Summary Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.

Highlights d diffTF estimates differential TF activity based on chromatin data (basic mode) d Integration with RNA-seq identifies activator/repressor TFs (classification mode) d We applied diffTF to subtypes of CLL and hematopoietic differentiation d The TF classification was experimentally validated in both case studies

The epigenetic regulator TET2 is frequently mutated in hematological diseases. Mutations have been shown to arise in hematopoietic stem cells early in disease development and lead to altered DNA methylation landscapes and an increased risk of hematopoietic malignancy. Here, we show by genome-wide mapping of TET2 binding sites in different cell types that TET2 localizes to regions of open chromatin and cell-type-specific enhancers. We find that deletion of Tet2 in native hematopoiesis as well as fully transformed acute myeloid leukemia (AML) results in changes in transcription factor (TF) activity within these regions, and we provide evidence that loss of TET2 leads to attenuation of chromatin binding of members of the basic helix-loop-helix (bHLH) TF family. Together, these findings demonstrate that TET2 activity shapes the local chromatin environment at enhancers to facilitate TF binding and provides an example of how epigenetic dysregulation can affect gene expression patterns and drive disease development.

Environmental and epigenetic factors often play an important role in polygenic disorders. However, how such factors affect disease-specific tissues at the molecular level remains to be understood. Here, we address this in pulmonary arterial hypertension (PAH). We obtain pulmonary arterial endothelial cells (PAECs) from lungs of patients and controls (n = 19), and perform chromatin, transcriptomic and interaction profiling. Overall, we observe extensive remodeling at active enhancers in PAH PAECs and identify hundreds of differentially active TFs, yet find very little transcriptomic changes in steady-state. We devise a disease-specific enhancer-gene regulatory network and predict that primed enhancers in PAH PAECs are activated by the differentially active TFs, resulting in an aberrant response to endothelial signals, which could lead to disturbed angiogenesis and endothelial-to-mesenchymaltransition. We validate these predictions for a selection of target genes in PAECs stimulated with TGF-β, VEGF or serotonin. Our study highlights the role of chromatin state and enhancers in disease-relevant cell types of PAH.