Serum Response Factor Is Critical for Terminal Erythropoiesis

Serum Response Factor (SRF) is a transcription factor that regulates gene expressions related to cytoskeleton organization, cell cycle, cell migration, and focal adhesion. Conditional SRF knockout in megakaryocyte results in reduced platelet count, and SRF deficiency in neutrophil leads to the failure of neutrophil migration to sites of inflammation. However, the role of SRF in erythropoiesis is still unknown.

To study the effect of SRF loss in erythropoiesis, we crossed Srf^fl/fl mice with Vav-Cre expressing mice and studied the erythroid differentiation in-vivo and ex-vivo. Vav-Cre driven SRF knockout mice died within 24 hours after birth due to server hemorrhage, which was possibly caused by the expression of Vav-Cre in endothelium. Complete blood count (CBC) in day 0 post-partum (P0) mice showed a dramatic reduction in red blood cell (RBC) count, hemoglobin, and hematocrit (HCT). We purified c-Kit positive cells from P0 liver and seeded the cells for in vitro culture and MethoCult multi-lineage differentiation assay. SRF null c-Kit cells showed impaired erythroid maturation characterized by decreased CD71⁺ Ter119⁺ population in EPO containing differentiation medium. In addition, the percentage of enucleated cells was decreased in SRF null derived erythroblasts. After ten days of differentiation in MethoCult gel, we counted the colony numbers and recovered the differentiated colonies by melting the gel in PBS, then profiled the differentiated cells with flowcytometry. SRF deficiency led to reduction in CD71⁺ population, and an increase in CD11b⁺ and c-Kit⁺ populations. However, no significant difference in total colony numbers was observed. These results indicate SRF plays an important role in erythropoiesis, to investigate how SRF regulates erythropoiesis, we performed CUT&RUN with in vitro erythroid differentiated erythrocytes on day 0, day 1 and day 2 post-differentiation. GO analysis showed the SRF binding sites throughout erythropoiesis were close to genes that regulate cytoskeleton organization and cell migration. Interestingly, mir101c, a microRNA that is upregulated in erythroblasts in myeloproliferative neoplasms (MPN), was also one of the most enriched gene loci where SRF was binding to during erythropoiesis, indicating SRF may promote erythroid differentiation and proliferation through regulating mir101c. GO analysis of the putative binding sites of mir101c showed "protein binding", "cell development", and "cell differentiation" are among the top enriched biological processes. To be noted, Tet2, a commonly mutated epigenetic regulator in MDS and MPN, was one of the top predicted targets of mir101c.

Both cytoskeleton organization and chromatin epigenetic remodeling are critical in terminal erythropoiesis. Together, our data indicate SRF may play a pivotal role in erythroid differentiation by trans regulating cytoskeleton organization and chromatin remolding at the same time.

No relevant conflicts of interest to declare.