Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans.

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.

Signal integration: a framework for understanding the efficacy of therapeutics targeting the human EGFR family.

The human EGFR (HER) family is essential for communication between many epithelial cancer cell types and the tumor microenvironment. Therapeutics targeting the HER family have demonstrated clinical success in the treatment of diverse epithelial cancers. Here we propose that the success of HER family-targeted monoclonal antibodies in cancer results from their ability to interfere with HER family consolidation of signals initiated by a multitude of other receptor systems. Ligand/receptor systems that initiate these signals include cytokine receptors, chemokine receptors, TLRs, GPCRs, and integrins. We further extrapolate that improvements in cancer therapeutics targeting the HER family are likely to incorporate mechanisms that block or reverse stromal support of malignant progression by isolating the HER family from autocrine and stromal influences.

Chromatin immunoprecipitation and multiplex sequencing (ChIP-Seq) to identify global transcription factor binding sites in the nematode Caenorhabditis elegans.

The global identification of transcription factor (TF) binding sites is a critical step in the elucidation of the functional elements of the genome. Several methods have been developed that map TF binding in human cells, yeast, and other model organisms. These methods make use of chromatin immunoprecipitation, or ChIP, and take advantage of the fact that formaldehyde fixation of living cells can be used to cross-link DNA sequences to the TFs that bind them in vivo. In ChIP, the cross-linked TF-DNA complexes are sheared by sonication, size fractionated, and incubated with antibody specific to the TF of interest to generate a library of TF-bound DNA sequences. ChIP-chip was the first technology developed to globally identify TF-bound DNA sequences and involves subsequent hybridization of the ChIP DNA to oligonucleotide microarrays. However, ChIP-chip proved to be costly, labor-intensive, and limited by the fixed number of probes available on the microarray chip. ChIP-Seq combines ChIP with massively parallel high-throughput sequencing (see Explanatory Chapter: Next Generation Sequencing) and has demonstrated vast improvement over ChIP-chip with respect to time and cost, signal-to-noise ratio, and resolution. In particular, multiplex sequencing can be used to achieve a higher throughput in ChIP-Seq analyses involving organisms with genomes of lower complexity than that of human (Lefrançois et al., 2009) and thereby reduce the cost and amount of time needed for each result. The multiplex ChIP-Seq method described in this section has been developed for Caenorhabditis elegans, but is easily adaptable for other organisms.

Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project.

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.

Src-Induced cisplatin resistance mediated by cell-to-cell communication.

Cisplatin-induced cell death can be triggered by cell-to-cell communication through gap junctions. Here, we show that activated src produces tyrosine phosphorylation of the gap junction protein connexin 43, decreases gap junction communication, and increases cell survival in response to cisplatin. Experiments with mixed cell populations show that src activity in one cell can confer increased cisplatin survival on neighboring cells, even when the neighboring cells lack such src activity. This work is the first demonstration that expression of an oncogene in one cell can affect the survival of a neighboring cell not expressing the oncogene in response to a chemotherapeutic drug. The trans-acting effect of activated src on neighboring cells can be blocked by inhibitors of src kinase or by siRNA-mediated knockdown of src expression, and it can be counteracted by forced up-regulation of connexin 43, via either gene transfer or proteasome inhibition. These results identify a novel pathway of cisplatin resistance that may be amenable to therapeutic intervention.

Targeted gene disruption of methionine aminopeptidase 2 results in an embryonic gastrulation defect and endothelial cell growth arrest.

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-2/p53 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.

A single amino acid residue defines the difference in ovalicin sensitivity between type I and II methionine aminopeptidases.

TNP-470, the first anti-angiogenic small molecule to enter clinical trials, targets methionine aminopeptidase-2 (MetAP-2), a metalloprotease that cleaves the N-terminal methionine of proteins. Previously, biochemical binding, in vivo yeast studies, and structural studies of human methionine aminopeptidase-2 bound to TNP-470 and its analogs fumagillin and ovalicin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1. To further elucidate the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer ovalicin resistance. Of the three resistant alleles, A362T appeared in the majority of clones and was found to be the most resistant to the ovalicin class of inhibitors. Alignment of human MetAP-2 with human MetAP-1, which is naturally ovalicin-resistant, revealed that the analogous residue in MetAP-1 is also a threonine. Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that an alanine at this position is critical for inhibition by ovalicin. These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1 and may prove useful in the development of additional MetAP-2-specific therapeutic agents.