How overall principles of cell-type–specific gene regulation (the “logic”) may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic, and three-dimensional (3D) genomic profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1, and Myb occupancy. EryP-/EryD-shared enhancers are highly correlated with red blood cell identity genes, whereas cell-type–specific regulation employs different cis elements in EryP and EryD cells. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes rely more on distal enhancers for regulation involving Myb-mediated enhancer activation. Gata1 HiChIP demonstrated an overall increased enhancer–promoter interactions at EryD-specific genes, whereas genome editing in selected loci confirmed distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer dependence of transcription, we observed a progressive reliance on cell-specific enhancers with increasing ontogenetic age among diverse tissues of mouse and human origin. Our findings highlight fundamental and conserved differences at distinct developmental stages, characterized by simpler promoter-centric regulation of cell-type–specific genes in embryonic cells and increased combinatorial enhancer-driven control in adult cells.

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Many enhancers exist as clusters in the genome and control cell identity and disease genes; however, the underlying mechanism remains largely unknown. Here, we introduce an algorithm, eNet, to build enhancer networks by integrating single-cell chromatin accessibility and gene expression profiles. The complexity of enhancer networks is assessed by two metrics: the number of enhancers and the frequency of predicted enhancer interactions (PEIs) based on chromatin co-accessibility. We apply eNet algorithm to a human blood dataset and find cell identity and disease genes tend to be regulated by complex enhancer networks. The network hub enhancers (enhancers with frequent PEIs) are the most functionally important. Compared with super-enhancers, enhancer networks show better performance in predicting cell identity and disease genes. eNet is robust and widely applicable in various human or mouse tissues datasets. Thus, we propose a model of enhancer networks containing three modes: Simple, Multiple and Complex, which are distinguished by their complexity in regulating gene expression. Taken together, our work provides an unsupervised approach to simultaneously identify key cell identity and disease genes and explore the underlying regulatory relationships among enhancers in single cells.

Running Title: Regulatory logic during ontogeny 2 Summary How overall principles of gene regulation (the "logic") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic and topological profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1 and Myb occupancy.In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes employ distal enhancers for long-range regulation through enhancerpromoter looping, confirmed by Gata1 HiChIP. Genome editing demonstrated distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancerdependence of transcription, we observed a progressive reliance on enhancer control with increasing age of ontogeny among diverse primary cells and tissues of mouse and human origin. Our findings highlight fundamental and conserved differences in regulatory logic at distinct developmental stages, characterized by simpler promoter-centric regulation in embryonic cells and combinatorial enhancer-driven control in adult cells. KeywordsActive enhancer, enhancer dependent-regulatory logic, HiChIP, enhancer-promoter interactions, Gata1, Myb, erythropoiesis Highlights• Regulation of embryonic-specific erythroid genes is promoter-centric through Gata1• Adult-specific control is combinatorial enhancer-driven and requires Myb• Adult specific genes have increased enhancer-promoter chromatin interactions• Enhancer-dependence increases progressively with increasing developmental age Scl/Tal1, Ldb1, Fog1 and Lmo2 (Palis, 2014). Knockout of each of these genes leads to defects in EryP and EryD cells Mead et al., 2001;Palis, 2014). In contrast, the loss of Myb, which is expressed selectively in definitive type cells, impairs proliferation and differentiation of EryD, sparing EryP cells (Mucenski et al., 1991). Given the different reliance of EyP and EryD cells on TFs for their development, we have asked whether these related, but distinct cell lineages in ontogeny differ in their fundamental regulatory organization and logic.To address this question, we isolated mouse EryP and EryD erythroblasts and characterized transcriptomes, chromatin accessibility, histone modifications, transcription factor (TF) occupancies, and 3D chromatin interactions. We observed that gene regulation in EryP is largely promoter-centric, whereas that in EryD was distal enhancer driven for activation. We hypothesized that these features reflect inherent differences between embryonic cells and more diverse, long-lived adult cell types. Analyses of available datasets of diverse mouse and human cells and tissues provided further support for the unexpected finding that regulatory logic changes with ontogeny. 6 ResultsEryP and EryD transcription correlates differently with distal chromatin accessibility CD71 + /Ter119 + EryP and EryD cells were isolated by FACS from E10.5 embryonic peripheral blood and E13...