Abstract
The expression level of transcription factor c-Myb oscillates during hematopoiesis. Fbw7 promotes ubiquitin-mediated degradation of c-Myb, which is dependent on phosphorylation of Thr572. To investigate the physiological relevance of Fbw7-mediated c-Myb degradation, we generated mutant mice carrying c-Myb-T572A (TA). Homozygous mutant (TA/TA) mice exhibited a reduction in the number of peripheral red blood cells and diminished erythroblasts in bone marrow, presumably as a result of failure during erythroblast differentiation. We found that c-Myb high-expressing cells converged in the Lin−CD71+ fraction, and the expression of c-Myb was higher in TA/TA mice than in wild-type mice. Moreover, TA/TA mice had an increased proportion of the CD71+ subset in Lin− cells. The c-Myb level in the Lin−CD71+ subset showed three peaks, and the individual c-Myb level was positively correlated with that of c-Kit, a marker of undifferentiated cells. Ultimately, the proportion of c-Mybhi subgroup was significantly increased in TA/TA mice compared with wild-type mice. These results indicate that a delay in reduction of c-Myb protein during an early stage of erythroid differentiation creates its obstacle in TA/TA mice. In this study, we showed the T572-dependent downregulation of c-Myb protein is required for proper differentiation in early-stage erythroblasts, suggesting the in vivo significance of Fbw7-mediated c-Myb degradation.
Highlights
The expression level of transcription factor c-Myb oscillates during hematopoiesis
By immunoblot analysis with mouse tissue lysates using clone 1–1 anti c-Myb antibodies, we observed that c-Myb expression was abundant in the thymus but was not detectable in the bone marrow (BM), which is mainly composed of L in+ cells (Supplementary Fig. S4a)
The sorted Lin−c-Kit+ subset, which belongs to BM-derived cells, showed higher expression of c-Myb than thymocytes, which we confirmed by immunoblot assay (Supplementary Fig. S4b)
Summary
The expression level of transcription factor c-Myb oscillates during hematopoiesis. Fbw[7] promotes ubiquitin-mediated degradation of c-Myb, which is dependent on phosphorylation of Thr[572]. The proportion of c-Mybhi subgroup was significantly increased in TA/TA mice compared with wild-type mice These results indicate that a delay in reduction of c-Myb protein during an early stage of erythroid differentiation creates its obstacle in TA/TA mice. Levels of c-Myb decrease during terminal differentiation to mature blood cells[4]; tetracycline-regulated expression of c-Myb in c-Myb−/− embryonic stem (ES) cells prevents the terminal differentiation of erythrocytes and megakaryocytes[5] These observations prove that appropriate levels of c-Myb protein are strictly defined in distinct stages of differentiation of the hematopoietic cell lineage to maintain normal proliferation and differentiation. Consistent with this role, several c-Myb target genes linked to proliferation and differentiation, such as c-Myc and c-Kit, have been identified in myeloid cells[2]. These findings indicate that Fbw[7] plays different roles in diverse situations
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