C-Myb Transactivates the Expression of Erythroid Hsa-miR16-2 Gene,

Abstract 3386

Deregulated expression of miRNAs is associated with neoplasia. We recently showed that miR-16-2 is overexpressed in CD34(+) cells of patients with polycythemia vera (PV) versus their normal counterparts and that deregulation of miR-16-2 contributes, in a way independent of JAK/STAT pathway activation, to the abnormal expansion of the erythroid lineage characterizing PV. In fact, forced expression of miR-16 in normal CD34+ cells stimulated erythroid maturation while exposure of PV CD34(+) cells to antagomirs against pre-miR-16-2 reduced erythroid colonies. Moreover, erythroid fate was impaired in mice injected with a miR-16 antagomir, indicating a role of miR-16 in normal erythropoiesis. Collectively these data identified miR-16-2 as a positive regulator of the erythropoiesis and linked the abnormal expansion toward the erythroid lineage to the overexpression of miR-16-2 in PV patients (Guglielmelli P, Blood, 117:6923–6927, 2011). However, the mechanisms underlying miR-16-2 overexpression are still unknown.

In this project we aim to identify the transcription factors regulating miR-16-2 expression in normal and PV erythropoiesis.

miR-16-2 is an intronic miRNA located within the SMC4 (Structural Maintenance of Chromosome 4) gene on chromosome 3. A functionally defined promoter of miR-16 has not been characterized yet. Therefore, (1) based on data from genome-wide studies of H3K4me3, H3K9/14Ac, RNA pol II-enrichment and nucleosome positioning identifying a RNA pol II-enriched region overlapping the SMC4 promoter for miR16-2 expression control and (2) based on the evidence of a correlation between the expression levels of this intronic miRNA and those of its host gene SMC4, we focused our attention on the transcriptional regulators of SMC4 gene.

We screened the SMC4 gene promoter region in order to identify putative binding sites for transcription factors already known to be involved in erythroid differentiation, such as c-myb, KLF1 and GATA1. Next, we cloned the SMC4 promoter region between ∼20 bp downstream and 1200 bp upstream the Transcription Start Site into the pXP1 plasmid, upstream to the promoterless firefly luciferase reporter gene. HEK293T cells were transfected with the pXP1 vector carrying the Luciferase reporter gene under the SMC4 promoter control and increasing amounts of plasmid coding for either c-myb or KLF1. Luciferase activity measurements were done in duplicate and signals were normalized for transfection efficiency to the internal Renilla control. At least 3 independent experiments were performed for each of the transcription factors tested.

Our data demonstrated that increasing levels of c-myb protein expression are able to transactivate SMC4 promoter-driven luciferase expression. In fact, increasing amounts of the c-myb-coding plasmid determined a dose-dependent increase in SMC4 promoter-driven luciferase activity (735+/−196, 995+/−286 and 1759+/−474 for 100, 200 and 400ng of c-myb-coding plasmid respectively, versus 590+/−190 for the empty plasmid control; average+/−SD values). Therefore, the c-myb-driven SMC4 promoter transactivation trend identified (P<0.01 in myb-coding versus empty vector transfected samples) points out the potential involvement of c-myb in SMC4/miR-16-2 upregulation during normal and/or pathologic erythroid differentiation. On the contrary, increasing levels of KLF1 expression failed to affect SMC4 promoter-driven luciferase gene expression, suggesting that a role for KLF1 in this process could be ruled out. Further experiments will elucidate the role of GATA1 in this process.

In conclusion, our data demonstrated that c-myb is able to transactivate SMC4/miR16-2 expression, by shedding for the first time some light on the molecular players involved in normal and PV erythropoiesis. Despite the overwhelming body of studies demonstrating the key role of c-myb in the erythropoiesis, little is known on the molecular mechanisms of c-myb-driven erythroid differentiation. We recently gained insights in this process by demonstrating that c-myb supports erythropoiesis by transactivating KLF1 and LMO2 expression (Bianchi E, Blood, 116:e99–110, 2010). The present data suggest SMC4/miR16 transactivation as a new pathway through which c-myb affects the erythroid differentiation. However, further studies need to be performed to more deeply unravel this mechanism and its relevance in normal and PV erythropoiesis.