1005 – A GENOME-WIDE RELAY OF SIGNALLING-RESPONSIVE ENHANCERS DRIVES HEMATOPOIETIC SPECIFICATION

Developmental control of gene expression critically depends on distal cis-regulatory elements including enhancers which interact with promoters to activate gene expression. To date no global experiments have been conducted that identify dynamic cell type and cell stage-specific enhancer activity within one developmental pathway and in a chromatin context. Using the differentiation of mouse embryonic stem cells as model, we previously conducted a multi-omics analysis which revealed dynamic gene regulatory networks specific for each of the major stages of blood cell specification (Goode et al., Dev Cell, 2016). However, whilst we correlated chromatin alterations with dynamic gene expression, our data did not provide functional evidence for (i) which cis-elements have enhancer activity, (ii) how extrinsic signals control their activity and (iii) which transcription factors mediate signalling responsiveness.Here, we describe a high-throughput method that identifies differentially active cis-elements able to stimulate a minimal promoter at five stages of hematopoietic specification. We show that blood cell-specific gene expression is controlled by thousands of differentiation stage-specific cis-elements and demonstrate that differential enhancer usage is directly correlated with differential gene expression. Many of these elements respond to specific cytokines and signalling responsiveness is developmentally regulated. Single cell RNA-Seq demonstrates that differential enhancer usage was directly correlated with an alteration in cell fate. Finally, we found that vascular endothelial growth factor (VEGF) impacts on enhancers regulating the finely tuned balance between endothelial and hematopoietic fate and is part of a signalling-responsive enhancer and transcription factor network that drives blood cell specification. We believe that our work presents a major advance in our understanding of developmental gene expression control in the hematopoietic system and beyond.