A Genetic Strategy for Single and Combinatorial Analysis of miRNA Function in Mammalian Hematopoietic Stem Cells

Abstract

The regulatory role of micro-RNAs (miRNAs) in hematopoietic development is increasingly appreciated. Reverse genetics strategies based on the targeted disruption of miRNAs offer a powerful tool to study miRNA functions in mammalian hematopoiesis. The miR-144/451 cluster comprises two miRNAs coexpressed from a common precursor transcript in an erythroid-specific manner. To decipher the contribution of each miRNA of the cluster in mammalian erythropoiesis, we developed a strategy for stable in vivo individual and combinatorial miRNA inhibition. We developed decoy target sequences for each miRNA expressed by lentiviral vectors marked with distinct fluorescent proteins and used them to probe the functions of miR-144 and miR-451 in the murine hematopoietic system in a competitive repopulation setting. Murine hematopoietic chimeras expressing lentiviral-encoded inhibitory sequences specific for miR-144 or miR-451 exhibited markedly reduced Ter119 1 erythroblast counts, with the combined knockdown showing additive effect. These chimeras showed abnormal patterns of erythroid differentiation primarily affecting the proerythroblast to basophilic erythroblast transition, coinciding with the stage where expression of the miRNA cluster is dramatically induced and posttranscriptional gene regulation becomes prominent. These results reveal a role for the miR-144/451 locus in mammalian erythropoiesis and provide the first evidence of functional cooperativity between clustered miRNAs in the hematopoietic system. The strategy described herein will prove useful in functional miRNA studies in mammalian hematopoietic stem cells.

miR-451 protects against erythroid oxidant stress by repressing 14-3-3ζ

“…Discussion miR-144/451 is strongly induced during erythropoiesis in zebrafish, mice, and humans (Kloosterman et al 2006;Choong et al 2007;Masaki et al 2007;Zhan et al 2007;Dore et al 2008;Du et al 2009), and artificial manipulation of these miRNAs alters erythroid maturation in several experimental models (Zhan et al 2007;Dore et al 2008;Pase et al 2009;Papapetrou et al 2010). Here we report the consequences of miR-144/451 ablation in mice, and further investigations into the effects of miR-451 depletion in zebrafish.…”

“…These miRNAs are strongly induced during zebrafish, mouse, and human erythroid maturation (Bruchova et al 2007;Choong et al 2007;Masaki et al 2007;Zhan et al 2007;Dore et al 2008;Merkerova et al 2008;Du et al 2009;Fu et al 2009;Pase et al 2009;Papapetrou et al 2010). miR-451 is also present in mature circulating red blood cells (Kloosterman et al 2006;Rathjen et al 2006).…”

“…Overexpression of miR-451 augments chemical-induced maturation of murine erythroleukemia (MEL) cells. Conversely, miR-451/miR-144 antagonists inhibit erythroid maturation in MEL cells, in hematopoietic stem cells reintroduced into mice (Papapetrou et al 2010), and in zebrafish embryos (Dore et al 2008;Du et al 2009;Pase et al 2009). In zebrafish, miR-451 targets mRNA encoding transcription factor gata2 (Pase et al 2009), and miR-144 regulates the production of embryonic a-globin indirectly by inhibiting klfd, a Kruppel-like DNA-binding protein Du et al 2009).…”

The Potential Role of miR-451 in Cancer Diagnosis, Prognosis, and Therapy

“…Overexpression of miR-451 augments dimethyl sulfoxide-induced erythroid differentiation in MEL cells (11). Conversely, loss-of-function of miR-144/451 impedes erythroid maturation in MEL cells (4), in zebrafish embryos (4,6,34), and in mice (9,35). miR-144 has been shown to negatively regulate a-hemoglobin expression and form a feedback circuitry in fine-turning of its expression (13).…”

microRNAs as Therapeutic Targets to Combat Diverse Human Diseases

“…(Barbato, et al, 2010) Both miR-144 and miR-451 were found to be required for erythropoiesis in bone marrow reconstitution experiments 3-4 weeks after transplantation of cells expressing miRNA sponges. (Papapetrou, et al, 2010) …”