Nadine L. Vastenhouw

Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis

Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies.

Chromatin Signature of Embryonic Pluripotency Is Established during Genome Activation

After fertilization the embryonic genome is inactive until transcription is initiated during the maternal–zygotic transition. This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing lysine trimethylation modifications before and after the maternal–zygotic transition in zebrafish. Histone H3 lysine 27 trimethylation (H3K27me3), which is repressive, and H3K4me3, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by H3K4me3, including many inactive developmental regulatory genes that were also marked by H3K27me3. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed. Furthermore, we found many inactive genes that were uniquely marked by H3K4me3. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published data sets revealed similar monovalent domains in embryonic stem cells. Moreover, H3K4me3 marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA polymerase II, as indicated by the analysis of an inducible transgene. These results indicate that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during the maternal–zygotic transition.

Gene expression: long-term gene silencing by RNAi.

Small RNA molecules participate in a variety of activities in the cell: in a process known as RNA interference (RNAi), double-stranded RNA triggers the degradation of messenger RNA that has a matching sequence; the small RNA intermediates of this process can also modify gene expression in the nucleus. Here we show that a single episode of RNAi in the nematode Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger. Our findings may prove useful in the ongoing development of RNAi to treat disease.

A Genome-Wide Screen Identifies 27 Genes Involved in Transposon Silencing in C. elegans

Transposon jumps are a major cause of genome instability. In the C. elegans strain Bristol N2, transposons are active in somatic cells, but they are silenced in the germline, presumably to protect the germline from mutations. Interestingly, the transposon-silencing mechanism shares factors with the RNAi machinery. To better understand the mechanism of transposon silencing, we performed a genome-wide RNAi screen for genes that, when silenced, cause transposition of Tc1 in the C. elegans germline. We identified 27 such genes, among which are mut-16, a mutator that was previously found but not identified at the molecular level, ppw-2, a member of the argonaute family, and several factors that indicate a role for chromatin structure in the regulation of transposition. Some of the newly identified genes are also required for cosuppression and therefore represent the shared components of the two pathways. Since most of the newly identified genes have clear homologs in other species, and since transposons are found from protozoa to human, it seems likely that they also protect other genomes against transposon activity in the germline.

Bivalent histone modifications in early embryogenesis

Histone modifications influence the interactions of transcriptional regulators with chromatin. Studies in embryos and embryonic stem (ES) cells have uncovered histone modification patterns that are diagnostic for different cell types and developmental stages. For example, bivalent domains consisting of regions of H3 lysine 27 trimethylation (H3K27me3) and H3 lysine 4 trimethylation (H3K4me3) mark lineage control genes in ES cells and zebrafish blastomeres. Such bivalent domains have garnered attention because the H3K27me3 mark might help repress lineage-regulatory genes during pluripotency while the H3K4me3 mark could poise genes for activation upon differentiation. Despite the prominence of the bivalent domain concept, studies in other model organisms have questioned its universal nature, and the function of bivalent domains has remained unclear. Histone marks are also associated with developmental regulatory genes in sperm. These observations have raised the possibility that specific histone modification patterns might persist from parent to offspring, but it is unclear whether histone marks are inherited or formed de novo. Here, we review the potential roles of H3K4me3 and H3K27me3 marks in embryos and ES cells and discuss how histone marks might be established, maintained and resolved during embryonic development.

Canonical nucleosome organization at promoters forms during genome activation

The organization of nucleosomes influences transcriptional activity by controlling accessibility of DNA binding proteins to the genome. Genome-wide nucleosome binding profiles have identified a canonical nucleosome organization at gene promoters, where arrays of well-positioned nucleosomes emanate from nucleosome-depleted regions. The mechanisms of formation and the function of canonical promoter nucleosome organization remain unclear. Here we analyze the genome-wide location of nucleosomes during zebrafish embryogenesis and show that well-positioned nucleosome arrays appear on thousands of promoters during the activation of the zygotic genome. The formation of canonical promoter nucleosome organization is independent of DNA sequence preference, transcriptional elongation, and robust RNA polymerase II (Pol II) binding. Instead, canonical promoter nucleosome organization correlates with the presence of histone H3 lysine 4 trimethylation (H3K4me3) and affects future transcriptional activation. These findings reveal that genome activation is central to the organization of nucleosome arrays during early embryogenesis.

B-myb rescues ras-induced premature senescence, which requires its transactivation domain

B-myb, a ubiquitously expressed member of the myb gene family, is highly regulated throughout the cell cycle and appears to be required for cell cycle progression. In contrast to its relatives A-myb, c-myb, and v-myb, no transforming activity of B-myb has been reported thus far. We report here that B-myb can rescue senescence induced by an activated ras oncogene in rodent cells in vitro. We show that transformation by B-Myb involves its ability to activate transcription. Similar to other oncogenic transcription factors, such as c-Myc and E2F, we show that B-Myb also has repression activity. We demonstrate that the C-terminus of B-Myb can function as a repressor of transcription, that B-Myb interacts with the repressor molecules BS69 and N-CoR and that the repression function, like the transactivation domain, contributes to B-myb transformation.

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo. DOI: http://dx.doi.org/10.7554/eLife.23326.001

The timing of zygotic genome activation.

After fertilization, the embryonic genome is inactive until transcription is initiated during the maternal-to-zygotic transition. How the onset of transcription is regulated in a precisely timed manner, however, is a long standing question in biology. Several mechanisms have been shown to contribute to the temporal regulation of genome activation but none of them can fully explain the general absence of transcription as well the gene specific onset that follows. Here we review the work that has been done toward elucidating the mechanisms underlying the temporal regulation of transcription in embryos.

A tunable refractive index matching medium for live imaging cells, tissues and model organisms

In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids. DOI: http://dx.doi.org/10.7554/eLife.27240.001