DamID data for SUUR binding in 12-14h Embryo, larval salivary glands and larval brain
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE33873 | GEO | 2011/11/23
SECONDARY ACCESSION(S): PRJNA148339
REPOSITORIES: GEO
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Project description:SUUR protein was found to be a major component of three repressive chromatin states named BLACK, BLUE and GREEN chromatins. Here we addressed a question of developmental dynamics of these chromatin states using SUUR as a marker. SUUR binding in larval salivary glands, brains and 12-14 hours embryos was determined using DamID technique. Comparison of Dam-SUUR binding to Dam-alone signal in duplicate for each sample type.
2011-11-23 | E-GEOD-33873 | biostudies-arrayexpress
Project description:Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SUUR protein is found at late-replicating intercalary heterochromatin sites and in pericentric heterochromatin. We performed a genome-wide mapping of SUUR target genes in the non-polytenic Drosophila Kc cells by using DamID. This approach is based on the ability of a chromatin protein fused to Escherichia coli DNA adenine methyltransferase (Dam) to methylate the native binding site of the chromatin protein. Dam-fusion proteins are expressed at very low levels to avoid mistargeting. Subsequently, methylated DNA fragments are isolated, labeled (using Cy3 or Cy5) and hybridized to a microarray. Methylated DNA fragments from cells transfected with Dam alone served as reference. Genomic binding sites of the protein can then be identified based on the targeted methylation pattern. For detailed background information on DamID, see: van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat Genet 27, 304-8 (2001); van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8 (2000). We generated both an N- and a C-terminal fusion of the full-length SuUR open reading frame with Dam (Dam-SUUR and SUUR-Dam, respectively). For each SUUR fusion protein we performed four independent replicates. We used for this study a cDNA array developed by the GeneCore facility in EMBL (Heidelberg, Germany), covering the DGC1 and DGC2 cDNA libraries from the Berkeley Drosophila Genome Project, which represents more than 70% of the coding Drosophila genome. We found that SUUR preferentially binds to genes that are transcriptionally silent and late replicated. We compared the SUUR binding profile to the binding profile of three PcG proteins, which are known to bind to many intercalary heterochromatin sites, and found that there is a significant overlap with Pc and esc, but less with Sce. A significant overlap is also detected with two markers of pericentric heterochromatin, the heterochromatin proteins HP1 and SU(VAR)3-9. Finally, we demonstrated that SUUR binding profile negatively correlates with DNA polytenization level in salivary gland polytene chromosomes. Taken together, these results suggest that SUUR modulates the level of underreplication by direct binding to intercalary and pericentric heterochromatin.
2007-07-04 | E-MEXP-863 | biostudies-arrayexpress
Project description:Heterochromatin contains repressively modified histones and replicates late in S phase of the cell cycle. Besides the shortage in replication origins, little is known about replication timing regulation in silenced regions. In Drosophila polytene cells, late replication results in under-replication and decreased DNA copy number in heterochromatic regions of the genome. The Suppressor of Under-replication (SUUR) protein controls this feature – in its absence the DNA polytenization level in most silenced regions is restored, however the repressive histone marks are lost. We hypothesized that SUUR regulates the re-establishment of repressive histone pattern during replication which results in delayed replication completion of heterochromatin. Measuring DNA copy number in mutants with disrupted repressive pathways, we found that under-replication is directly linked to repressive histone marks supply. DamID-seq and ChIP-seq experiments revealed that SuUR mutation does not affect the establishment of heterochromatin domains. Here, we identified a novel SUUR protein interaction (CG12018) that supports SUUR association with replication complex. SUUR loads onto replication forks shortly after the origin firing and participates in chromatin maintenance rather than its establishment. Thus, our findings provide comprehensive evidence that late replication in Drosophila is caused by the time-consuming process of replication-coupled repressive chromatin renewal.
2016-01-10 | GSE74907 | GEO
Project description:Heterochromatin contains repressively modified histones and replicates late in S phase of the cell cycle. Besides the shortage in replication origins, little is known about replication timing regulation in silenced regions. In Drosophila polytene cells, late replication results in under-replication and decreased DNA copy number in heterochromatic regions of the genome. The Suppressor of Under-replication (SUUR) protein controls this feature â?? in its absence the DNA polytenization level in most silenced regions is restored, however the repressive histone marks are lost. We hypothesized that SUUR regulates the re-establishment of repressive histone pattern during replication which results in delayed replication completion of heterochromatin. Measuring DNA copy number in mutants with disrupted repressive pathways, we found that under-replication is directly linked to repressive histone marks supply. DamID-seq and ChIP-seq experiments revealed that SuUR mutation does not affect the establishment of heterochromatin domains. Here, we identified a novel SUUR protein interaction (CG12018) that supports SUUR association with replication complex. SUUR loads onto replication forks shortly after the origin firing and participates in chromatin maintenance rather than its establishment. Thus, our findings provide comprehensive evidence that late replication in Drosophila is caused by the time-consuming process of replication-coupled repressive chromatin renewal. Examination of three chromatin proteins in slivary glands in presence or absence of SuUR mutation using DamID technique.
2016-01-10 | E-GEOD-74907 | biostudies-arrayexpress
Project description:ChIP was performed to identify regions of gDNA bound by RNA polymerase II in third instar salivary glands of Drosophila WT and SuUR mutants. This demonstrated that RNAPolII does not localize to regions that are under-replicated in SuUR mutant third instar salivary glands.
2011-10-18 | GSE31896 | GEO
Project description:ChIP was performed to identify regions of gDNA bound by H3K27me3 in third instar salivary glands of Drosophila WT and SuUR mutants. This demonstrated that H3K27me3 binds differently in under-replicated in SuUR mutant third instar salivary glands.
2011-10-18 | GSE31897 | GEO
Project description:Heterochromatin contains repressively modified histones and replicates late in S phase of the cell cycle. Besides the shortage in replication origins, little is known about replication timing regulation in silenced regions. In Drosophila polytene cells, late replication results in under-replication and decreased DNA copy number in heterochromatic regions of the genome. The Suppressor of Under-replication (SUUR) protein controls this feature â in its absence the DNA polytenization level in most silenced regions is restored, however the repressive histone marks are lost. We hypothesized that SUUR regulates the re-establishment of repressive histone pattern during replication which results in delayed replication completion of heterochromatin. Measuring DNA copy number in mutants with disrupted repressive pathways, we found that under-replication is directly linked to repressive histone marks supply. DamID-seq and ChIP-seq experiments revealed that SuUR mutation does not affect the establishment of heterochromatin domains. Here, we identified a novel SUUR protein interaction (CG12018) that supports SUUR association with replication complex. SUUR loads onto replication forks shortly after the origin firing and participates in chromatin maintenance rather than its establishment. Thus, our findings provide comprehensive evidence that late replication in Drosophila is caused by the time-consuming process of replication-coupled repressive chromatin renewal. Examination of H3K27me3 histone modification in 3 cell types in presence or absence of SuUR mutation.
2016-01-10 | E-GEOD-74908 | biostudies-arrayexpress
Project description:ChIP was performed to identify regions of gDNA bound by RNA polymerase II in third instar salivary glands of Drosophila WT and SuUR mutants. This demonstrated that RNAPolII does not localize to regions that are under-replicated in SuUR mutant third instar salivary glands. Comparison of RNAPolII ChIP from third instar salivary glands relative to control IgG pulldown for two different Drosophila strains
2011-10-17 | E-GEOD-31896 | biostudies-arrayexpress
Project description:ChIP was performed to identify regions of gDNA bound by H3K27me3 in third instar salivary glands of Drosophila WT and SuUR mutants. This demonstrated that H3K27me3 binds differently in under-replicated in SuUR mutant third instar salivary glands. Comparison of H3K27me3 ChIP sample relative to input DNA or control IgG pulldown for two different Drosophila strains
2011-10-17 | E-GEOD-31897 | biostudies-arrayexpress
Project description:We analyzed gamma-H2Av ChIP-Seq from hand dissected salivary glands of wandering third instar larvae from wild-type (OrR) or suppressor of under-replication (SuUR) mutant Drosophila. Goals were to determine the DNA damage profile relative to underreplicated domains. We analyzed SUUR and Cdc45 ChIP-Seq from hand dissected early (stage 10) and late (stage 12/13) egg chambers from adult Drosophila ovaries. The goals was to determine the localization of SUUR relative to replication forks.
2014-11-10 | GSE55932 | GEO