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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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Related in: MedlinePlus

Uba5 is indispensable for the development of erythroid cells.(a) Representative FACS analysis. Mouse fetal liver cells were freshly isolated from E11.5 embryos and labelled with a 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 fetal liver at E11.5. Uba5 deletion was associated with decreased erythroid progenitor activity. Data are means±s.d. of five mice from each group. Statistical analysis was performed using the unpaired t-test. *P<0.05 (Welch test). (d) Representative FACS analysis. The myeloid progenitors (Lin−IL-7R−Sca-1−c-Kit+) prepared from fetal liver cells of the indicated genotype mice 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. (e) TUNEL staining of fetal livers of Uba5+/− and Uba5−/− mice at E12.5. Scale bars: top panel, 100 μm; bottom panel, 20 μm. (f) Quantification of mRNA levels of erythroid-related genes, including Gata1, Fog1 and EPO, in Uba5-deficient liver at E11.5. Total RNAs were prepared from wild-type or Uba5-deficient livers and reverse transcribed into their respective cDNAs, which were used as templates for quantitative real-time PCR. Values were expressed relative to the amount of the respective mRNA in the wild-type livers. The experiments were conducted three times. Data are means±s.d. of three experiments. Statistical analysis was carried out by the unpaired t-test. *P<0.05 and **P<0.01 (Welch test).
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f2: Uba5 is indispensable for the development of erythroid cells.(a) Representative FACS analysis. Mouse fetal liver cells were freshly isolated from E11.5 embryos and labelled with a 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 fetal liver at E11.5. Uba5 deletion was associated with decreased erythroid progenitor activity. Data are means±s.d. of five mice from each group. Statistical analysis was performed using the unpaired t-test. *P<0.05 (Welch test). (d) Representative FACS analysis. The myeloid progenitors (Lin−IL-7R−Sca-1−c-Kit+) prepared from fetal liver cells of the indicated genotype mice 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. (e) TUNEL staining of fetal livers of Uba5+/− and Uba5−/− mice at E12.5. Scale bars: top panel, 100 μm; bottom panel, 20 μm. (f) Quantification of mRNA levels of erythroid-related genes, including Gata1, Fog1 and EPO, in Uba5-deficient liver at E11.5. Total RNAs were prepared from wild-type or Uba5-deficient livers and reverse transcribed into their respective cDNAs, which were used as templates for quantitative real-time PCR. Values were expressed relative to the amount of the respective mRNA in the wild-type livers. The experiments were conducted three times. Data are means±s.d. of three experiments. Statistical analysis was carried out by the unpaired t-test. *P<0.05 and **P<0.01 (Welch test).

Mentions: Haematopoietic progenitor cells can be accurately classified on the basis of their developmental potential, as schematically represented in Supplementary Figure S3a. Each type of erythroblast can be distinguished by examining the expression profiles of certain surface proteins such as CD71 and Ter119 (ref. 20; Supplementary Fig. S3b). At E11.5, although Ter119medCD71high proerythroblasts and Ter119highCD71high basophilic erythroblasts were present in wild-type fetal livers, the numbers of both types of cells were diminished in Uba5−/− fetal livers (10.0±1.2% versus 4.2±3.2% and 23.6±1.6% versus 1.0±0.6%, respectively, n=3, P<0.05, Fig. 2a). Next, we compared erythroid colony-forming units (CFU-Es) and more immature erythroid burst-forming units (BFU-Es) in wild-type and Uba5−/− fetal livers at E11.5. Both types of colonies were formed even in the absence of Uba5. However, the number of Uba5−/− CFU-Es (Fig. 2b) and BFU-Es (Fig. 2c) was significantly lower than in wild-type CFU-Es and BFU-Es (58.9% and 33.0%, respectively, P<0.05), suggesting reduced numbers of erythroid progenitors. Furthermore, the colony formation assay for megakaryocyte (CFU-Megs) indicated that cells derived from Uba5-knockout fetal livers had a reduced ability to form colonies (see Fig. 4d). These results indicate that loss of Uba5 is associated with defective differentiation of both megakaryocytes aned erythrocytes.


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)

Uba5 is indispensable for the development of erythroid cells.(a) Representative FACS analysis. Mouse fetal liver cells were freshly isolated from E11.5 embryos and labelled with a 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 fetal liver at E11.5. Uba5 deletion was associated with decreased erythroid progenitor activity. Data are means±s.d. of five mice from each group. Statistical analysis was performed using the unpaired t-test. *P<0.05 (Welch test). (d) Representative FACS analysis. The myeloid progenitors (Lin−IL-7R−Sca-1−c-Kit+) prepared from fetal liver cells of the indicated genotype mice 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. (e) TUNEL staining of fetal livers of Uba5+/− and Uba5−/− mice at E12.5. Scale bars: top panel, 100 μm; bottom panel, 20 μm. (f) Quantification of mRNA levels of erythroid-related genes, including Gata1, Fog1 and EPO, in Uba5-deficient liver at E11.5. Total RNAs were prepared from wild-type or Uba5-deficient livers and reverse transcribed into their respective cDNAs, which were used as templates for quantitative real-time PCR. Values were expressed relative to the amount of the respective mRNA in the wild-type livers. The experiments were conducted three times. Data are means±s.d. of three experiments. Statistical analysis was carried out by the unpaired t-test. *P<0.05 and **P<0.01 (Welch test).
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f2: Uba5 is indispensable for the development of erythroid cells.(a) Representative FACS analysis. Mouse fetal liver cells were freshly isolated from E11.5 embryos and labelled with a 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 fetal liver at E11.5. Uba5 deletion was associated with decreased erythroid progenitor activity. Data are means±s.d. of five mice from each group. Statistical analysis was performed using the unpaired t-test. *P<0.05 (Welch test). (d) Representative FACS analysis. The myeloid progenitors (Lin−IL-7R−Sca-1−c-Kit+) prepared from fetal liver cells of the indicated genotype mice 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. (e) TUNEL staining of fetal livers of Uba5+/− and Uba5−/− mice at E12.5. Scale bars: top panel, 100 μm; bottom panel, 20 μm. (f) Quantification of mRNA levels of erythroid-related genes, including Gata1, Fog1 and EPO, in Uba5-deficient liver at E11.5. Total RNAs were prepared from wild-type or Uba5-deficient livers and reverse transcribed into their respective cDNAs, which were used as templates for quantitative real-time PCR. Values were expressed relative to the amount of the respective mRNA in the wild-type livers. The experiments were conducted three times. Data are means±s.d. of three experiments. Statistical analysis was carried out by the unpaired t-test. *P<0.05 and **P<0.01 (Welch test).
Mentions: Haematopoietic progenitor cells can be accurately classified on the basis of their developmental potential, as schematically represented in Supplementary Figure S3a. Each type of erythroblast can be distinguished by examining the expression profiles of certain surface proteins such as CD71 and Ter119 (ref. 20; Supplementary Fig. S3b). At E11.5, although Ter119medCD71high proerythroblasts and Ter119highCD71high basophilic erythroblasts were present in wild-type fetal livers, the numbers of both types of cells were diminished in Uba5−/− fetal livers (10.0±1.2% versus 4.2±3.2% and 23.6±1.6% versus 1.0±0.6%, respectively, n=3, P<0.05, Fig. 2a). Next, we compared erythroid colony-forming units (CFU-Es) and more immature erythroid burst-forming units (BFU-Es) in wild-type and Uba5−/− fetal livers at E11.5. Both types of colonies were formed even in the absence of Uba5. However, the number of Uba5−/− CFU-Es (Fig. 2b) and BFU-Es (Fig. 2c) was significantly lower than in wild-type CFU-Es and BFU-Es (58.9% and 33.0%, respectively, P<0.05), suggesting reduced numbers of erythroid progenitors. Furthermore, the colony formation assay for megakaryocyte (CFU-Megs) indicated that cells derived from Uba5-knockout fetal livers had a reduced ability to form colonies (see Fig. 4d). These results indicate that loss of Uba5 is associated with defective differentiation of both megakaryocytes aned erythrocytes.

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