Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Identification of SUUR protein binding sites in Drosophila Kc cells using DamID

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Alexey Pindyurin

PROVIDER: E-MEXP-863 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

SUUR joins separate subsets of PcG, HP1 and B-type lamin targets in Drosophila.

Pindyurin Alexey V AV Moorman Celine C de Wit Elzo E Belyakin Stepan N SN Belyaeva Elena S ES Christophides George K GK Kafatos Fotis C FC van Steensel Bas B Zhimulev Igor F IF

Journal of cell science 20070701 Pt 14

Drosophila melanogaster Suppressor of Under-Replication (SuUR) gene encodes a protein that modulates replicative properties of heterochromatin in endocycles of polytene cells. The SuUR mutation abolishes underreplication of intercalary heterochromatin and results in partial underreplication of pericentric heterochromatin. We performed a genome-wide mapping of SUUR target genes in non-polytenic Drosophila Kc cells by using the DamID approach. We show that SUUR preferentially binds genes that are ...[more]

PMID: 17606990

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Project description:Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem-cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila Kc cells. We found that Pc associates with large genomic regions of up to ~150kb in size, hereafter referred to as âPc-domainsâ. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 lysine 27 and in general transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains. Keywords: DamID, Chromatin immunoprecipitation, ChIP-chip To study PcG binding profiles we used DamID, which is based on the ability of a chromatin protein fused to E.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 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).

Project description:The local protein composition of chromatin is important for the regulation of transcription and other functions. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, we show that the genome is segmented into five principal chromatin types that are defined by unique, yet overlapping combinations of proteins, and form domains that can extend over >100 kb. We identify a novel repressive chromatin type that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two distinct types that differ in molecular organization and H3K36 methylation, and regulate distinct classes of genes. Finally, we provide evidence that the different chromatin types act as guides that help to target DNA-binding factors to specific subsets of their recognition motifs. These results uncover basic principles of chromatin organization in a higher eukaryote. For this study, we generated whole-genome DamID binding profiles of 45 chromatin proteins in Drosophila Kc167 cells. Additionally, we perused published binding data of 8 chromatin proteins and generated a binding profile of one exogenous (yeast) DNA binding factor in Kc167 cells. On the same array platform, we obtained ChIP-on-chip profiles of histone H3, H1, H3K9me2, H3K27me3, H3K4me2, and H3K79me3. See supplementary files below. Gene expression was measured by RNA tag profiling. See GeneCounts supplementary file below. [1] RNA tag sequences were optained on an Illumina GAII with the digital gene expression (DGE) module from duplicate RNA samples. [2] All DamID and ChIP experiments were done in Drosophila Kc167 cells in duplicate. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Every experiment was done in duplicate in the reverse dye orientation, where Dam-fusion material was hybridized over Dam-only material. For ChIP, immunoprecipitated material was hybridized over ChIP input material. 18 previously-submitted Samples were included in this study. 10 of 18 Samples have been renormalized for the GSE22069 study: GSM509087, GSM509088, GSM509089, GSM509090, GSM509091, GSM509092, GSM509093, GSM509094, GSM509095, GSM509096 New GSM accession numbers have been issued for these 10 samples. 8 of 18 Samples are identical in the original studies and in GSE22069: GSM423290, GSM423291, GSM423298, GSM423299, GSM493592, GSM493593, GSM509085, GSM509086 [3] The genomic locations in files GSE22069_norm_aggregated_discretized_tiling_arrays.txt and GSE22069_norm_aggregated_tiling_arrays.txt are relative to FlyBase release 5 (BDGP R5/dm3).

Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.

Project description:Stem cells reside in a specialized microenvironment, called niche, which provides essential signals controlling stem cell behavior. Proper niche architecture is a key for normal stem cell function, yet only few upstream regulators are known. Here we report that the Hox transcription factor Abd-B, active in pre-meiotic spermatocytes, affects niche positioning in the Drosophila testis by regulating integrin localization in differentiated somatic cyst cells. Loss of Abd-B results in cell non-autonomous effects within the niche including centrosome misorientation in germline stem cells (GSCs) and reduced GSC divisions in larval testis, leading to a dramatic reduction of pre-meiotic stages in adult testes. By identifying Abd-B binding regions throughout the genome, we find that Abd-B mediates its effects on niche function by directly controlling at multiple levels the localization and thus signaling activity of the Sevenless (Sev) ligand, Bride of Sevenless (Boss), via its direct targets src42A and sec63. In sum, our data show for the first time that Abd-B through local signaling provides positional cues for integrin localization, which is critical for niche localization and architecture, and ensures proper niche function and GSC activity. DamID (DNA adenine methyltransferase identification) method was used to identify direct Abd-B target genes in the Drosophila 3rd instar larval testis Dam was fused to the N terminus of Abd-B and transgenic flies were generated. For identifying Abd-B targets in the Drosophila testis the fusion protein was expressed from the uninduced minimal Hsp70 promoter of the UAS vector pUAST (Brand and Perrimon, 1993). As a control for nonspecific Dam activity, transgenic flies expressing the Dam alone were used (Choksi et al., 2006). Subsequently, genomic DNA was extracted from 3rd instar larval testes, expressing either the Dam-Abd-B fusion protein or the Dam protein alone using a specific protocol (Tolhuis et al., 2011); and van Steensel personal communication). Two individual replicates, for Dam-AbdB and Dam alone, have been generated. Following a methylation-sensitive DNA digestion and PCR amplification, DNA fragments from Dam-Abd-B and control DNA were labeled and hybridized to genomic Affymetrix arrays in duplicates (Protocol available at M-bM-^@M-^\www.flychip.org.ukM-bM-^@M-^]).

Dataset Information

Identification of SUUR protein binding sites in Drosophila Kc cells using DamID

Publications

SUUR joins separate subsets of PcG, HP1 and B-type lamin targets in Drosophila.

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