Abstract
The ubiquitination or SUMOylation of hematopoietic-related factors plays pivotal roles in hematopoiesis. RNF111, known as a ubiquitin ligase, is a newly discovered SUMO-targeted ubiquitin ligase involved in multiple signaling pathways mediated by transforming growth factor (TGF)-β family members. However, its role in hematopoiesis remains unclear. Herein, a heritable Rnf111 mutant zebrafish line was generated by CRISPR/Cas9-mediated genome editing. Impairment of hematopoietic stem and progenitor cells (HSPC) of definitive hematopoiesis was found in Rnf111-deficient mutants. Ablation of Rnf111 resulted in decreased phosphorylation of Smad2/3 in HSPC. Definitive endoderm 2 inducer (IDE2), which specifically activates TGF-β signaling and downstream Smad2 phosphorylation, could restore definitive hematopoiesis in Rnf111-deficient embryos. Further molecular mechanism studies revealed that Gcsfr/NO signaling was an important target pathway of Smad2/3 involved in Rnf111-mediated HSPC development. In conclusion, our study demonstrated that Rnf111 contributes to the development of HSPC by maintaining Smad2/3 phosphorylation and activation of the Gcsfr/NO signaling pathway.
Introduction
In vertebrates, hematopoiesis occurs in multiple waves.1,2 The earliest wave, namely primitive hematopoiesis, mainly produces erythroid and myeloid cells.2 The second wave of hematopoiesis - intermediate hematopoiesis - occurs in the posterior blood island and gives rise to erythromyeloid progenitors.3 The last wave, also called definitive hematopoiesis, generates hematopoietic stem cells (HSC) that are multipotent and can give rise to all lineages of blood cells for the whole life span.4 The hematopoietic regulatory network of zebrafish (Danio rerio) is highly conserved with that of mammals. Almost all transcription factors and key genes involved in hematopoietic regulation in mammals have orthologues in zebrafish.5 In zebrafish, definitive hematopoietic stem and progenitor cells (HSPC) emerge from the ventral wall of the dorsal aorta, an area functionally equivalent to the mammalian aorta-gonad-mesonephros,6,7 at approximately 33 to 35 hours post-fertilization (hpf) and then the nascent HSPC migrate to the caudal hematopoietic tissue, which is analogous to the mammalian fetal liver.8 Beginning at around 4 days post-fertilization (dpf), HSPC migrate through caudal hematopoietic tissue to the adult hematopoietic tissue-thymus and kidney marrow, which is similar to the process by which HSC migrate through fetal liver and home to bone marrow in mammals.8
The development of HSC is precisely regulated by a variety of transcription factors and signaling pathways. Dysregulation of this process leads to serious developmental defects or diseases. It has been reported that the ubiquitination and SUMOylation of hematopoietic-related factors also play important roles in the hematologic system.9-11 Arkadia (also known as RING finger 111, RNF111) is a nuclear E3 ubiquitin ligase (UbL) that ubiquitinates intracellular effectors and regulators of transforming growth factor (TGF)-β/nodal-related signaling, leading to their proteasome-dependent degradation. The characteristic RING finger domain of RNF111 in its C-terminus is required for degradation of the three major negative regulators of TGF-β signaling - Smad7, c-Ski and SnoN - in a small ubiquitin-like modifier (SUMO)-independent manner.12-15 RNF111 specifically regulates induced regulatory T-cell differentiation through facilitating the degradation of SKI/SnoN proteins.16 Knockdown of RNF111 promotes the differentiation of C2C12 myoblasts through reducing myostatin/TGF-β signaling.17 RNF111 is also involved in atrial fibrillation-induced myocardial fibrosis through mediating poly-ubiquitination and degradation of Smad7.18 Besides, RNF111 has been recognized as a SUMO-targeted ubiquitin ligase (STUbL) in the past several years. STUbL recognizes polysumoylated proteins through N-terminal SUMO-interacting motifs (SIM), mediating the ubiquitination degradation of target sumoylated substrates.19 RNF111 was shown to ubiquitinate polysumoylated promyelocytic leukemia in a SIM-dependent manner and was required for subsequent degradation of the polyubiquitinated product in promyelocytic leukemia nuclear bodies.20,21 Meanwhile, RNF111 has been discovered to enhance the ubiquitylation of SUMOylated xeroderma pigmentosum C protein, a pivotal DNA damage recognition factor of nucleotide excision repair, to facilitate the DNA damage response.22 However, the physiological function of RNF111 in hematopoiesis currently remains unknown.
To determine the role of RNF111 in hematopoiesis, we generated a heritable Rnf111 zebrafish mutant line by CRISPR/ Cas9-mediated knockout technology. The Rnf111-deficient embryos showed defects in definitive hematopoiesis, manifested by a reduction in HSPC, whereas primitive hematopoiesis was unaffected. Mechanistic studies indicated that Rnf111 plays an important role in the development of HSPC through maintaining the phosphorylation of Smad2/3 and activating the granulocyte colony-stimulating factor receptor/nitric oxide (Gcsfr/NO) signaling pathway.
Methods
Zebrafish maintenance and mutant generation
Zebrafish were maintained and staged under standard conditions as described before.23 All animal-related procedures were approved by the Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. The methods for generating Rnf111 knockout zebrafish and identifying the genotypes are described in the Online Supplementary Methods. Heritable fish with 4-base deletions were preserved and utilized for subsequent phenotypic analysis.
Whole-mount in situ hybridization
Whole-mount in situ hybridization (WISH) was carried out with probes including cmyb, scl, mpx, hbae1, runx1 and rag1, which have already been used and reported before,24 and the gcsfr, rnf111 and gata2b probes, which were newly cloned into pGEM-T Easy vector (Promega). Details of the procedure are described in the Online Supplementary Methods.
Morpholinos and mRNA microinjection
The sequences of the morpholinos and details about mRNA microinjection are provided in the Online Supplementary Methods.
Quantitative real-time polymerase chain reaction
The procedures for the reverse transcription and quantitative polymerase chain reaction (PCR) are described in the Online Supplementary Methods. Each sample was tested in triplicate. The Online Supplementary Table details the primers employed for real-time quantitative PCR.
Immunofluorescence assay, bromodeoxyuridine and enhanced green fluorescent protein double immunostaining and confocal fluorescent imaging
The 3 dpf transgenic Tg (cmyb:EGFP)25 embryos were used for immunofluorescence assays. Double staining with bromodeoxyuridine (Brdu) and enhanced green fluorescent protein (EGFP) was carried out on the 3 dpf Tg (runx1: EGFP)26 and Tg (cmyb:EGFP) embryos. Details of the procedures are described in the Online Supplementary Methods. An FV 1000 confocal microscope (Olympus, Tokyo, Japan) was used for observation and taking images.24
Immunohistochemistry, cell cultures and assays
The embryos were treated with 4% paraformaldehyde overnight at 4°C, followed by incubation with Sudan black (Sigma-Aldrich) solution for 25 minutes. Subsequently, they were thoroughly washed in 70% ethanol to facilitate the detection of the granules of granulocytes.
HEK293T cells were used for plasmid transfections according to the manufacturer’s instructions. Detailed procedures are provided in the Online Supplementary Methods.
The 3 dpf embryos were subjected to immunohistochemical studies according to procedures described in the Online Supplementary Methods.
The preparation of samples, information about antibodies and other relevant details on western blot analysis and chromatin immunoprecipitation (ChIP) PCR are described in the Online Supplementary Methods.
Chemical treatment
The concentrations of drugs and duration of treatments are listed in the Online Supplementary Methods.
DAF-FM assay
Embryos at 28 hpf, 2 dpf and 3 dpf were exposed to 5 μM DAF-FM DA (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) for 2 hours in the dark at 28.5°C, rinsed in fish water and then observed under a fluorescence microscope.27 The NO fluorescence intensity was quantified by ImageJ software and the relative intensity was calculated and presented as mean ± standard deviation.
Statistical analysis
All experiments were independently replicated at least three times. SPSS software (version 20) was used for the statistical analysis. Comparisons between two groups were conducted using a Student unpaired two-tailed t test, while one-way analysis of variance (ANOVA) was employed for the comparison of multiple groups. Statistical significance was established at a threshold of P<0.05.
Results
Evolutionary conservation of Rnf111 and generation of a heritable Rnf111-mutant zebrafish line
Zebrafish Rnf111 contains all the structural motifs, including nuclear localization signals, a RING finger domain, SUMO-interacting motifs (SIM) and Axin1 interaction domain (AID), which were originally described for mouse RNF111.28 Zebrafish rnf111 has a highly degree of synteny with the RNF111 locus in human (Online Supplementary Figure S1A) and its protein shares a 53.46% sequence similarity with human RNF111 (Online Supplementary Figure S1B). Subsequently, both antisense and sense probes were used to detect the mRNA expression pattern of rnf111 in zebrafish embryos with a WISH assay. The results showed that rnf111 is a maternal gene that exhibits ubiquitous expression, with high expression in the nervous system and relatively weak expression in hematopoietic tissues (Online Supplementary Figure S1C), consistent with the single-cell gene expression data on the Daniocell website.
To address the roles of Rnf111 in hematopoiesis, a Rnf111 mutant line was generated using the CRISPR/Cas9 system. The target site was in exon 5 and four nucleotides were deleted (Figure 1A, B), which led to a frameshift and formed a truncated protein containing only 552 amino acids (Figure 1C). The wild-type and mutant coding sequences were cloned into PCS2+ vector and transfected into 293T cells, and a shorter protein corresponding to the truncated mutant was detected by western blot analysis (Figure 1D). To further confirm the function of the truncated protein, the CAGA12-luc transcriptional reporter construct,15,29 stimulated by TGF-β and activin, was used to test whether the truncated protein can promote activation of TGF-β-inducible DNA elements detectable by dual luciferase reporter assay. As expected, Rnf111 mutants failed to promote the luciferase reporter gene expression (Figure 1E), indicating that the rnf111-mutated allele was indeed loss-of-function.
Figure 1.Generation of the Rnf111 mutant line. (A) The Cas9 target site was in the fifth exon; 4 base pair (bp) nucleotides were deleted. (B) The wild-type and mutant sequencing peak maps. (C) Schematic representation of wild-type and mutant Rnf111 proteins. (D) Western blot of HA-tagged wild-type and mutant Rnf111 proteins expressed in HEK293T cells. (E) CAGA12-luciferase transcriptional reporter assay of wild-type and mutant Rnf111. The data are presented as mean ± standard deviation with **P<0.01. WT: wild-type; MU: Rnf111 mutants with deletion of 4 bp; SIM: SUMO-interacting motifs.
Meanwhile, we found that the homozygous mutants cannot grow into adult fish and died within 8 to 10 days, which was consistent with the fate of Rnf111 knockout mice.
Ablation of Rnf111 impairs the development of definitive hematopoietic stem and progenitor cells
We analyzed the hematopoietic phenotypes of Rnf111 mutated embryos. Firstly, a series of markers involved in primitive hematopoiesis, such as the hematopoietic precursor cell marker scl,30 mature erythrocyte marker hbae131 and myeloid-specific marker mpx32 were examined at 22 hpf (Online Supplementary Figure S2A); no overt changes were observed, suggesting that primitive hematopoiesis was not affected in Rnf111 mutants. Then WISH analysis of the definitive wave was carried out. The expression of HSPC markers cmyb33 and runx11 was normal at 36 hpf, but the expression of cmyb was decreased in Rnf111 mutants from 2 dpf (Figure 2A, B), suggesting that definitive HSPC was impaired. Moreover, the expression of the mature erythrocyte marker hbae1, myeloid-specific marker mpx and Sudan black staining, and lymphoid specific marker rag134 were all diminished in Rnf111 mutants (Figure 2C), which further indicated that the deficiency of HSPC occurred in the Rnf111 mutants.
To further confirm the phenotype of hematopoiesis, a translation-blocking antisense morpholino oligonucleotide, which can sharply knock down the level of Rnf111 protein (Online Supplementary Figure S2B), was used to inhibit the function of Rnf111. The phenotypes of both primitive and definitive waves of rnf111 morpholino-injected embryos (rnf111 morphants) were detected by WISH using markers for each blood lineage involved in hematopoietic development. Consistent with the results in Rnf111 mutants, the scl, mpx and hbae1 markers were normal in primitive hematopoiesis (Online Supplementary Figure S2C). The expression of definitive HSPC markers cmyb33 and runx1 in rnf111 morphants was comparable with that in wild-type embryos at 33 hpf (Online Supplementary Figure S3A, B), which indicated that deficiency of Rnf111 had no effect on the generation of HSPC. The number of HSPC was decreased from 2 dpf in all subsequent developmental stages (Online Supplementary Figure S3A), suggesting that definitive HSPC was impaired. We then injected a morpholino to Tg (cmyb:EGFP) embryos (a stable zebrafish transgenic line expressing EGFP under the control of the cmyb promoter).25 As expected, the number of EGFP-positive cells was significantly decreased in the caudal hematopoietic tissue at 2 dpf and 3 dpf (Online Supplementary Figure S3C). Finally, the markers of downstream lineages were all diminished in rnf111 morphants (Online Supplementary Figure S3D). These results further confirmed that HSPC were specifically affected in Rnf111-deficient embryos.
The functional domain of ubiquitin ligase is required for Rnf111 to regulate hematopoietic stem and progenitor cell development
Rnf111 is a multifunctional protein acting as both an UbL and STUbL. In order to elucidate the functional contribu tion of Rnf111 in the regulation of HSPC development, four vectors - Rnf111 WT, Rnf111 SIM MU, Rnf111 RING MU and Rnf111-4bp MU - were constructed and the rescue effects of corresponding mRNA on HSPC defects were compared in Rnf111-deficient embryos (Figure 3A). The amino acids of the three SUMO-interacting motifs (SIM) were all mutated to alanine in the Rnf111 SIM mutant (Rnf111 SIM MU), which abolishes its binding to SUMO-modified substrates.21 Four cysteine residues within the RING domain of the active center of the UbL activity were mutated in the Rnf111 RING mutant (Rnf111 RING MU), which enabled it to still bind to but not to degrade the substrates.15 The result showed that Rnf111 WT effectively rescued the deficient HSPC in Rnf111 mutants, while the mRNA, whose sequence was consistent with the sequence of the Rnf111-4bp mutant line (-4bp MU), failed to do so (Figure 3B), demonstrating that the phenotype observed in Rnf111 mutants was indeed Rnf111 dependent. Interestingly, while Rnf111 SIM MU could rescue the defect of HSPC, Rnf111 RING MU lost its rescue effect (Figure 3B). The same rescue assays were carried out in rnf111 morphants, and similar results were obtained (Online Supplementary Figure S4A-C). All these results suggest that Rnf111 regulates HSPC development depending on its UbL function rather than STUbL activity.
Figure 2.Impairment of definitive hematopoiesis in Rnf111 mutants. (A) Whole-mount in situ hybridization (WISH) analysis of cmyb expression from 36 hpf to 8 dpf. (B) WISH assay of runx1 in mutants at 36 hpf. (C) WISH analysis of key hematopoietic markers and Sudan black analysis. All experiments were independently replicated at least three times. MU: Rnf111 mutants with deletion of 4 base pair nucleotides; hpf: hours post-fertilization; dpf: days post-fertilization.
Decreased proliferation capacity of hematopoietic stem and progenitor cells was observed in rnf111 morphants
TGF-β signaling, which was the main pathway that Rnf111 regulated,12-15 can affect HSPC development by regulating proliferation.35,36 Therefore, Brdu incorporation assays were carried out to detect the proliferation ability of HSPC in Tg (runx1: EGFP) and Tg (cmyb: EGFP) rnf111 morphants. The results showed that the number of Brdu+/GFP+ cells in both transgenic lines decreased sharply in the caudal hematopoietic tissue of rnf111 morphants (Figure 4A, B, Online Supplementary Figure S4D), indicating that the proliferation capacity of HSPC was severely impaired, which may be related to impaired TGF-β signaling.
The decrease of p-Smad2/3 protein leads to hematopoietic stem and progenitor cell defects in Rnf111-deficient embryos
In line with our finding that Rnf111 regulated the development of HSPC depending on its UbL function, it was reported previously that this function was also required for Rnf111 to regulate the TGF-β signaling pathway. Rnf111 degrades Smad7, an inhibitory protein of the TGF-β signaling pathway, in a SUMO-independent manner and promotes the phosphorylation of receptor Smad (Smad2 and Smad3). It was also reported that TGF-β at low doses (picogram levels) stimulated colony formation from CD34+ cells.36 Considering the reduced proliferation capacity of HSPC observed in rnf111 morphants, we speculated that Rnf111 may regulate the development of HSPC by affecting the phosphorylation of Smad2/3. We, therefore, performed anti-p-Smad2/3 and EGFP double immunostaining assays in Tg (cmyb: EGFP) embryos. The results showed a basal level of Smad2/3 phosphorylation in HSPC of wild-type embryos (Figure 5A, A’, A’’), while Smad2/3 phosphorylation was significantly reduced in HSPC of rnf111 morphants (Figure 5B, B’, B’’, C). Immunohistochemistry and western blot analysis of whole embryos demonstrated the same downward trend of p-Smad2/3 (Figure 5D-F) with no obvious changes in general Smad2 and Smad3 protein levels. Meanwhile, the protein level of Smad7 was upregulated (Figure 5G), which may be responsible for the reduction of p-Smad2/3. Then, to further investigate whether the observed Rnf111 ablation causing the HSPC defective phenotype was due to the decrease of p-Smad2/3, definitive endoderm 2 inducer (IDE2), a drug that specifically activates TGF-β signaling and downstream Smad2 phosphorylation with no effect on hemogenic endothelium and HSPC emergence (Online Supplementary Figure S5A, B), was used to treat Rnf111 mutants. As expected, IDE2 effectively rescued cmyb expression (Figure 5H). We also carried out the IDE2 rescue assay in rnf111 morphants, obtaining the same result (Online Supplementary Figure S5C, D). All these results demonstrated that the reduction of p-Smad2/3 was the key cause of the HSPC defect in Rnf111-deficient embryos.
Figure 3.Schematic representation and rescue assay of the four Rnf111 constructs. (A) Schematic diagram of the Rnf111 wild-type, Rnf111 RING mutant, Rnf111 SIM mutant and -4 base pair (bp) mutant proteins. (B) Whole-mount in situ hybridization analysis of the rescue efficiency of Rnf111 wild-type, Rnf111 RING mutant, Rnf111 SIM mutant and Rnf111-4 bp mutant RNA. Red arrows indicate cmyb-positive hematopoietic stem and progenitor cells. All experiments were independently replicated at least three times. WT: wild-type; RING MU: Rnf111 RING mutant with the RING domain mutated; SIM MU: Rnf111 SIM mutant with three SUMO-interacting motifs all mutated; SIM: SUMO-interacting motifs; -4bp MU: Rnf111-4 bp mutant with deletion of 4 bp nucleotides; dpf: days post-fertilization; MU: Rnf111 mutants with deletion of 4 bp nucleotides.
The Gcsf signaling pathway was the downstream target of Rnf111-Smad2/3
As an important regulator of classical TGF-β signaling pathway, Rnf111 promotes TGF-β signaling by degrading the repressor proteins and facilitates the phosphorylation of receptor-Smad2/3. It has been reported that TGF-β can regulate the amount of granulocyte-macrophage colony-stimulating factor receptors (GM-CSFR), interleukin-3 receptors (IL-3R), and granulocyte colony-stimulating factor receptors (GCSFR) in mouse hematopoietic progenitor cell lines without affecting the receptor affinity.37 Gcsf signaling is required for HSPC emergence and expansion in zebrafish.38
Figure 4.Bromodeoxyuridine incorporation assay. (A) Double immunostaining of runx1-EGFP with green fluorescent protein (GFP) and anti-bromodeoxyuridine (Brdu) in the caudal hematopoietic tissue of the transgenic Tg (runx1-EGFP) line at 3 dpf. White arrows indicate Brdu and runx1-EGFP double-positive cells. (B) Statistics of Brdu and runx1-GFP double-positive cells. The data are presented as mean ± standard deviation. with **P<0.01. dpf: days post-fertilization; WT: wild-type; MO rnf111: rnf111 morphants.
Based on our results, we hypothesized that Rnf111 regulates phosphorylation of Smad2/3 to promote Gcsf signaling in HSPC development. To test this hypothesis, quantitative RT-PCR was performed to detect the expression of gcsfr, gcsfa and gcsfb in 3 dpf embryos. All these three genes exhibited decreased expression in Rnf111 mutants and rnf111 morphants (Figure 6A, Online Supplementary Figure S6A). The WISH results further confirmed that gcsfr expression was reduced (Figure 6B). However, the expression of gcsfr in Rnf111 mutants (36 hpf) and the aorta-gonad-mesonephros region of rnf111 morphants (31 hpf) was unchanged (Online Supplementary Figure S6B, C). Then, cmyb-GPF positive HSPC at 3 dpf were sorted to detect gcsfr expression with quantitative RT-PCR assay. The result showed that gcsfr expression was reduced in HSPC (Figure 6C).
Then the -2.5k promotor sequence of gcsfr was cloned into the PGL3-basic vector and luciferase activity assay was performed. The results indicated that IDE2, Rnf111 WT and Rnf111 SIM MU could activate the luciferase expression, while Rnf111 RING MU failed (Figure 6D). These results were in line with those of the in vivo rescue assay (Figure 3). Then, GFP and Smad2-GFP RNA were injected in zebrafish embryos and chromatin immunoprecipitation PCR (ChIP-PCR) analysis was carried out to detect the binding ability of Smad2 to endogenous gcsfr promotor. The results showed that the promoter region of gcsfr could be specifically co-immunoprecipitated with Smad2-GFP (Figure 6E).
In order to further confirm the pivotal role of Gcsf signaling in Rnf111 regulation of HSPC development, the RNA rescue assay of both ligand (Gcsfb) and receptor (Gcsfr) was implemented. As expected, both gcsfb and gcsfr RNA rescued the developmental defects of HSPC in both Rnf111 mutants and rnf111 morphants (Figure 6F, Online Supplementary Figure S6D, F). Moreover, when the expression of Gcsfr was knocked down by gcsfr morpholinos, the rescue effect of IDE2 was abolished (Online Supplementary Figure S6E, F), suggesting that Gcsfr was a pivotal target of Smad2.
Decreased Gcsfr-NO signaling leads to the defect of hematopoietic stem and progenitor cell development in Rnf111-deficient embryos
It was reported that infection-induced Gcsfr-NO signaling can enhance the expansion of HSPC in zebrafish and that Cebpb-Nos2a acts downstream of Gcsf signaling.39 To verify whether Cebpb is involved in the HSPC defect induced by Rnf111 deficiency, cebpb RNA was injected into the Rnf111 mutants. Notably, the defect of HSPC could be effectively rescued (Figure 7A). Since our RT-qPCR results revealed decreased expression of the downstream gene nos2a (inosa) in both Rnf111 mutants (2 dpf and 3 dpf) and rnf111 morphants (3 dpf) (Figure 7B, Online Supplementary Figure S7A), the cell-permeable DAF-FM DA fluorescent NO probe was used to examine the active NO production.40 The results showed that NO production was decreased in rnf111 morphants at 2 dpf and 3 dpf (Figure 7C, D). It is reported that blood flow-induced Klf2a-NO signaling can regulate NO production.27 Therefore, we detected the expression of klf2a in Rnf111-deficient embryos by WISH assay and quantitative RT-PCR assay. klf2a expression was not decreased in Rnf111-deficient embryos compared to control ones (Online Supplementary Figure S7B, C), suggesting that Klf2a-NO signaling did not contribute to the defects of HSPC in Rnf111-deficient embryos. In addition, we also employed the rescue assay by treating Rnf111 mutants and rnf111 morphants with NO agonist, S nitroso N-acetylpenicillamine (SNAP). The WISH results showed that the expression of cmyb was partially restored (Figure 7E, Online Supplementary Figure S7D). And the rescue effect of SNAP was also proved in Tg(cmyb:EGFP) line (Online Supplementary Figure S7F). Next, we used the NOS2-specific inhibitor 1400W to block the production of NO and found that the rescue effect of gcsfr RNA on HSPC was abolished (Online Supplementary Figure S7E, F). Taken together, these data suggest that Rnf111 regulates HSPC expansion, at least in part, through Gcsfr-Cebpb-NO signaling.
Figure 5.Analysis of p-Smad2/3, Smad7 and IDE2 rescue assay in Rnf111-deficient embryos. (A, A’, A’’, B, B’, B’’) Double immunostaining of anti-GFP (A, B) and anti-p-Smad2/3 (A’, B’) in the caudal hematopoietic tissue of the transgenic Tg (cmyb-EGFP) line at 3 dpf. The bottom panel shows merged images (A’’, B’’). White arrows indicate p-Smad2/3 and cmyb-EGFP double-positive cells. (C) Statistics of p-Smad2/3 and cmyb-EGFP double-positive cells. The data are presented as mean ± standard deviation with ***P<0.001. (D, E) Immunohistochemistry of p-Smad2/3. (F) Western blot analysis of p-Smad2, Smad2, p-Smad3 and Smad3. (G) Western blot analysis of Smad7. (H) Whole-mount in situ hybridization analysis of rescue efficiency of IDE2. Red arrows indicate cmyb-positive hematopoietic stem and progenitor cells in the caudal hematopoietic tissue. All experiments were independently replicated at least three times. WT: wild-type; MO rnf111: rnf111 morphants; GFP: green fluorescent protein; MU: Rnf111 mutants with deletion of 4 base pair nucleotides; IDE2: definitive endoderm 2 inducer; EGFP: enhanced green fluorescent protein; dpf: days post-fertilization.
Figure 6.Gcsf signaling was the downstream target of Rnf111-Smad2/3. (A) Real-time quantitative polymerase chain reaction (qPCR) of gcsfa, gcsfb and gcsfr in siblings and mutants at 3 dpf. The data are presented as mean ± standard deviation (SD) with *P<0.05, **P<0.01. (B) Whole-mount in situ hybridization analysis of gcsfr. (C) Real-time qPCR of gcsfr in HSPC of rnf111 morphants. The data are presented as mean ± SD with ***P<0.01. (D) Luciferase assay of gcsfr promotor in HEK293T cells. The data are presented as mean ± SD with *P<0.05, **P<0.01, ***P<0.001, NS: no significant difference. (E) Chromatin immunoprecipitation qPCR assay of Smad2-GFP binding to gcsfr promotor at 2 dpf. The data are presented as mean ± SD with *P<0.05, NS: no significant difference. (F) Rescue assay of gcsfb and gcsfr RNA in Rnf111 mutants. Red arrows indicate cmyb-positive hematopoietic stem and progenitor cells in the caudal hematopoietic tissue. All experiments were independently replicated at least three times. WT: wild-type; MO rnf111: rnf111 morphants; HSPC: hematopoietic stem and progenitor cell; IDE2: definitive endoderm 2 inducer; RING MU: Rnf111 RING mutant with the RING domain mutated; SIM MU: Rnf111 SIM mutant with three SUMO-interacting motifs all mutated; GFP: green fluorescent protein; MU: Rnf111 mutants with deletion of 4 base pair nucleotides; hpf: hours post-fertilization; dpf: days post-fertilization.
Discussion
RNF111 is an UbL that enhances TGF-β signaling by promoting the degradation of repressors (SnoN, Ski and Smad7) that inhibit the expression of target genes.13, 14 RNF111 also was the second STUbL identified; its targets substrates with SUMO1-capped SUMO2 chains significantly superior to other identical substrates with homogeneous SUMO2 or SUMO1 chains.21 Rnf111 was responsible for induction of the node through enhancing nodal signaling,41,42 as well as progression of tissue fibrosis and cancer through regulating TGF-β signaling.43-45 In this study, we found a novel biological role of Rnf111 in zebrafish hematopoiesis. Both Rnf111 mutants and rnf111 morphants showed impaired definitive hematopoiesis without defects in the primitive wave, manifested by a decrease of definitive HSPC and downstream lineages. Although we could not exclude the possibility that erythromyeloid progenitors partially contributed to decreased mpx expression and Sudan black staining in mutants at 2 dpf, the number of myeloid cells of HSPC origin was indeed reduced, as shown by reduced mpx expression and Sudan black staining in Rnf111 mutants and rnf111 morphants at 2 dpf, 3 dpf, and 4 dpf. The homozygous mutants died within 8 to 10 days, which was consistent with the recessive lethal results in Rnf111 gene-trap insertion mutation mice.42 The regulatory role of Rnf111 in HSPC development depends on its UbL function rather than STUbL one. Our mechanism studies unraveled that the decrease of p-Smad2/3 due to stabilization of Smad7 protein caused by Rnf111 deletion was involved in the developmental defect of HSPC and the decrease of downstream Gcsfr/NO signaling results in an attenuated proliferative ability of HSPC (Figure 8). The behavior of HSPC such as self-renewal and quiescence is determined by a huge variety of factors, including external signaling cues present in the microenvironment of bone marrow. In the hematopoietic system, TGF-β signaling controls a wide range of biological processes, from immune system homeostasis to hematopoietic stem cell dormancy and self-renewal.46 The effect of TGF-β on HSC is bidirectional, with high levels of TGF-β blocking proliferation and low levels of TGF-β promoting proliferation.36,47 It is also reported that TGF-β at the dose of 0.01 ng/mL can expand normal HSC, and the phosphorylated Smad2/3 may contribute to this proliferative response of HSC under low concentrations of TGF-β.48 Therefore, during the development of normal HSC, an appropriate concentration of TGF-β is required to maintain the quiescence, self-renewal and differentiation functions. A suitable level of phosphorylation of Smad2/3 in HSC is crucial for the maintenance of their normal function in response to stimulation by low levels of TGF-β. Consistently, our results demonstrated that basal levels of Smad2/3 phosphorylation are present in the HSPC of wild-type embryos (Figure 5 A, A’, A’’, B, B’, B’’). However, p-Smad2/3 was downregulated in HSPC of rnf111 morphants, which may be the main cause leading to the HSPC defect. The Brdu incorporation assay showed the proliferative ability of HSPC in rnf111 morphants was decreased compared with that in WT embryos (Figure 4), indicating that the proliferative response of HSPC to normal concentrations of TGF-β was impaired in the absence of Rnf111, most likely due to the reduction of downstream effector p-Smad2/3. The rescue assay of IDE2 confirmed this notion. These data suggest that Rnf111 is present to maintain HSPC in response to TGF-β signals at normal concentrations.
Figure 7.Cebpb-Nos2a acts downstream of Gcsf signaling. (A) Rescue assay of cebpb RNA. (B) Real-time quantitative polymerase chain reaction results for inosa in Rnf111 mutants compared with wild-type embryos at 36 hpf, 2 dpf and 3 dpf. The data are presented as mean ± standard deviation (SD) with **P<0.01, NS: no significant difference. (C) DAF-FM assay showed the decreased production of nitric oxide (NO) morphants at 2 dpf and 3 dpf. (D) Statistics of NO fluorescence intensity. The data are presented as mean ± SD with **P<0.01, ***P<0.001, NS: no significant difference. (E) A rescue effect of the NO agonist S nitroso N-acetylpenicillamine (SNAP) on cmyb expression was observed in Rnf111 mutants. Red arrows indicate cmyb-positive hematopoietic stem and progenitor cells in caudal hematopoietic tissue. All experiments were independently replicated at least three times. dpf: days post-fertilization; MU: Rnf111 mutants with deletion of 4 base pair nucleotides; hpf: hours post-fertilization; WT: wild-type; MO rnf111: rnf111 morphants; DAF-FM: 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate.
It has been reported that TGF-β can regulate the amount of GM-CSFR, IL-3R, and GCSFR in mouse hematopoietic progenitor cell lines without significant changes in receptor affinity.37 It is also known that Gcsf signaling promotes the expansion of HSPC in zebrafish embryos.38 Indeed, we did find that the expression of gcsfr was downregulated in Rnf111 mutants and morphants at 3 dpf, but unchanged in Rnf111 mutants at 36 hpf and i the aorta-gonad-mesonephros region of rnf111 morphants at 31 hpf (Online Supplementary Figure S6B, C). This may be responsible for the unaffected generation and impaired proliferation of HSPC in Rnf111-deficient embryos. Meanwhile, the HSPC defect could be rescued by gcsfb and gcsfr RNA. A luciferase reporter assay and ChIP-qPCR further confirmed the direct binding of p-Smad2 on the gcsfr promotor. The rescue effect of IDE2 was blocked by the gcsfr morpholino, further confirming that Gcsfr was the target of Smad2/3.
Further experiments confirmed that the Cebpb/NO pathway was involved in the downstream of Gcsf signaling, evidenced by the rescue effect of cebpb RNA and NO donor, and the blocking of the rescue effect of gcsfr RNA by a NOS2-specific inhibitor, 1400W. It is worth noting that NO signaling at an earlier developmental stage was not decreased, as shown by the relatively normal DAF-FM signal intensity at 28 hpf in rnf111 morphants and nos2a expression level at 36 hpf in Rnf111 mutants. However, both DAF-FM signal intensity and nos2a expression level were significantly reduced at 2 dpf and 3 dpf (Figure 7B-D, Online Supplementary Figure S7A), which was consistent with the pattern of HSPC reduction in Rnf111-deficient embryos. The proliferation defect of HSPC caused by decreased NO in Rnf111-deficient embryos was consistent with the reported function of NO on HSPC proliferation at a physiological concentration,49 which further confirmed the pivotal role of NO in Rnf111 regulation of HSPC development. Taken together, we revealed a fine-tuned regulation of HSPC development by UbL.
Figure 8.Schema of Rnf111 regulating the development of hematopoietic stem and progenitor cells by maintaining Smad2/3 phosphorylation and activating the Gcsfr/NO signaling pathway. Left. At a physiological concentration of transforming growth factor-beta (TGF-β), ARK/Rnf111 maintains Smad2/3 phosphorylation by promoting the degradation of Smad7 and further facilitates activation of the Gcsfr/NO signaling pathway to ensure the proliferative response of HSPC to TGF-β. Right. Deletion of ARK/Rnf111 in hematopoietic stem and progenitor cells (HSPC) leads to a weakening of TGF-β signaling output, which is manifested by a decrease in the Gcsfr/NO signal, resulting in an attenuated proliferative response of HSPC to TGF-β.
Footnotes
- Received March 11, 2024
- Accepted September 23, 2024
Correspondence
Disclosures
No conflicts of interest to disclose.
Funding
This work was supported by the Shanghai Natural Science Foundation of China (19ZR1431600).
Acknowledgements
We thank Professor Feng Liu for providing the Tg (runx1: EGFP) transgenic line.
References
- Jagannathan-Bogdan M, Zon LI. Hematopoiesis. Development. 2013; 140(12):2463-2467. Google Scholar
- Chen AT, Zon LI. Zebrafish blood stem cells. J Cell Biochem. 2009; 108(1):35-42. Google Scholar
- Bertrand JY, Kim AD, Violette EP, Stachura DL, Cisson JL, Traver D. Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo. Development. 2007; 134(23):4147-4156. Google Scholar
- Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 2008; 132(4):631-644. Google Scholar
- Song HD, Sun XJ, Deng M. Hematopoietic gene expression profile in zebrafish kidney marrow. Proc Natl Acad Sci U S A. 2004; 101(46):16240-16245. Google Scholar
- Kissa K, Herbomel P. Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature. 2010; 464(7285):112-115. Google Scholar
- Kissa K, Murayama E, Zapata A. Live imaging of emerging hematopoietic stem cells and early thymus colonization. Blood. 2008; 111(3):1147-1156. Google Scholar
- Murayama E, Kissa K, Zapata A. Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development. Immunity. 2006; 25(6):963-975. Google Scholar
- Yu L, Ji W, Zhang H. SENP1-mediated GATA1 deSUMOylation is critical for definitive erythropoiesis. J Exp Med. 2010; 207(6):1183-1195. Google Scholar
- Yuan H, Zhou J, Deng M. Sumoylation of CCAAT/enhancerbinding protein alpha promotes the biased primitive hematopoiesis of zebrafish. Blood. 2011; 117(26):7014-7020. Google Scholar
- Yuan H, Zhang T, Liu X. Sumoylation of CCAAT/enhancerbinding protein alpha is implicated in hematopoietic stem/ progenitor cell development through regulating runx1 in zebrafish. Sci Rep. 2015; 5:9011. Google Scholar
- Mavrakis KJ, Andrew RL, Lee KL. Arkadia enhances Nodal/ TGF-beta signaling by coupling phospho-Smad2/3 activity and turnover. PLoS Biol. 2007; 5(3):e67. Google Scholar
- Koinuma D, Shinozaki M, Komuro A. Arkadia amplifies TGF-beta superfamily signalling through degradation of Smad7. EMBO J. 2003; 22(24):6458-6470. Google Scholar
- Nagano Y, Mavrakis KJ, Lee KL. Arkadia induces degradation of SnoN and c-Ski to enhance transforming growth factor-beta signaling. J Biol Chem. 2007; 282(28):20492-20501. Google Scholar
- Levy L, Howell M, Das D, Harkin S, Episkopou V, Hill CS. Arkadia activates Smad3/Smad4-dependent transcription by triggering signal-induced SnoN degradation. Mol Cell Biol. 2007; 27(17):6068-6083. Google Scholar
- Xu H, Wu L, Nguyen HH. Arkadia-SKI/SnoN signaling differentially regulates TGF-beta-induced iTreg and Th17 cell differentiation. J Exp Med. 2021; 218(11):e20210777. Google Scholar
- Yuzawa H, Koinuma D, Maeda S, Yamamoto K, Miyazawa K, Imamura T. Arkadia represses the expression of myoblast differentiation markers through degradation of Ski and the Ski-bound Smad complex in C2C12 myoblasts. Bone. 2009; 44(1):53-60. Google Scholar
- He X, Gao X, Peng L. Atrial fibrillation induces myocardial fibrosis through angiotensin II type 1 receptor-specific Arkadiamediated downregulation of Smad7. Circ Res. 2011; 108(2):164-175. Google Scholar
- Sriramachandran AM, Dohmen RJ. SUMO-targeted ubiquitin ligases. Biochim Biophys Acta. 2014; 1843(1):75-85. Google Scholar
- Erker Y, Neyret-Kahn H, Seeler JS, Dejean A, Atfi A, Levy L. Arkadia, a novel SUMO-targeted ubiquitin ligase involved in PML degradation. Mol Cell Biol. 2013; 33(11):2163-2177. Google Scholar
- Sriramachandran AM, Meyer-Teschendorf K, Pabst S. Arkadia/RNF111 is a SUMO-targeted ubiquitin ligase with preference for substrates marked with SUMO1-capped SUMO2/3 chain. Nat Commun. 2019; 10(1):3678. Google Scholar
- Poulsen SL, Hansen RK, Wagner SA. RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response. J Cell Biol. 2013; 201(6):797-807. Google Scholar
- Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn. 1995; 203(3):253-310. Google Scholar
- Liu X, Jia X, Yuan H. DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish. J Hematol Oncol. 2015; 8:15. Google Scholar
- North TE, Goessling W, Walkley CR. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature. 2007; 447(7147):1007-1011. Google Scholar
- Zhang P, He Q, Chen D. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition. Cell Res. 2015; 25(10):1093-1107. Google Scholar
- Wang L, Zhang P, Wei Y, Gao Y, Patient R, Liu F. A blood flow-dependent klf2a-NO signaling cascade is required for stabilization of hematopoietic stem cell programming in zebrafish embryos. Blood. 2011; 118(15):4102-4110. Google Scholar
- Bessarab DA, Mathavan S, Jones CM, Ray Dunn N. Zebrafish Rnf111 is encoded by multiple transcripts and is required for epiboly progression and prechordal plate development. Differentiation. 2015; 89(1-2):22-30. Google Scholar
- Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 1998; 17(11):3091-3100. Google Scholar
- Dooley KA, Davidson AJ, Zon LI. Zebrafish scl functions independently in hematopoietic and endothelial development. Dev Biol. 2005; 277(2):522-536. Google Scholar
- Brownlie A, Hersey C, Oates AC. Characterization of embryonic globin genes of the zebrafish. Dev Biol. 2003; 255(1):48-61. Google Scholar
- Lieschke GJ, Oates AC, Crowhurst MO, Ward AC, Layton JE. Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish. Blood. 2001; 98(10):3087-3096. Google Scholar
- Thompson MA, Ransom DG, Pratt SJ. The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Dev Biol. 1998; 197(2):248-269. Google Scholar
- Willett CE, Cherry JJ, Steiner LA. Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish. Immunogenetics. 1997; 45(6):394-404. Google Scholar
- Sitnicka E, Ruscetti FW, Priestley GV, Wolf NS, Bartelmez SH. Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells. Blood. 1996; 88(1):82-88. Google Scholar
- Kale VP. Differential activation of MAPK signaling pathways by TGF-beta1 forms the molecular mechanism behind its dosedependent bidirectional effects on hematopoiesis. Stem Cells Dev. 2004; 13(1):27-38. Google Scholar
- Jacobsen SE, Ruscetti FW, Dubois CM, Lee J, Boone TC, Keller JR. Transforming growth factor-beta trans-modulates the expression of colony stimulating factor receptors on murine hematopoietic progenitor cell lines. Blood. 1991; 77(8):1706-1716. Google Scholar
- Stachura DL, Svoboda O, Campbell CA. The zebrafish granulocyte colony-stimulating factors (Gcsfs): 2 paralogous cytokines and their roles in hematopoietic development and maintenance. Blood. 2013; 122(24):3918-3928. Google Scholar
- Hall CJ, Flores MV, Oehlers SH. Infection-responsive expansion of the hematopoietic stem and progenitor cell compartment in zebrafish is dependent upon inducible nitric oxide. Cell Stem Cell. 2012; 10(2):198-209. Google Scholar
- North TE, Goessling W, Peeters M. Hematopoietic stem cell development is dependent on blood flow. Cell. 2009; 137(4):736-748. Google Scholar
- Niederlander C, Walsh JJ, Episkopou V, Jones CM. Arkadia enhances nodal-related signalling to induce mesendoderm. Nature. 2001; 410(6830):830-834. Google Scholar
- Episkopou V, Arkell R, Timmons PM, Walsh JJ, Andrew RL, Swan D. Induction of the mammalian node requires Arkadia function in the extraembryonic lineages. Nature. 2001; 410(6830):825-830. Google Scholar
- Liu FY, Li XZ, Peng YM, Liu H, Liu YH. Arkadia-Smad7-mediated positive regulation of TGF-beta signaling in a rat model of tubulointerstitial fibrosis. Am J Nephrol. 2007; 27(2):176-183. Google Scholar
- Le Scolan E, Zhu Q, Wang L. Transforming growth factorbeta suppresses the ability of Ski to inhibit tumor metastasis by inducing its degradation. Cancer Res. 2008; 68(9):3277-3285. Google Scholar
- Nagano Y, Koinuma D, Miyazawa K, Miyazono K. Context-dependent regulation of the expression of c-Ski protein by Arkadia in human cancer cells. J Biochem. 2010; 147(4):545-554. Google Scholar
- Blank U, Karlsson S. TGF-beta signaling in the control of hematopoietic stem cells. Blood. 2015; 125(23):3542-3550. Google Scholar
- Kale VP, Vaidya AA. Molecular mechanisms behind the dosedependent differential activation of MAPK pathways induced by transforming growth factor-beta1 in hematopoietic cells. Stem Cells Dev. 2004; 13(5):536-547. Google Scholar
- Park SM, Deering RP, Lu Y. Musashi-2 controls cell fate, lineage bias, and TGF-beta signaling in HSCs. J Exp Med. 2014; 211(1):71-87. Google Scholar
- Hummer J, Kraus S, Brandle K, Lee-Thedieck C. Nitric oxide in the control of the in vitro proliferation and differentiation of human hematopoietic stem and progenitor cells. Front Cell Dev Biol. 2020; 8:610369. Google Scholar
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