Keng Po Lai, Nathan Tam, Simon Yuan Wang, Xiao Lin, Ting Fung Chan, Doris Wai Ting Au, Rudolf Shiu Sun Wu, and Richard Yuen Chong Kong. 2020. “Hypoxia causes sex-specific hepatic toxicity at the transcriptome level in marine medaka (Oryzias melastigma).” Aquatic Toxicology, Pp. 105520. Publisher's VersionAbstract
Hypoxia, a low environmental oxygen level, is a common problem in the ocean globally. Hypoxia has been known to cause disruption to the endocrine system of marine organisms in both laboratory and field studies. Our previous studies have demonstrated the sex-specific response to hypoxia in the neural and reproductive systems of marine fish. In the current report, we aim to study the sex-specific hepatic response of fish at the transcriptome level to hypoxic stress. By using a comparative transcriptome analysis, followed by a systematic bioinformatics analysis including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA), we found that hypoxia altered expression of genes related to cell proliferation and apoptosis of hepatocytes, which are associated with human pathologies, such as liver inflammation hepatic steatosis and steatohepatitis. Furthermore, we observed sex-specific responses in the livers of fish through different cell signaling pathways. In female fish, hypoxia causes dysregulation of expression of genes related to impairment in endoplasmic reticulum structure and liver metabolism. In male fish, genes associated with redox homeostasis and fatty acid metabolism were altered by hypoxic stress. The findings of this study support the notion that hypoxia could cause sex-specific changes (hepatic toxicity and changes) in marine fish.
Noa Liberman*, O’Brown Zach K.*, Andrew Scott Earl*, Konstantinos Boulias, Maxim V. Gerashchenko, Simon Yuan Wang, Colette Fritsche, Paul-Enguerrand Fady, Anna Dong, Vadim N. Gladyshev, and Eric Lieberman Greer. 2020. “N6-adenosine methylation of ribosomal RNA affects lipid oxidation and stress resistance.” Science Advances. Publisher's VersionAbstract
During stress, global translation is reduced, but specific transcripts are actively translated. How stress-responsive mRNAs are selectively translated is unknown. We show that METL-5 methylates adenosine 1717 on 18S ribosomal RNA in C. elegans, enhancing selective ribosomal binding and translation of specific mRNAs. One of these mRNAs, CYP-29A3, oxidizes the omega-3 polyunsaturated fatty acid eicosapentaenoic acid to eicosanoids, key stress signaling molecules. While metl-5–deficient animals grow normally under homeostatic conditions, they are resistant to a variety of stresses. metl-5 mutant worms also show reduced bioactive lipid eicosanoids and dietary supplementation of eicosanoid products of CYP-29A3 restores stress sensitivity of metl-5 mutant worms. Thus, methylation of a specific residue of 18S rRNA by METL-5 selectively enhances translation of cyp-29A3 to increase production of eicosanoids, and blocking this pathway increases stress resistance. This study suggests that ribosome methylation can facilitate selective translation, providing another layer of regulation of the stress response.
Transgenerational epigenetic inheritance: from phenomena to molecular mechanisms
Noa Liberman*, Simon Yuan Wang*, and Eric Lieberman Greer. 10/17/2019. “Transgenerational epigenetic inheritance: from phenomena to molecular mechanisms.” Current Opinion in Neurobiology. Publisher's VersionAbstract

Inherited information not encoded in the DNA sequence can regulate a variety of complex phenotypes. However, how this epigenetic information escapes the typical epigenetic erasure that occurs upon fertilization and how it regulates behavior is still unclear. Here we review recent examples of brain related transgenerational epigenetic inheritance and delineate potential molecular mechanisms that could regulate how non-genetic information could be transmitted.

Dissected Caenorhabditis elegans germlines stained with DAPI (blue) and double strand break repair protein RAD-51 (Red) reveal increased levels of DNA damage or delayed DNA damage repair in mutants for the demethylating enzyme NMAD-1
Simon Yuan Wang*, Mao Hui*, Hiroki Shibuya*, Satoru Uzawa, Zach Klapholz O'Brown, Sage Wesenberg, Nara Shin, Takamune Saito, Jinmin Gao, Barbara J. Meyer, Monica P Colaiacovo, and Eric Lieberman Greer. 7/8/2019. “The demethylase NMAD-1 regulates DNA replication and repair in the C. elegans germline.” PLOS Genetics. Publisher's VersionAbstract
The biological roles of nucleic acid methylation, other than at the C5-position of cytosines in CpG dinucleotides, are still not well understood. Here, we report genetic evidence for a critical role for the putative DNA demethylase NMAD-1 in regulating meiosis in C. elegans. nmad-1mutants have reduced fertility. They show defects in prophase I of meiosis, which leads to reduced embryo production and an increased incidence of males due to defective chromosomal segregation. In nmad-1 mutant worms, nuclear staging beginning at the leptotene and zygotene stages is disorganized, the cohesin complex is mislocalized at the diplotene and diakinesis stages, and chromosomes are improperly condensed, fused, or lost by the end of diakinesis. RNA sequencing of the nmad-1 germline revealed reduced induction of DNA replication and DNA damage response genes during meiosis, which was coupled with delayed DNA replication, impaired DNA repair and increased apoptosis of maturing oocytes. To begin to understand how NMAD-1 regulates DNA replication and repair, we used immunoprecipitation and mass spectrometry to identify NMAD-1 binding proteins. NMAD-1 binds to multiple proteins that regulate DNA repair and replication, including topoisomerase TOP-2 and co-localizes with TOP-2 on chromatin. Moreover, the majority of TOP-2 binding to chromatin depends on NMAD-1. These results suggest that NMAD-1 functions at DNA replication sites to regulate DNA replication and repair during meiosis.
Zach K. O’Brown, Konstantinos Boulias, Jie Wang, Simon Yuan Wang, Natasha M. O’Brown, Ziyang Hao, Hiroki Shibuya, Paul-Enguerrand Fady, Yang Shi, Chuan He, Sean G. Megason, Tao Liu, and Eric Lieberman Greer. 6/3/2019. “Sources of artifact in measurements of6mA and 4mC abundance in eukaryoticgenomic DNA.” BMC Genomics, 20, Pp. 445. Publisher's VersionAbstract

Background: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa.

Results: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background.

Conclusions: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA.pdf
Hypoxia causes transgenerational impairment of ovarian development and hatching success in fish
Keng Po Lai*, Simon Yuan Wang*, Jing Woei Li, Yin Tong, Ting-Fung Chan, Nana Jin, Anna Tse, Jiang Wen Zhang, Miles Teng Wan, Nathan Tam, Doris Wai Ting Au, Bo Young Lee, Jae-Seong Lee, Alice Sze Tsai Wong, Richard Yuen Chong Kong, and Rudolf Shiu Sun Wu. 3/7/2019. “Hypoxia causes transgenerational impairment of ovarian development and hatching success in fish.” Environmental Science & Technology, 53, 7, Pp. 3917-3928. Publisher's VersionAbstract
Hypoxia is a pressing environmental problem in both marine and freshwater ecosystems globally, and this problem will be further exacerbated by global warming in the coming decades. Recently, we reported that hypoxia can cause transgenerational impairment of sperm quality and quantity in fish (in F0, F1, and F2 generations) through DNA methylome modifications. Here, we provide evidence that female fish (Oryzias melastigma) exposed to hypoxia exhibit reproductive impairments (follicle atresia and retarded oocyte development), leading to a drastic reduction in hatching success in the F2 generation of the transgenerational group, although they have never been exposed to hypoxia. Further analyses show that the observed transgenerational impairments in ovarian functions are related to changes in the DNA methylation and expression pattern of two gene clusters that are closely associated with stress-induced cell cycle arrest and cell apoptosis. The observed epigenetic and transgenerational alterations suggest that hypoxia may pose a significant threat to the sustainability of natural fish populations.
Hypoxia Causes Transgenerational Impairment of Ovarian Development and Hatching Success in Fish.pdf
Keng Po Lai, Jing Woei Li, Simon Yuan Wang, Miles Teng Wan, Ting Fung Chan, Wing Yee Lui, Doris Wai-Ting Au, Rudolf Shiu-Sun Wu, and Richard Yuen-Chong Kong. 2/2018. “Transcriptomic analysis reveals transgenerational effect of hypoxia on the neural control of testicular functions.” Aquatic Toxicology, 195, Pp. 41-48. Publisher's VersionAbstract
There are over 400 hypoxic zones in the ocean worldwide. Both laboratory and field studies have shown that hypoxia causes endocrine disruption and reproductive impairments in vertebrates. More importantly, our recent study discovered that parental (F0) hypoxia exposure resulted in the transgenerational impairment of sperm quality in the F2 generation through the epigenetic regulation of germ cells. In the present study, we aim to test the hypothesis that the brain, as the major regulator of the brain-pituitary-gonad (BPG) axis, is also involved in the observed transgenerational effect. Using comparative transcriptomic analysis on brain tissues of marine medaka Oryzias melastigma, 45 common differentially expressed genes caused by parental hypoxia exposure were found in the hypoxic group of the F0 and F2 generations, and the transgenerational groups of the F2 generation. The bioinformatic analysis on this deregulated gene cluster further highlighted the possible involvement of the brain in the transgenerational effect of hypoxia on testicular structure, including abnormal morphologies of the epididymis and the seminal vesicle, and degeneration of the seminiferous tubule. This finding is concordant to the result of hematoxylin and eosin staining, which showed the reduction of testicular lobular diameter in the F0 and F2 generations. Our study demonstrated for the first time the involvement of the brain in the transgenerational effect of hypoxia.
Anna Chung-Kwan Tse, Jing-Woei Li, Simon Yuan Wang, Ting-Fung Chan, Keng Po Lai, and Rudolf Shiu-Sun Wu. 11/30/2016. “Hypoxia alters testicular functions of marine medaka through microRNAs regulation.” Aquatic Toxicology, 180, Pp. 266-273. Publisher's VersionAbstract
Hypoxia is a global environmental concern and poses a significant threat to aquatic ecosystems, including the sustainability of natural fish populations. The deleterious effects of hypoxia on fish reproductive fitness, as mediated by disruption of sex hormones and gene expression along the Brain-Pituitary-Gonad axis, have been well documented. Recently, we further demonstrated that the observed disruption of steroidogenesis in the ovary of marine medaka Oryzias melastigma is mediated through microRNAs (miRNAs). More importantly, we reported the transgenerational epigenetic effect of hypoxia on the male reproductive impairment of marine medaka. This study attempts to elucidate the function of miRNAs and its potential role in the transgenerational effect of hypoxia in the male medaka testis, using small RNA sequencing. A total of 558 miRNAs were found in the testis, of which 9 were significant upregulated and 5 were downregulated by hypoxia. Bioinformatics analysis further revealed that among the 2885 genes targeted by the hypoxia-responsive miRNAs, many are closely related to stress response, cell cycle, epigenetic modification, sugar metabolism and cell motion. Furthermore, the integrated analysis of transcriptome data and the result of target gene prediction demonstrated 108 genes and 65 genes were concordantly upregulated and downregulated, respectively. In which, euchromatic histone-lysine N-methyltransferase 2, the epigenetic regulator of transgenerational reproductive impairment caused by hypoxia, is found to be targeted by miR-125-5p. The present findings not only reveal that miRNAs are crucial downstream mediators of hypoxic stress in fish male gonad, but also shed light on the underlying epigenetic mechanism for the reproductive impairments of hypoxia on male fish, including the observed transgenerational effects.
Keng Po Lai, Jing Woei Li, Anna Chung Kwan Tse, Angela Cheung, Simon Yuan Wang, Ting Fung Chan, Richard Yuen Chong Kong, and Rudolf Shiu Sun Wu. 8/31/2016. “Transcriptomic responses of marine medaka’s ovary to hypoxia.” Aquatic Toxicology, 177, Pp. 476-483. Publisher's VersionAbstract
Hypoxia, an endocrine disruptor, is pressing global problem affecting marine organisms in over 400 “Dead Zones” worldwide. There is growing evident demonstrated the disruptive effect of hypoxia on reproductive systems of marine fish through the impairments of steroidogenic gene expression, leading to the alteration of sex hormone production in gonads. But the detailed molecular mechanism underlying the responses of female reproductive systems to hypoxic stress remains largely unknown. In the present report, we used marine medaka Oryzias melastigma as a model, together with high-throughput transcriptome sequencing and bioinformatics analysis, aiming to determine the changes in transcriptional signature in the ovary of marine fish under hypoxic stress. Our result discovered over two hundred differential expressed genes in ovary in response to hypoxia. The bioinformatics analysis together with quantitative RT-PCR validation on the deregulated genes highlighted the dysregulations of a number of female reproductive functions including interruptions of ovarian follicle development, gonad development and steroid metabolic process. Additionally, we revealed that these deregulations are through the modulation of leukemia inhibitory factor (LIF), insulin-like growth factor 1 receptor (IGF1R) and follicle stimulating hormone (FSH). The result of this work complements previous studies and provides additional insights into the underlying molecular mechanism of hypoxia-induced impairment of female reproductive system.
Hypoxia causes epigenetic changes and transgenerational reproductive impairments
Simon Yuan Wang, Karen Lau, Keng-Po Lai, Jiang-Wen Zhang, Anna Chung-Kwan Tse, Jing-Woei Li, Yin Tong, Ting-Fung Chan, Chris Kong-Chu Wong, Jill Man-Ying Chiu, Doris Wai-Ting Au, Alice Sze-Tsai Wong, Richard Yuen-Chong Kong, and Rudolf Shiu-Sun Wu. 7/4/2016. “Hypoxia causes epigenetic changes and transgenerational reproductive impairments.” Nature Communications, 7. Publisher's VersionAbstract
Hypoxia is amongst the most widespread and pressing problems in aquatic environments. Here we demonstrate that fish (Oryzias melastigma) exposed to hypoxia show reproductive impairments (retarded gonad development, decrease in sperm count and sperm motility) in F1 and F2 generations despite these progenies (and their germ cells) having never been exposed to hypoxia. We further show that the observed transgenerational reproductive impairments are associated with a differential methylation pattern of specific genes in sperm of both F0 and F2 coupled with relevant transcriptomic and proteomic alterations, which may impair spermatogenesis. The discovered transgenerational and epigenetic effects suggest that hypoxia might pose a dramatic and long-lasting threat to the sustainability of fish populations. Because the genes regulating spermatogenesis and epigenetic modifications are highly conserved among vertebrates, these results may also shed light on the potential transgenerational effects of hypoxia on other vertebrates, including humans.
Hypoxia causes transgenerational impairments in reproduction of fish.pdf
Keng-Po Lai, Jing-Woei Li, Anna Chung-Kwan Tse, Simon Yuan Wang, Ting-Fung Chan, and Rudolf Shiu-Sun Wu. 3/2016. “Differential responses of female and male brains to hypoxia in the marine medaka Oryzias melastigma.” Aquatic Toxicology, 172, Pp. 36-43. Publisher's VersionAbstract
Hypoxia, an endocrine disruptor, affects synthesis and balance of sex steroid hormones, leading to reproductive impairment in both female and male fish. Cumulating reports demonstrated the alternation of hypothalamus-pituitary-gonad axis (HPG-axis) by hypoxia. However, the detail mechanism underlying how hypoxia may alter other brain functions remains largely unknown. In this report, we used marine medaka as a model and conducted a high-throughput RNA sequencing followed by bioinformatics analysis on hypoxia-exposed brain tissues, aiming to determine the change of transcriptional signature and to unravel the pathways that are induced by hypoxia. We found that hypoxia lead to dysregulation of brain functions (including synaptic transmission, axon guidance, potassium ion transport, neuron differentiation, and development of brain and pituitary gland), and also signaling pathways (e.g., gap junction, calcium signaling pathway, and GnRH signaling pathway). Our results further demonstrate gender-specific responses to hypoxia in female and male fish’s brains, which provides novel insights into the mechanism underlying the hypoxia induced sex specific brain functions impairments.
Keng Po Lai, Jing-Woei Li, Simon Yuan Wang, Jill Man-Ying Chiu, Anna Tse, Karen Lau, Si Lok, Doris Wai-Ting Au, William Ka-Fai Tse, Chris Kong-Chu Wong, Ting-Fung Chan, Richard Yuen-Chong Kong, and Rudolf Shiu-Sun Wu. 2/27/2015. “Tissue-specific transcriptome assemblies of the marine medaka Oryzias melastigma and comparative analysis with the freshwater medaka Oryzias latipes.” BMC Genomics, 16, 1, Pp. 135. Publisher's VersionAbstract


The marine medaka Oryzias melastigma has been demonstrated as a novel model for marine ecotoxicological studies. However, the lack of genome and transcriptome reference has largely restricted the use of O. melastigma in the assessment of in vivo molecular responses to environmental stresses and the analysis of biological toxicity in the marine environment. Although O. melastigma is believed to be phylogenetically closely related to Oryzias latipes, the divergence between these two species is still largely unknown. Using Illumina high-throughput RNA sequencing followed by de novo assembly and comprehensive gene annotation, we provided transcriptomic resources for the brain, liver, ovary and testis of O. melastigma. We also investigated the possible extent of divergence between O. melastigma and O. latipes at the transcriptome level.


More than 14,000 transcripts across brain, liver, ovary and testis in marine medaka were annotated, of which 5880 transcripts were orthologous between O. melastigma and O. latipes. Tissue-enriched genes were identified in O. melastigma, and Gene Ontology analysis demonstrated the functional specificity of the annotated genes in respective tissue. Lastly, the identification of marine medaka-enriched transcripts suggested the necessity of generating transcriptome dataset of O. melastigma.


Orthologous transcripts between O. melastigma and O. latipes, tissue-enriched genes and O. melastigma-enriched transcripts were identified. Genome-wide expression studies of marine medaka require an assembled transcriptome, and this sequencing effort has generated a valuable resource of coding DNA for a non-model species. This transcriptome resource will aid future studies assessing in vivo molecular responses to environmental stresses and those analyzing biological toxicity in the marine environment.