To develop a catalog of regulatory sites in two major model organisms, and, 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.

BackgroundThe tumor suppressor Rb/E2F regulates gene expression to control differentiation in multiple tissues during development, although how it directs tissue-specific gene regulation in vivo is poorly understood.ResultsWe determined the genome-wide binding profiles for Caenorhabditis elegans Rb/E2F-like components in the germline, in the intestine and broadly throughout the soma, and uncovered highly tissue-specific binding patterns and target genes. Chromatin association by LIN-35, the C. elegans ortholog of Rb, is impaired in the germline but robust in the soma, a characteristic that might govern differential effects on gene expression in the two cell types. In the intestine, LIN-35 and the heterochromatin protein HPL-2, the ortholog of Hp1, coordinately bind at many sites lacking E2F. Finally, selected direct target genes contribute to the soma-to-germline transformation of lin-35 mutants, including mes-4, a soma-specific target that promotes H3K36 methylation, and csr-1, a germline-specific target that functions in a 22G small RNA pathway.ConclusionsIn sum, identification of tissue-specific binding profiles and effector target genes reveals important insights into the mechanisms by which Rb/E2F controls distinct cell fates in vivo.

The nucleolus has shown to be integral for many processes related to cell growth and proliferation. Stem cells in particular are likely to depend upon nucleolus-based processes to remain in a proliferative state. A highly conserved nucleolar factor named nucleostemin is proposed to be a critical link between nucleolar function and stem-cell–specific processes. Currently, it is unclear whether nucleostemin modulates proliferation by affecting ribosome biogenesis or by another nucleolus-based activity that is specific to stem cells and/or highly proliferating cells. Here, we investigate nucleostemin (nst-1) in the nematode C. elegans, which enables us to examine nst-1 function during both proliferation and differentiation in vivo. Like mammalian nucleostemin, the NST-1 protein is localized to the nucleolus and the nucleoplasm; however, its expression is found in both differentiated and proliferating cells. Global loss of C. elegans nucleostemin (nst-1) leads to a larval arrest phenotype due to a growth defect in the soma, while loss of nst-1 specifically in the germ line causes germline stem cells to undergo a cell cycle arrest. nst-1 mutants exhibit reduced levels of rRNAs, suggesting defects in ribosome biogenesis. However, NST-1 is generally not present in regions of the nucleolus where rRNA transcription and processing occurs, so this reduction is likely secondary to a different defect in ribosome biogenesis. Transgenic studies indicate that NST-1 requires its N-terminal domain for stable expression and both its G1 GTPase and intermediate domains for proper germ line function. Our data support a role for C. elegans nucleostemin in cell growth and proliferation by promoting ribosome biogenesis.

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.

The complex cellular events that occur in response to fertilization are essential for mediating the oocyte-to-embryo transition. Here, we describe a comprehensive small-molecule screen focused on identifying compounds that affect early embryonic events in Caenorhabditis elegans. We identify a single novel compound that disrupts early embryogenesis with remarkable stage and species specificity. The compound, named C22, primarily impairs eggshell integrity, leading to osmotic sensitivity and embryonic lethality. The C22-induced phenotype is dependent upon the upregulation of the LET-607/CREBH transcription factor and its candidate target genes, which primarily encode factors involved in diverse aspects of protein trafficking. Together, our data suggest that in the presence of C22, one or more key components of the eggshell are inappropriately processed, leading to permeable, inviable embryos. The remarkable specificity and reversibility of this compound will facilitate further investigation into the role and regulation of protein trafficking in the early embryo, as well as serve as a tool for manipulating the life cycle for other studies such as those involving aging.

SummaryProtein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer’s and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases.

28Chromatin immunoprecipitation (IP) followed by sequencing (ChIP-seq) is the gold standard to 29 detect genome-wide DNA-protein binding. The binding sites of transcription factors facilitate many 30 biological studies. Of emerging concern is the abundance of spurious sites in ChIP-seq, which are mainly 31 caused by uneven genomic sonication and nonspecific interactions between chromatin and antibody. A 32 "mock" IP is designed to correct for both factors, whereas a DNA input control corrects only for uneven 33 sonication. However, a mock IP is more susceptible to technical noise than a DNA input, and empirically, 34 these two controls perform similarly for ChIP-seq. Therefore, DNA input is currently being used almost 35 exclusively. With a large dataset, we demonstrate that using a DNA input control results in a definable set 36 of spurious sites, and their abundance is tightly associated with the intrinsic properties of the ChIP-seq 37 sample. For example, compared to human cell lines, samples such as human tissues and whole worm and 38 fly have more accessible genomes, and thus have more spurious sites. The large and varying abundance of 39 spurious sites may impede comparative studies across multiple samples. In contrast, using a mock IP as 40 control substantially removes these spurious sites, resulting in high-quality binding sites and facilitating 41 their comparability across samples. Although outperformed by mock IP, DNA input is still informative 42 and has unique advantages. Therefore, we have developed a method to use both controls in combination 43 to further improve binding site detection. 44 45 46 47 48 49 50 51 52 53 3 INTRODUCTION 54ChIP-seq was developed to profile in vivo protein-DNA binding and histone modifications on a 55 genomic scale (Barski et al. 2007; Johnson et al. 2007; Mikkelsen et al. 2007; Robertson et al. 2007). 56Compared to its predecessors, ChIP-seq has less noise and higher resolution (Schones and Zhao 2008; Ho 57 et al. 2011), and thus is currently the standard technique to identify the binding sites of a transcription 58 factor in the genome. ChIP-seq protocols typically begin with cross-linking DNA and its adjacent proteins 59 using formaldehyde, followed by shearing DNA into small fragments by sonication. Next in the IP step, 60 an antibody that binds specifically to the transcription factor (TF) of interest is used to enrich the TF-61 DNA complexes. Finally, the precipitated DNA fragments are sequenced and mapped back to a reference 62 genome for binding site detection. The genomic regions with significantly more reads than controls are 63 likely to be TF binding sites. 64As with many high-throughput techniques, ChIP-seq is also susceptible to technical and 65 biological biases (Park 2009; Kidder et al. 2011). In ChIP-seq, one bias arises during genome sonication, 66 in which open chromatin regions are more easily sheared than other regions, and thus these open regions 67 yield more protein-DNA complexes. Consequently, the IP step immunoprecipitates more complexes from 68...

All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments as well as their coordinated activity during the catalysis of homologous recombination.The bacterial RecA protein is the founding member of a family of enzymes that catalyzes recombination between homologous DNA substrates, a family which includes the phage T4 UvsX, yeast Rad51, archael RadA and human Rad51 proteins (1,2). To perform this function these recombinase enzymes form a helical filament that assembles cooperatively on a singlestranded region of DNA resulting in the formation of an active nucleoprotein complex that subsequently interacts with a dsDNA substrate to catalyze a search for homology and DNA strand exchange (1,3,4). Early biochemical studies of RecA revealed that in the absence of substrate DNA the protein exists in solution as a complex heterogeneous mix of various oligomers (5-8) and later work showed that this mix included monomers, ring-shaped hexamers/heptamers, short filaments ranging in length from 0.03 to 0.15 μm as well as bundles of filaments (9-12).The RecA nucleoprotein filament assembles in an ATP-dependent, highly cooperative manner (13-16). Kinetic analysis of nucleoprotein filament assembly has shown that RecA polymerization onto ssDNA can be divided into two major steps, i. nucleation, which is rate limiting, and ii. filament extension (17). However, the oligomeric heterogeneity of RecA in these studies precluded identification of a specific RecA oligomer, e.g. monomers or dimers, as being an obligatory component of the assembly process, or whether various larger RecA oligomers, e.g. trimers or hexamers, could participate in filament assembly. It was also found that RecA oligomers formed in the absence of ATP or ssDNA are not directly interconvertible tel. -(508) EXPERIMENTAL PROCEDURES Expression ConstructsThe gene encoding wild type recA was expressed using a previously described construct, pTRecA420, in which recA is regulated by the tac promoter (20,21). The gene encoding the fused RecA dimer (recA-FD) was constructed as follows. Two point mutations, Lys6Ala and Arg28Ala, were introduced into the wild type recA gene carried in plasmid pTRecA420 using the QuikChange protocol (Stratagene). This mutant recA gene was PCR amplified using a top strand primer that overlapped the NcoI restriction site (underlined) at the fMet initiation codon (5′-GG AGT GAT GCC ATG GCT ATC GAC G -3′) and a bottom strand primer that adds a five-residue linker (Gly 3 Ser 2 )...