Characterization of human erythropoiesis using ex vivo differentiation of CD34 HSC (ChIP-seq)

Genome Wides Association Studies (GWAS) have identified tens of thousands of associations between human genetic variation and common disease. Despite the abundance of GWAS associations, functional identification and characterization of causative variants and effector genes remains a challenging prospect. Human erythropoiesis provides a highly tractable model system for the development of tools for GWAS analysis. We have developed a three-phase protocol for differentiation of peripheral CD34+ Haematopoietic Stem Cells (HSC) into mature enucleating erythrocytes. This protocol has been characterized for it’s the changes observed morphologically, immunologically (by FACS) and epigenetically (by ATAC-seq, ChIP-seq, and RNA-seq). We have used this protocol to study the effects of common GWAS variants affecting red blood cell traits, and more severe mutations causing Type-1 Congenital Dyserythropoietic Anemia (CDA-I). To characterise the epigenetic landscape and identify chromatin modifications associated with nucleosome depleted regions, ChIP-seq was carried out on differentiating erythroid cells at Day 10. CD34+ cells from normal leukocyte cones (NCO) were differentiated simultaneously to Day 10 and ChIP was performed for enhancer (H3K4me1), promoter (H3K4me3) and active transcription (H3K27ac) marks, as well as for the boundary protein CTCF. All ChIPs were performed on the same material simultaneously and a 5% input was stored prior to immunoprecipitation as a control.