New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project.

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.

RNA polymerase II transcription with partially assembled TFIID complexes.

The recognition of core promoter sequences by the general transcription factor TFIID is the first step in the process of RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Inducible Taf7 knock out (KO) results in the formation of a Taf7-less TFIID complex, while Taf10 KO leads to serious defects within the TFIID assembly pathway. Either TAF7 or TAF10 depletions correlate with the detected TAF occupancy changes at promoters, and with the distinct phenotype severities observed in mouse embryonic stem cells or mouse embryos. Surprisingly however, under either Taf7 or Taf10 deletion conditions, TBP is still associated to the chromatin, and no major changes are observed in nascent Pol II transcription. Thus, partially assembled TFIID complexes can sustain Pol II transcription initiation, but cannot replace holo-TFIID over several cell divisions and/or development.

Intra-promoter switch of transcription initiation sites in proliferation signaling-dependent RNA metabolism.

Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5' terminal oligopyrimidine (5'TOP) sequence, dramatically increasing the range of genes potentially subjected to 5'TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K-Akt-mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.

The miR-430 locus with extreme promoter density forms a transcription body during the minor wave of zygotic genome activation.

In anamniote embryos, the major wave of zygotic genome activation starts during the mid-blastula transition. However, some genes escape global genome repression, are activated substantially earlier, and contribute to the minor wave of genome activation. The mechanisms underlying the minor wave of genome activation are little understood. We explored the genomic organization and cis-regulatory mechanisms of a transcription body, in which the minor wave of genome activation is first detected in zebrafish. We identified the miR-430 cluster as having excessive copy number and the highest density of Pol-II-transcribed promoters in the genome, and this is required for forming the transcription body. However, this transcription body is not essential for, nor does it encompasse, minor wave transcription globally. Instead, distinct minor-wave-specific promoter architecture suggests that promoter-autonomous mechanisms regulate the minor wave of genome activation. The minor-wave-specific features also suggest distinct transcription initiation mechanisms between the minor and major waves of genome activation.

CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals.

Cap methyltransferases (CMTrs) O methylate the 2' position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. Here, we show that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation.

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

Germ cell differentiation requires Tdrd7-dependent chromatin and transcriptome reprogramming marked by germ plasm relocalization.

In many animal models, primordial germ cell (PGC) development depends on maternally deposited germ plasm, which prevents somatic cell fate. Here, we show that PGCs respond to regulatory information from the germ plasm in two distinct phases using two distinct mechanisms in zebrafish. We demonstrate that PGCs commence zygotic genome activation together with the somatic blastocysts with no demonstrable differences in transcriptional and chromatin opening. Unexpectedly, both PGC and somatic blastocysts activate germ-cell-specific genes, which are only stabilized in PGCs by cytoplasmic germ plasm determinants. Disaggregated perinuclear relocalization of germ plasm during PGC migration is regulated by the germ plasm determinant Tdrd7 and is coupled to dramatic divergence between PGC and somatic transcriptomes. This transcriptional divergence relies on PGC-specific cis-regulatory elements characterized by promoter-proximal distribution. We show that Tdrd7-dependent reconfiguration of chromatin accessibility is required for elaboration of PGC fate but not for PGC migration.

Visualization of Transcriptional Activity in Early Zebrafish Primordial Germ Cells.

Here, we describe a fast and straightforward methodology to in vivo detect transcriptional activity in the early zebrafish germ line. We report how fluorescently labeled morpholinos, targeted to nascent early transcripts, can be used to track the onset of transcriptional events during early embryogenesis. This method could be applied to any tagged cell line in a developing early zebrafish embryo as long as the gene of interest is expressed at high enough level for morpholino detection and is expressed at the first and main wave of genome activation, for which the protocol has been verified. The protocol, in combination with genetic manipulation, allows studies of mechanisms driving zygotic genome activation (ZGA) in individual cells. The reported procedures apply to a broad range of purposes for zebrafish embryo manipulation in view of imaging nuclear molecules in specific cell types.

Ancestrally Duplicated Conserved Noncoding Element Suggests Dual Regulatory Roles of HOTAIR in cis and trans.

HOTAIR was proposed to regulate either HoxD cluster genes in trans or HoxC cluster genes in cis, a mechanism that remains unclear. We have identified a 32-nucleotide conserved noncoding element (CNE) as HOTAIR ancient sequence that likely originated at the root of vertebrate. The second round of whole-genome duplication resulted in one copy of the CNE within HOTAIR and another copy embedded in noncoding transcript of HOXD11. Paralogous CNEs underwent compensatory mutations, exhibit sequence complementarity with respect to transcripts directionality, and have high affinity in vitro. The HOTAIR CNE resembled a poised enhancer in stem cells and an active enhancer in HOTAIR-expressing cells. HOTAIR expression is positively correlated with HOXC11 in cis and negatively correlated with HOXD11 in trans. We propose a dual modality of HOTAIR regulation where transcription of HOTAIR and its embedded enhancer regulates HOXC11 in cis and sequence complementarity between paralogous CNEs suggests HOXD11 regulation in trans.

Dual-initiation promoters with intertwined canonical and TCT/TOP transcription start sites diversify transcript processing.

Variations in transcription start site (TSS) selection reflect diversity of preinitiation complexes and can impact on post-transcriptional RNA fates. Most metazoan polymerase II-transcribed genes carry canonical initiation with pyrimidine/purine (YR) dinucleotide, while translation machinery-associated genes carry polypyrimidine initiator (5'-TOP or TCT). By addressing the developmental regulation of TSS selection in zebrafish we uncovered a class of dual-initiation promoters in thousands of genes, including snoRNA host genes. 5'-TOP/TCT initiation is intertwined with canonical initiation and used divergently in hundreds of dual-initiation promoters during maternal to zygotic transition. Dual-initiation in snoRNA host genes selectively generates host and snoRNA with often different spatio-temporal expression. Dual-initiation promoters are pervasive in human and fruit fly, reflecting evolutionary conservation. We propose that dual-initiation on shared promoters represents a composite promoter architecture, which can function both coordinately and divergently to diversify RNAs.

A cell cycle-coordinated Polymerase II transcription compartment encompasses gene expression before global genome activation.

Most metazoan embryos commence development with rapid, transcriptionally silent cell divisions, with genome activation delayed until the mid-blastula transition (MBT). However, a set of genes escapes global repression and gets activated before MBT. Here we describe the formation and the spatio-temporal dynamics of a pair of distinct transcription compartments, which encompasses the earliest gene expression in zebrafish. 4D imaging of pri-miR430 and zinc-finger-gene activities by a novel, native transcription imaging approach reveals transcriptional sharing of nuclear compartments, which are regulated by homologous chromosome organisation. These compartments carry the majority of nascent-RNAs and active Polymerase II, are chromatin-depleted and represent the main sites of detectable transcription before MBT. Transcription occurs during the S-phase of increasingly permissive cleavage cycles. It is proposed, that the transcription compartment is part of the regulatory architecture of embryonic nuclei and offers a transcriptionally competent environment to facilitate early escape from repression before global genome activation.

Estrogen sensitive liver transgenic zebrafish (Danio rerio) line (Tg(vtg1:mCherry)) suitable for the direct detection of estrogenicity in environmental samples.

Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations. The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites. A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos. Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen.

Publisher Correction: Enhancers active in dopamine neurons are a primary link between genetic variation and neuropsychiatric disease.

In the version of this article initially published, the legends for Supplementary Figs. 4-8 and 10-14 contained errors. The Supplementary Figure legends have been corrected in the HTML and PDF versions of the article.

Enhancers active in dopamine neurons are a primary link between genetic variation and neuropsychiatric disease.

Enhancers function as DNA logic gates and may control specialized functions of billions of neurons. Here we show a tailored program of noncoding genome elements active in situ in physiologically distinct dopamine neurons of the human brain. We found 71,022 transcribed noncoding elements, many of which were consistent with active enhancers and with regulatory mechanisms in zebrafish and mouse brains. Genetic variants associated with schizophrenia, addiction, and Parkinson's disease were enriched in these elements. Expression quantitative trait locus analysis revealed that Parkinson's disease-associated variants on chromosome 17q21 cis-regulate the expression of an enhancer RNA in dopamine neurons. This study shows that enhancers in dopamine neurons link genetic variation to neuropsychiatric traits.

The Tetraodon nigroviridis reference transcriptome: developmental transition, length retention and microsynteny of long non-coding RNAs in a compact vertebrate genome.

Pufferfish such as fugu and tetraodon carry the smallest genomes among all vertebrates and are ideal for studying genome evolution. However, comparative genomics using these species is hindered by the poor annotation of their genomes. We performed RNA sequencing during key stages of maternal to zygotic transition of Tetraodon nigroviridis and report its first developmental transcriptome. We assembled 61,033 transcripts (23,837 loci) representing 80% of the annotated gene models and 3816 novel coding transcripts from 2667 loci. We demonstrate the similarities of gene expression profiles between pufferfish and zebrafish during maternal to zygotic transition and annotated 1120 long non-coding RNAs (lncRNAs) many of which differentially expressed during development. The promoters for 60% of the assembled transcripts result validated by CAGE-seq. Despite the extreme compaction of the tetraodon genome and the dramatic loss of transposons, the length of lncRNA exons remain comparable to that of other vertebrates and a small set of lncRNAs appears enriched for transposable elements suggesting a selective pressure acting on lncRNAs length and composition. Finally, a set of lncRNAs are microsyntenic between teleost and vertebrates, which indicates potential regulatory interactions between lncRNAs and their flanking coding genes. Our work provides a fundamental molecular resource for vertebrate comparative genomics and embryogenesis studies.

Testing of Cis-Regulatory Elements by Targeted Transgene Integration in Zebrafish Using PhiC31 Integrase.

Herein we present several strategies for testing the function of cis-regulatory elements using the PhiC31 integrase system. Firstly, we present two different strategies to analyze the activity of candidate enhancer elements. Targeted integration of candidate enhancers into the same genomic location circumvents the variability-associated random integration and position effects. This method is suitable for testing of candidate enhancers identified through computational or other analyses a priori. Secondly, we present methodology for targeted integration of BACs into the same genomic location(s). By using additional reporters integrated into a BAC, this enables experimental testing whether cis-regulatory elements are functional in the sequence inserted in the BAC.

Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is a dominantly inherited familial cancer syndrome characterised by the development of benign skin fibrofolliculomas, multiple lung and kidney cysts, spontaneous pneumothorax and susceptibility to renal cell carcinoma. BHD is caused by mutations in the gene encoding Folliculin (FLCN). Little is known about what FLCN does in a healthy individual and how best to treat those with BHD. As a first approach to developing a vertebrate model for BHD we aimed to identify the temporal and spatial expression of flcn transcripts in the developing zebrafish embryo. To gain insights into the function of flcn in a whole organism system we generated a loss of function model of flcn by the use of morpholino knockdown in zebrafish. RESULTS: flcn is expressed broadly and upregulated in the fin bud, somites, eye and proliferative regions of the brain of the Long-pec stage zebrafish embryos. Together with knockdown phenotypes, expression analysis suggest involvement of flcn in zebrafish embryonic brain development. We have utilised the zFucci system, an in vivo, whole organism cell cycle assay to study the potential role of flcn in brain development. We found that at the 18 somite stage there was a significant drop in cells in the S-M phase of the cell cycle in flcn morpholino injected embryos with a corresponding increase of cells in the G1 phase. This was particularly evident in the brain, retina and somites of the embryo. Timelapse analysis of the head region of flcn morpholino injected and mismatch control embryos shows the temporal dynamics of cell cycle misregulation during development. CONCLUSIONS: In conclusion we show that zebrafish flcn is expressed in a non-uniform manner and is likely required for the maintenance of correct cell cycle regulation during embryonic development. We demonstrate the utilisation of the zFucci system in testing the role of flcn in cell proliferation and suggest a function for flcn in regulating cell proliferation in vertebrate embryonic brain development.

Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs.

MicroRNAs (miRNAs) play a major role in the post-transcriptional regulation of target genes, especially in development and differentiation. Our understanding about the transcriptional regulation of miRNA genes is limited by inadequate annotation of primary miRNA (pri-miRNA) transcripts. Here, we used CAGE-seq and RNA-seq to provide genome-wide identification of the pri-miRNA core promoter repertoire and its dynamic usage during zebrafish embryogenesis. We assigned pri-miRNA promoters to 152 precursor-miRNAs (pre-miRNAs), the majority of which were supported by promoter associated post-translational histone modifications (H3K4me3, H2A.Z) and RNA polymerase II (RNAPII) occupancy. We validated seven miR-9 pri-miRNAs by in situ hybridization and showed similar expression patterns as mature miR-9. In addition, processing of an alternative intronic promoter of miR-9-5 was validated by 5' RACE PCR. Developmental profiling revealed a subset of pri-miRNAs that are maternally inherited. Moreover, we show that promoter-associated H3K4me3, H2A.Z and RNAPII marks are not only present at pri-miRNA promoters but are also specifically enriched at pre-miRNAs, suggesting chromatin level regulation of pre-miRNAs. Furthermore, we demonstrated that CAGE-seq also detects 3'-end processing of pre-miRNAs on Drosha cleavage site that correlates with miRNA-offset RNAs (moRNAs) production and provides a new tool for detecting Drosha processing events and predicting pre-miRNA processing by a genome-wide assay.

Description of embryonic development of spotted green pufferfish (Tetraodon nigroviridis).

Pufferfish species of the Tetraodontidae family carry the smallest genomes among vertebrates. Their compressed genomes are thought to be enriched for functional DNA compared to larger vertebrate genomes, and they are important models for comparative genomics. The significance of pufferfish as model organisms in comparative genomics is due to the availability of two sequenced genomes, that of spotted green pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes). However, there is only a very limited utilization of pufferfish as an experimental model organism, due to the lack of established husbandry and developmental genetics protocols. In this study, we provide the first description of the normal embryonic development of Tetraodon nigroviridis. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored by brightfield microscopy at constant temperature. Tetraodon development was divided into distinct stages based on diagnostic morphological features, which were adopted from published literature on normal development of other fish species like medaka (Oryzias latipes), zebrafish (Danio rerio), and fugu. Tetraodon embryos show more similar morphologies to medaka than to zebrafish, reflecting its phylogenetic position. The early developmental stage series described in this study forms the foundation for the utilization of tetraodon as an experimental model organism for comparative developmental studies.

Two independent transcription initiation codes overlap on vertebrate core promoters.

A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs and recruits general transcription factors to initiate transcription. The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle. Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition. This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression, and determined the positions of H3K4me3-marked promoter-associated nucleosomes. Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (+1) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts.