb region surrounding Cebpa. We had also inferred the TFs that regulate each CRM by fitting a sequence-based thermodynamic model to a comprehensive reporter activity dataset. Here, we report the cis-regulatory logic of Cebpa CRMs at the resolution of individual binding sites. 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 in a myeloid cell line. The enhancers were confirmed to be activated by combinations of PU.1, C/EBP family TFs, Egr1, and Gfi1 as predicted by the model. 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. These results indicate 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 model, this work highlights the utility of this computational approach for understanding mammalian gene regulation.
CCAAT/Enhancer binding protein, α (Cebpa) encodes a TF that is necessary for neutrophil development  as well as the specification of hepatocytes and adipocytes [2, 3]. During hematopoiesis, Cebpa is expressed in hematopoietic stem cells, granulocyte-monocyte progenitors (GMPs), neutrophils, and macrophages (http://biogps.org/gene/12606; [4, 5]). Although the most apparent hematopoietic phenotype of Cebpa−/− mice is neutropenia , Cebpa also has a role in specifying macrophages. Cebpa is expressed at intermediate and high levels in macrophages and neutrophils respectively and the cell-fate decision is thought to depend on the ratio of PU.1, a TF necessary for white-blood cell lineages , and C/EBPα expression levels . Correspondingly, the cell-fate decision has been modeled as a bistable switch in which PU.1 and C/EBPα activate the mutual antagonists Egr1/2 and Gfi1 respectively . Cebpa is also sufficient for specifying macrophages, since B-cells can be transdifferentiated into them by expressing Cebpa ectopically .
We have comprehensively analyzed the regulation of 7 CRMs neighboring Cebpa at the resolution of individual binding sites. In the process of doing so, we have also verified the predictive ability of a thermodynamic model of mammalian gene regulation that we developed recently . It is worth noting that prior to our efforts, thermodynamic modeling was limited to Drosophila gene regulation [16, 22, 24, 60–64], with a single gene, even-skipped, as the focus of most of the work. Our model is closely related to its Drosophila counterparts, incorporating just one additional mechanism, long-distance dominant repression, lacking in the latter. The ability of models with shared mechanisms of gene regulation to predict reporter activity in these divergent species supports the view that the rules of transcriptional regulation are universal.