Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells, and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, while others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.

To elucidate cellular machinery on a global scale, we performed a multiple comparison of the recently available protein-protein interaction networks of Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae. This comparison integrated protein interaction and sequence information to reveal 71 network regions that were conserved across all three species and many exclusive to the metazoans. We used this conservation, and found statistically significant support for 4,645 previously undescribed protein functions and 2,609 previously undescribed protein interactions. We tested 60 interaction predictions for yeast by two-hybrid analysis, confirming approximately half of these. Significantly, many of the predicted functions and interactions would not have been identified from sequence similarity alone, demonstrating that network comparisons provide essential biological information beyond what is gleaned from the genome.comparative analysis ͉ multiple alignment ͉ protein network ͉ yeast two-hybrid A major challenge of postgenomic biology is to understand the complex networks of interacting genes, proteins, and small molecules that give rise to biological form and function. Advances in whole-genome approaches are now enabling us to characterize these networks systematically, by using procedures such as the two-hybrid assay (1) and protein coimmunoprecipitation (2) to screen for protein-protein interactions. To date, these technologies have generated large interaction networks for bacteria (3), yeast (4-7), and, recently, fruit fly (8) and nematode worm (9).The large amount of protein interaction data now available presents opportunities and challenges in understanding evolution and function. Such challenges involve assigning functional roles to interactions (10), separating true protein-protein interactions from false positives (11), and, ultimately, organizing large-scale interaction data into models of cellular signaling and regulatory machinery. As is often the case in biology, an approach based on evolutionary cross-species comparisons provides a valuable framework for addressing these challenges. However, although methods for comparing DNA and protein sequences have been a mainstay of bioinformatics over the past 30 years, development of similar tools at other levels of biological information, including protein interactions (12-14), metabolic networks (15-17), or gene expression data (18)(19)(20), is just beginning.Recently, we devised a method called PATHBLAST (13) for comparing the protein interaction networks of two species. Just as BLAST performs rapid pairwise alignment of protein sequences (21), PATHBLAST is based on efficient alignment of two protein networks to identify conserved network regions. Here, we extend this approach to present a computational framework for alignment and comparison of more than two protein networks. We apply this multiple network alignment strategy to compare the recently available protein networks for worm, fly, and yeast, and show that although any single net...

Sonic hedgehog (Shh) acts as a morphogen to mediate the specification of distinct cell identities in the ventral neural tube through a Gli-mediated (Gli1-3) transcriptional network. Identifying Gli targets in a systematic fashion is central to the understanding of the action of Shh. We examined this issue in differentiating neural progenitors in mouse. An epitope-tagged Gli-activator protein was used to directly isolate cis-regulatory sequences by chromatin immunoprecipitation (ChIP). ChIP products were then used to screen custom genomic tiling arrays of putative Hedgehog (Hh) targets predicted from transcriptional profiling studies, surveying 50-150 kb of non-transcribed sequence for each candidate. In addition to identifying expected Gli-target sites, the data predicted a number of unreported direct targets of Shh action. Transgenic analysis of binding regions in Nkx2.2, Nkx2.1 (Titf1) and Rab34 established these as direct Hh targets. These data also facilitated the generation of an algorithm that improved in silico predictions of Hh target genes. Together, these approaches provide significant new insights into both tissue-specific and general transcriptional targets in a crucial Shh-mediated patterning process.

Failure of cells to respond to DNA damage is a primary event associated with mutagenesis and environmental toxicity. To map the transcriptional network controlling the damage response, we measured genomewide binding locations for 30 damage-related transcription factors (TFs) after exposure of yeast to methyl-methanesulfonate (MMS). The resulting 5272 TF-target interactions revealed extensive changes in the pattern of promoter binding and identified damage-specific binding motifs. As systematic functional validation, we identified interactions for which the target changed expression in wild-type cells in response to MMS but was nonresponsive in cells lacking the TF. Validated interactions were assembled into causal pathway models that provide global hypotheses of how signaling, transcription, and phenotype are integrated after damage.Exposure of cells to chemical and physical damaging agents can result in DNA lesions that contribute to the onset of cancer, aging, immune deficiencies, and other degenerative diseases (1). DNA damage is sensed by a highly conserved mechanism involving the ATM/ATR protein kinases in humans (ataxia-telangiectasia mutated/ataxia-telangiectasia and Rad3-related; homologous to Tel1 and Mec1 in yeast). These aggregate at DNA lesions (2) and activate signaling cascades that include the Chk protein kinases (Chk1, Rad53, and Dun1 in yeast). Chk kinases, in turn, trigger both transcriptional and transcription-independent responses, including activation of DNA repair machinery and cell-cycle arrest (1).Beyond the known DNA repair genes, genomewide expression profiling in yeast has identified several hundred genes (3-5) whose expression is increased or decreased in response to alkylation damage by methyl-methanesulfonate (MMS). At the level of growth phenotype, systematic deletion studies have also identified several hundred genes that are required for normal recovery from alkylation damage (6)(7)(8). Surprisingly, the set of genes that, when deleted, affect damage recovery is not enriched for genes whose transcript levels change upon damage exposure (7,9). Thus, neither transcriptional profiling alone, nor genomic phenotyping alone, adequately defines the cellular response to DNA-damaging agents. However, these studies do suggest that the DNA damage response involves multiple levels of regulation, †To whom correspondence should be addressed. trey@bioeng.ucsd.edu. * These authors contributed equally to this work. affecting not only DNA repair genes but also genes that influence protein and lipid turnover, cytoskeleton remodeling, and general stress pathways.To construct a global model of yeast transcriptional networks activated by MMS, we applied a systems approach (10) that integrated data from genomewide chromatin immunoprecipitation (ChIP) assays, expression profiling, systematic phenotyping, and protein interaction databases (Fig. 1). First, we performed a systematic screen for transcription factors (TFs) involved in the MMS response. TFs were chosen from a set of 141 yeast DNA bind...

Hox genes controlling motor neuron subtype identity are expressed in rostro-caudal patterns that are spatially and temporally collinear with their chromosomal organization. Here we demonstrate that Hox chromatin is subdivided into discrete domains, controlled by rostro-caudal patterning signals that trigger rapid, domain-wide clearance of repressive H3K27me3 Polycomb modifications. Treatment of differentiating mouse neural progenitors with retinoic acid (RA) leads to activation and binding of RA receptors (RARs) to Hox1-5 chromatin domains, followed by a rapid domain-wide removal of H3K27me3 and acquisition of cervical spinal identity. Wnt and FGF signals induce expression of Cdx2 transcription factor that binds and clears H3K27me3 from Hox1-9 chromatin domains, leading to specification of brachial/thoracic spinal identity. We propose that rapid clearance of repressive modifications in response to transient patterning signals encodes global rostro-caudal neural identity and that maintenance of these chromatin domains ensures transmission of the positional identity to postmitotic motor neurons later in development.