ATM-dependent activation of the gene encoding MAP kinase phosphatase 5 by radiomimetic DNA damage.

Cellular responses to DNA damage are mediated by an extensive network of signaling pathways. The ATM protein kinase is a master regulator of the response to double-strand breaks (DSBs), the most cytotoxic DNA lesion caused by ionizing radiation. ATM is the protein missing or inactive in patients with the pleiotropic genetic disorder ataxia-telangiectasia (A-T). A major response to DNA damage is altered expression of numerous genes. While studying gene expression in control and A-T cells following treatment with the radiomimetic chemical neocarzinostatin (NCS), we identified an expressed sequence tag that represented a gene that was induced by DSBs in an ATM-dependent manner. The corresponding cDNA encoded a dual specificity phosphatase of the MAP kinase phosphatase family, MKP-5. MKP-5 dephosphorylates and inactivates the stress-activated MAP kinases JNK and p38. The phosphorylation-dephosphorylation cycle of JNK and p38 by NCS was attenuated in A-T cells. Thus, ATM modulates this cycle in response to DSBs. These results further highlight ATM as a link between the DNA damage response and major signaling pathways involved in proliferative and apoptotic processes.

Parallel profiling of the transcriptome, cistrome, and epigenome in the cellular response to ionizing radiation.

The DNA damage response (DDR) is a vast signaling network that is robustly activated by DNA double-strand breaks, the critical lesion induced by ionizing radiation (IR). Although much of this response operates at the protein level, a critical component of the network sustains many DDR branches by modulating the cellular transcriptome. Using deep sequencing, we delineated three layers in the transcriptional response to IR in human breast cancer cells: changes in the expression of genes encoding proteins or long noncoding RNAs, alterations in genomic binding by key transcription factors, and dynamics of epigenetic markers of active promoters and enhancers. We identified protein-coding and previously unidentified noncoding genes that were responsive to IR, and demonstrated that IR-induced transcriptional dynamics was mediated largely by the transcription factors p53 and nuclear factor κB (NF-κB) and was primarily dependent on the kinase ataxia-telangiectasia mutated (ATM). The resultant data set provides a rich resource for understanding a basic, underlying component of a critical cellular stress response.

Transcriptional modulation induced by ionizing radiation: p53 remains a central player.

The cellular response to DNA damage is vital for maintaining genomic stability and preventing undue cell death or cancer formation. The DNA damage response (DDR), most robustly mobilized by double-strand breaks (DSBs), rapidly activates an extensive signaling network that affects numerous cellular systems, leading to cell survival or programmed cell death. A major component of the DDR is the widespread modulation of gene expression. We analyzed together six datasets that probed transcriptional responses to ionizing radiation (IR) - our novel experimental data and 5 published datasets - to elucidate the scope of this response and identify its gene targets. According to the mRNA expression profiles we recorded from 5 cancerous and non-cancerous human cell lines after exposure to 5 Gy of IR, most of the responses were cell line-specific. Computational analysis identified significant enrichment for p53 target genes and cell cycle-related pathways among groups of up-regulated and down-regulated genes, respectively. Computational promoter analysis of the six datasets disclosed that a statistically significant number of the induced genes contained p53 binding site signatures. p53-mediated regulation had previously been documented for subsets of these gene groups, making our lists a source of novel potential p53 targets. Real-time qPCR and chromatin immunoprecipitation (ChIP) assays validated the IR-induced p53-dependent induction and p53 binding to the respective promoters of 11 selected genes. Our results demonstrate the power of a combined computational and experimental approach to identify new transcriptional targets in the DNA damage response network.

Dissection of a DNA-damage-induced transcriptional network using a combination of microarrays, RNA interference and computational promoter analysis.

BACKGROUND: Gene-expression microarrays and RNA interferences (RNAi) are among the most prominent techniques in functional genomics. The combination of the two holds promise for systematic, large-scale dissection of transcriptional networks. Recent studies, however, raise the concern that nonspecific responses to small interfering RNAs (siRNAs) might obscure the consequences of silencing the gene of interest, throwing into question the ability of this experimental strategy to achieve precise network dissections. RESULTS: We used microarrays and RNAi to dissect a transcriptional network induced by DNA damage in a human cellular system. We recorded expression profiles with and without exposure of the cells to a radiomimetic drug that induces DNA double-strand breaks (DSBs). Profiles were measured in control cells and in cells knocked-down for the Rel-A subunit of NFkappaB and for p53, two pivotal stress-induced transcription factors, and for the protein kinase ATM, the major transducer of the cellular responses to DSBs. We observed that NFkappaB and p53 mediated most of the damage-induced gene activation; that they controlled the activation of largely disjoint sets of genes; and that ATM was required for the activation of both pathways. Applying computational promoter analysis, we demonstrated that the dissection of the network into ATM/NFkappaB and ATM/p53-mediated arms was highly accurate. CONCLUSIONS: Our results demonstrate that the combined experimental strategy of expression arrays and RNAi is indeed a powerful method for the dissection of complex transcriptional networks, and that computational promoter analysis can provide a strong complementary means for assessing the accuracy of this dissection.

Transcription profiling of inner ears from Pou4f3(ddl/ddl) identifies Gfi1 as a target of the Pou4f3 deafness gene.

Pou4f3 (Brn3.1, Brn3c) is a class IV POU domain transcription factor that has a central function in the development of all hair cells in the human and mouse inner ear sensory epithelia. A mutation of POU4F3 underlies human autosomal dominant non-syndromic progressive hearing loss DFNA15. Through a comparison of inner ear gene expression profiles of E16.5 wild-type and Pou4f3 mutant deaf mice using a high density oligonucleotide microarray, we identified the gene encoding growth factor independence 1 (Gfi1) as a likely in vivo target gene regulated by Pou4f3. To validate this result, we performed semi-quantitative RT-PCR and in situ hybridizations for Gfi1 on wild-type and Pou4f3 mutant mice. Our results demonstrate that a deficiency of Pou4f3 leads to a statistically significant reduction in Gfi1 expression levels and that the dynamics of Gfi1 mRNA abundance closely follow the pattern of expression for Pou4f3. To examine the role of Gfi1 in the pathogenesis of Pou4f3-related deafness, we performed comparative analyses of the embryonic inner ears of Pou4f3 and Gfi1 mouse mutants using immunohistochemistry and scanning electron microscopy. The loss of Gfi1 results in outer hair cell degeneration, which appears comparable to that observed in Pou4f3 mutants. These results identify Gfi1 as the first downstream target of a hair cell specific transcription factor and suggest that outer hair cell degeneration in Pou4f3 mutants is largely or entirely a result of the loss of expression of Gfi1.