C-Myb Regulates Mir-101 Expression During the Early Stages of Erythropoiesis
Authors: Brito Giselle , Li Ningkun , Shetzline Susan E. 
Published: October 2019

Abstract
Abstract 3876
The proto-oncogene c-myb encodes the transcription factor c-Myb, which is predominantly expressed in immature hematopoietic cells where it plays an obligate role in definitive hematopoiesis. Given the critical functions of c-Myb in lineage commitment, proliferation, and differentiation, c-Myb regulatory factors are of great interest but remain incompletely defined. In recent years, c-Myb has been shown to regulate the expression of microRNA (miRNA) molecules in hematopoietic cells. MiRNA molecules are noncoding RNA molecules that are 21–23 nucleotides in length and function to hybridize to the 3′UTR region of its target mRNA to stimulate/repress translation or induce mRNA degradation. For example, in hematopoietic cells, miR-15a and c-Myb form an autoregulatory negative feedback loop in that over-expression of miR-15a in hematopoietic cells was determined to block erythroid and myeloid colony formation. In megakaryocytes, the hormone thrombopoietin induced miR-150 expression which subsequently functioned to degrade c-myb mRNA through direct interaction with c-myb's 3′-UTR. Our studies have focused on determining the physiologic function of the neuroendocrine Neuromedin U (NmU) during the early stages of erythropoiesis, because we recently determined that silencing NmU in primary human CD34+ cells impairs burst-forming units-erythroid and colony-forming unit-erythroid formation. In subsequent studies, we determined that c-Myb directly interacts with the NmU promoter at Myb Response Elements (MREs) distal to its transcription start site. Also, the expression profiles of NmU and c-myb are similar in CD34+ cells cultured under erythroid inducing conditions for 10 days, and silencing c-myb expression in hematopoietic cells inhibits NmU expression. To gain insight into the regulatory mechanism involved in NmU expression during the early stages of erythropoiesis, we hypothesized that miRNA molecules regulated by c-Myb would inhibit NmU expression through a negative feedback loop. To address this hypothesis, we first scanned the 3′-UTR of NmU and identified 24 different miRNA molecules predicted to interact with NmU's 3′-UTR. Second, we used luciferase reporter assays to determine which of the miRNA molecules interacted with NmU's 3′-UTR. Of the three miRNA molecules we tested, miR-101 directly interacted with NmU's 3′-UTR in a dose-dependent manner. Third, we determined the expression profile of miR-101 in primary CD34+ cells cultured under erythroid inducing conditions. The gene expression of miR-101 was inversely correlated with NmU and c-myb. Finally, because miR-101 contained 6 MREs, we determined the ability of c-Myb to directly interact with the promoter of miR-101 using chromatin immunoprecipitation (ChIP) assays. Using primers that flank the MREs proximal to miR-101's transcription start site, we observed a greater than 2-fold increase in the amplification of DNA recovered from ChIP assays completed with c-Myb antibody compared to ChIP assays completed with irrelevant antibody. Studies are underway to confirm by luciferase-reporter assays that c-Myb directly binds to and transactivates the miR-101 promoter. Collectively, these data identify a regulatory loop comprised of c-Myb, NmU, and miR-101 that could be of potential importance during human erythropoiesis.

Disclosures:
No relevant conflicts of interest to declare.