DamID of Dam::LMN-1 and Dam::EMR-1 in Caenorhabditis elegans adult worms
ABSTRACT: Laminopathies are caused by mutations in components of the nuclear envelope (NE). While most NE components are widely expressed, laminopathies affect only a subset of tissues. However, the understanding of the molecular mechanisms that explain this phenomenon is still elusive. Here we have performed a genome wide DamID analysis in adult C. elegans nematodes comparing the DNA association profile of two components of the NE, Lamin/LMN-1 and Emerin/EMR-1. Although both proteins were associated to silent DNA, EMR-1 showed a predominant role in the anchoring of muscle and neuronal promoters to the nuclear periphery. Deletion of either EMR-1 or LEM-2, another integral NE protein, caused local changes in nuclear architecture with both increased and decreased LMN-1 association. Comparison of Dam::LMN-1 and Dam::EMR-1 DNA assotiation in wild type strains and Dam::LMN-1 DNA association in wild type, lem-2(tm1582) and emr-1(gk119) mutant backgrounds.
Project description:We show there is minimal genome-wide chromatin rearrangements (as measured by DNA accessibility) during tissue differentiation in C. elegans transgenic expression of E. coli DAM (DNA Adenine Methyltransferase) from tissue-specific promoters followed by mapping of methylated sites using a procedure that captures 20bp sequences flanking methylated sites for Illumina sequencing PD3994 = transgenic line expressing DAM from myo-3 promoter PD3995 = transgenic line expressing DAM from rol-6 promoter PD3997 = transgenic line expressing DAM from vit-2 promoter N2dam = wildtype (N2) line; its genomic DNA was in vitro DAM-methylated parsed = Solexa reads in which linker sequences were successfully parsed out by the authors WS170DAMtags_ALL.txt = the set of all in_silico-generated DAM tags from C. elegans genome version WS170
Project description:Profiling of DAF-16 binding by comparison of DNA methylation of a C. elegans control strain expressing the DNA adenine methyltransferase (DAM) and an experimental strain expressing a DAF-16::DAM. Both strains were fed daf-2 RNAi to identify DAF-16 binding associated with long lived worms.
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 “www.flychip.org.uk”).
Project description:The nuclear lamina (NL) is a filamentous layer lining the inner-nuclear-membrane (INM) that aids in the organization of the genome in large domains of low transcriptional activity. Recently, it was shown that the single-cell genome-NL interactions are much more dynamic than previously anticipated, which challenges the concept of the NL as a safe guard for transcriptional repressed genes. Here we discuss the role of the NL in light of these new findings and introduce Lamin A and BAF as potential modulators of LAD positioning BAF-chromatin and Lamin B2-chromatin interactions were assayed in human HT1080 by DamID on Nimblegen microarrays, with two biological replicates each, that were hybridized in a dye-swap design.
Project description:We have adapted the DamID protocol for use with high throughput sequencing. We have used DamID to identify the positions within the Drosophila genome where the transcription factor DSX is bound. We sequenced DpnI-digested genomic DNA from fly tissues containing UAS-Dam (control) or UAS-Dam-DsxF or UAS-Dam-DsxM. We have performed DamID-seq on adult male and female fatbody and on ovary. We used two biological replicates for each tissue and sex.
Project description:Trisomy 21 (T21) is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in T21, and to eliminate the noise of the genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for T21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either up- or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins’ fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of DS and wild-type, also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall LADs position was not altered in trisomic cells. However, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results suggest that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome and that GEDDs may therefore contribute to some T21 phenotypes. LaminB1-chromatin interactions in a pair of monozygotic twins discordant for trisomy21
Project description:We asked if the perinuclear position of chromosome arms in C. elegans depends on the histone methyltransferases MET-2 and SET-25. To this end, we performed LMN-1-DamID in wild-type (N2) and mutant (set-25 met-2) strains. LMN-1-DamID signal on chromosome arms was significantly reduced in the mutant. Three biological replicas were performed for each genotype. For one replica dyes were swapped. For each replica methylated DNA amplified from a strain expressing LMN-1-Dam was competitively hybridized against DNA amplified from a strain expressing GFP-Dam.
Project description:The nuclear lamina (NL) interacts with hundreds of large genomic regions termed lamina-associated domains (LADs). The dynamics of these interactions and the relation to epigenetic modifications are poorly understood. We visualized the fate of LADs in single cells using a novel 'molecular contact memory' approach. In each interphase nucleus, only ~30% of LADs are positioned at the periphery; these LADs are in intermittent molecular contact with the NL but remain constrained to the periphery. Upon mitosis, LAD positioning is not detectably inherited but instead is stochastically reshuffled. Contact of individual LADs with the NL correlates with their degree of H3K9 dimethylation in single cells, and inactivation of the H3K9 methyltransferase G9a reduces the NL contact frequencies. These results indicate that nuclear positioning and histone modification of LADs are both stochastic yet linked in single cells. Collectively, these results highlight principles of the dynamic spatial architecture of chromosomes. LaminB1-chromatin interactions were assayed in human HT1080 cells by induction of Dam_LMNB1 expression in a stable cell line with shield1.
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.  RNA tag sequences were optained on an Illumina GAII with the digital gene expression (DGE) module from duplicate RNA samples.  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  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:Specific interactions of the genome with the nuclear lamina (NL) are thought to assist chromosome folding inside the nucleus and to contribute to the regulation of gene expression. High-resolution mapping has recently identified hundreds of large, sharply defined lamina-associated domains (LADs) in the human genome, and suggested that the insulator protein CTCF may help to demarcate these domains. Here, we report the detailed structure of LADs in Drosophila cells, and investigate the putative roles of five insulator proteins in LAD organization. We found that of these five proteins, only SU(HW) binds preferentially at LAD borders and at specific positions inside LADs, while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By knockdown and overexpression studies we demonstrate that SU(HW) weakens LAD – NL interactions by a local antagonistic effect. Our results provide insights into the evolution of LAD organization and reveal a role for SU(HW) in the regulation of genome – NL interactions. DamID experiments for Lamin, CTCF, SU(HW), GAF, DWG, and BEAF-32, and for Lamin after overexpression and after knockdown of SU(HW), were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Every experiment was done in duplicate in the reverse dye orientation. The supplementary file 'GSE20311_DamID_norm_mean.txt' contains the mean log2(Dam-fusion/Dam-only) values of two replicates.