Abstract
Lineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. Here we perform dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis uncovers differential fate commitment dynamics between the two lineages as they exit the stem and progenitor stage. Collectively, these data provide insights into the temporally regulated synergy of the cis- and the trans-regulatory components underlying hematopoietic lineage commitment and differentiation.
Highlights
1, Tannishtha Som[1], Lineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites
Characteristic features of developing erythroblast cells were confirmed by immunophenotyping using canonical cell-surface markers of early (CD117, C-Kit) and late (CD235a, Glycophorin A) erythropoiesis as well as morphologically by hematoxylin-eosin staining of cell smears (Supplementary Fig. 1)
Biological replicates from CD34+ hematopoietic stem and progenitor cell (HSPC) from 3 donors were highly reproducible for both chromatin accessibility and gene expression profiles where the majority of the observed variability was accounted for by developmental trajectory (Fig. 1b, c and Supplementary Fig. 2a), as biological replicates were highly correlated (Supplementary Fig. 2b, c)
Summary
1, Tannishtha Som[1], Lineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Over the course of cellular differentiation programmed shifts in the global transcription factor milieu drive extensive re-organization of chromatin[1,2], where silencing of regulatory DNA associated with alternate lineage and the de novo activation of lineage-restricted elements result in the narrowing of the epigenetic and functional landscape[3] It is unclear how and when regulatory DNA is dynamically activated and silenced during cell state transitions to establish lineage restricted gene expression programs and how these epigenetic changes relate to developmental potential. Single-cell chromatin and transcriptional profiling assays have attempted to resolve the spatio-temporal cis- and trans- dynamics in different stages of hematopoiesis[13–16] These studies have largely relied on the analysis of either bulk or immunophenotypically isolated populations of steady-state peripheral blood or bone marrow derived cells, whereby hierarchical relationships and developmental trajectories between cell states are predicted computationally. Because erythrocytes share their developmental origins with other myeloid lineages (granulocytic/monocytic and megakaryocytic), erythropoiesis represents an ideal system to study how lineage choice is genetically and epigenetically encoded
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