EPC1/2 regulate hematopoietic stem and progenitor cell proliferation by modulating H3 acetylation and DLST.

Enhancers of polycomb 1 (EPC1) and 2 (EPC2) are involved in multiple biological processes as components of histone acetyltransferases/deacetylase complexes and transcriptional cofactors, and their dysfunction was associated with developmental defects and diseases. However, it remains unknown how their dysfunction induces hematopoietic stem and progenitor cell (HSPC) defects. Here, we show that depletion of EPC1/2 significantly reduced the number of hematopoietic stem and progenitor cells (HSPCs) in the aorta-gonad mesonephros and caudal hematopoietic tissue regions by impairing HSPC proliferation, and consistently downregulated the expression of HSPC genes in K562 cells. This study demonstrates the functions of EPC1/2 in regulating histone H3 acetylation, and in regulating DLST (dihydrolipoamide S-succinyltransferase) via H3 acetylation and cooperating with transcription factors serum response factor and FOXR2 together, and in the subsequent HSPC emergence and proliferation. Our results demonstrate the essential roles of EPC1/2 in regulating H3 acetylation, and DLST as a linkage between EPC1 and EPC2 with mitochondria metabolism, in HSPC emergence and proliferation.

Copper nanoparticles and silver nanoparticles impair lymphangiogenesis in zebrafish.

Lymphatic system distributes in almost all vertebrate tissues and organs, and plays important roles in the regulation of body fluid balance, lipid absorption and immune monitoring. Although CuNPs or AgNPs accumulation has been reported to be closely associated with delayed hatching and motor dysfunction in zebrafish embryos, their biological effects on lymphangiogenesis remain unknown. In this study, thoracic duct was observed to be partially absent in both CuNPs and AgNPs stressed zebrafish larvae. Specifically, CuNPs stress induced hypermethylation of E2F7/8 binding sites on CCBE1 promoters via their producing ROS, thereby leading to the reduction of binding enrichment of E2F7/8 on CCBE1 promoter and its subsequently reduced expression, then resulting in defective lymphatic vessel formation. Differently, AgNPs stress induced down-regulated CCBE1 expression via down-regulating mRNA and protein levels of E2F7/8 transcription factors, thereby resulting in defective lymphatic vessel formation. This study may be the first to demonstrate that CuNPs and AgNPs damaged lymphangiogenesis during zebrafish embryogenesis, mechanistically, CuNPs epigenetically regulated the expression of lymphangiogenesis regulator CCBE1 via hypermethylating its promoter binding sites of E2F7/8, while AgNPs via regulating E2F7/8 expression. Meanwhile, overexpression of ccbe1 mRNA effectively rescued the lymphangiogenesis defects in both AgNPs and CuNPs stressed larvae, while overexpression of e2f7/8 mRNA effectively rescued the lymphangiogenesis defects in AgNPs rather than CuNPs stressed larvae. The results in this study will shed some light on the safety assessment of nanomaterials applied in medicine and on the ecological security assessments of nanomaterials. Video Abstract.

Copper overload impairs hematopoietic stem and progenitor cell proliferation via prompting HSF1/SP1 aggregation and the subsequently downregulating FOXM1-Cytoskeleton axis.

Unbalanced Cu homeostasis has been suggested to be associated with hematopoietic disease, but the roles of Cu overload in the hematopoietic system and the potential mechanisms are obscure. Here, we report a novel association and the novel potential pathways for Cu overload to induce proliferation defects in zebrafish embryonic hematopoietic stem and progenitor cells (HSPCs) via down-regulating expression of foxm1-cytoskeleton axis, which is conserved from fish to mammals. Mechanistically, we show the direct binding of Cu to transcriptional factors HSF1 and SP1 and that Cu overload induces the cytoplasmic aggregation of proteins HSF1 and SP1. These result in the reduced transcriptional activities of HSF1 and SP1 on their downstream FOXM1 as well as the FOXM1 transcriptional activities on cytoskeletons in HSPCs, which leads to ultimately cell proliferation impairment. These findings unveil the novel linkage of Cu overload with specific signaling transduction as well as the subsequent HSPC proliferation defects.

Atp7b deficiency induces zebrafish eye developmental defects.

As a copper (Cu) transport ATPase, ATP7B plays an important role in maintaining Cu homeostasis in the body and its dysfunction is associated with retinal disease. How ATP7B dysfunction and the subsequent Cu overload induce retinal damage, however, are unknown. Here, we show that atp7b-/- homozygous zebrafish larvae are insensitive to light stimulation, with a reduction in retinal cells but normal like morphological phenotypes. Additionally, a series of differentially expressed genes are unveiled in atp7b-/- mutated larvae, which enrich in photo-transduction, structural constituent of eye lens, sensory perception of light stimulus, oxidative phosphorylation, and ATPase activity. Moreover, we show the Cu accumulation in retinal cells in atp7b-/- mutated larvae, which results in endoplasmic reticulum (ER) stress and retinal cell apoptosis and subsequent retinal defects. The integral data in this study demonstrate that atp7b mutation leads to Cu accumulation in zebrafish retinal cells and the consequence ER stress and retinal cell death. These data may give some possible hints to explain retinal disease occurred in the Cu dysregulation syndromes Wilson's disease with ATP7B mutation.

Zebrafish ELL-associated factors Eaf1/2 modulate erythropoiesis via regulating gata1a expression and WNT signaling to facilitate hypoxia tolerance.

EAF1 and EAF2, the eleven-nineteen lysine-rich leukemia (ELL)-associated factors which can assemble to the super elongation complex (AFF1/4, AF9/ENL, ELL, and P-TEFb), are reported to participate in RNA polymerase II to actively regulate a variety of biological processes, including leukemia and embryogenesis, but whether and how EAF1/2 function in hematopoietic system related hypoxia tolerance during embryogenesis remains unclear. Here, we unveiled that deletion of EAF1/2 (eaf1-/- and eaf2-/-) caused reduction in hypoxia tolerance in zebrafish, leading to reduced erythropoiesis during hematopoietic processes. Meanwhile, eaf1-/- and eaf2-/- mutants showed significant reduction in the expression of key transcriptional regulators scl, lmo2, and gata1a in erythropoiesis at both 24 h post fertilization (hpf) and 72 hpf, with gata1a downregulated while scl and lmo2 upregulated at 14 hpf. Mechanistically, eaf1-/- and eaf2-/- mutants exhibited significant changes in the expression of epigenetic modified histones, with a significant increase in the binding enrichment of modified histone H3K27me3 in gata1a promoter rather than scl and lmo2 promoters. Additionally, eaf1-/- and eaf2-/- mutants exhibited a dynamic expression of canonical WNT/ß-catenin signaling during erythropoiesis, with significant reduction in p-ß-Catenin level and in the binding enrichment of both scl and lmo2 promoters with the WNT transcriptional factor TCF4 at 24 hpf. These findings demonstrate an important role of Eaf1/2 in erythropoiesis in zebrafish and may have shed some light on regeneration medicine for anemia and related diseases and on molecular basis for fish economic or productive traits, such as growth, disease resistance, hypoxia tolerance, and so on.

Copper stress impairs angiogenesis and lymphangiogenesis during zebrafish embryogenesis by down-regulating pERK1/2-foxm1-MMP2/9 axis and epigenetically regulating ccbe1 expression.

Molecular transport and cell circulation between tissues and organs through blood and lymphatic vessels are essential for physiological homeostasis in vertebrates. Despite the report of its association with vessel formation in solid tumors, the biological effects of Copper (Cu) accumulation on angiogenesis and lymphangiogenesis during embryogenesis are still unknown. In this study, we unveiled that intersegmental blood circulation was partially blocked in Cu2+-stressed zebrafish embryos and cell migration and tube formation were impaired in Cu2+-stressed mammalian HUVECs. Specifically, Cu2+-stressed embryos showed down-regulation in the expression of amotl2 and its downstream pERK1/2-foxm1-MMP2/9 regulatory axis, and knockdown/knockout of foxm1 in zebrafish embryos phenocopied angiogenesis defects, while FOXM1 knockdown HUVECs phenocopied cell migration and tube formation defects, indicating that excessive Cu2+-induced angiogenesis defects and blocked cell migration via down-regulating amotl2-pERK1/2-foxm1-MMP2/9 regulatory axis in both embryos and mammalian cells. Additionally, thoracic duct was revealed to be partially absent in Cu2+-stressed zebrafish embryos. Specifically, Cu2+-stressed embryos showed down-regulation in the expression of ccbe1 (a gene with pivotal function in lymphangiogenesis) due to the hypermethylation of the E2F7/8 binding sites on ccbe1 promoter to reduce their binding enrichment on the promoter, contributing to the potential mechanisms for down-regulation of ccbe1 and the formation of lymphangiogenesis defects in Cu2+-stressed embryos and mammalian cells. These integrated data demonstrate that Cu2+ stress impairs angiogenesis and lymphangiogenesis via down-regulation of pERK1/2-foxm1-MMP2/9 axis and epigenetic regulation of E2F7/8 transcriptional activity on ccbe1 expression, respectively.

Effects of parental environmental copper stress on offspring development: DNA methylation modification and responses of differentially methylated region-related genes in transcriptional expression.

Parental environmental copper (Cu) exposure is widespread, causing problems for sustainability of fish populations, and epigenetics is suggested to be fundamental during the process, but the mechanism is scarcely reported. Here, we describe the effects of parental environmental Cu exposure on zebrafish developmental abnormality in subsequent generation. This study demonstrated for the first time that: 1. offspring from Cu-stressed paternal adult zebrafish showed developmental defects in the nervous and digestive system and changes in transcriptome; 2. Cu-induced alterations in sperm methylome and transcriptome could induce loci-specific alterations in DNA methylome and corresponding changes in the related gene transcription in offspring; 3. differentially methylated regions in pmpcb, crebl2 and tab2 promoters acted pivotally in their transcription; 4. pmpcb, crebl2 and tab2 are key individual contributors to parental Cu exposure-induced developmental defects in the nervous system, retina and digestive system of the offspring. Those data revealed that Cu-induced alterations in sperm methylome and transcriptome can be passed down to their fertilized offspring, reprogramming the epigenetic and transcriptional regulation of embryogenesis and causing embryonic developmental defects, suggesting that environmental Cu might pose a huge threat to the sustainability of fish populations.

Copper ions impair zebrafish skeletal myofibrillogenesis via epigenetic regulation.

Unbalanced copper (Cu2+ ) homeostasis is associated with the developmental defects of vertebrate myogenesis, but the underlying molecular mechanisms remain elusive. In this study, it was found that Cu2+ stressed zebrafish embryos and larvae showed reduced locomotor speed as well as loose and decreased myofibrils in skeletal muscle, coupled with the downregulated expression of muscle fiber markers mylpfa and smyhc1l and the irregular arrangement of myofibril and sarcomere. Meanwhile, the Cu2+ stressed zebrafish embryos and larvae also showed significant reduction in the expression of H3K4 methyltransferase smyd1b transcripts and H3K4me3 protein as well as in the binding enrichment of H3K4me3 on gene mylpfa promoter in skeletal muscle cells, suggesting that smyd1b-H3K4me3 axis mediates the Cu2+ -induced myofibrils specification defects. Additionally, whole genome DNA methylation sequencing unveiled that the gene smyd5 exhibited significant promoter hyper-methylation and increased expression in Cu2+ stressed embryos, and the ectopic expression of smyd5 in zebrafish embryos also induced the myofibrils specification defects as those observed in Cu2+ stressed embryos. Moreover, Cu2+ was shown to suppress myofibrils specification and smyd1b promoter transcriptional activity directly independent of the integral function of copper transporter cox17 and atp7b. All these data may shed light on the linkage of unbalanced copper homeostasis with specific gene promoter methylation and epigenetic histone protein modification as well as the resultant signaling transduction and the myofibrillogenesis defects.

Common responses of fish embryos to metals: an integrated analysis of transcriptomes and methylomes in zebrafish embryos under the stress of copper ions or silver nanoparticles.

Recently, the responses of embryos to Cu2+ or AgNP stresses have been investigated, but few studies have been performed on the common responses of embryos to both Cu2+ and AgNPs, the same kind of stressor metal. In this study, a large number of commonly down-regulated and up-regulated differentially expressed genes (DEGs) were revealed in both Cu2+- and AgNP-stressed embryos. The down-regulated DEGs were enriched in myosin complex and muscle structure development, ion transport and metal ion binding, transmission of nerve impulses, etc., and the up-regulated DEGs were enriched in heart development, iron ion binding, etc. Based on the whole-genome bisulfite sequencing (WGBS) in both Cu2+- and AgNP-stressed embryos, a total of 57 and 64 differentially methylated genes (DMGs) were identified in Cu2+ embryos and AgNP embryos, with 15 and 12 of them being common ion-relevant genes, respectively. The correlation of the gene transcriptional expression and the methylated status of some common DMGs were further verified. The integrated analysis of transcriptomes and methylomes in zebrafish embryos stressed with Cu2+ or AgNPs revealed for the first time their common transcriptional and methylomic responses to the same kind of stressor metals, and revealed that ion-relevant genes were mostly differentially expressed and methylated genes in both Cu2+- and AgNP-stressed embryos.

Identification of five genes in endoplasmic reticulum (ER) stress-apoptosis pathways in yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to dietary lipid levels.

The activating transcription factor 4 (ATF4), DNA damage-inducible transcript 3 (DDIT3), growth arrest, and DNA damage-inducible protein 34 (GADD34), endoplasmic reticulum oxidoreductin 1α (ERO1α), and tumor necrosis factor receptor associated factor 2 (TRAF2) cDNAs were first characterized from yellow catfish Pelteobagrus fulvidraco. Compared to corresponding genes of mammals, all of these proteins shared similar conserved domains. Their mRNAs were widely expressed in various tissues, but at variable levels. Dietary lipid levels did not significantly influence ATF4 mRNA expression. mRNA expression of DDIT3 and GADD34 was highest for fish fed the low-lipid diets and lowest for fish fed middle-lipid diets. The mRNA levels of ERO1α and TRAF2 declined with increasing dietary lipid levels. For the first time, we characterized the full-length cDNA sequences of ATF4, DDIT3, GADD34, ERO1α, and TRAF2 and determined their tissue expression profiles and transcriptional responses to dietary lipid levels, which would contribute to our exploration into their biological functions, and providing new insights on relations between ER stress and lipid metabolism in fish.

Identification of full-length cDNA sequences for three development-relevant genes from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to high fat diet.

The goal of this study was to clone and characterize complete cDNA sequences of three important development-relevant genes of yellow catfish Pelteobagrus fulvidraco, including lrp6, sox9a1 and fgfr2c, and explore their transcriptional responses in several tissues of P. fulvidraco to high fat diet. The predicted amino acid sequences of P. fulvidraco Lrp6, Sox9a1 and Fgfr2c contained all of the conserved structural features that were characteristic of these genes in other species, including YWTD domains, EGF-like repeats, LDLR ligand binding repeats, PPSP repeats motifs, HMG box, TA-binding functional domain, Ig I-III and PTK I-II. The mRNAs of the three genes were expressed in various tissues, but their mRNA levels varied among tissues. Compared to the control, high fat diet tended to down-regulate the mRNA expression of sox9a1 and fgfr2c in mesenteric fat, liver and ovary, and up-regulate their mRNA levels in muscle and kidney; in contrast, high fat diet down-regulated lrp6 mRNA levels in the ovary and muscle, but had no significant effects on lrp6 mRNA expression in mesenteric fat, liver and kidney. Our findings provide the first data about their expression responses to dietary lipid in teleosts and reinforce the multiple functions at the molecular level.

Simultaneous enhancement of magnetic and mechanical properties in Ni-Mn-Sn alloy by Fe doping.

Both magnetic-field-induced reverse martensitic transformation (MFIRMT) and mechanical properties are crucial for application of Ni-Mn-Sn magnetic shape memory alloys. Here, we demonstrate that substitution of Fe for Ni can simultaneously enhance the MFIRMT and mechanical properties of Ni-Mn-Sn, which are advantageous for its applications. The austenite in Ni44Fe6Mn39Sn11 shows the typical ferromagnetic magnetization with the highest saturation magnetization of 69 emu/g at 223 K. The result shows that an appropriate amount of Fe substitution can really enhance the ferromagnetism of Ni50Mn39Sn11 alloy in austenite, which directly leads to the enhancement of MFIRMT. Meanwhile, the mechanical property significantly improves with Fe doping. When there is 4 at.% Fe added, the compressive and maximum strain reach the maximum value (approximately 725.4 MPa and 9.3%). Furthermore, using first-principles calculations, we clarify the origin of Fe doping on martensitic transformation and magnetic properties.