Genetic analysis of gene silencing in Arabidopsis thaliana

Gene silencing frequently occurs in transgenic plants, the mechanism of which underlines an epigenetic control of plant genome structure and expression, but is poorly understood. A system of gene silencing was established in Arabidopsis thaliana. Transgenic plants expressing a fusion protein between either beta-glucuronidase (GUS) or green fluorescent protein (GFP) and CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COPl) displayed three types of gene silencing phenotypes, type L (late onset), type E (early onset) and type C (complex), which could be distinguished based on the gene dosage dependence and developmental timing of silencing, post-transcriptional versus transcriptional control, and the extent of endogene cosuppression. Chromosomal flanking sequences of seventeen transgene loci were isolated and characterized. Type Land type E loci had one and two T-DNAs, respectively, and were silenced post-transcriptionally. Gene dosage alone was important and allelic interaction was not required for type L silencing. On the other hand, the early onset endogene silencing in type E lines was determined by both gene dosage and the arrangement of T-DNAs in the transgene loci. Type C loci contained two or more T-DNAs, and were transcriptionally silenced. Two type C loci were able to heritably suppress cosuppression in type L and type E loci, a phenomenon reminiscent of paramutation. The paramutated state of a type Land a type E locus was reversible, and was not correlated with DNA methylation on the transgene coding sequence. One paramutated type L locus acquired the ability to suppress silencing of a paramutable locus in trans and might have become paramutagenic. In summary, gene silencing phenotypes were strongly correlated with transgene locus structure, while the effect of genomic environment on gene silencing was not significant. The possibility that PTGS could mature into TGS during plant development or through generations was suggested by the epigenetic transition of type C lines and of transgenic lines harboring more than two transgene loci. Ecotype effects on PTGS in the system were discovered between Nossen and Enkheim ecotypes, and between Columbia and Landsberg ecotypes, which leads to a new way of identifying genes in charge of PTGS

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

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

<p>Abstract</p> <p>Background</p> <p>The TGF-β/SMAD pathway is part of a broader signaling network in which crosstalk between pathways occurs. While the molecular mechanisms of TGF-β/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.</p> <p>Results</p> <p>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-β/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-β 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).</p> <p>Conclusion</p> <p>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.</p

Aberrant Transforming Growth Factor β1 Signaling and SMAD4 Nuclear Translocation Confer Epigenetic Repression of ADAM19 in Ovarian Cancer12

Transforming growth factor-beta (TGF-β)/SMAD signaling is a key growth regulatory pathway often dysregulated in ovarian cancer and other malignancies. Although loss of TGF-β-mediated growth inhibition has been shown to contribute to aberrant cell behavior, the epigenetic consequence(s) of impaired TGF-β/SMAD signaling on target genes is not well established. In this study, we show that TGF-β1 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-β1 target gene. Conversely, induction and nuclear translocation of SMAD4 were negligible in ovarian cancer cells refractory to TGF-β1 stimulation, and ADAM19 expression was greatly reduced. Furthermore, in the TGF-β1 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-β1 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