Abstract
The c-Myb transcription factor, a key regulator of proliferation and differentiation in hematopoietic and other cell types, has an N-terminal DNA binding domain and a large C-terminal domain responsible for transcriptional activation, negative regulation and determining target gene specificity. Overexpression and rearrangement of the c-myb gene (MYB) has been reported in some patients with leukemias and other types of cancers, implicating activated alleles of c-myb in the development of human tumors. Alternative RNA splicing can produce variants of c-myb with qualitatively distinct transcriptional activities that may be involved in transformation and leukemogenesis. Here, by performing a detailed, single molecule assay we found that c-myb alternative RNA splicing was elevated and much more complex in leukemia samples than in cell lines or CD34+ hematopoietic progenitor cells from normal donors. The results revealed that leukemia samples express more than 60 different c-myb splice variants, most of which have multiple alternative splicing events and were not detectable by conventional microarray or PCR approaches. For example, the single molecule assay detected 21 and 22 splice variants containing the 9B and 9S exons, respectively, most of which encoded unexpected variant forms of c-Myb protein. Furthermore, the detailed analysis identified some splice variants whose expression correlated with poor survival in a small cohort of precursor B-ALL samples. Our findings indicate that single molecule assays can reveal complexities in c-myb alternative splicing that have potential as novel biomarkers and could help explain the role of c-Myb variants in the development of human leukemia.
Highlights
We set out to determine whether the shift in the patterns of c-myb alternative splicing could be a novel mechanism for activating c-Myb oncogenicity in leukemias
The c-myb gene is organized in way that the vast majority of alternative RNA splicing occurs in exons encoding the C-terminal domains of c-Myb protein, generating a family of transcription factors with the same N-terminal DNA binding domains but differences affecting transcriptional activity, negative regulation and target gene selection [8,16,17,18]
We found that c-myb alternative splicing is greatly increased in precursor B-ALL samples, that the pattern of c-myb alternative splicing is more complex in leukemia patients than in normal CD34+ cells, and that the pattern is distinct in each patient sample, suggesting that activation of alternative RNA splicing is a mechanism that could contribute to leukemogenesis
Summary
The c-myb gene (MYB) encodes a transcription factor that is absolutely required for normal hematopoiesis [1] including the differentiation of stem cells into committed progenitors [2], the normal development of myeloid and erythroid lineages [1,3] and for B-cell and T-cell differentiation [4,5]. (Note: we use the designations c-myb and c-Myb for the gene and protein, respectively, and to distinguish the normal cellular versions from the oncogenic variants v-myb and v-Myb) The c-Myb protein has a highly conserved DNA binding domain near its N-terminus and a large C-terminal domain required for transcriptional activation as well as negative regulation [6,7,8]. The mechanisms leading to activation of the c-myb gene, and its conversion from a normal regulator to an oncogene, remain largely unexplained [13]. Most of the differences in the activity of v-Myb are due to changes in the large C-terminal domain, which appears to control target gene specificity by affecting protein-protein interactions [8,17]. The implication is that the activity of c-Myb is highly variable and subject to change through mechanisms that alter its C-terminal domain, such as post-translational modifications, mutations or deletions. Such modifications can target c-Myb to different promoters and convert it from a normal regulator into a potent transforming protein
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