An integrative ChIP-chip and gene expression profiling to model SMAD regulatory modules.

BACKGROUND: The TGF-beta/SMAD pathway is part of a broader signaling network in which crosstalk between pathways occurs. While the molecular mechanisms of TGF-beta/SMAD signaling pathway have been studied in detail, the global networks downstream of SMAD remain largely unknown. The regulatory effect of SMAD complex likely depends on transcriptional modules, in which the SMAD binding elements and partner transcription factor binding sites (SMAD modules) are present in specific context. RESULTS: To address this question and develop a computational model for SMAD modules, we simultaneously performed chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and mRNA expression profiling to identify TGF-beta/SMAD regulated and synchronously coexpressed gene sets in ovarian surface epithelium. Intersecting the ChIP-chip and gene expression data yielded 150 direct targets, of which 141 were grouped into 3 co-expressed gene sets (sustained up-regulated, transient up-regulated and down-regulated), based on their temporal changes in expression after TGF-beta activation. We developed a data-mining method driven by the Random Forest algorithm to model SMAD transcriptional modules in the target sequences. The predicted SMAD modules contain SMAD binding element and up to 2 of 7 other transcription factor binding sites (E2F, P53, LEF1, ELK1, COUPTF, PAX4 and DR1). CONCLUSION: Together, the computational results further the understanding of the interactions between SMAD and other transcription factors at specific target promoters, and provide the basis for more targeted experimental verification of the co-regulatory modules.

Aberrant transforming growth factor beta1 signaling and SMAD4 nuclear translocation confer epigenetic repression of ADAM19 in ovarian cancer.

Transforming growth factor-beta (TGF-beta)/SMAD signaling is a key growth regulatory pathway often dysregulated in ovarian cancer and other malignancies. Although loss of TGF-beta-mediated growth inhibition has been shown to contribute to aberrant cell behavior, the epigenetic consequence(s) of impaired TGF-beta/SMAD signaling on target genes is not well established. In this study, we show that TGF-beta1 causes growth inhibition of normal ovarian surface epithelial cells, induction of nuclear translocation SMAD4, and up-regulation of ADAM19 (a disintegrin and metalloprotease domain 19), a newly identified TGF-beta1 target gene. Conversely, induction and nuclear translocation of SMAD4 were negligible in ovarian cancer cells refractory to TGF-beta1 stimulation, and ADAM19 expression was greatly reduced. Furthermore, in the TGF-beta1 refractory cells, an inactive chromatin environment, marked by repressive histone modifications (trimethyl-H3K27 and dimethyl-H3K9) and histone deacetylase, was associated with the ADAM19 promoter region. However, the CpG island found within the promoter and first exon of ADAM19 remained generally unmethylated. Although disrupted growth factor signaling has been linked to epigenetic gene silencing in cancer, this is the first evidence demonstrating that impaired TGF-beta1 signaling can result in the formation of a repressive chromatin state and epigenetic suppression of ADAM19. Given the emerging role of ADAMs family proteins in growth factor regulation in normal cells, we suggest that epigenetic dysregulation of ADAM19 may contribute to the neoplastic process in ovarian cancer.

Cytogenetic damage in buccal epithelia and peripheral lymphocytes of young healthy individuals exposed to ozone.

Ozone (O(3)) is an important component of air pollution and a potent oxidant of biomolecules. To address the hypothesis that elevated ambient O(3) can induce cytogenetic damage in healthy people, we collected buccal cells from two groups of students (N = 126) from University of California, Berkeley, in the spring and again in the fall. One group spent their summer in the Los Angeles (LA) area where summer O(3) concentrations are significantly higher than in the San Francisco Bay (SF) area, and another remained in SF. During the school year, all students were exposed to low O(3) levels in SF. The micronucleus assay in a total of 611,000 buccal cells demonstrated that, in the fall, micronuclei (MN) in normal cells for the LA group had increased 39% relative to levels in the spring (1.52 and 0.87 MN/1,000 cells, respectively, P = 0.001). Students who spent the summer in SF had a 12.7% increase (P = 0.48). A similar effect of season was seen in degenerated buccal cells for the LA group (3.23 versus 1.88 MN/1,000 cells, P = 0.003). LA but not SF subjects also had more degenerated cells in the fall sample (P = 0.003). These findings were paralleled by an increase in MN and nucleoplasmic bridges in lymphocytes and MN in buccal cells in a sub-group of 15 students who underwent a 4-h controlled exposure to 200 p.p.b. O(3). This cytogenetic evidence, along with recent studies linking O(3) exposure to elevated lung cancer risk and mortality, suggest potential public health implications from exposures to high oxidant environments.

Epigenetic interactions between Arabidopsis transgenes: characterization in light of transgene integration sites.

The stochastic variability of expression that is a characteristic of eukaryotic nuclear transgenes is often attributed to epigenetic mechanisms that are triggered by repetitive transgene locus structures and influenced by chromosomal position effects. In order to address the contribution of chromosomal position effects in the context of a fully sequenced genome, a novel set of transgene loci was established in the compact genome of Arabidopsis thaliana. Transgenes expressing GFP-tagged or GUS-tagged fusion proteins of Arabidopsis COP1 collectively displayed three types of gene silencing, which are distinguished by their developmental timing, gene dosage dependence, (post)transcriptional control, and extent of endogene co-suppression. Subsequently, the heritability of epistatic interactions between allelic and non-allelic transgene loci was investigated in light of both intrinsic transgene features, in particular T-DNA copy number per locus, and chromosomal insertion sites. The notion that chromosomal flanking sequences underlie the ability of transgenes to function as masters or targets of epigenetically heritable trans-silencing interactions was generally not favored by our data. Moreover, among single T-DNA loci at different chromosomal locations the great majority showed homozygosity-dependent posttranscriptional silencing. However, spontaneous silencing (in cis) may be promoted by a pericentromeric location. Instead, intrinsic transgene features correlated with all major aspects of silencing behavior tested.

Epigenetic history of an Arabidopsis trans-silencer locus and a test for relay of trans-silencing activity.

BACKGROUND: Meiotically heritable epimutations affecting transgene expression are not well understood, even and in particular in the plant model species, Arabidopsis thaliana. The Arabidopsis trans-silencer locus, C73, which encodes a fusion protein between the repressor of photomorphogenesis, COP1, and green fluorescent protein (GFP-COP1), heritably modifies the expression pattern and cop1-like cosuppression phenotypes of multiple GFP-COP1 target loci by transcriptional gene silencing. RESULTS: Here we describe three additional features of trans-silencing by the C73 locus. First, the silencing phenotype of C73 and of similar complex loci was acquired epigenetically over the course of no more than two plant generations via a stage resembling posttranscriptional silencing. Second, imprints imposed by the C73 locus were maintained heritably for at least five generations in the absence of the silencer with only sporadic spontaneous reversion. Third, the pairing of two other GFP-COP1 transgene loci, L91 and E82, showed an increased tendency for epigenetic modification when L91 carried an epigenetic imprint from C73, but not when E82 bore the imprint. CONCLUSIONS: The latter data suggest a transfer of trans-silencing activity from one transgene locus, C73, to another, namely L91. These results extend our operational understanding of interactions among transgenes in Arabidopsis.