Transcription profiling of lung adenocarcinomas of c-myc-transgenic mice: Identification of the c-myc regulatory gene network

<p>Abstract</p> <p>Background</p> <p>The transcriptional regulator c-Myc is the most frequently deregulated oncogene in human tumors. Targeted overexpression of this gene in mice results in distinct types of lung adenocarcinomas. By using microarray technology, alterations in the expression of genes were captured based on a female transgenic mouse model in which, indeed, c-Myc overexpression in alveolar epithelium results in the development of bronchiolo-alveolar carcinoma (BAC) and papillary adenocarcinoma (PLAC). In this study, we analyzed exclusively the promoters of induced genes by different in silico methods in order to elucidate the c-Myc transcriptional regulatory network.</p> <p>Results</p> <p>We analyzed the promoters of 361 transcriptionally induced genes with respect to c-Myc binding sites and found 110 putative binding sites in 94 promoters. Furthermore, we analyzed the flanking sequences (+/- 100 bp) around the 110 c-Myc binding sites and found Ap2, Zf5, Zic3, and E2f binding sites to be overrepresented in these regions. Then, we analyzed the promoters of 361 induced genes with respect to binding sites of other transcription factors (TFs) which were upregulated by c-Myc overexpression. We identified at least one binding site of at least one of these TFs in 220 promoters, thus elucidating a potential transcription factor network. The analysis correlated well with the significant overexpression of the TFs Atf2, Foxf1a, Smad4, Sox4, Sp3 and Stat5a. Finally, we analyzed promoters of regulated genes which where apparently not regulated by c-Myc or other c-Myc targeted TFs and identified overrepresented Oct1, Mzf1, Ppargamma, Plzf, Ets, and HmgIY binding sites when compared against control promoter background.</p> <p>Conclusion</p> <p>Our in silico data suggest a model of a transcriptional regulatory network in which different TFs act in concert upon c-Myc overexpression. We determined molecular rules for transcriptional regulation to explain, in part, the carcinogenic effect seen in mice overexpressing the c-Myc oncogene.</p

Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation

UVSSA and USP7, a new couple in transcription-coupled DNA repair

Transcription-coupled nucleotide excision repair (TC-NER) specifically removes transcription-blocking lesions from our genome. Defects in this pathway are associated with two human disorders: Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS). Despite a similar cellular defect in the UV DNA damage response, patients with these syndromes exhibit strikingly distinct symptoms; CS patients display severe developmental, neurological, and premature aging features, whereas the phenotype of UVSS patients is mostly restricted to UV hypersensitivity. The exact molecular mechanism behind these clinical differences is still unknown; however, they might be explained by additional functions of CS proteins beyond TC-NER. A short overview of the current hypotheses addressing possible molecular mechanisms and the proteins involved are presented in this review. In addition, we will focus on two new players involved in TC-NER which were recently identified: UV-stimulated scaffold protein A (UVSSA) and ubiquitin-specific protease 7 (USP7). UVSSA has been found to be the causative gene for UVSS and, together with USP7, is implicated in regulating TC-NER activity. We will discuss the function of UVSSA and USP7 and how the discovery of these proteins contributes to a better understanding of the molecular mechanisms underlying the clinical differences between UVSS and the more severe CS

Computational Identification of Transcriptional Regulators in Human Endotoxemia

One of the great challenges in the post-genomic era is to decipher the underlying principles governing the dynamics of biological responses. As modulating gene expression levels is among the key regulatory responses of an organism to changes in its environment, identifying biologically relevant transcriptional regulators and their putative regulatory interactions with target genes is an essential step towards studying the complex dynamics of transcriptional regulation. We present an analysis that integrates various computational and biological aspects to explore the transcriptional regulation of systemic inflammatory responses through a human endotoxemia model. Given a high-dimensional transcriptional profiling dataset from human blood leukocytes, an elementary set of temporal dynamic responses which capture the essence of a pro-inflammatory phase, a counter-regulatory response and a dysregulation in leukocyte bioenergetics has been extracted. Upon identification of these expression patterns, fourteen inflammation-specific gene batteries that represent groups of hypothetically ‘coregulated’ genes are proposed. Subsequently, statistically significant cis-regulatory modules (CRMs) are identified and decomposed into a list of critical transcription factors (34) that are validated largely on primary literature. Finally, our analysis further allows for the construction of a dynamic representation of the temporal transcriptional regulatory program across the host, deciphering possible combinatorial interactions among factors under which they might be active. Although much remains to be explored, this study has computationally identified key transcription factors and proposed a putative time-dependent transcriptional regulatory program associated with critical transcriptional inflammatory responses. These results provide a solid foundation for future investigations to elucidate the underlying transcriptional regulatory mechanisms under the host inflammatory response. Also, the assumption that coexpressed genes that are functionally relevant are more likely to share some common transcriptional regulatory mechanism seems to be promising, making the proposed framework become essential in unravelling context-specific transcriptional regulatory interactions underlying diverse mammalian biological processes

Structural basis for the initiation of eukaryotic transcription-coupled DNA repair

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity1. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II–CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II–Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation