Abstract
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.
Highlights
CCAAT/Enhancer binding protein, α (Cebpa) encodes a transcription factor (TF) that is necessary for neutrophil development [1] as well as the specification of hepatocytes and adipocytes [2, 3]
The function and regulation of silencers was investigated in Gata1-deficient megakaryocyteerythrocyte (G1ME) cells [36], which are derived from Gata1−/− mice and represent the red-blood cell lineage by virtue of being blocked at the megakaryocyte-erythrocyte progenitor (MEP) stage
Uninduced PUER cells function like myeloid progenitors and can be induced to differentiate by treatment with 4-hydroxy-tamoxifen (OHT) into either macrophages or neutrophils in the presence of IL3 or Granulocyte Colony Stimulating Factor (GCSF) respectively (Fig 1B, Fig A in S1 Text, and [7])
Summary
CCAAT/Enhancer binding protein, α (Cebpa) encodes a TF that is necessary for neutrophil development [1] as well as the specification of hepatocytes and adipocytes [2, 3]. Cebpa is regulated in 32Dcl myeloid cells by PU., other Ets TFs, SCL, Gata, Myb, and C/EBPα, which bind to an enhancer located 37kb downstream of the gene [11, 12]. It is not known whether, like other pleiotropic TFs [13], Cebpa is regulated by multiple CRMs. It is not known whether, like other pleiotropic TFs [13], Cebpa is regulated by multiple CRMs It is not understood how the regulatory contributions of these and other TFs modulate Cebpa’s gene expression during differentiation. The function and regulation of silencers was investigated in G1ME cells [36], which are derived from Gata1−/− mice and represent the red-blood cell lineage by virtue of being blocked at the megakaryocyte-erythrocyte progenitor (MEP) stage
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