Introduction
Epigenetic modifications, notably histone demethylation, participate in regulating developmental gene transcription via chromatin remodeling (Bhat et al., Reference Bhat, Ümit Kaniskan, Jin and Gozani2021; Mudbhary and Sadler, Reference Mudbhary and Sadler2011). Histone lysine-specific demethylase 4A (KDM4A) catalyzes the removal of trimethylation modifications on histone H3 lysine 9 (H3K9me3) as well as histone H3 lysine 36 (H3K36me3), then regulating gene transcription (Young and Dere, Reference Young and Dere2021). KDM4A plays essential roles in a variety of biological processes including chromatin activity, development, and metabolism senescence (Ji et al., Reference Ji, Yan, Han and Zhang2021). The Drosophila KDM4 ortholog dKDM4 (CG15835) establishes heterochromatin-euchromatin boundaries through H3K36 methylation-dependent chromatin remodeling (Lloret-Llinares et al., Reference Lloret-Llinares, Carré, Vaquero, de Olano and Azorín2008), dKDM4A can also localize to heterochromatin and regulate repetitive DNA regions within heterochromatic domains, as well as DNA damage repair (Crona et al., Reference Crona, Dahlberg, Lundberg, Larsson and Mannervik2013). KDM4A suppresses astrocyte differentiation by inducing H3K36 demethylation and RNA polymerase II disassembly at glial fibrillary acidic protein (GFAP) locus and promotes osteogenic differentiation of bone mesenchymal stem cells (BMSCs) (Cascante et al., Reference Cascante, Klum, Biswas, Antolin-Fontes, Barnabé-Heider and Hermanson2014; Qin et al., Reference Qin, Li, Wang, Yang, Chen, Tang, Wang, Zhang, Jiang, Lin and Huo2020a). KDM4A downregulation enhances pyruvate dehydrogenase (PDH) activity and mitochondrial oxidation, triggering excessive accumulation of ROS (Zhuang et al., Reference Zhuang, Ma, Guo and Sun2017).
Emerging evidence underscores the conserved role of KDM4A in embryonic development across species, including mice (Sankar et al., Reference Sankar, Kooistra, Gonzalez, Ohlsson, Poutanen and Helin2017), goats (Wu et al., Reference Wu, Wary, Revskoy, Gao, Tsang, Komarova, Rehman and Malik2015), and pigs (Weng et al., Reference Weng, Cai, Zhang, Liu, Liu and Liu2020). Despite these advances, the functional conservation of KDM4A in non-mammalian vertebrates remains unexplored. Notably, zebrafish -exhibiting 87% genetic homology with humans, high fecundity, and rapid embryogenesis-serves as a powerful model for dissecting conserved regulatory mechanisms in vertebrate development (Wang et al., Reference Wang, Zhang, He, Wang and Liu2021). However, the roles of the zebrafish kdm4a paralogs-kdm4aa and kdm4ab-in embryogenesis, particularly their paralog-specific functions, remain unclear. Our previous work showed that knockout of kdm4aa function leads to reduced survival rate during the early stages of zebrafish development (Lijie et al., Reference Lijie, Juntao, Songya, Xueling, Junfang and Bingshe2023). Building on this initial finding, our subsequent research has further demonstrated that kdm4aa is ubiquitously expressed, with the highest mRNA levels in the male gonads of adult zebrafish (Y et al., Reference Yao, Juntao, Mengran, Lijie, Bingshe and Junfang2024), whereas kdm4ab expression is more spatially restricted (Zhang et al., Reference Zhang, Luo, Zhang, Han and Zhang2024). These divergent expression patterns suggest potential subfunctionalization between the two paralogs. Furthermore, knockout of kdm4aa but not kdm4ab leads to significant skeletal defects in adult zebrafish, implying a specific role for kdm4aa in development (Zhang et al., Reference Zhang, Luo, Zhang, Han and Zhang2024). However, the precise molecular mechanisms underlying their functional specialization, particularly during the critical window of early embryogenesis, are still unclear. This study aims to dissect the paralog-specific contributions of kdm4aa and kdm4ab to zebrafish embryonic development, with a focus on elucidating the epigenetic and metabolic pathways involved.
RNA binding motif (RBM) proteins contain multiple RNA-binding domains (Qin et al., Reference Qin, Ni, Liu, Yuan, Xi, Li and Zheng2020b) and function in various RNA processes including pre-mRNA splicing, RNA stability, mRNA translation, and RNA editing (Li et al., Reference Li, Guo, Zhang, Fu, Wang, Wang and Tang2021), thereby influencing a variety of physiological processes including spermatogonia differentiation (Qian et al., Reference Qian, Li, Yan, Han, Bu, Gong, Zheng, Yuan, Ren, He, Zhang, Xu, Wang, Sun, Lin, Zhou and Ye2022), association complex formation (Peart et al., Reference Peart, Johnson, Lee, Sears, Yang, Quesnel-Vallières, Feng, Recinos, Barash, Zhang, Hermann, Wang, Geyer and Carstens2022), and mitosis-meiosis transition (Lv et al., Reference Lv, Lu, Cai, Su, Liang, Wang, Mu, He, Huang, Ma, Zhao, Chen, Xue, Liu and Chan2023). Among them RNA-binding motif protein 46 (RBM46) plays a critical role in facilitating the transition from mitotic to meiotic programs during mammalian spermatogenesis (Qian et al., Reference Qian, Li, Yan, Han, Bu, Gong, Zheng, Yuan, Ren, He, Zhang, Xu, Wang, Sun, Lin, Zhou and Ye2022). RBM46 expression steadily increases with gestational age in human fetal ovaries (Houmard et al., Reference Houmard, Small, Yang, Naluai-Cecchini, Cheng, Hassold and Griswold2009). RBM46 is also highly expressed in oocytes of early follicle stages in juvenile and aged cynomolgus monkey ovaries (Wang et al., Reference Wang, Zheng, Li, Yu, Zhang, Song, Liu, Min, Hu, Jing, He, Sun, Ma, Esteban, Chan, Qiao, Zhou, Izpisua Belmonte, Qu, Tang and Liu2020). Rbm46 regulates trophectoderm specification through stabilizing caudal type homeobox 2 (Cdx2) mRNA in early mouse embryos (Wang et al., Reference Wang, Chen, Deng, Gao and Li2015). RBM46 deficiency in mouse embryonic stem cells (mESCs) leads to ROS accumulation, impaired adenosine triphosphate (ATP) production, and ultimately apoptosis (Zhong et al., Reference Zhong, Xu, Chen, Li, Li, Deng, Feng, Ling, Wang, Zhou and Li2024). In zebrafish, rbm46 is initially characterized as essential for spermatogenesis (Dai et al., Reference Dai, Cheng, Huang, Gao, Wang, Feng, Zhai, Lou, He, Wang and Yin2021).
In this study, we investigated the mechanism by which kdm4aa knockout disturbs zebrafish development, and demonstrated that kdm4aa knockout severely disturbs the early embryonic development of zebrafish via downregulating rbm46, providing mechanistic insights into the functional role of kdm4aa during zebrafish embryogenesis.
Materials and methods
Experimental animals
The AB strain zebrafish were raised at 28°C under a 10/14 h dark/light cycle. Embryos were collected after natural spawning and rinsed in system water to remove contaminants. Embryos/larvae were developmentally staged by hours/days/months post fertilization (hpf/dpf/mpf).
Generation of kdm4aa and kdm4ab mutant Zebrafish
kdm4aa and kdm4ab mutant zebrafish were generated via CRISPR/Cas9, targeting exon 2 (5’-GGAGTCGCAGGGGGCGCACA-3’) of kdm4aa and exon 9 (5’-GGTACGAGCTGTGGTTGGAG-3’) of kdm4ab (Lijie et al., Reference Lijie, Juntao, Songya, Xueling, Junfang and Bingshe2023; Y et al., Reference Yao, Juntao, Mengran, Lijie, Bingshe and Junfang2024). Gene-specific gRNAs were transcribed using the MAXIscript T7 Transcription Kit (AM1314, Thermo Fisher Scientific, USA) and co-injected with Cas9 (Z03389-100, GenScript) protein into one-cell stage embryos. Founder (F0) mutants, identified via T7 Endonuclease I assay (M0302s, NEB, USA), were outcrossed to establish heterozygous F1 lines. Homozygous F2 mutants were then generated by intercrossing, with genotypes confirmed by PCR (primers in Supplementary Table 1) and Sanger sequencing. Two mutant lines for kdm4aa (kdm4aa Δ10 and kdm4aa Δ11) with 10-bp and 11-bp deletions, and one for kdm4ab (kdm4ab Δ13) with a 13-bp deletion, were established, all of which resulted in frameshift mutations and premature stop codons.
Evaluation of survival rate
Six pairs of zebrafish were set to spawn, and approximately 300 embryos per genotype were analyzed. All embryos were collected 2 hpf, then the survival rate was determined and expressed in the percentage of fertilized eggs. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Log-rank test (Mantel-Cox test).
RNA-seq sequencing, and bioinformatic analysis
Total RNA was extracted from a pool of 50 embryos per replicate at 5.25 hpf. RNA-seq libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain at least 22 million 150 bp paired-end reads per sample. Raw reads were quality-checked using FastQC (v0.12.1), trimmed with Trimmomatic (v0.39), and aligned to the zebrafish reference genome (Danio rerio, GRCz11, ENSEMBL 113) using HISAT2 (v2.2.0). The resulting SAM files were sorted by read name (@RG tags) and converted to BAM format with SAMtools (v1.18). Gene-level read counts were generated using featureCounts (v2.0.6) and imported into R, where differential expression analysis was performed with DESeq2 (v1.44.0) employing an empirical Bayes approach and the Wald test to calculate log2 fold changes (LFC). Transcript abundance was further estimated as FPKM values using the dplyr (v1.1.4) package in R.
RNA isolation, cDNA synthesis, and real-time quantitative PCR (RT-qPCR)
Total RNA was extracted using Trizol™ reagent (AT341-01; ABclonal, USA), and cDNA was synthesized from 1 μg of RNA in a 20 μL reaction using PrimeScript™ RT reagent Kit with gDNA Eraser (AT341-01; TransGen Biotech, China). After that, RT-qPCR was performed using iTaq Univer SYBR Green Supermix (AQ601-02-V2; TransGen Biotech, China). Zebrafish β-actin was used as an internal reference gene, and all primers are shown in Supplementary Table 1. For each replicate, total RNA was extracted from a pool of 80 embryos, and the gene expression was calculated by the relative quantification method (2−ΔΔCt). Each experiment included three biological replicates (n = 3).
Cleavage under targets and tagmentation (CUT&Tag) assay
CUT&Tag was performed with 50 embryos (5.25 hpf) using Hyperactive Universal CUT&Tag Assay Kit for Illumina protocol (TD904, Vazyme, China). Following the manufacturer’s protocol, CUT&Tag procedure included ConA bead activation, incubation with primary antibodies against H3K4me3 (ab8580, Abcam, UK), H3K9me3 (ab176919, Abcam, UK), and H3K36me3 (ab9050, Abcam, UK) and a secondary antibody (goat anti-rabbit IgG H&L, Ab207-01, Abcam, UK), pA/G-Tnp-mediated tagmentation at 37°C for 1 h, and final DNA purification using DNA Extract Beads Pro. DNA spike-in control was added at a concentration of 1 pg per 100,000 cells prior to allow normalization among samples. Libraries were amplified by 14 PCR cycles, purified with VAHTS DNA Clean Beads (Vazyme). Final libraries were quantified by Qubit.
For CUT&Tag-qPCR, experiments were conducted following Vazyme TD904-1 protocol using 1 µL of purified DNA as template in 10 µL reactions containing 2× PerfectStart® Green qPCR SuperMix (AQ601-02-V2, TransGen Biotech, China). Spike-in normalization was applied to calculate relative enrichment. Primer sequences are listed in Supplementary Table 1. Each experiment included three biological replicates (n = 3).
CUT&Tag databioinformatic
CUT&Tag libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain at least 26 million 150-bp paired-end reads per sample. Raw reads were quality-checked using FastQC (v0.12.1), trimmed with Trimmomatic (v0.39) using the parameters HEADCROP:20 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:52, and aligned to the zebrafish reference genome (Danio rerio, GRCz11, ENSEMBL 113) using Bowtie2 (v2.5.2). The resulting SAM files were sorted and converted to BAM format using SAMtools (v1.18). Peak calling was performed using MACS3 (v3.0.1) with default parameters.
mRNA synthesis and microinjection
The full-length coding sequences (CDS) of rbm46 was amplified via PCR with the forward primer (Supplementary Table 1) incorporating the SP6 promoter sequence. In vitro transcription was performed using MAXIscript™ SP6 Transcription Kit (AM1314, Thermo Fisher Scientific, USA), followed by mRNA purification via LiCl precipitation. Approximately 1 nL of mRNA solution (150 ng/μL) was microinjected into one-cell stage zebrafish embryos.
Detection of ROS production
ROS levels were determined using Reactive Oxygen Species Assay Kit (S0033s, Beyotime, China) according to the manufacturer’s instructions. Each replicate used a pool of 100 embryos homogenized in ice-cold 0.5 M Tris-HCl buffer using a 1:6 weight-to-volume ratio. 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) were used to detect the intracellular ROS. The ROS levels were normalized against protein concentrations. Each experiment included three biological replicates (n = 3).
Measurement of ATP production
ATP levels in embryos were determined using ATP Assay Kit (S0026, Beyotime, China) according to the manufacturer’s protocols. Briefly, tissue lysates of 50 embryos were centrifuged at 12,000 g at 4°C for 10 min, and the supernatant was incubated in the ATP-detection buffer containing firefly luciferin and firefly luciferase enzyme reagent for 5 min, the Relative Light Unit (RLU) was then detected. The ATP levels were normalized against protein concentrations. Each experiment included three biological replicates (n = 3).
Examination of total antioxidation capability (T-AOC)
T-AOC was determined using Total Antioxidant Capability Assay Kit with FRAP method (A015-3-1, Jiancheng Bioengineering Institute, China). Briefly, 100 embryos were homogenized in ice-cold PBS using a 1:4 weight-to-volume ratio. And 5 μL of the supernatants was incubated with 180 μL of chromophoric substrate for 5 min at 37°C. The reduction of Fe3+-TPTZ was then detected at 593 nm. The T-AOC levels were normalized against protein concentrations. Each experiment included three biological replicates (n = 3).
Statistical analysis
All statistical analyses were executed with GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by student t-test. The p-value in the figure legends is shown as: no significant (ns); *: P < 0.05; **: P < 0.01; ***: P< 0.001; ****: P < 0.0001.
Results
kdm4aa knockout downregulates rbm46 during Zebrafish development
Our previous study indicated that kdm4aa knockout impairs embryo survival of zebrafish, primarily by compromising oocyte quality (Lijie et al., Reference Lijie, Juntao, Songya, Xueling, Junfang and Bingshe2023). To delineate the role of kdm4aa and kdm4ab in zebrafish development, we compared the survival of WT, kdm4aa –/–, and kdm4ab –/–embryos (Figure 1A), and examined their morphological development via bright-field microscopy at key developmental stages (Supplementary Fig. 1). At 26 hpf kdm4aa –/– zebrafish exhibited markedly reduced survival (22.32%) during gastrulation, when kdm4ab –/– embryos only showed moderately increased embryo death (17.11%). Furthermore,the kdm4aa –/– embryos displayed noticeable developmental retardation. Then RNA-seq revealed that rbm46 was downregulated after kdm4aa knockout (Supplementary Table 2), and RT-qPCR further confirmed that rbm46, but not other rbm family members, decreased only in kdm4aa –/– embryos at 5.25 hpf (Figure 1B).
kdm4aa knockout downregulates rbm46 mRNA expression during early embryonic development. (A) The survival rates of WT, kdm4aa –/–, kdm4ab –/– embryos were determined at indicated timepoints and expressed in the percentage using 300 embryos per genotype and expressed as a percentage of fertilized eggs. (B) RT-qPCR showed the mRNA expression of indicated rbm family genes in WT, kdm4aa –/–, kdm4ab –/– zebrafish embryos at 5.25 hpf. The mRNA expression levels of rbm46 (C), kdm4aa (D), and kdm4ab (E) in zebrafish embryos of the indicated genotypes at various developmental stages, as measured using RT-qPCR at indicated stages of development, with 80 embryos per replicate per genotype. Data for graphing were obtained from the means ± SD of three biological replicates, *: P < 0.05, ****: P < 0.0001.
Then rbm46 expression was examined in above embryos using RT-qPCR at six developmental stages(4-cell, 1,000-cell, dome, 50% epiboly, 10 hpf, and 24 hpf). As shown in Figure 1C, rbm46 was consistently downregulated in kdm4aa –/– embryos, with the most significant difference occurring at 5.25 hpf. In contrast, rbm46 in kdm4ab –/– embryos remained comparable to WT embryos throughout the development (Figure 1C). Notably, kdm4aa and rbm46 mRNA expression showed highly synchronized expression profiles (Figure 1D), suggesting a potential direct regulatory relationship. Importantly, the loss of kdm4aa also produced an indirect suppression of rbm46 in the kdm4ab mutant background, further supporting a direct regulatory link between kdm4aa and rbm46, especially pronounced at 5.25 hpf. Figure 1E shows the temporal pattern of kdm4ab expression and indicates that kdm4ab exhibits a decline over these stages that does not closely mirror rbm46; this contrast emphasizes that, although both paralogs can influence early transcriptional programs, the impact of kdm4aa loss on rbm46 is more substantial.
These findings demonstrated that rbm46 participated in early zebrafish development, and that kdm4aa knockout specifically suppressed rbm46 mRNA expression during this critical period.
Kdm4aa regulates histone modifications of rbm46 promoter
KDM4A can catalyze the demethylation of H3K9 and H3K36 (Couture et al., Reference Couture, Collazo, Ortiz-Tello, Brunzelle and Trievel2007). It has been reported that H3K36me3 associates with transcriptional elongation, H3K9me3 marks gene repression, and H3K4me3 designates active promoters (Guenther et al., Reference Guenther, Levine, Boyer, Jaenisch and Young2007). Here CUT&Tag (Figure 2A) and CUT&Tag qPCR (Figure 2B) of 5.25 hpf kdm4aa –/– embryos showed that H3K4me3 levels at the rbm46 promoter were dramatically reduced compared to those in WT embryos. In kdm4aa –/– embryos, H3K9me3 and H3K36me3 levels at rbm46 promoter remarkably increased (Figure 2C–F). These results supported that kdm4aa modulated rbm46 transcription by reshaping the histone modification landscape at its promoter.
kdm4aa regulates histone modifications of rbm46 promoter. CUT&Tag (A, C, E) and CUT&Tag-qPCR (B, D, F) were performed using 50 and 80 embryos per replicate, respectively, to detect H3K4me3 (A, B), H3K9me3 (C, D), H3K36me3 (E, F) enrichment at rbm46 promoter of 5.25 hpf embryos. Data for graphing were obtained from the means ± SD of three biological replicates, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.
kdm4aa –/– zebrafish embryos exhibit redox imbalance and mitochondrial dysfunction
RBM46 can regulate the development of embryonic stem cells in mice, where its knockout leads to elevated ROS and reduced ATP levels (Zhong et al., Reference Zhong, Xu, Chen, Li, Li, Deng, Feng, Ling, Wang, Zhou and Li2024). Here we investigated the effect of kdm4aa knockout on ROS and ATP levels. At 5.25 and 10 hpf, kdm4aa –/– embryos, not kdm4ab –/– embryos, showed significantly increased ROS (Figure 3A) compared to WT embryos. Excessive ROS accumulation causes oxidative stress that impairs mitochondrial function, ultimately reducing ATP production (Yang et al., Reference Yang, Karakhanova, Hartwig, D’Haese, Philippov, Werner and Bazhin2016). Here we showed that at 5.25 hpf and 10 hpf, kdm4aa –/– embryos displayed progressive antioxidant dysfunction, with reduction of T-AOC levels at 5.25 hpf, and sustained downregulation of superoxide dismutase (sod), catalase (cat) and glutathione peroxidase (gpx) at 5.25 hpf and 10 hpf (Figure 3B, C, D). We also observed markedly decreased ATP levels (Figure 3E) and reduced mRNA expression of mitochondrial transcription factor A (tfam), nuclear respiratory factor 1(nrf1) and nuclear respiratory factor 1(nrf2), accompanied by upregulated expression of uncoupling Protein 2 (ucp2) (Figure 3F). These findings suggested that kdm4aa knockout suppress rbm46, leading to impaired antioxidant defenses and mitochondrial dysfunction.
kdm4aa –/– zebrafish embryos exhibit redox imbalance and mitochondrial dysfunction. ROS (A), T-AOC (B), and ATP (E) levels were detected using 100, 100, and 50 embryos per replicate, respectively, in WT and mutant zebrafish embryos at 5.25 hpf or 10 hpf. The mRNA expression of indicated genes was detected using 80 embryos per replicate with RT-qPCR in WT and mutant embryos at 5.25 hpf (C, F and 10 hpf (D). Data for graphing were obtained from the means ± SD of three biological replicates, *: P < 0.05, **: P< 0.01, ***: P < 0.001.
rbm46 enhances Zebrafish embryo survival
The RBM family members participate in ROS regulation post-transcriptionally and are implicated in mitochondrial pathology and oxidative damage(Lv et al., Reference Lv, Lu, Cai, Su, Liang, Wang, Mu, He, Huang, Ma, Zhao, Chen, Xue, Liu and Chan2023) (Ravanidis and Doxakis, Reference Ravanidis and Doxakis2020). And above-mentioned findings support that RBM46 modulates mitochondrial pathology and oxidative damage in zebrafish embryos during gastrulation. Here we injected rbm46 mRNA into one-cell stage WT zebrafish embryos. Embryos overexpressing rbm46 exhibited a 59.24% survival rate within 26 hpf, significantly higher than the 44.9% in controls (Figure 4A). rbm46 overexpression also significantly reduced ROS levels (Figure 4B) and upregulated antioxidant genes (sod, cat, gpx) (Figure 4D), accompanied with enhanced T-AOC levels in 5.25 hpf embryos (Figure 4C). Meanwhile, ATP levels and mitochondrial biogenesis-related genes (tfam, nrf1 and nrf2) increased significantly, while mitochondrial metabolic-related gene ucp2 decreased (Figure 4E, F). Our findings demonstrated that rbm46 coordinates antioxidant defenses with mitochondrial energy metabolism, playing a critical protective role during early embryonic development.
rbm46 regulates redox homeostasis and mitochondrial function in zebrafish embryos. (A) One-cell stage WT zebrafish embryos were injected with rbm46 mRNA or H2O, then the survival rates were determined at indicated timepoints and expressed in the percentage of fertilized eggs, with 300 embryos per genotype. ROS (B), T-AOC (C), and ATP (E) levels were detected using 100, 100, and 50 embryos per replicate, respectively, in control and rbm46-overexpressing WT zebrafish embryos at 5.25 hpf. The mRNA expression of indicated genes was detected using 80 embryos per replicate with RT-qPCR in WT and mutant embryos at 5.25 hpf (D, F). Data for graphing were obtained from the means ± SD of three biological replicates, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.
rbm46 mRNA improves the survival of kdm4aa –/– Zebrafish embryos
To further investigate the role of rbm46 in kdm4aa –/– zebrafish embryos, we injected rbm46 mRNA into one-cell stage kdm4aa –/– embryos. Embryos overexpressing rbm46 exhibited a 53.92% survival rate within 26 hpf, significantly higher than the 21.33% in controls (Figure 5A). rbm46 overexpression reduced ROS levels (Figure 5B). Corroborating this finding, fluorescence microscopy revealed a marked reduction in the ROS fluorescence area in the rescued embryos (Supplementary Fig. 2). Furthermore, the rescue led to enhanced T-AOC levels (Figure 5C), and upregulated sod, cat, gpx (Figure 5D). Meanwhile, ATP levels, tfam and nrf2 increased significantly, while ucp2 decreased (Figure 5E, F). These results showed that rbm46 improves the development of kdm4aa –/– embryos by restoring redox equilibrium and optimizing mitochondrial energy metabolism, acting as a crucial factor in kdm4aa-mediated embryonic survival.
rbm46 mRNA improves the survival of kdm4aa –/– zebrafish embryos. (A) kdm4aa –/– zebrafish embryos were injected with rbm46 mRNA or H2O, then the survival rates were determined at indicated timepoints and expressed in the percentage of fertilized eggs, with 300 embryos per genotype. ROS (B), T-AOC (C), and ATP (E) levels were detected using 100, 100, and 50 embryos per replicate, respectively, at 5.25 hpf. The mRNA expression of indicated genes was detected using 80 embryos per replicate with RT-qPCR at 5.25 hpf (D, F). Data for graphing were obtained from the means ± SD of three biological replicates, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.
Discussion
KDM4A, as a critical epigenetic enzyme, is essential for epigenetic reprogramming and pre-implantation development of embryos (Ji et al., Reference Ji, Yan, Han and Zhang2021). Variable KDM4A expression in human MII oocytes may contribute to the defective zygotic genome activation (ZGA) in human embryos (Sankar et al., Reference Sankar, Lerdrup, Manaf, Johansen, Gonzalez, Borup, Blanshard, Klungland, Hansen, Andersen, Dahl, Helin and Hoffmann2020). Overexpression of KDM4A significantly improves the blastocyst formation rate in human somatic cell nuclear transfer (SCNT) embryos by facilitating transcriptional reprogramming (Chung et al., Reference Chung, Matoba, Liu, Eum, Lu, Jiang, Lee, Sepilian, Cha, Lee and Zhang2015). In cloned embryos, KDM4A enhances reprogramming efficiency in goat and pig embryos by antagonizing the epigenetic barrier mediated by H3K9me3 (Deng et al., Reference Deng, Liu, Chen, Wan, Yang, Zhang, Cai, Zhou and Wang2020; Weng et al., Reference Weng, Cai, Zhang, Liu, Liu and Liu2020). Furthermore, KDM4A and KDM4C synergistically regulate angiogenesis-related genes to drive the directed differentiation of mESCs, and their combined loss leads to early embryonic lethality (Pedersen et al., Reference Pedersen, Kooistra, Radzisheuskaya, Laugesen, Johansen, Hayward, Nilsson, Agger and Helin2016). These findings reveal the conserved role of KDM4A in embryonic development across mammals, while their evolutionary preservation in zebrafish remains unvalidated. Intriguingly, our previous study found that zebrafish genome harbors two kdm4a paralogs, kdm4aa and kdm4ab (Wei et al., Reference Wei, Juntao and Tingting2020), and showed that knockout of either kdm4aa or kdm4ab affected embryonic survival, with kdm4aa being essential for this process (Lijie et al., Reference Lijie, Juntao, Songya, Xueling, Junfang and Bingshe2023; Zhang et al., Reference Zhang, Luo, Zhang, Han and Zhang2024). However, the precise molecular mechanisms underlying this phenomenon remain unclear.
In this study, we further showed that kdm4aa knockout elicited markedly more severe embryonic lethality than kdm4ab knockout during early zebrafish development, particularly at gastrulation. This phenotypic disparity correlated with kdm4aa’s specific suppression of rbm46 mRNA expression, a key regulator of redox homeostasis and mitochondrial function, while kdm4ab knockout did not alter rbm46 expression or its dependent pathways, highlighting the paralog-specific regulatory dominance of kdm4aa in zebrafish embryogenesis. Similar to mammalian KDM4A, zebrafish Kdm4aa and Kdm4ab both have domain-based functions like Jumonji N-terminal domain (JmjN), Jumonji C-terminal domain (JmjC) and Tudor domains (Young and Dere, Reference Young and Dere2021), which are involved in histone demethylation and chromatin-related regulation (Guerra-Calderas et al., Reference Guerra-Calderas, González-Barrios, Herrera, Cantú de León and Soto-Reyes2015), yet differ in structure details due to evolutionary distinctions. kdm4aa contains a 6,039 bp CDS translating to a 2,012 amino acid polypeptide, whereas kdm4ab possesses a shorter 3,138 bp CDS that encodes a 1,045 amino acid protein, these structural divergences imply distinct functional constraints and evolutionary trajectories. The truncated kdm4ab CDS lacking 967 aa relative to kdm4aa likely reflects subfunctionalization through domain loss. This paralog-specific architecture may underlie differential transcriptional regulation in zebrafish embryogenesis. Nevertheless, the mechanistic basis for these differences has yet to be further validated.
RBMs, a family of proteins defined by RNA-binding motifs (Jayasena and Bronner, Reference Jayasena and Bronner2012), regulate mRNA stability and translation through interactions with the 3’ UTR of target transcripts (Qin et al., Reference Qin, Ni, Liu, Yuan, Xi, Li and Zheng2020b). In this study, we revealed that Rbm46, as a member of RBMs, plays a critical role in zebrafish embryonic development and demonstrated that kdm4aa knockout induces the downregulation of rbm46, and this downregulation contributes causally, at least in part, to the developmental abnormalities observed in kdm4aa –/– embryos. The post-transcriptional regulation of ROS by RNA-binding proteins (RBPs) has been shown to be implicated in mitochondrial pathology and oxidative damage (Ravanidis and Doxakis, Reference Ravanidis and Doxakis2020). Among these RBPs, Rbm46 has recently been highlighted as a direct post-transcriptional regulator of ROS levels through the modulation of BCL2 interacting protein 3 (Bnip3) mRNA expression, where it plays an indispensable role in mESCs development (Zhong et al., Reference Zhong, Xu, Chen, Li, Li, Deng, Feng, Ling, Wang, Zhou and Li2024). Although rbm46 was initially characterized as essential for zebrafish spermatogenesis (Dai et al., Reference Dai, Cheng, Huang, Gao, Wang, Feng, Zhai, Lou, He, Wang and Yin2021), we demonstrated for the first time that rbm46 maintains normal development of zebrafish embryos through restoring redox equilibrium and optimizing mitochondrial energy metabolism, consistent with the results of the study in mESCs. Collectively, rbm46 provides novel insights into molecular pathways regulating ROS accumulation and ATP homeostasis during zebrafish embryogenesis.
As a key member of the histone demethylase family, KDM4A mediates the demethylation of H3K9me3 and H3K36me3, specifically influences H3K4me3-targeted point mutations (Pedersen et al., Reference Pedersen, Kooistra, Radzisheuskaya, Laugesen, Johansen, Hayward, Nilsson, Agger and Helin2016). In this study, we demonstrated markedly decreased H3K4me3 enrichment and increased H3K9me3/H3K36me3 enrichment at rbm46 promoter in kdm4aa –/– zebrafish embryos. H3K4me3 marks active promoters (Guenther et al., Reference Guenther, Levine, Boyer, Jaenisch and Young2007) and H3K9me3 mediates transcriptional repression (Becker et al., Reference Becker, Nicetto and Zaret2016), proper H3K36me3 epigenome function plays a critical role in maintaining genomic stability (Cohen and Meshorer, Reference Cohen and Meshorer2023). KDM4A plays a major role in preventing the formation of a H3K9me3 reprogramming barrier during normal development, overexpression reduces H3K9me3 enrichment and activate H3K9me3-mediated repressive genes (Matoba et al., Reference Matoba, Liu, Lu, Iwabuchi, Shen, Inoue and Zhang2014; Yang et al., Reference Yang, Li, Tao, Liu, Zhu, Du, Fei, She and Chen2024). KDM4A localizes to H3K4me3-positive promoters, where they have widespread and redundant roles in preventing accumulation of H3K9me3 and H3K36me3 (Pedersen et al., Reference Pedersen, Kooistra, Radzisheuskaya, Laugesen, Johansen, Hayward, Nilsson, Agger and Helin2016). The loss of KDM4A in oocytes causes aberrant H3K9me3 deposition across H3K4me3 broad domains, resulting in insufficient transcriptional activation of genes (Sankar et al., Reference Sankar, Lerdrup, Manaf, Johansen, Gonzalez, Borup, Blanshard, Klungland, Hansen, Andersen, Dahl, Helin and Hoffmann2020), providing a mechanistic example of crosstalk between histone modifications with an impact on epigenome. Collectively, our findings demonstrated that kdm4aa knockout reduced rbm46 mRNA levels, likely through transcriptional suppression mediated by altered histone methylation at its promoter. While future work using direct methods, such as 4-thiouridine (4sU) labeling-seq or global run-on sequencing (GRO-seq), is needed to precisely quantify transcription, our data provided strong mechanistic support for this regulatory axis.
In summary, our results showed that knockout of kdm4aa inhibits the transcriptional level of rbm46 in early zebrafish embryonic development, causing oxidative damage and mitochondrial dysfunction in zebrafish embryos, resulting in high mortality rate of kdm4aa –/– mutants, especially in gastrulation. This present study provides new insights into mechanisms of developmental disorders in zebrafish embryogenesis.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0967199425100257
Data availability
All relevant data and details of resources can be found within the article and its supplementary information.
Author contributions
Hui Jiang: Conceptualization, Writing-original draft. Xinqing Cai: Data curation, Methodology. Rui Deng: Methodology. Jiamin Wang: Methodology. Hongkuan Song: Methodology. Weiran Chai: Methodology. Bingshe Han: Methodology, Writing-review & editing. Junfang Zhang: Funding acquisition, Writing-review & editing.
Funding statement
This work was supported by the Shanghai Agricultural Science and Technology Innovation Program (Grant No. 2024-02-08-00-12-F00047).
Competing interests
All authors declare that there are no conflicts of interest for this paper.
Ethical standard
The experimental protocol was approved by the Animal Ethics committee of Shanghai Ocean University and abides by the Guidelines on Ethical Treatment of Experimental Animals established by the Ministry of Science and Technology, China.
