Genomic Determinants of Human Fetal and Adult Erythroid Gene Expression

Abstract 1227

Erythropoiesis in mammals occurs in three waves consisting of primitive progenitors in the yolk sac, definitive erythroid precursors in the fetal liver and later in the postnatal bone marrow. The molecular determinants of developmental stage-specific gene expression programs remain largely unknown. Several transcription factors, including GATA1 and TAL1, are essential for normal erythroid development in vivo and are recognized as ‘master’ regulators. These lineage-specifying master regulators, together with other transcriptional co-regulators, act within complexes on chromatin, establish transcriptional networks, and orchestrate the differentiation process. However, it is less clear how master regulators control gene expression programs at different stages of development within the same cell lineage.

We reasoned that comparative transcriptome, transcription factor, and epigenetic profiling of closely related cell types corresponding to distinct developmental stages should delineate the regulatory networks that are directly related to the associated gene expression programs. Classification of the trans- and cis-regulatory elements that are either shared or stage-specific should clarify their relative importance and prioritize functional candidates. To explore this approach, we focused on an ex vivo maturation system for human fetal and adult erythropoiesis. Primary human hematopoietic stem/progenitor cells (HSPCs) are propagated and induced for erythroid differentiation ex vivo. We first determined the mRNA expression profiles in both fetal and adult HSPCs and differentiating proerythroblasts (ProEs). Comparative transcriptome profiling revealed distinct gene expression programs at different stages of erythroid maturation. For example, 1039 and 1291 genes linked to distinct functional annotations were differentially expressed (fold change > 1.5, FDR < 0.05) in fetal and adult ProEs, respectively.

To investigate the underlying basis of these distinct gene expression programs, we generated genome-wide maps for chromatin state and transcription factor occupancy by a ChIP-seq approach. Specifically, we profiled 9 histone modifications (H3K4me1/me2/me3, H3K9me3, H3K37me3, H3K36me2/me3, H3K9ac, and H3K27ac) and 6 transcription factors (GATA1, TAL1, NFE2, CTCF, RAD21, and RNA polymerase II) in both fetal and adult ProEs. Contrasting the similarities and differences between human fetal and adult erythropoiesis provides important insights into the erythroid gene expression programs and gene regulatory networks operating at different stages of development. We find that gene-distal enhancers, rather than promoters, are marked with highly stage-specific histone modifications and DNase I hypersensitivity, strongly correlate to developmental stage-specific gene expression changes, and are functionally active in a stage-specific manner. The master regulators GATA1 and TAL1 act cooperatively within active enhancers but have little predictive value for stage-specific transcriptional activity. Differential enrichment of consensus motifs for binding of transcription factors within fetal or adult stage-specific enhancers provides a strategy for identifying candidate co-regulators that drive differential gene expression and stage-specificity. By this computational approach and subsequent functional validation, we demonstrate that the interferon regulatory factors IRF2 and IRF6 are essential for activation of adult erythroid gene expression programs in cooperation with master regulators and cohesin-mediator complexes at distal enhancers. Thus, the comparative profiling of red cell development provides critical insights into the ontogeny of human erythropoiesis and temporal regulation of transcriptional networks in a mammalian genome.