Eivind Valen

An atlas of active enhancers across human cell types and tissues

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.

JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update

JASPAR is a popular open-access database for matrix models describing DNA-binding preferences for transcription factors and other DNA patterns. With its third major release, JASPAR has been expanded and equipped with additional functions aimed at both casual and power users. The heart of the JASPAR database—the JASPAR CORE sub-database—has increased by 12% in size, and three new specialized sub-databases have been added. New functions include clustering of matrix models by similarity, generation of random matrices by sampling from selected sets of existing models and a language-independent Web Service applications programming interface for matrix retrieval. JASPAR is available at http://jaspar.genereg.net.

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.

CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing

Major advances in genome editing have recently been made possible with the development of the TALEN and CRISPR/Cas9 methods. The speed and ease of implementing these technologies has led to an explosion of mutant and transgenic organisms. A rate-limiting step in efficiently applying TALEN and CRISPR/Cas9 methods is the selection and design of targeting constructs. We have developed an online tool, CHOPCHOP (https://chopchop.rc.fas.harvard.edu), to expedite the design process. CHOPCHOP accepts a wide range of inputs (gene identifiers, genomic regions or pasted sequences) and provides an array of advanced options for target selection. It uses efficient sequence alignment algorithms to minimize search times, and rigorously predicts off-target binding of single-guide RNAs (sgRNAs) and TALENs. Each query produces an interactive visualization of the gene with candidate target sites displayed at their genomic positions and color-coded according to quality scores. In addition, for each possible target site, restriction sites and primer candidates are visualized, facilitating a streamlined pipeline of mutant generation and validation. The ease-of-use and speed of CHOPCHOP make it a valuable tool for genome engineering.

Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5′ adenine were improved by rescuing 5′ end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

Toddler: An Embryonic Signal That Promotes Cell Movement via Apelin Receptors

Introduction Embryogenesis is thought to be directed by a small number of signaling pathways with most if not all embryonic signals having been identified. However, the molecular control of some embryonic processes is still poorly understood. For example, it is unclear how cell migration is regulated during gastrulation, when mesodermal and endodermal germ layers form. The goal of our study was to identify and characterize previously unrecognized signals that regulate embryogenesis. Toddler promotes gastrulation movements via Apelin receptor signaling. Toddler is an essential, short, conserved embryonic signal that promotes cell migration during zebrafish gastrulation. The internalization movement highlighted by the colored cell tracks requires Toddler signaling. Toddler signals via the G-protein–coupled APJ/Apelin receptor and may be one of several uncharacterized embryonic signals. Methods To identify uncharacterized signaling molecules, we mined zebrafish genomic data sets for previously non-annotated translated open reading frames (ORFs). One such ORF encoded a putative signaling protein that we call Toddler (also known as Apela/Elabela/Ende). We analyzed expression, production, and secretion of Toddler using RNA in situ hybridization, mass spectrometry, and Toddler-GFP fusion proteins, respectively. We used transcription activator-like effector (TALE) nucleases to generate frame-shift mutations in the toddler gene. To complement loss-of-function analyses with gain-of-function studies, Toddler was misexpressed through mRNA or peptide injection. We characterized phenotypes using marker gene expression analysis and in vivo imaging, using confocal and lightsheet microscopy. Toddler mutants were rescued thorugh global or localized toddler production. The relationship between Toddler and APJ/Apelin receptors was studied through genetic interaction and receptor internalization experiments. Results We identified several hundred non-annotated candidate proteins, including more than 20 putative signaling proteins. We focused on the functional importance of the short, conserved, and secreted peptide Toddler. Loss or overproduction of Toddler reduced cell movements during zebrafish gastrulation; mesodermal and endodermal cells were slow to internalize and migrate. Both the local and ubiquitous expression of Toddler were able to rescue gastrulation movements in toddler mutants, suggesting that Toddler acts as a motogen, a signal that promotes cell migration. Toddler activates G-protein–coupled APJ/Apelin receptor signaling, as evidenced by Toddler-induced internalization of APJ/Apelin receptors and rescue of toddler mutants through expression of the known receptor agonist Apelin. Discussion These findings indicate that Toddler promotes cell movement during zebrafish gastrulation by activation of APJ/Apelin receptor signaling. Toddler does not seem to act as a chemo-attractant or -repellent, but rather as a global signal that promotes the movement of mesendodermal cells. Both loss and overproduction of Toddler reduce cell movement, revealing that Toddler levels need to be tightly regulated during gastrulation. The discovery of Toddler helps explain previous genetic studies that found a broader requirement for APJ/Apelin receptors than for Apelin. We propose that in these cases, Toddler—not Apelin—activates APJ/Apelin receptor signaling. Our genomics analysis identifying a large number of candidate proteins that function during embryogenesis suggests the existence of other previously uncharacterized embryonic signals. Applying similar genomic approaches to adult tissues might identify additional signals that regulate physiological and behavioral processes. It has been assumed that most, if not all, signals regulating early development have been identified. Contrary to this expectation, we identified 28 candidate signaling proteins expressed during zebrafish embryogenesis, including Toddler, a short, conserved, and secreted peptide. Both absence and overproduction of Toddler reduce the movement of mesendodermal cells during zebrafish gastrulation. Local and ubiquitous production of Toddler promote cell movement, suggesting that Toddler is neither an attractant nor a repellent but acts globally as a motogen. Toddler drives internalization of G protein–coupled APJ/Apelin receptors, and activation of APJ/Apelin signaling rescues toddler mutants. These results indicate that Toddler is an activator of APJ/Apelin receptor signaling, promotes gastrulation movements, and might be the first in a series of uncharacterized developmental signals. A conserved signal is identified that activates G protein–coupled receptors to promote zebrafish gastrulation. Toddler Welcome It has been assumed that most, if not all, major signals that control vertebrate embryogenesis have been identified. Using genomics, Pauli et al. (10.1126/science.1248636, published online 9 January) have now identified several new candidate signals expressed during early zebrafish development. One of these signals, Toddler, is a short, conserved, and secreted peptide that promotes the movement of cells during zebrafish gastrulation. Toddler signals through G protein–coupled receptors to drive internalization of the Apelin receptor, and activation of Apelin signaling can rescue toddler mutants.

Polyadenylation site–induced decay of upstream transcripts enforces promoter directionality

Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3′ ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

Ribosome profiling reveals resemblance between long non-coding RNAs and 5′ leaders of coding RNAs

Large-scale genomics and computational approaches have identified thousands of putative long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of these RNAs is truly non-coding. Here, we combine ribosome profiling with a machine-learning approach to validate lncRNAs during zebrafish development in a high throughput manner. We find that dozens of proposed lncRNAs are protein-coding contaminants and that many lncRNAs have ribosome profiles that resemble the 5′ leaders of coding RNAs. Analysis of ribosome profiling data from embryonic stem cells reveals similar properties for mammalian lncRNAs. These results clarify the annotation of developmental lncRNAs and suggest a potential role for translation in lncRNA regulation. In addition, our computational pipeline and ribosome profiling data provide a powerful resource for the identification of translated open reading frames during zebrafish development.

CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering

In just 3 years CRISPR genome editing has transformed biology, and its popularity and potency continue to grow. New CRISPR effectors and rules for locating optimum targets continue to be reported, highlighting the need for computational CRISPR targeting tools to compile these rules and facilitate target selection and design. CHOPCHOP is one of the most widely used web tools for CRISPR- and TALEN-based genome editing. Its overarching principle is to provide an intuitive and powerful tool that can serve both novice and experienced users. In this major update we introduce tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases. We support a number of new features that improve the targeting power, usability and efficiency of CHOPCHOP. To increase targeting range and specificity we provide support for custom length sgRNAs, and we evaluate the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies. These and other new features, coupled with an updated interface for increased usability and support for a continually growing list of organisms, maintain CHOPCHOP as one of the leading tools for CRISPR genome editing. CHOPCHOP v2 can be found at http://chopchop.cbu.uib.no

PROMoter uPstream Transcripts share characteristics with mRNAs and are produced upstream of all three major types of mammalian promoters

PROMoter uPstream Transcripts (PROMPTs) were identified as a new class of human RNAs, which are heterologous in length and produced only upstream of the promoters of active protein-coding genes. Here, we show that PROMPTs carry 3′-adenosine tails and 5′-cap structures. However, unlike mRNAs, PROMPTs are largely nuclear and rapidly turned over by the RNA exosome. PROMPT-transcribing DNA is occupied by RNA polymerase II (RNAPII) complexes with serine 2 phosphorylated C-terminal domains (CTDs), mimicking that of the associated genic region. Thus, the inefficient elongation capacity of PROMPT transcription cannot solely be assigned to poor CTD phosphorylation. Conditions that reduce gene transcription increase RNAPII occupancy of the upstream PROMPT region, suggesting that they reside in a common transcription compartment. Surprisingly, gene promoters that are actively transcribed by RNAPI or RNAPIII also produce PROMPTs that are targeted by the exosome. RNAPIII PROMPTs bear hallmarks of RNAPII promoter-associated RNAs, explaining the physical presence of RNAPII upstream of many RNAPIII-transcribed genes. We propose that RNAPII activity upstream gene promoters are wide-spread and integral to the act of gene transcription.