The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation.

During gene regulation, DNA accessibility is thought to limit the availability of transcription factor (TF) binding sites, while TFs can increase DNA accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events in the modulation of gene expression remain unknown for the vast majority of genes. We utilized deeply sequenced ATAC-Seq data and site-specific knock-in reporter genes to investigate the relationship between the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of Cebpa during macrophage-neutrophil differentiation. While the enhancers upregulate reporter expression during the earliest stages of differentiation, there is little corresponding increase in their total accessibility. Conversely, total accessibility peaks during the last stages of differentiation without any increase in enhancer activity. The accessibility of positions neighboring C/EBP-family TF binding sites, which indicates TF occupancy, does increase significantly during early differentiation, showing that the early upregulation of enhancer activity is driven by TF binding. These results imply that a generalized increase in DNA accessibility is not sufficient, and binding by enhancer-specific TFs is necessary, for the upregulation of gene expression. Additionally, high-coverage ATAC-Seq combined with time-series expression data can infer the sequence of regulatory events at binding-site resolution.

The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation.

The upregulation of gene expression by enhancers depends upon the interplay between the binding of sequence-specific transcription factors (TFs) and DNA accessibility. DNA accessibility is thought to limit the ability of TFs to bind to their sites, while TFs can increase accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events underlying the modulation of gene expression during cellular differentiation remain unknown for the vast majority of genes. We investigated the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of an important neutrophil gene, Cebpa, during macrophage-neutrophil differentiation. Reporter genes were integrated in a site-specific manner in PUER cells, which are progenitors that can be differentiated into neutrophils or macrophages in vitro by activating the pan-leukocyte TF PU.1. Time series data show that two enhancers upregulate reporter expression during the first 48 hours of neutrophil differentiation. Surprisingly, there is little or no increase in the total accessibility, measured by ATAC-Seq, of the enhancers during the same time period. Conversely, total accessibility peaks 96 hrs after PU.1 activation-consistent with its role as a pioneer-but the enhancers do not upregulate gene expression. Combining deeply sequenced ATAC-Seq data with a new bias-correction method allowed the profiling of accessibility at single-nucleotide resolution and revealed protected regions in the enhancers that match all previously characterized TF binding sites and ChIP-Seq data. Although the accessibility of most positions does not change during early differentiation, that of positions neighboring TF binding sites, an indicator of TF occupancy, did increase significantly. The localized accessibility changes are limited to nucleotides neighboring C/EBP-family TF binding sites, showing that the upregulation of enhancer activity during early differentiation is driven by C/EBP-family TF binding. These results show that increasing the total accessibility of enhancers is not sufficient for upregulating their activity and other events such as TF binding are necessary for upregulation. Also, TF binding can cause upregulation without a perceptible increase in total accessibility. Finally, this study demonstrates the feasibility of comprehensively mapping individual TF binding sites as footprints using high coverage ATAC-Seq and inferring the sequence of events in gene regulation by combining with time-series gene expression data.

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

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.