Project description:Heterochromatin is important for the maintenance of genome stability and regulation of gene expression, yet our knowledge of heterochromatin structure and function is incomplete. We identified four novel Drosophila heterochromatin proteins. Three of these proteins (HP3, HP4, and HP5) interact directly with HP1, while HP6 in turn binds to each of these three proteins. Immunofluorescence microscopy and genome-wide mapping of in vivo binding sites shows that all four proteins are components of heterochromatin. Depletion of HP1 causes redistribution of all four proteins, indicating that HP1 is essential for their heterochromatic targeting. Finally, mutants of HP4 and HP5 are dominant suppressors of position effect variegation, demonstrating their importance in heterochromatic gene silencing. These results indicate that HP1 acts as a docking platform for several mediator proteins that contribute to heterochromatin function. Keywords: DamID knock-down
Project description:Gene expression profiling revealed that hundreds of genes were differentially expressed among these Y introgression lines, and that global gene expression pattern diverged between the low-PEV and high-PEV (defined in the accompanying publication) flies. The results further expand our understanding of the role of the Y chromosome in modulating global gene expression, and suggest a link with modifications of chromatin state. Keywords: position-effect variegation (PEV) The Y chromosomes of H15 and H23 have lower level of suppression on the expression of white gene, therefore show lower level of eye color variegation. In contrast, the Y chromosomes of H5 and H7 have higher level of suppression on the expression of white gene resulting higher level of eye variegation.
Project description:Gene expression profiling revealed that hundreds of genes were differentially expressed among these Y introgression lines, and that global gene expression pattern diverged between the low-PEV and high-PEV (defined in the accompanying publication) flies. The results further expand our understanding of the role of the Y chromosome in modulating global gene expression, and suggest a link with modifications of chromatin state. Keywords: position-effect variegation (PEV) The Y chromosomes of H15 and H23 have lower level of suppression on the expression of white gene, therefore show lower level of eye color variegation. In contrast, the Y chromosomes of H5 and H7 have higher level of suppression on the expression of white gene resulting higher level of eye variegation. A loop design was used with all the Y introgression lines. In addition to biological replicates, a Cy5-Cy3 dye swap was performed.
Project description:Heavy metals and organic compounds, such as pesticides and plasticizers, exert toxicity through their ability to perturb molecular mechanisms. We investigated the ability of 18 compounds to modify the epigenetic state of the white locus in a Drosophila model of position-effect variegation (PEV). Our data indicate that cadmium chloride (CdCl2) is a potent enhancers of variegation. We demonstrate that genes differentially expressed upon CdCl2 exposure are enriched for genes that have heterochromatic states associated with them.
Project description:Position-effect variegation (PEV) is the stochastic transcriptional silencing of a gene positioned adjacent to heterochromatin. white-mottled X-chromosomal inversions in Drosophila are classic PEV models that show variegation of the eye color gene white due to its relocation next to pericentric heterochromatin. To obtain insight into the mechanism of PEV, we constructed detailed binding maps of Heterochromatin Protein 1 (HP1), a major component of heterochromatin, on white-mottled chromosomes. We find that HP1 invades euchromatin across the inversion breakpoints over ~175kb and ~30kb, causing de novo association of HP1 with 20 genes. However, HP1 binding levels in these regions show substantial local variation; white is most strongly bound by HP1 and is one of only two genes that are substantially repressed by heterochromatin. HP1 binding to the invaded region is exceptionally sensitive to the dosage of the histone methyltransferase Su(var)3-9, indicating that the de novo formed heterochromatin is relatively unstable. Our molecular maps demonstrate that heterochromatin can invade a normally euchromatic region, yet the strength of HP1 binding and effects on gene expression are highly dependent on local context. Keywords: DamID, gene expression, genetic modification.
Project description:Topoisomerases solve topological problems during DNA metabolism, but whether they participate in RNA metabolism remains unclear. Top3b represents a family of topoisomerases carrying activities for both DNA and RNA. Here we show that in Drosophila, Top3b interacts biochemically and genetically with the RNAi-induced silencing complex (RISC) containing AGO2, p68 RNA helicase, and FMRP. Top3b and RISC mutants are similarly defective in heterochromatin formation and transcriptional silencing by position-effect variegation assay. Moreover, both Top3b and AGO2 mutants exhibit reduced levels of heterochromatin protein HP1 in pericentric heterochromatin. Furthermore, expression of several genes and transposable elements in heterochromatin is increased in the Top3b mutant. Notably, Top3b mutants defective in either RNA binding or catalytic activity are deficient in promoting HP1 recruitment and silencing of transposable elements. Our data suggest that Top3b may act as an RNA topoisomerase in siRNA-guided heterochromatin formation and transcriptional silencing.