Comprehensive Identification of Cis- and Trans- Elements Driving Human Hematopoiesis

The conventional depiction of the hematopoietic development is a linear and hierarchical process occurring in discrete steps mediated by the expression of lineage specific transcription factors. While many of the key transcription factors that mediate hematopoietic lineage specification are known, it has remained a significant challenge to temporally link these with functional cis elements and their direct downstream targets in the regulatory circuitry. Here, we aim to uncover temporal dynamics at the regulatory level underlying the differentiation of adult HSPCs towards the red blood cell and megakaryocyte fates. We performed dense profiling of chromatin accessibility during the ex vivo differentiation of primary human mobilized CD34+ cells towards the erythroid and the megakaryocytic lineage, identifying thousands of individual cis-elements (i.e., regulatory DNA) that show quantitative activation or silencing during early development. Profiling the transcriptional changes throughout the progression of each lineage allowed us to identify factors with diverging trajectories. By timing the onset of the divergence, we unveil a fine temporal structure directing the decision between the erythroid or megakaryocytic fate and the differentiation onwards. Overlaying single-cell RNA-seq data along the ex vivo differentiation, we couple the temporal structure with subpopulation dynamics to construct a detailed transcriptional program. By linking the regulatory elements identified by DNase I-seq to transcriptional changes, we enable the direct deconvolution of both cis- and trans-regulatory circuitry that dictates the progenitor fate choice decision and differentiation program. Furthermore, we discover hundreds red blood cell trait-associated genetic variants within these compact developmentally regulated cis-elements that appear to directly modulate transcription factor occupancy during various phases of cellular maturation.