No abstract

The gene activity underlying cell differentiation is regulated by a diverse set of transcription factors (TFs), histone modifications, chromatin structures and more. Although definitive hematopoietic stem cells (HSCs) are known to emerge via endothelial-to-hematopoietic transition (EHT), how the multi-layered epigenome is sequentially unfolded in a small portion of endothelial cells (ECs) transitioning into the hematopoietic fate remains elusive. With optimized low-input itChIP-seq and Hi-C assays, we performed multi-omics dissection of the HSC ontogeny trajectory across early arterial ECs (eAECs), hemogenic endothelial cells (HECs), pre-HSCs and long-term HSCs (LT-HSCs) in mouse embryos. Interestingly, HSC regulatory regions are already pre-configurated with active histone modifications as early as eAECs, preceding chromatin looping dynamics within topologically associating domains. Chromatin looping structures between enhancers and promoters only become gradually strengthened over time. Notably, RUNX1, a master TF for hematopoiesis, enriched at half of these loops is observed early from eAECs through pre-HSCs but its enrichment further increases in HSCs. RUNX1 and co-TFs together constitute a central, progressively intensified enhancer-promoter interactions. Thus, our study provides a framework to decipher how temporal epigenomic configurations fulfill cell lineage specification during development.

Sculpting the epigenome with a combination of histone modifications and transcription factor (TF) occupancy determines gene transcription and cell fate specification. Here we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultra-high throughput single-cell joint profiling of multiple epigenetic proteins. Through extensive optimization for sensitivity and multimodality resolution, we demonstrate that uCoTarget enables simultaneous detection of five histone modifications (H3K27ac, H3K4me3, H3K4me1, H3K36me3 and H3K27me3) in 19,860 single cells. We applied uCoTarget to the in vitro generation of hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells, presenting multimodal epigenomic profiles in 26,418 single cells. uCoTarget with high sensitivity per modality reveals establishment of pairing of HSPC enhancers (H3K27ac) and promoters (H3K4me3) along the differentiation trajectory and RUNX1 engagement priming for the H3K27ac activation along the HSPC path. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets. Together, our methods enable generalizable, versatile multi-modal profiles for reconstructing comprehensive epigenome and transcriptome landscapes and analyzing the regulatory interplay at single-cell level.