The c-myb proto-oncogene and microRNA-15a comprise an active autoregulatory feedback loop in human hematopoietic cells.
The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment, proliferation, and differentiation. Given its critical functions, c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay, we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic, functionality of binding was shown. The mimic decreased c-Myb expression, and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest, the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally, in studies using normal human CD34(+) cells, we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation, and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate, these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis.
Zhao
Huiwu
H
Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Kalota
Anna
A
Jin
Shenghao
S
Gewirtz
Alan M
AM
D020413:3' Untranslated Regions / Q000235:genetics; D018952:Antigens, CD34 / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001773:Blood Cells / Q000166:cytology / Q000378:metabolism*; D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology; D025461:Feedback, Physiological / Q000502:physiology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D020570:Genes, myb / Q000502:physiology*; D006410:Hematopoiesis / Q000502:physiology*; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D047468:Immunoprecipitation; D020014:K562 Cells; D007938:Leukemia / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D020413:3' Untranslated Regions; D018952:Antigens, CD34; C000715470:MAS1 protein, human; C505361:MIRN15 microRNA, human; D035683:MicroRNAs; D000090063:Proto-Oncogene Mas; D020598:Proto-Oncogene Proteins c-myb