We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.

Regulation of gene expression by sequence-specific transcription factors is central to developmental programs and depends on the binding of transcription factors with target sites in the genome. To date, most such analyses in Caenorhabditis elegans have focused on the interactions between a single transcription factor with one or a few select target genes. As part of the modENCODE Consortium, we have used chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) to determine the genome-wide binding sites of 22 transcription factors (ALR-1, BLMP-1, CEH-14, CEH-30, EGL-27, EGL-5, ELT-3, EOR-1, GEI-11, HLH-1, LIN-11, LIN-13, LIN-15B, LIN-39, MAB-5, MDL-1, MEP-1, PES-1, PHA-4, PQM-1, SKN-1, and UNC-130) at diverse developmental stages. For each factor we determined candidate gene targets, both coding and non-coding. The typical binding sites of almost all factors are within a few hundred nucleotides of the transcript start site. Most factors target a mixture of coding and non-coding target genes, although one factor preferentially binds to non-coding RNA genes. We built a regulatory network among the 22 factors to determine their functional relationships to each other and found that some factors appear to act preferentially as regulators and others as target genes. Examination of the binding targets of three related HOX factors-LIN-39, MAB-5, and EGL-5-indicates that these factors regulate genes involved in cellular migration, neuronal function, and vulval differentiation, consistent with their known roles in these developmental processes. Ultimately, the comprehensive mapping of transcription factor binding sites will identify features of transcriptional networks that regulate C. elegans developmental processes.

SummaryDespite the large evolutionary distances, metazoan species show remarkable commonalities, which has helped establish fly and worm as model organisms for human biology1,2. Although studies of individual elements and factors have explored similarities in gene regulation, a large-scale comparative analysis of basic principles of transcriptional regulatory features is lacking. We mapped the genome-wide binding locations of 165 human, 93 worm, and 52 fly transcription-regulatory factors (RFs) generating a total of 1,019 data sets from diverse cell-types, developmental stages, or conditions in the three species, of which 498 (48.9%) are presented here for the first time. We find that structural properties of regulatory networks are remarkably conserved and that orthologous RF families recognize similar binding motifs in vivo and show some similar co-associations. Our results suggest that gene-regulatory properties previously observed for individual factors are general principles of metazoan regulation that are remarkably well-preserved despite extensive functional divergence of individual network connections. The comparative maps of regulatory circuitry provided here will drive an improved understanding in the regulatory underpinnings of model organism biology and how these relate to human biology, development, and disease.

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The antiangiogenic agent fumagillin (Fg) and its analog TNP-470 bind to intracellular metalloprotease methionine aminopeptidase-2 (MetAP-2) and inhibit endothelial cell growth in a p53-dependent manner. To confirm the role of MetAP-2 in endothelial cell proliferation and to validate it as a physiological target for the Fg class of antiangiogenic agents, we have generated a conditional MetAP-2 knockout mouse. Ubiquitous deletion of the MetAP-2 gene (MAP2) resulted in an early gastrulation defect, which is bypassed in double MetAP-2͞p53 knockout embryos. Targeted deletion of MAP2 specifically in the hemangioblast lineage resulted in abnormal vascular development, and these embryos die at the midsomite stage. In addition, knockdown of MetAP-2 using small interfering RNA or homologous recombination specifically suppresses the proliferation of cultured endothelial cells. Together, these results demonstrate an essential role for MetAP-2 in angiogenesis and indicate that MetAP-2 is responsible for the endothelial cell growth arrest induced by Fg and its derivatives.angiogenesis ͉ TNP-470 T he methionine aminopeptidase (MetAPs) family of cytosolic metalloproteases are responsible for the cleavage of the initial methionine from the N termini of nascent proteins (1, 2). Eukaryotes express two forms of MetAPs, types 1 and 2, which possess similar in vitro substrate specificities. Removal of the N-terminal methionine by MetAP activity is important for subsequent Nterminal modifications, such as myristoylation (3) and acetylation (4). In addition, MetAP activity may affect protein stability according to the N-end rule proposed by Varshavsky and coworkers (5). It has been shown that MetAP function is essential for growth and survival of prokaryotes (which express only MetAP-1) and of the budding yeast Saccharomyces cerevisiae (6, 7). However, the physiological roles of MetAPs have not yet been identified in vertebrates.MetAP-2 is specifically inhibited by TNP-470, a synthetic derivative of the Aspergillus fumigatus natural product fumagillin (Fg; see refs. 8 and 9). TNP-470 selectively inhibits endothelial cell growth in vitro with picomolar efficacy and has a potent antiangiogenic effect in vivo (10, 11). Because of the requirement of angiogenesis for solid tumor growth, MetAP-2 is currently being targeted with small-molecule inhibitors for anticancer and other angiogenesisrelated diseases therapy, even though the mode of action of TNP-470 is not entirely clear (12-16). Several lines of evidence suggest that MetAP-2 is important for the antiangiogenic effect of TNP-470. First, we have recently demonstrated that an A362T variant of human MetAP-2 confers resistance to the cytostatic effect of a Fg analog in MAP1-null yeast (17). In addition, it has been shown that the ability of various Fg analogues to inhibit endothelial cell growth correlates with their MetAP-2 enzymatic inhibitory activity in vitro (9). However, a recent report using RNA interference (RNAi)-mediated MetAP-2 down-regulation contradicts our results and thus r...