A Systems Approach to Mapping DNA Damage Response Pathways

Abstract

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...

A systems approach to delineate functions of paralogous transcription factors: Role of the Yap family in the DNA damage response

“…Yap-associated chromatin was immunoprecipitated by using an anti-TAP antibody and analyzed with an Agilent yeast genome tiling array (Materials and Methods). The reproducibility of these data were Ϸ50% (average overlap of the sets of bound promoters identified by replicate experiments), which is comparable with other large-scale chIP and expression datasets (16,21,22) (SI Fig. 8).…”

“…In this context, when a gene that is differentially expressed under MMS or CDDP becomes unresponsive in a specific Yap TF knockout background, the TF is said to be ''regulatory epistatic'' to the gene. For each possible TF/gene pair, we scored the significance of this effect, using a Bayesian scoring function (16). At P Ͻ 0.005, the five Yaps were epistatic to a total of 170 genes under MMS, corresponding to 49 genes on average and a range of 26 genes for YAP6 to 128 genes for YAP1 ( Fig.…”

“…As many as five Yaps (1, 2, 4, 5, and 6) have been implicated in the cellular response to methylmethanesulfonate (MMS), a DNA alkylating agent (12)(13)(14)(15)(16), and cis-diamminedichloroplatinum (CDDP), a DNA cross-linking agent (17,18). With regard to other stresses, Yap TFs carry out overlapping but distinct biological functions with Yap1 being the major player in oxidative stress, Yap2 in cadmium stress, Yap4 and Yap6 in osmotic stress, and Yap8 in arsenic stress (5,19).…”

Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair

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Improved Bayesian Network inference using relaxed gene ordering

“…We demonstrate inferring static signal transduction hierarchy using BNs and yeast single point mutation microarray data generated in Workman et al (2006). Around 6000 gene expression were profiled over 27 knockout conditions that are relevant to DNA damage response.…”