Transcriptome- Based Analysis of EPO Dependent Cfu-e to Erythroblast Development

Abstract 3188

To provide molecular insight into erythroid developmental programs, including EPO- regulated aspects, we have employed transcriptome-based approaches to analyze the stage-wise development of purified murine bone marrow- derived CFUe, proerythroblasts and maturing erythroblasts. In vivo and ex vivo, these progenitors develop as Kit^posCD71^highTer119^neg; Kit^negCD71^highTer119^neg; and Kit^negCD71^highTer119^pos cohorts (designated as E1, E2, E3 stages, respectively). In the context of EPO- modulation, stage E1 cells exhibited ∼250 EPO- regulated target genes. In stage-E2 proerythroblasts, in contrast, 750 transcripts proved to be EPO- regulated while stage-E3 erythroblasts exhibited only select EPO- modulated genes (<50). At E1 and E2 stages, EPO- regulated targets included overlapping yet distinct sets, and this was reflected in functional sets of GO terms. Major EPO- targets at stage E1 included cell cycle and cell biogenesis genes, while in E2 proerythroblasts, negative regulators of protein kinases (and kinase activity) constituted a major EPO/EPOR target set. As E1 CFUe transitioned to E2 proerythroblasts, an unexpected transient narrowing in the complexity of overall expressed genes was exhibited. Stage- modulated global sets of transcripts included myeloid cell differentiation factors, cell number homeostasis factors and heme biosynthetic processes. As E2 proerythroblasts transitioned to E3 erythroblasts, functional GO sets were associated predominantly with dynamics in organelle and cellular compartments. In addition, HomoloGene and Connectivity Mapping approaches were applied to compare transcriptomes of stage E1, E2 and E3 murine bone marrow erythroid progenitors with four recently studied stages of human erythroid progenitor cell development (here, termed H1, H2, H3 and H4). High correlation of stage E1 m-CFUe with not only human H1 CFUe but also H2 proerythroblasts was observed (0.59 and 0.57 correlations). Stage E2 murine proerythroblasts best corresponded to H2 human proerythroblasts (0.36 correlation score), while E3 murine erythroblasts aligned closely with human H4 late stage erythroblasts (0.58 correlation score). These latter analyses provide novel transcriptome- based comparisons, and transcript specific insight, into conserved vs distinct features of murine and human erythroid development at CFUe to maturing erythroblast stages.