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Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish.

Yuan H, Zhang T, Liu X, Deng M, Zhang W, Wen Z, Chen S, Chen Z, de The H, Zhou J, Zhu J - Sci Rep (2015)

Bottom Line: Impairment of sumoylation attenuates HSPC generation and proliferation.The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent.While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis.

No MeSH data available.


HSPCs are depleted in SUMO-deficient embryos.(A–O) WISH assay of cmyb at 30 hpf (A, D, G, J, M), 72 hpf (B, E, H, K, N) and 5 dpf (C, F, I, L, O). Boxed regions indicate the AGM or CHT, respectively. (A′–O′) Magnified images of corresponding boxed regions from A to O, respectively. Red arrows identify cmyb-positive cells in the AGM or CHT, respectively. MO, morpholino oligonucleotides.
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f1: HSPCs are depleted in SUMO-deficient embryos.(A–O) WISH assay of cmyb at 30 hpf (A, D, G, J, M), 72 hpf (B, E, H, K, N) and 5 dpf (C, F, I, L, O). Boxed regions indicate the AGM or CHT, respectively. (A′–O′) Magnified images of corresponding boxed regions from A to O, respectively. Red arrows identify cmyb-positive cells in the AGM or CHT, respectively. MO, morpholino oligonucleotides.

Mentions: As our previous studies demonstrated that lack of sumoylation biased primitive hematopoiesis9, here we questioned its role in definitive hematopoiesis. To evaluate the effects of sumoylation on definitive hematopoietic development, the temporal and spatial expression patterns of a panel of HSPC and lineage-specific markers were examined by whole-mount mRNA in situ hybridization (WISH) after antisense MO-mediated knock-down. The MOs efficacy has been successfully validated previously1011. Compared with control embryos (Fig. 1 A–C), the expression of cmyb, a marker of HSPCs, was strikingly diminished in the AGM from 30 hpf onward in SUMO-deficient embryos (Fig. 1D), and almost no cmyb signals were detected in the CHT at 5 dpf (Fig. 1F), consistent with the phenotypes observed in the sae1 mutant, a SUMO E1 mutant line12. Similarly, the expression of another HSPCs marker, runx1, was also markedly decreased in the AGM in SUMOs morphants (Supplementary Fig. 1A). Two Ubc9 genes (Ubc9.1 and Ubc9.2), encoding a unique SUMO-conjugating enzyme of sumoylation pathway, have been identified in zebrafish1113. As expected, knockdown of Ubc9 also led to depleted HSPCs in the CHT, as indicated by reduced cmyb expression at 72 hpf and 5 dpf (Fig. 1 H–I). The cmyb expression level was relatively unchanged in Ubc9 morphants up to 30 hpf (Fig. 1G), possibly due to maternal Ubc9 protein11. Consistent with WISH data, loss of SUMOs or Ubc9 resulted in considerably reduced cmyb-EGFP positive cells in the CHT of a Tg (cmyb-EGFP) transgenic line (Supplementary Fig. 1 B–C). To rule out the MOs off-target effects mediated through p53 activation14, SUMOs or Ubc9 MO was injected with p53 MO or into p53 mutants, respectively. Similar phenotypes were observed in the co-injected embryos or p53 mutants (Fig. 1 J–O and Supplementary Fig. 1D). Collectively, these data indicate that impairment of sumoylation leads to depletion of HSPCs during definitive hematopoiesis.


Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish.

Yuan H, Zhang T, Liu X, Deng M, Zhang W, Wen Z, Chen S, Chen Z, de The H, Zhou J, Zhu J - Sci Rep (2015)

HSPCs are depleted in SUMO-deficient embryos.(A–O) WISH assay of cmyb at 30 hpf (A, D, G, J, M), 72 hpf (B, E, H, K, N) and 5 dpf (C, F, I, L, O). Boxed regions indicate the AGM or CHT, respectively. (A′–O′) Magnified images of corresponding boxed regions from A to O, respectively. Red arrows identify cmyb-positive cells in the AGM or CHT, respectively. MO, morpholino oligonucleotides.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355724&req=5

f1: HSPCs are depleted in SUMO-deficient embryos.(A–O) WISH assay of cmyb at 30 hpf (A, D, G, J, M), 72 hpf (B, E, H, K, N) and 5 dpf (C, F, I, L, O). Boxed regions indicate the AGM or CHT, respectively. (A′–O′) Magnified images of corresponding boxed regions from A to O, respectively. Red arrows identify cmyb-positive cells in the AGM or CHT, respectively. MO, morpholino oligonucleotides.
Mentions: As our previous studies demonstrated that lack of sumoylation biased primitive hematopoiesis9, here we questioned its role in definitive hematopoiesis. To evaluate the effects of sumoylation on definitive hematopoietic development, the temporal and spatial expression patterns of a panel of HSPC and lineage-specific markers were examined by whole-mount mRNA in situ hybridization (WISH) after antisense MO-mediated knock-down. The MOs efficacy has been successfully validated previously1011. Compared with control embryos (Fig. 1 A–C), the expression of cmyb, a marker of HSPCs, was strikingly diminished in the AGM from 30 hpf onward in SUMO-deficient embryos (Fig. 1D), and almost no cmyb signals were detected in the CHT at 5 dpf (Fig. 1F), consistent with the phenotypes observed in the sae1 mutant, a SUMO E1 mutant line12. Similarly, the expression of another HSPCs marker, runx1, was also markedly decreased in the AGM in SUMOs morphants (Supplementary Fig. 1A). Two Ubc9 genes (Ubc9.1 and Ubc9.2), encoding a unique SUMO-conjugating enzyme of sumoylation pathway, have been identified in zebrafish1113. As expected, knockdown of Ubc9 also led to depleted HSPCs in the CHT, as indicated by reduced cmyb expression at 72 hpf and 5 dpf (Fig. 1 H–I). The cmyb expression level was relatively unchanged in Ubc9 morphants up to 30 hpf (Fig. 1G), possibly due to maternal Ubc9 protein11. Consistent with WISH data, loss of SUMOs or Ubc9 resulted in considerably reduced cmyb-EGFP positive cells in the CHT of a Tg (cmyb-EGFP) transgenic line (Supplementary Fig. 1 B–C). To rule out the MOs off-target effects mediated through p53 activation14, SUMOs or Ubc9 MO was injected with p53 MO or into p53 mutants, respectively. Similar phenotypes were observed in the co-injected embryos or p53 mutants (Fig. 1 J–O and Supplementary Fig. 1D). Collectively, these data indicate that impairment of sumoylation leads to depletion of HSPCs during definitive hematopoiesis.

Bottom Line: Impairment of sumoylation attenuates HSPC generation and proliferation.The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent.While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis.

View Article: PubMed Central - PubMed

Affiliation: CNRS-LIA124, Sino-French Research Center for Life Sciences and Genomics, State Key Laboratory of Medical Genomics, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT
The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis.

No MeSH data available.