Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve With Raphe Formation.

BACKGROUND: A better understanding of the molecular mechanism of aortic valve development and bicuspid aortic valve (BAV) formation would significantly improve and optimize the therapeutic strategy for BAV treatment. Over the past decade, the genes involved in aortic valve development and BAV formation have been increasingly recognized. On the other hand, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family members have been reported to be able to modulate cardiovascular development and diseases. The present study aimed to further investigate the roles of ADAMTS family members in aortic valve development and BAV formation. METHODS: Morpholino-based ADAMTS family gene-targeted screening for zebrafish heart outflow tract phenotypes combined with DNA sequencing in a 304 cohort BAV patient registry study was initially carried out to identify potentially related genes. Both ADAMTS gene-specific fluorescence in situ hybridization assay and genetic tracing experiments were performed to evaluate the expression pattern in the aortic valve. Accordingly, related genetic mouse models (both knockout and knockin) were generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method to further study the roles of ADAMTS family genes. The lineage-tracing technique was used again to evaluate how the cellular activity of specific progenitor cells was regulated by ADAMTS genes. Bulk RNA sequencing was used to investigate the signaling pathways involved. Inducible pluripotent stem cells derived from both BAV patients and genetic mouse tissue were used to study the molecular mechanism of ADAMTS. Immunohistochemistry was performed to examine the phenotype of cardiac valve anomalies, especially in the extracellular matrix components. RESULTS: ADAMTS genes targeting and phenotype screening in zebrafish and targeted DNA sequencing on a cohort of patients with BAV identified ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs 16) as a BAV-causing gene and found the ADAMTS16 p. H357Q variant in an inherited BAV family. Both in situ hybridization and genetic tracing studies described a unique spatiotemporal pattern of ADAMTS16 expression during aortic valve development. Adamts16+/- and Adamts16+/H355Q mouse models both exhibited a right coronary cusp-noncoronary cusp fusion-type BAV phenotype, with progressive aortic valve thickening associated with raphe formation (fusion of the commissure). Further, ADAMTS16 deficiency in Tie2 lineage cells recapitulated the BAV phenotype. This was confirmed in lineage-tracing mouse models in which Adamts16 deficiency affected endothelial and second heart field cells, not the neural crest cells. Accordingly, the changes were mainly detected in the noncoronary and right coronary leaflets. Bulk RNA sequencing using inducible pluripotent stem cells-derived endothelial cells and genetic mouse embryonic heart tissue unveiled enhanced FAK (focal adhesion kinase) signaling, which was accompanied by elevated fibronectin levels. Both in vitro inducible pluripotent stem cells-derived endothelial cells culture and ex vivo embryonic outflow tract explant studies validated the altered FAK signaling. CONCLUSIONS: Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components.

The genetic compensation response (GCR) has recently been proposed as a possible explanation for the phenotypic discrepancies between gene-knockout and gene-knockdown1,2; however, the underlying molecular mechanism of the GCR remains uncharacterized. Here, using zebrafish knockdown and knockout models of the capn3a and nid1a genes, we show that mRNA bearing a premature termination codon (PTC) promptly triggers a GCR that involves Upf3a and components of the COMPASS complex. Unlike capn3a-knockdown embryos, which have small livers, and nid1a-knockdown embryos, which have short body lengths2, capn3a-null and nid1a-null mutants appear normal. These phenotypic differences have been attributed to the upregulation of other genes in the same families. By analysing six uniquely designed transgenes, we demonstrate that the GCR is dependent on both the presence of a PTC and the nucleotide sequence of the transgene mRNA, which is homologous to the compensatory endogenous genes. We show that upf3a (a member of the nonsense-mediated mRNA decay pathway) and components of the COMPASS complex including wdr5 function in GCR. Furthermore, we demonstrate that the GCR is accompanied by an enhancement of histone H3 Lys4 trimethylation (H3K4me3) at the transcription start site regions of the compensatory genes. These findings provide a potential mechanistic basis for the GCR, and may help lead to the development of therapeutic strategies that treat missense mutations associated with genetic disorders by either creating a PTC in the mutated gene or introducing a transgene containing a PTC to trigger a GCR.

Hhex and Prox1a synergistically dictate the hepatoblast to hepatocyte differentiation in zebrafish.

The specification of endoderm cells to prospective hepatoblasts is the starting point for hepatogenesis. However, how a prospective hepatoblast gains the hepatic fate remains elusive. Previous studies have shown that loss-of-function of either hhex or prox1a alone causes a small liver phenotype but without abolishing the hepatocyte differentiation, suggesting that absence of either Hhex or Prox1a alone is not sufficient to block the hepatoblast differentiation. Here, via genetic studies of the zebrafish two single (hhex-/- and prox1a-/-) and one double (hhex-/-prox1a-/-) mutants, we show that simultaneous loss-of-function of the hhex and prox1a two genes does not block the endoderm cells to gain the hepatoblast potency but abolishes the hepatic differentiation from the prospective hepatoblast. Consequently, the hhex-/-prox1a-/- double mutant displays a liverless phenotype that cannot be rescued by the injection of bmp2a mRNA. Taken together, we provide strong evidences showing that Hhex teams with Prox1a to act as a master control of the differentiation of the prospective hepatoblasts towards hepatocytes.

Capturing Protein-Protein Interactions with Acidic Amino Acids Reactive Cross-Linkers.

Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here a previously reported "plant-and-cast" strategy is extended to develop heterobifunctional cross-linkers. These cross-linkers first react rapidly with Lys sidechains and then react with Asp and Glu sidechains, in a proximity-enhanced fashion. The cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents. The efficiency and robustness of cross-linking using model proteins, ranging from small monomeric proteins to large protein complexes are demonstrated. Importantly, it is shown that this type of cross-linkers are efficient at identifying protein-protein interactions involving acidic domains. The Cross-linking mass spectrometry (XL-MS) study with p53 identified 87 putative binders of the C-terminal domain of p53. Among them, SARNP, ZRAB2, and WBP11 are shown to regulate the expression and alternative splicing of p53 target genes. Thus, these carboxylate-reactive cross-linkers will further expand the power of XL-MS in the analysis of protein structures and protein-protein interactions.

Rcl1 depletion impairs 18S pre-rRNA processing at the A1-site and up-regulates a cohort of ribosome biogenesis genes in zebrafish.

Yeast Rcl1 is a potential endonuclease that mediates pre-RNA cleavage at the A2-site to separate 18S rRNA from 5.8S and 25S rRNAs. However, the biological function of Rcl1 in opisthokonta is poorly defined. Moreover, there is no information regarding the exact positions of 18S pre-rRNA processing in zebrafish. Here, we report that zebrafish pre-rRNA harbours three major cleavage sites in the 5'ETS, namely -477nt (A'-site), -97nt (A0-site) and the 5'ETS and 18S rRNA link (A1-site), as well as two major cleavage regions within the ITS1, namely 208-218nt (site 2) and 20-33nt (site E). We also demonstrate that depletion of zebrafish Rcl1 mainly impairs cleavage at the A1-site. Phenotypically, rcl1-/- mutants exhibit a small liver and exocrine pancreas and die before 15 days post-fertilization. RNA-seq analysis revealed that the most significant event in rcl1-/- mutants is the up-regulated expression of a cohort of genes related to ribosome biogenesis and tRNA production. Our data demonstrate that Rcl1 is essential for 18S rRNA maturation at the A1-site and for digestive organogenesis in zebrafish. Rcl1 deficiency, similar to deficiencies in other ribosome biogenesis factors, might trigger a common mechanism to upregulate the expression of genes responsible for ribosome biogenesis.

An 86 amino acids motif in CAPN3 is essential for formation of the nucleolus-localized Def-CAPN3 complex.

Digestive-organ expansion factor (Def) is a nucleolar protein that recruits cysteine proteinase Calpain3 (CAPN3) into the nucleolus to form the Def-CAPN3 complex in both human and zebrafish. This complex mediates the degradation of the tumor suppressor p53 and ribosome biogenesis factor mitotic phosphorylated protein 10 (Mpp10) in nucleolus, demonstrating the importance of this complex in regulating cell cycle and ribosome biogenesis. However, the Def and CAPN3 interacting motifs have yet been identified. In this report, by using a series of truncated or internally deleted human CAPN3 (hCAPN3) derivatives we identify that an essential motif of 86 amino acids (86-aa) (430-515aa) in hCAPN3 for its interaction with human Def (hDef), and this 86-aa motif is highly conserved in zebrafish Capn3b (zCapn3b) and is also required for the interaction between zebrafish Def (zDef) and zCapn3b. We further identify the 2/3 C-terminus of hDef is responsible for mediating the hDef-hCAPN3 interaction, and the corresponding region is conserved for the zDef and zCapn3b interaction. Our results lay the ground to resolve the structure of the Def-CAPN3 complex in the future.

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.

Difference in an intermolecular disulfide-bond between two highly homologous serum proteins Leg1a and Leg1b implicates their functional differentiation.

Zebrafish Liver-enriched gene 1a (Leg1a) and Leg1b are liver-produced serum proteins encoded by two adjacently linked homologous genes leg1a and leg1b, respectively. We previously showed that maternal-zygotic (MZ) leg1a null mutant developed a small liver at 3.5 days post-fertilization (dpf) during winter-time or under UV-treatment and displayed an abnormal stature at its adulthood. It is puzzling why Leg1b, which shares 89.3% identity with Leg1a and co-expressed with Leg1a, cannot fully compensate for the loss-of-function of Leg1a in the leg1azju1 MZ mutant. Here we report that Leg1a and Leg1b share eight cysteine residues but differ in amino acid residue 358, which is a serine in Leg1a but cysteine (C358) in Leg1b. We find that Leg1b forms an intermolecular disulfide bond through C358. Mutating C358 to Methionine (M358) does not affect Leg1b secretion whereas mutating other conserved cysteine residues do. We propose that the intermolecular disulfide bond in Leg1b might establish a rigid structure that makes it functionally different from Leg1a under certain oxidative conditions.

Excessive supply of glucose elicits an NF-κB2-dependent glycolysis in lactating goat mammary glands.

During lactation, an improper glucose supply often threatens mammary gland (MG) health. However, information is limited on the metabolic trajectories and molecules that regulate lactating MGs with an excessive glucose supply. Based on the network analysis of transcriptome and microRNAs, we found that the oversupply of glucose-induced severe glucose metabolic disorders in MGs of lactating goats, shifting lactose synthesis to acute fermentative glycolysis which caused increased flux of glucose metabolism into lactate. Moreover, NF-κB2 played a key role in regulating glycolysis, exhibiting a metabolic shift when MGs had an excessive supply of glucose. In primary mammary epithelial cells, fermentative glycolysis, and intracellular concentration of reactive oxygen species (ROS) were reduced by ganoderic acid A through blocking NF-κB2, while activation of NF-κB2 with phorbol myristate acetate (PMA) upregulated fermentative glycolysis and increased cellular ROS accumulation under excessive glucose. Thus, we established an NF-κB2-targeting method to reform the metabolic shift toward glycolysis caused by glucose oversupply by integrating NF-κB2 blockade and intracellular ROS scavenging.

Sas10 controls ribosome biogenesis by stabilizing Mpp10 and delivering the Mpp10-Imp3-Imp4 complex to nucleolus.

Mpp10 forms a complex with Imp3 and Imp4 that serves as a core component of the ribosomal small subunit (SSU) processome. Mpp10 also interacts with the nucleolar protein Sas10/Utp3. However, it remains unknown how the Mpp10-Imp3-Imp4 complex is delivered to the nucleolus and what biological function the Mpp10-Sas10 complex plays. Here, we report that the zebrafish Mpp10 and Sas10 are conserved nucleolar proteins essential for the development of the digestive organs. Mpp10, but not Sas10/Utp3, is a target of the nucleolus-localized Def-Capn3 protein degradation pathway. Sas10 protects Mpp10 from Capn3-mediated cleavage by masking the Capn3-recognition site on Mpp10. Def interacts with Sas10 to form the Def-Sas10-Mpp10 complex to facilitate the Capn3-mediated cleavage of Mpp10. Importantly, we found that Sas10 determines the nucleolar localization of the Mpp10-Imp3-Imp4 complex. In conclusion, Sas10 is essential not only for delivering the Mpp10-Imp3-Imp4 complex to the nucleolus for assembling the SSU processome but also for fine-tuning Mpp10 turnover in the nucleolus during organogenesis.

rDNA subtypes and their transcriptional expression in zebrafish at different developmental stages.

Eukaryotic 18S, 5.8S and 28S rRNAs are processed from a single transcript transcribed from the 45S rDNA gene, which is normally tandemly arrayed over hundred copies in a genome. Recently, a maternal (M) subtype and a somatic (S) subtype of rDNA were identified in zebrafish, with M-subtype on chromosome 4 and S-subtype on chromosome 5. It appears that the M-subtype is only expressed in eggs whilst the expression of the S-subtype is coupled with the initiation of zygotic gene expression. In this report, we identified three novel but transcriptionally inactive 18S variants in zebrafish genome with chromosome location different from the M- and S-subtype, suggesting translocation of 18S rDNA fragment during zebrafish evolution. Furthermore, we confirmed that the unfertilized eggs only have the M-subtype transcripts while brain, heart and liver have only the S-subtype transcripts. Both the M- and S-subtype transcripts were detected in female gonad. Our results support that the expression of different subtypes of rDNA is differentially regulated to meet the requirement for 'specialized ribosomes' during different developmental stages.

ABNORMAL INFLORESCENCE MERISTEM1 Functions in Salicylic Acid Biosynthesis to Maintain Proper Reactive Oxygen Species Levels for Root Meristem Activity in Rice.

Root meristem activity determines root growth and root architecture and consequently affects water and nutrient uptake in plants. However, our knowledge about the regulation of root meristem activity in crop plants is very limited. Here, we report the isolation and characterization of a short root mutant in rice (Oryza sativa) with reduced root meristem activity. This root growth defect is caused by a mutation in ABNORMAL INFLORESCENCE MERISTEM1 (AIM1), which encodes a 3-hydroxyacyl-CoA dehydrogenase, an enzyme involved in ß-oxidation. The reduced root meristem activity of aim1 results from reduced salicylic acid (SA) levels and can be rescued by SA application. Furthermore, reduced SA levels are associated with reduced levels of reactive oxygen species (ROS) in aim1, likely due to increased expression of redox and ROS-scavenging-related genes, whose increased expression is (at least in part) caused by reduced expression of the SA-inducible transcriptional repressors WRKY62 and WRKY76. Like SA, ROS application substantially increased root length and root meristem activity in aim1 These results suggest that AIM1 is required for root growth in rice due to its critical role in SA biosynthesis: SA maintains root meristem activity through promoting ROS accumulation by inducing the activity of WRKY transcriptional repressors, which repress the expression of redox and ROS-scavenging genes.

Centrosomal protein FOR20 is essential for cilia-dependent development in zebrafish embryos.

Centrosomal proteins play critical roles in ciliogenesis. Mutations in many centrosomal proteins have been documented to contribute to developmental defects and cilium-related diseases. Centrosomal protein fibroblast growth factor receptor 1 oncogene partner-related protein of 20 kDa (FOR20) is crucial for ciliogenesis in mammalian cells and the unicellular eukaryote Paramecium; however, the biologic significance of FOR20 in vertebrate development remains unclear. We cloned the zebrafish homolog of the for20 gene and found that for20 mRNA is enriched in ciliated tissues during early zebrafish development. Knockdown of for20 by morpholino oligonucleotides in zebrafish results in multiple ciliary phenotypes, including curved body, hydrocephaly, pericardial edema, kidney cysts, and left-right asymmetry defects. for20 morphants show reduced number and length of cilia in Kupffer's vesicle and pronephric ducts. High-speed video microscopy reveals that cilia in most for20 morphants are consistently paralyzed or beat arrhythmically. To confirm the ciliary phenotypes of for20 morphants, we used the CRISPR/Cas9 system to disrupt for20 gene in zebrafish. for20 mutants exhibit multiple ciliary phenotypes resembling the defects in for20 morphants. All of these phenotypes in for20 morphants and mutants are significantly reversed by exogenous expression of for20 mRNA. Taken together, these data suggest that FOR20 is required for cilium-mediated processes during zebrafish embryogenesis.-Xie, S., Jin, J., Xu, Z., Huang, Y., Zhang, W., Zhao, L., Lo, L. J., Peng, J., Liu, W., Wang, F., Shu, Q., Zhou, T. Centrosomal protein FOR20 is essential for cilia-dependent development in zebrafish embryos.

Advanced biomaterials for cancer immunotherapy.

Immunotherapy, as a powerful strategy for cancer treatment, has achieved tremendous efficacy in clinical trials. Despite these advancements, there is much to do in terms of enhancing therapeutic benefits and decreasing the side effects of cancer immunotherapy. Advanced nanobiomaterials, including liposomes, polymers, and silica, play a vital role in the codelivery of drugs and immunomodulators. These nanobiomaterial-based delivery systems could effectively promote antitumor immune responses and simultaneously reduce toxic adverse effects. Furthermore, nanobiomaterials may also combine with each other or with traditional drugs via different mechanisms, thus giving rise to more accurate and efficient tumor treatment. Here, an overview of the latest advancement in these nanobiomaterials used for cancer immunotherapy is given, describing outstanding systems, including lipid-based nanoparticles, polymer-based scaffolds or micelles, inorganic nanosystems, and others.

Erythrocyte Membrane-Camouflaged IR780 and DTX Coloading Polymeric Nanoparticles for Imaging-Guided Cancer Photo-Chemo Combination Therapy.

Conventional systemic chemotherapy leads to poor therapeutic outcomes at moments in cancer therapy because the nontargeting anticancer drug release results in adverse effects and consequently drug resistance. The combination therapeutic strategy provides an alternative way to solve the conundrums. Herein, drug delivery systems with a rational design and tumor-targeting abilities become the ideal carriers for combinatorial therapy. IR780 iodide possesses near-infrared fluorescence intensity for fluorescence imaging (FI) and photothermal conversion for photoacoustic imaging (PAI), which also can be employed for tumor phototherapy (including photothermal therapy and photodynamic therapy). However, hydrophobicity and rapid elimination in vivo limit its biomedical applications. Furthermore, the hydrophobicity and high crystallization of IR780 result in poor drug-loading capacity and low stability. In this study, the high-pressure homogenization method was utilized for hydrophobic molecular IR780 and DTX coloading to construct IR780/DTX-PCEC nanoparticles which exhibit narrow size distribution and satisfactory drug-loading capacity. With further erythrocyte membrane [red blood cell (RBC)] camouflaging, the obtained IR780/DTX-PCEC@RBC nanoparticles present desired stability and prolonged circulation time in vivo. Additionally, the IR780/DTX-PCEC@RBC nanoparticles not only can be employed as a FI/PAI dual model imaging probe but also exhibit the property for phototherapy and chemotherapy of tumors. Based on the therapeutic outcome of combination therapy, the IR780/DTX-PCEC@RBC nanoparticles can serve as promising FI- and PAI-guided photo-chemo combination therapy agents for the future treatment of breast cancer.

Phosphorylation of Def Regulates Nucleolar p53 Turnover and Cell Cycle Progression through Def Recruitment of Calpain3.

Digestive organ expansion factor (Def) is a nucleolar protein that plays dual functions: it serves as a component of the ribosomal small subunit processome for the biogenesis of ribosomes and also mediates p53 degradation through the cysteine proteinase calpain-3 (CAPN3). However, nothing is known about the exact relationship between Def and CAPN3 or the regulation of the Def function. In this report, we show that CAPN3 degrades p53 and its mutant proteins p53A138V, p53M237I, p53R248W, and p53R273P but not the p53R175H mutant protein. Importantly, we show that Def directly interacts with CAPN3 in the nucleoli and determines the nucleolar localisation of CAPN3, which is a prerequisite for the degradation of p53 in the nucleolus. Furthermore, we find that Def is modified by phosphorylation at five serine residues: S50, S58, S62, S87, and S92. We further show that simultaneous phosphorylations at S87 and S92 facilitate the nucleolar localisation of Capn3 that is not only essential for the degradation of p53 but is also important for regulating cell cycle progression. Hence, we propose that the Def-CAPN3 pathway serves as a nucleolar checkpoint for cell proliferation by selective inactivation of cell cycle-related substrates during organogenesis.

Liver-Enriched Gene 1, a Glycosylated Secretory Protein, Binds to FGFR and Mediates an Anti-stress Pathway to Protect Liver Development in Zebrafish.

Unlike mammals and birds, teleost fish undergo external embryogenesis, and therefore their embryos are constantly challenged by stresses from their living environment. These stresses, when becoming too harsh, will cause arrest of cell proliferation, abnormal cell death or senescence. Such organisms have to evolve a sophisticated anti-stress mechanism to protect the process of embryogenesis/organogenesis. However, very few signaling molecule(s) mediating such activity have been identified. liver-enriched gene 1 (leg1) is an uncharacterized gene that encodes a novel secretory protein containing a single domain DUF781 (domain of unknown function 781) that is well conserved in vertebrates. In the zebrafish genome, there are two copies of leg1, namely leg1a and leg1b. leg1a and leg1b are closely linked on chromosome 20 and share high homology, but are differentially expressed. In this report, we generated two leg1a mutant alleles using the TALEN technique, then characterized liver development in the mutants. We show that a leg1a mutant exhibits a stress-dependent small liver phenotype that can be prevented by chemicals blocking the production of reactive oxygen species. Further studies reveal that Leg1a binds to FGFR3 and mediates a novel anti-stress pathway to protect liver development through enhancing Erk activity. More importantly, we show that the binding of Leg1a to FGFR relies on the glycosylation at the 70th asparagine (Asn(70) or N(70)), and mutating the Asn(70) to Ala(70) compromised Leg1's function in liver development. Therefore, Leg1 plays a unique role in protecting liver development under different stress conditions by serving as a secreted signaling molecule/modulator.

LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root system architecture in rice.

Root system architecture is very important for plant growth and crop yield. It is essential for nutrient and water uptake, anchoring, and mechanical support. Root growth angle (RGA) is a vital constituent of root system architecture and is used as a parameter for variety evaluation in plant breeding. However, little is known about the underlying molecular mechanisms that determine root growth angle in rice (Oryza sativa). In this study, a rice mutant large root angle1 (lra1) was isolated and shown to exhibit a large RGA and reduced sensitivity to gravity. Genome resequencing and complementation assays identified OsPIN2 as the gene responsible for the mutant phenotypes. OsPIN2 was mainly expressed in roots and the base of shoots, and showed polar localization in the plasma membrane of root epidermal and cortex cells. OsPIN2 was shown to play an important role in mediating root gravitropic responses in rice and was essential for plants to produce normal RGAs. Taken together, our findings suggest that OsPIN2 plays an important role in root gravitropic responses and determining the root system architecture in rice by affecting polar auxin transport in the root tip.

TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis.

In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II ß binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.

A naturally occurring 4-bp deletion in the intron 4 of p53 creates a spectrum of novel p53 isoforms with anti-apoptosis function.

p53 functions as a tumor suppressor by transcriptionally regulating the expression of genes involved in controlling cell proliferation or apoptosis. p53 and its isoform Δ133p53/Δ113p53 form a negative regulation loop in that p53 activates the expression of Δ133p53/Δ113p53 while Δ133p53/Δ113p53 specifically antagonizes p53 apoptotic activity. This pathway is especially important to safeguard the process of embryogenesis because sudden activation of p53 by DNA damage signals or developmental stress is detrimental to a developing embryo. Here we report the identification of five novel p53 isoforms. p53ß is generated due to alternative splicing of the intron 8 of p53 while the other four, namely, TA2p53, TA3p53, TA4p53 and TA5p53, result from the combination of alternative splicing of intron 1 (within intron 4 of the p53 gene) of the Δ113p53 gene and a naturally occurring CATT 4 bp deletion within the alternative splicing product in zebrafish. The CATT 4 bp deletion creates four translation start codons which are in-frame to the open reading frame of Δ113p53. We also show that TAp53 shares the same promoter with Δ113p53 and functions to antagonize p53 apoptotic activity. The identification of Δ113p53/TA2/3/4/5p53 reveals a pro-survival mechanism which operates robustly during embryogenesis in response to the DNA-damage condition.