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Celebrating, educating and mobilizing the global community to achieve equality for women and girls in science is recognized worldwide each February 11 on the International Day of Women and Girls in Science. This day serves as a rousing call for people of all genders to gain unfettered access to opportunities to become successful, engaged and productive scientists. One strategy to achieve this is to develop policies to ensure that all people can pursue science education, training and careers, unimpeded by their sex, gender and gender identity. Another strategy is to assure representation of women and non-binary people at every level of the scientific workforce. Collectively, these strategies and other important efforts are shifting the global mindset to one where women and girls are recognized as vital, capable and innovative contributors to disciplines that have historically been dominated by men. These male-dominated disciplines have evolved cultural norms rooted in masculine stereotypes and the next challenge is to continue this shift away from a scientific culture in which hypercompetitive, individualistic, solo artists work alone in single-minded pursuit of a breakthrough. Instead, the reality is that contemporary biomedical scientific research is a cosmopolitan culture; one where breakthroughs depend on collaboration, where data sharing is the norm and where all are encouraged to contribute their best ideas to help solve science's most vexing and exciting problems.

We hypothesized that the highly controlled pattern of gene expression that is essential for liver regeneration is encoded by an epigenetic code set in quiescent hepatocytes. Here we report that epigenetic and transcriptomic profiling of quiescent and regenerating mouse livers define chromatin states that dictate gene expression and transposon repression. We integrate ATACseq and DNA methylation profiling with ChIPseq for the histone marks H3K4me3, H3K27me3 and H3K9me3 and the histone variant H2AZ to identify 6 chromatin states with distinct functional characteristics. We show that genes involved in proliferation reside in active states, but are marked with H3K27me3 and silenced in quiescent livers. We find that during regeneration, H3K27me3 is depleted from their promoters, facilitating their dynamic expression. These findings demonstrate that hepatic chromatin states in quiescent livers predict gene expression and that pro-regenerative genes are maintained in active chromatin states, but are restrained by H3K27me3, permitting a rapid and synchronized response during regeneration. Few studies have provided functional analysis of the epigenetic landscape in the regenerating liver. Here the authors define chromatin states in the quiescent vs. regenerating mouse liver through integration of genome wide profiles of DNA methylation, histone modifications, and chromatin accessibility, identifying H3K27me3 as an epigenetic mark conferring regenerative potential.

Background Epigenetic regulatory mechanisms are divergent across the animal kingdom, yet these mechanisms are not well studied in non-model organisms. Unique features of cephalopods make them attractive for investigating behavioral, sensory, developmental, and regenerative processes, and recent studies have elucidated novel features of genome organization and gene and transposon regulation in these animals. However, it is not known how epigenetics regulates these interesting cephalopod features. We combined bioinformatic and molecular analysis of Octopus bimaculoides to investigate the presence and pattern of DNA methylation and examined the presence of DNA methylation and 3 histone post-translational modifications across tissues of three cephalopod species. Results We report a dynamic expression profile of the genes encoding conserved epigenetic regulators, including DNA methylation maintenance factors in octopus tissues. Levels of 5-methyl-cytosine in multiple tissues of octopus, squid, and bobtail squid were lower compared to vertebrates. Whole genome bisulfite sequencing of two regions of the brain and reduced representation bisulfite sequencing from a hatchling of O. bimaculoides revealed that less than 10% of CpGs are methylated in all samples, with a distinct pattern of 5-methyl-cytosine genome distribution characterized by enrichment in the bodies of a subset of 14,000 genes and absence from transposons. Hypermethylated genes have distinct functions and, strikingly, many showed similar expression levels across tissues while hypomethylated genes were silenced or expressed at low levels. Histone marks H3K27me3, H3K9me3, and H3K4me3 were detected at different levels across tissues of all species. Conclusions Our results show that the DNA methylation and histone modification epigenetic machinery is conserved in cephalopods, and that, in octopus, 5-methyl-cytosine does not decorate transposable elements, but is enriched on the gene bodies of highly expressed genes and could cooperate with the histone code to regulate tissue-specific gene expression.

Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.

Fatty liver disease (FLD) is characterized by lipid accumulation in hepatocytes and is accompanied by secretory pathway dysfunction, resulting in induction of the unfolded protein response (UPR). Activating transcription factor 6 (ATF6), one of three main UPR sensors, functions to both promote FLD during acute stress and reduce FLD during chronic stress. There is little mechanistic understanding of how ATF6, or any other UPR factor, regulates hepatic lipid metabolism to cause disease. We addressed this using zebrafish genetics and biochemical analyses and demonstrate that Atf6 is necessary and sufficient for FLD. atf6 transcription is significantly upregulated in the liver of zebrafish with alcoholic FLD and morpholino-mediated atf6 depletion significantly reduced steatosis incidence caused by alcohol. Moreover, overexpression of active, nuclear Atf6 (nAtf6) in hepatocytes caused FLD in the absence of stress. mRNA-Seq and qPCR analyses of livers from five day old nAtf6 transgenic larvae revealed upregulation of genes promoting glyceroneogenesis and fatty acid elongation, including fatty acid synthase (fasn), and nAtf6 overexpression in both zebrafish larvae and human hepatoma cells increased the incorporation of 14C-acetate into lipids. Srebp transcription factors are key regulators of lipogenic enzymes, but reducing Srebp activation by scap morpholino injection neither prevented FLD in nAtf6 transgenics nor synergized with atf6 knockdown to reduce alcohol-induced FLD. In contrast, fasn morpholino injection reduced FLD in nAtf6 transgenic larvae and synergistically interacted with atf6 to reduce alcoholic FLD. Thus, our data demonstrate that Atf6 is required for alcoholic FLD and epistatically interacts with fasn to cause this disease, suggesting triglyceride biogenesis as the mechanism of UPR induced FLD.

Toxicology – the study of how chemicals interact with biological systems – has clear relevance to human health and disease. Persistent exposure to natural and synthetic chemicals is an unavoidable part of living on our planet; yet, we understand very little about the effects of exposure to the vast majority of chemicals. While epidemiological studies can provide strong statistical inference linking chemical exposure to disease, research in model systems is essential to elucidate the mechanisms of action and to predict outcomes. Most research in toxicology utilizes a handful of mammalian models that represent a few distinct branches of the evolutionary tree. This narrow focus constrains the understanding of chemical-induced disease processes and systems that have evolved in response to exposures. We advocate for casting a wider net in environmental toxicology research to utilize diverse model systems, including zebrafish, and perform more mechanistic studies of cellular responses to chemical exposures to shift the perception of toxicology as an applied science to that of a basic science. This more-inclusive perspective will enrich the field and should remain central to research on chemical-induced disease.

The rapid increase in fatty liver disease (FLD) incidence is attributed largely to genetic and lifestyle factors; however, environmental toxicants are a frequently overlooked factor that can modify the effects of more common causes of FLD. Chronic exposure to inorganic arsenic (iAs) is associated with liver disease in humans and animal models, but neither the mechanism of action nor the combinatorial interaction with other disease-causing factors has been fully investigated. Here, we examined the contribution of iAs to FLD using zebrafish and tested the interaction with ethanol to cause alcoholic liver disease (ALD). We report that zebrafish exposed to iAs throughout development developed specific phenotypes beginning at 4 days post-fertilization (dpf), including the development of FLD in over 50% of larvae by 5 dpf. Comparative transcriptomic analysis of livers from larvae exposed to either iAs or ethanol revealed the oxidative stress response and the unfolded protein response (UPR) caused by endoplasmic reticulum (ER) stress as common pathways in both these models of FLD, suggesting that they target similar cellular processes. This was confirmed by our finding that arsenic is synthetically lethal with both ethanol and a well-characterized ER-stress-inducing agent (tunicamycin), suggesting that these exposures work together through UPR activation to cause iAs toxicity. Most significantly, combined exposure to sub-toxic concentrations of iAs and ethanol potentiated the expression of UPR-associated genes, cooperated to induce FLD, reduced the expression of as3mt, which encodes an arsenic-metabolizing enzyme, and significantly increased the concentration of iAs in the liver. This demonstrates that iAs exposure is sufficient to cause FLD and that low doses of iAs can potentiate the effects of ethanol to cause liver disease. This article has an associated First Person interview with the first author of the paper.

Activation of transposable elements (TEs) can cause cellular damage. Cytoplasmic nucleic acid sensing pathways evolved to detect pathogens, but can also serve to cull cells with inappropriate TE activation as TEs can be viral mimetics. Epigenetic silencing of TEs is mediated in part by DNA methylation, but it is not clear if TE activation or the immune system contribute to the cellular damage caused by loss of DNA methylation. Here, we provide mechanistic insight into the observation of an activated interferon response in the liver of zebrafish larvae with deletion in critical components of the DNA methylation machinery, and . We focus on dissecting the relationship between DNA methylation, TE activation and induction of an immune response through cytoplasmic DNA and double stranded RNA sensing pathways and identify as a mediator of cell death in the liver of these mutants. Integrated RNAseq and methylome analysis identified LTR transposons as the most upregulated in these mutants and also the most methylated in control larvae, indicating a direct role of DNA methylation in suppressing this TE subclass. RNAseq analysis from these same samples revealed expression signatures of a type-I interferon response and of activation, mimicking the pattern of gene expression in virally infected cells. CRISPR/Cas9 mediated depletion of the cellular antiviral sensors and reduced expression of interferon response genes and depletion dramatically reduced cell death in mutant livers. This suggests that the antiviral response induced by DNA hypomethylation and TE activation in the liver is mediated by the signaling pathways activated by both cytoplasmic double stranded RNA and DNA and that mediates cell death as a potential mechanism to eliminate these damaged cells.

Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 μmol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes.

We have generated several hundred lines of zebrafish (Danio rerio), each heterozygous for a recessive embryonic lethal mutation. Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now.