A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification

Abstract

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 their cell type and cell stage-specific activity within one developmental pathway and in a chromatin context. Here, we describe a high-throughput method that identifies thousands of differentially active cis-elements able to stimulate a minimal promoter at five stages of hematopoietic progenitor development from embryonic stem (ES) cells, which can be adapted to any ES cell derived cell type. We show that blood cell-specific gene expression is controlled by the concerted action of thousands of differentiation stage-specific sets of cis-elements which respond to cytokine signals terminating at signalling responsive transcription factors. Our work provides an important resource for studies of hematopoietic specification and highlights the mechanisms of how and where extrinsic signals program a cell type-specific chromatin landscape driving hematopoietic differentiation.

Chromatin priming elements direct tissue-specific gene activity prior to hematopoietic specification

“…In our previous work we used a mouse embryonic stem cell (ESC) based in vitro differentiation system spanning 5 different stages along the path to blood cell specification (Edginton-White et al, 2023; Goode et al, 2016; Obier et al, 2016) (depicted in Fig 1A): (1) ESCs, (2) FLK1+ hemangioblast cells (HB) which give rise to hematopoietic and endothelial cells, (3) the first stage of the hemogenic endothelium (HE1) which expresses a low level of the master regulator of hematopoiesis, RUNX1; (4) the second stage of the HE, HE2 where RUNX1 is upregulated and cells show full hematopoietic commitment; and (5) the final stage, hematopoietic progenitors (HP) which are cells budding off from the HE capable of forming mature blood cells. To identify functional enhancer elements, we isolated genomic fragments from open chromatin regions and re-integrated them into the genome at a safe habour reporter site embedded in the ES cell genome as depicted schematically in Fig 1B.…”

“…In our previous work we used chromatin accessibility assays (ATAC-Seq) performed with cells isolated from a serum-free ES cell differentiation system which allowed us to examine the effect of specific cytokines on enhancer activation (Edginton-White et al, 2023; Maytum et al, 2023; Pearson et al, 2008). We found that one cytokine, VEGF, was instrumental in activating enhancers driving the expression of endothelial genes, but then needed to be withdrawn to enable the activation of hematopoietic enhancers, notably those of Runx1 which is essential for the expression of blood cell specific genes such as SPI1 (PU.1) (Huang et al, 2008).…”

Cell Type-Specific Regulation by a Heptad of Transcription Factors in Human Hematopoietic Stem and Progenitor Cells

“…However, identifying enhancers with the appropriate spatiotemporal activity for use in gene therapy vectors is not trivial. While various experimental techniques have been developed to allow massively parallel interrogation of enhancer activity and specificity (Edginton-White et al, 2023;Hrvatin et al, 2019;Xu et al, 2022), our data provides a catalogue of putative enhancers already annotated with respect to spatiotemporal activity that could fast track functional studies.…”

From Genotype to Phenotype: How Enhancers Control Gene Expression and Cell Identity in Hematopoiesis

“…53,54 Moreover, use of a Venus-YFP reporter in embryonic stem cells (ESCs) undergoing hematopoietic specification provided functional evidence that tissue-specific enhancers were associated with expression of genes in the same stage. 55 A recent publication using bacterial methylation labeling showed coordinated enhancer and gene activity throughout enterocyte differentiation. 56…”

“…In recent years, strategies that overcome some of these drawbacks have been developed. For example, in site‐specific integration fluorescence‐activated cell sorting followed by sequencing, putative enhancers coupled with a reporter are integrated into the HPRT locus of ESCs, ensuring a constant and accessible chromatin environment 55 , 135 . Differentiating cells are next sorted based on reporter expression and candidate regions enriched in populations with high reporter signal are considered functionally active enhancers.…”