Binding profiles for 954 Drosophila and C. elegans transcription factors reveal tissue specific regulatory relationships.

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here we present the culmination of the modERN (model organism Encyclopedia of Regulatory Networks) consortium that systematically assayed TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). We describe key features of these datasets, comprising 604 TFs identifying 3.6M sites in the fly and 350 TFs identifying 0.9 M sites in the worm. Applying a machine learning model to these data identifies sets of TFs with a prominent role in promoting target gene expression in specific cell types. TF binding data are available through the ENCODE Data Coordinating Center and at https://epic.gs.washington.edu/modERNresource, which provides access to processed and summary data, as well as widgets to probe cell type-specific TF-target relationships. These data are a rich resource that should fuel investigations into TF function during development.

To mock or not: a comprehensive comparison of mock IP and DNA input for ChIP-seq.

Chromatin immunoprecipitation (IP) followed by sequencing (ChIP-seq) is the gold standard to detect transcription-factor (TF) binding sites in the genome. Its success depends on appropriate controls removing systematic biases. The predominantly used controls, i.e. DNA input, correct for uneven sonication, but not for nonspecific interactions of the IP antibody. Another type of controls, 'mock' IP, corrects for both of the issues, but is not widely used because it is considered susceptible to technical noise. The tradeoff between the two control types has not been investigated systematically. Therefore, we generated comparable DNA input and mock IP experiments. Because mock IPs contain only nonspecific interactions, the sites predicted from them using DNA input indicate the spurious-site abundance. This abundance is highly correlated with the 'genomic activity' (e.g. chromatin openness). In particular, compared to cell lines, complex samples such as whole organisms have more spurious sites-probably because they contain multiple cell types, resulting in more expressed genes and more open chromatin. Consequently, DNA input and mock IP controls performed similarly for cell lines, whereas for complex samples, mock IP substantially reduced the number of spurious sites. However, DNA input is still informative; thus, we developed a simple framework integrating both controls, improving binding site detection.

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

The ModERN Resource: Genome-Wide Binding Profiles for Hundreds of Drosophila and Caenorhabditis elegans Transcription Factors.

To develop a catalog of regulatory sites in two major model organisms, Drosophila melanogaster and Caenorhabditis elegans, the modERN (model organism Encyclopedia of Regulatory Networks) consortium has systematically assayed the binding sites of transcription factors (TFs). Combined with data produced by our predecessor, modENCODE (Model Organism ENCyclopedia Of DNA Elements), we now have data for 262 TFs identifying 1.23 M sites in the fly genome and 217 TFs identifying 0.67 M sites in the worm genome. Because sites from different TFs are often overlapping and tightly clustered, they fall into 91,011 and 59,150 regions in the fly and worm, respectively, and these binding sites span as little as 8.7 and 5.8 Mb in the two organisms. Clusters with large numbers of sites (so-called high occupancy target, or HOT regions) predominantly associate with broadly expressed genes, whereas clusters containing sites from just a few factors are associated with genes expressed in tissue-specific patterns. All of the strains expressing GFP-tagged TFs are available at the stock centers, and the chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center and also through a simple interface (http://epic.gs.washington.edu/modERN/) that facilitates rapid accessibility of processed data sets. These data will facilitate a vast number of scientific inquiries into the function of individual TFs in key developmental, metabolic, and defense and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks and globally across the life spans of these two key model organisms.

Diverse patterns of genomic targeting by transcriptional regulators in Drosophila melanogaster.

Annotation of regulatory elements and identification of the transcription-related factors (TRFs) targeting these elements are key steps in understanding how cells interpret their genetic blueprint and their environment during development, and how that process goes awry in the case of disease. One goal of the modENCODE (model organism ENCyclopedia of DNA Elements) Project is to survey a diverse sampling of TRFs, both DNA-binding and non-DNA-binding factors, to provide a framework for the subsequent study of the mechanisms by which transcriptional regulators target the genome. Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of 84 TRFs at various stages of development. This regulatory map reveals a variety of genomic targeting patterns, including factors with strong preferences toward proximal promoter binding, factors that target intergenic and intronic DNA, and factors with distinct chromatin state preferences. The data also highlight the stringency of the Polycomb regulatory network, and show association of the Trithorax-like (Trl) protein with hotspots of DNA binding throughout development. Furthermore, the data identify more than 5800 instances in which TRFs target DNA regions with demonstrated enhancer activity. Regions of high TRF co-occupancy are more likely to be associated with open enhancers used across cell types, while lower TRF occupancy regions are associated with complex enhancers that are also regulated at the epigenetic level. Together these data serve as a resource for the research community in the continued effort to dissect transcriptional regulatory mechanisms directing Drosophila development.

Isolation of highly persistent mutants of Salmonella enterica serovar typhimurium reveals a new toxin-antitoxin module.

Bacterial persistence is characterized by the ability of a subpopulation within bacterial cultures to survive exposure to antibiotics and other lethal treatments. The surviving persisters are not the result of genetic changes but represent epigenetic variants that are in a physiological state where growth is inhibited. Since characterization of persisters has been performed mainly in Escherichia coli K-12, we sought to identify mechanisms of persistence in the pathogen Salmonella enterica serovar Typhimurium. Isolation of new highly persistent mutants revealed that the shpAB locus (Salmonella high persistence) imparted a 3- to 4-order-of-magnitude increase in survival after ampicillin exposure throughout its growth phase and protected the population against exposure to multiple antibiotics. Genetic characterization revealed that shpAB is a newly discovered toxin-antitoxin (TA) module. The high-persistence phenotype was attributed to a nonsense mutation in the 3' end of the shpB gene encoding an antitoxin protein. Characteristic of other TA modules, shpAB is autoregulated, and high persistence depends on the Lon protease.

Evidence of abundant stop codon readthrough in Drosophila and other metazoa.

While translational stop codon readthrough is often used by viral genomes, it has been observed for only a handful of eukaryotic genes. We previously used comparative genomics evidence to recognize protein-coding regions in 12 species of Drosophila and showed that for 149 genes, the open reading frame following the stop codon has a protein-coding conservation signature, hinting that stop codon readthrough might be common in Drosophila. We return to this observation armed with deep RNA sequence data from the modENCODE project, an improved higher-resolution comparative genomics metric for detecting protein-coding regions, comparative sequence information from additional species, and directed experimental evidence. We report an expanded set of 283 readthrough candidates, including 16 double-readthrough candidates; these were manually curated to rule out alternatives such as A-to-I editing, alternative splicing, dicistronic translation, and selenocysteine incorporation. We report experimental evidence of translation using GFP tagging and mass spectrometry for several readthrough regions. We find that the set of readthrough candidates differs from other genes in length, composition, conservation, stop codon context, and in some cases, conserved stem-loops, providing clues about readthrough regulation and potential mechanisms. Lastly, we expand our studies beyond Drosophila and find evidence of abundant readthrough in several other insect species and one crustacean, and several readthrough candidates in nematode and human, suggesting that functionally important translational stop codon readthrough is significantly more prevalent in Metazoa than previously recognized.

A cis-regulatory map of the Drosophila genome.

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide has successfully identified specific subtypes of regulatory elements. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements, chromatin states, transcription factor binding sites, RNA polymerase II regulation and insulator elements; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.