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The Ufm1-activating enzyme Uba5 is indispensable for erythroid differentiation in mice.

Tatsumi K, Yamamoto-Mukai H, Shimizu R, Waguri S, Sou YS, Sakamoto A, Taya C, Shitara H, Hara T, Chung CH, Tanaka K, Yamamoto M, Komatsu M - Nat Commun (2011)

Bottom Line: In this study, we report the essential role of Uba5, a specific activating enzyme for the ubiquitin-like modifier, Ufm1, in erythroid development.Although Uba5 was dispensable for the production of erythropoietin, its genetic loss led to impaired development of megakaryocyte and erythroid progenitors from common myeloid progenitors.Our results suggest that one of the ubiquitin-like protein modification systems, the Ufm1 system, is involved in the regulation of haematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa 2-1-6, Setagaya-ku, Tokyo 156-8506, Japan.

ABSTRACT
Post-translational protein modifications are systems designed to expand restricted genomic information through functional conversion of target molecules. Ubiquitin-like post-translational modifiers regulate numerous cellular events through their covalent linkages to target protein(s) by an enzymatic cascade analogous to ubiquitylation consisting of E1 (activating), E2 (conjugating) and E3 (ligating) enzymes. In this study, we report the essential role of Uba5, a specific activating enzyme for the ubiquitin-like modifier, Ufm1, in erythroid development. Mice lacking Uba5 exhibited severe anaemia, followed by death in utero. Although Uba5 was dispensable for the production of erythropoietin, its genetic loss led to impaired development of megakaryocyte and erythroid progenitors from common myeloid progenitors. Intriguingly, transgenic expression of Uba5 in the erythroid lineage rescued the Uba5-deficient embryos from anaemia and prolonged their survival, demonstrating the importance of Uba5 in cell-autonomous erythroid differentiation. Our results suggest that one of the ubiquitin-like protein modification systems, the Ufm1 system, is involved in the regulation of haematopoiesis.

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Suppression of defective differentiation of both erythrocytes and megakaryocytes following expression of Uba5 in erythroid lineage.(a) FACS analysis. Mouse fetal liver cells were freshly isolated from control and rescued mice at E11.5 and labelled with an FITC-conjugated anti-CD71 monoclonal antibody and PE-conjugated anti-Ter119 antibody. Left gate: proerythroblast population (Ter119med CD71high), right gate: basoerythroblast population (Ter119high CD71high). Axes mean log10 fluorescent intensity. (b) Quantification of erythroid colony-forming units (CFU-Es) and (c) erythroid burst-forming units (BFU-Es). Colony assays were carried out with cells prepared from control and rescued fetal liver at E11.5. The number of CFU-E (b) and BFU-E (c) colonies remained unchanged in both genotypes. Data are means±s.d. of five mice from each group. (d) Colony formation assay for megakaryocytes (CFU-Meg). The assays were carried out with cells prepared from the fetal liver of the indicated genotypes at E11.5. Uba5 deletion was accompanied by a weaker megakaryocytic differentiation activity, and this defect was suppressed by forced expression of Uba5 in megakaryocytic lineage. Data are means±s.d. of Uba5+/− (n=3), Uba5−/− (n=4) and Uba5−/−;TgUba5 (n=3). Statistical analysis was carried out using the unpaired t-test. **P<0.01 (Welch test). (e) FACS analysis. The myeloid progenitors (Lin−IL-7R−Sca-1−c-Kit+) prepared from liver cells of control and rescued fetuses at E11.5 were subfractionated into presumptive common myeloid progenitors (CD34highFcγRII/IIIlow), granulocyte/macrophage progenitors (CD34highFcγRII/IIIhigh) and megakaryocyte/erythroid progenitors (CD34lowFcγRII/IIIlow) by FACS. Axes mean log10 fluorescent intensity. (f) TUNEL staining of the fetal livers of control and rescue mice at E12.5. Scale bars: top panel, 100 μm; bottom panel, 20 μm.
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f4: Suppression of defective differentiation of both erythrocytes and megakaryocytes following expression of Uba5 in erythroid lineage.(a) FACS analysis. Mouse fetal liver cells were freshly isolated from control and rescued mice at E11.5 and labelled with an FITC-conjugated anti-CD71 monoclonal antibody and PE-conjugated anti-Ter119 antibody. Left gate: proerythroblast population (Ter119med CD71high), right gate: basoerythroblast population (Ter119high CD71high). Axes mean log10 fluorescent intensity. (b) Quantification of erythroid colony-forming units (CFU-Es) and (c) erythroid burst-forming units (BFU-Es). Colony assays were carried out with cells prepared from control and rescued fetal liver at E11.5. The number of CFU-E (b) and BFU-E (c) colonies remained unchanged in both genotypes. Data are means±s.d. of five mice from each group. (d) Colony formation assay for megakaryocytes (CFU-Meg). The assays were carried out with cells prepared from the fetal liver of the indicated genotypes at E11.5. Uba5 deletion was accompanied by a weaker megakaryocytic differentiation activity, and this defect was suppressed by forced expression of Uba5 in megakaryocytic lineage. Data are means±s.d. of Uba5+/− (n=3), Uba5−/− (n=4) and Uba5−/−;TgUba5 (n=3). Statistical analysis was carried out using the unpaired t-test. **P<0.01 (Welch test). (e) FACS analysis. The myeloid progenitors (Lin−IL-7R−Sca-1−c-Kit+) prepared from liver cells of control and rescued fetuses at E11.5 were subfractionated into presumptive common myeloid progenitors (CD34highFcγRII/IIIlow), granulocyte/macrophage progenitors (CD34highFcγRII/IIIhigh) and megakaryocyte/erythroid progenitors (CD34lowFcγRII/IIIlow) by FACS. Axes mean log10 fluorescent intensity. (f) TUNEL staining of the fetal livers of control and rescue mice at E12.5. Scale bars: top panel, 100 μm; bottom panel, 20 μm.

Mentions: Next, we examined the effect of Uba5 expression in the erythroid lineage on the differentiation of proerythroblasts and basophilic erythroblasts in Uba5−/− mice. The decreased population of proerythroblasts and basophilic erythroblasts in Uba5-deficient fetal liver (4.2±3.2% and 1.0±0.6%, respectively, n=3, Fig. 2a) was rescued by forced expression of Uba5 (9.7±1.6% and 18.2±8.7%, respectively, n=3, Fig. 4a). Subsequently, we confirmed both CFU-E and BFU-E activities in cells derived from Uba5-rescued mouse fetal livers (Fig. 4b,c, and compare with Fig. 2b,c) as well as the sufficient ability to form CFU-Megs (Fig. 4d). As expected, the decreased population of MEPs in Uba5-deficient fetal liver (4.1±3.5%, n=3, Fig. 2d) was recovered in the rescued fetal liver (10.2±1.6%, n=3, Fig. 4e). Moreover, unlike the increase in TUNEL-positive cells in Uba5-deficient fetal livers, this phenomenon was hardly detected in the rescued mouse livers (Fig. 4f and Supplementary Fig. S5b). Taken together, these results indicate that Uba5 expression in erythroid and megakaryocytic progenitors grants functional rescue of defective erythropoiesis and megakaryopoiesis in Uba5−/− mice.


The Ufm1-activating enzyme Uba5 is indispensable for erythroid differentiation in mice.

Tatsumi K, Yamamoto-Mukai H, Shimizu R, Waguri S, Sou YS, Sakamoto A, Taya C, Shitara H, Hara T, Chung CH, Tanaka K, Yamamoto M, Komatsu M - Nat Commun (2011)

Suppression of defective differentiation of both erythrocytes and megakaryocytes following expression of Uba5 in erythroid lineage.(a) FACS analysis. Mouse fetal liver cells were freshly isolated from control and rescued mice at E11.5 and labelled with an FITC-conjugated anti-CD71 monoclonal antibody and PE-conjugated anti-Ter119 antibody. Left gate: proerythroblast population (Ter119med CD71high), right gate: basoerythroblast population (Ter119high CD71high). Axes mean log10 fluorescent intensity. (b) Quantification of erythroid colony-forming units (CFU-Es) and (c) erythroid burst-forming units (BFU-Es). Colony assays were carried out with cells prepared from control and rescued fetal liver at E11.5. The number of CFU-E (b) and BFU-E (c) colonies remained unchanged in both genotypes. Data are means±s.d. of five mice from each group. (d) Colony formation assay for megakaryocytes (CFU-Meg). The assays were carried out with cells prepared from the fetal liver of the indicated genotypes at E11.5. Uba5 deletion was accompanied by a weaker megakaryocytic differentiation activity, and this defect was suppressed by forced expression of Uba5 in megakaryocytic lineage. Data are means±s.d. of Uba5+/− (n=3), Uba5−/− (n=4) and Uba5−/−;TgUba5 (n=3). Statistical analysis was carried out using the unpaired t-test. **P<0.01 (Welch test). (e) FACS analysis. The myeloid progenitors (Lin−IL-7R−Sca-1−c-Kit+) prepared from liver cells of control and rescued fetuses at E11.5 were subfractionated into presumptive common myeloid progenitors (CD34highFcγRII/IIIlow), granulocyte/macrophage progenitors (CD34highFcγRII/IIIhigh) and megakaryocyte/erythroid progenitors (CD34lowFcγRII/IIIlow) by FACS. Axes mean log10 fluorescent intensity. (f) TUNEL staining of the fetal livers of control and rescue mice at E12.5. Scale bars: top panel, 100 μm; bottom panel, 20 μm.
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f4: Suppression of defective differentiation of both erythrocytes and megakaryocytes following expression of Uba5 in erythroid lineage.(a) FACS analysis. Mouse fetal liver cells were freshly isolated from control and rescued mice at E11.5 and labelled with an FITC-conjugated anti-CD71 monoclonal antibody and PE-conjugated anti-Ter119 antibody. Left gate: proerythroblast population (Ter119med CD71high), right gate: basoerythroblast population (Ter119high CD71high). Axes mean log10 fluorescent intensity. (b) Quantification of erythroid colony-forming units (CFU-Es) and (c) erythroid burst-forming units (BFU-Es). Colony assays were carried out with cells prepared from control and rescued fetal liver at E11.5. The number of CFU-E (b) and BFU-E (c) colonies remained unchanged in both genotypes. Data are means±s.d. of five mice from each group. (d) Colony formation assay for megakaryocytes (CFU-Meg). The assays were carried out with cells prepared from the fetal liver of the indicated genotypes at E11.5. Uba5 deletion was accompanied by a weaker megakaryocytic differentiation activity, and this defect was suppressed by forced expression of Uba5 in megakaryocytic lineage. Data are means±s.d. of Uba5+/− (n=3), Uba5−/− (n=4) and Uba5−/−;TgUba5 (n=3). Statistical analysis was carried out using the unpaired t-test. **P<0.01 (Welch test). (e) FACS analysis. The myeloid progenitors (Lin−IL-7R−Sca-1−c-Kit+) prepared from liver cells of control and rescued fetuses at E11.5 were subfractionated into presumptive common myeloid progenitors (CD34highFcγRII/IIIlow), granulocyte/macrophage progenitors (CD34highFcγRII/IIIhigh) and megakaryocyte/erythroid progenitors (CD34lowFcγRII/IIIlow) by FACS. Axes mean log10 fluorescent intensity. (f) TUNEL staining of the fetal livers of control and rescue mice at E12.5. Scale bars: top panel, 100 μm; bottom panel, 20 μm.
Mentions: Next, we examined the effect of Uba5 expression in the erythroid lineage on the differentiation of proerythroblasts and basophilic erythroblasts in Uba5−/− mice. The decreased population of proerythroblasts and basophilic erythroblasts in Uba5-deficient fetal liver (4.2±3.2% and 1.0±0.6%, respectively, n=3, Fig. 2a) was rescued by forced expression of Uba5 (9.7±1.6% and 18.2±8.7%, respectively, n=3, Fig. 4a). Subsequently, we confirmed both CFU-E and BFU-E activities in cells derived from Uba5-rescued mouse fetal livers (Fig. 4b,c, and compare with Fig. 2b,c) as well as the sufficient ability to form CFU-Megs (Fig. 4d). As expected, the decreased population of MEPs in Uba5-deficient fetal liver (4.1±3.5%, n=3, Fig. 2d) was recovered in the rescued fetal liver (10.2±1.6%, n=3, Fig. 4e). Moreover, unlike the increase in TUNEL-positive cells in Uba5-deficient fetal livers, this phenomenon was hardly detected in the rescued mouse livers (Fig. 4f and Supplementary Fig. S5b). Taken together, these results indicate that Uba5 expression in erythroid and megakaryocytic progenitors grants functional rescue of defective erythropoiesis and megakaryopoiesis in Uba5−/− mice.

Bottom Line: In this study, we report the essential role of Uba5, a specific activating enzyme for the ubiquitin-like modifier, Ufm1, in erythroid development.Although Uba5 was dispensable for the production of erythropoietin, its genetic loss led to impaired development of megakaryocyte and erythroid progenitors from common myeloid progenitors.Our results suggest that one of the ubiquitin-like protein modification systems, the Ufm1 system, is involved in the regulation of haematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Protein Metabolism Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa 2-1-6, Setagaya-ku, Tokyo 156-8506, Japan.

ABSTRACT
Post-translational protein modifications are systems designed to expand restricted genomic information through functional conversion of target molecules. Ubiquitin-like post-translational modifiers regulate numerous cellular events through their covalent linkages to target protein(s) by an enzymatic cascade analogous to ubiquitylation consisting of E1 (activating), E2 (conjugating) and E3 (ligating) enzymes. In this study, we report the essential role of Uba5, a specific activating enzyme for the ubiquitin-like modifier, Ufm1, in erythroid development. Mice lacking Uba5 exhibited severe anaemia, followed by death in utero. Although Uba5 was dispensable for the production of erythropoietin, its genetic loss led to impaired development of megakaryocyte and erythroid progenitors from common myeloid progenitors. Intriguingly, transgenic expression of Uba5 in the erythroid lineage rescued the Uba5-deficient embryos from anaemia and prolonged their survival, demonstrating the importance of Uba5 in cell-autonomous erythroid differentiation. Our results suggest that one of the ubiquitin-like protein modification systems, the Ufm1 system, is involved in the regulation of haematopoiesis.

Show MeSH
Related in: MedlinePlus