Project description:In order to map levels of genome-wide HP1a occupancy we applied DamID (van Steensel & Henikoff, Nat Biotech, 2000; PMID: 10748524) in combination with next-generation sequencing of methylated GATC fragments. Mapping by next-generation sequencing makes it possible to examine heterochromatic regions that were not covered by earlier datasets which were generated using microarrays.
Project description:In most mammalian cell lines, chromatin located at the nuclear periphery is represented by condensed heterochromatin as observed by both microscopy observations and DamID mapping of lamina-associated domains (LADs), enriched in dimethylated Lys9 of histone H3 (H3K9me2). In Drosophila Kc167 cell culture, where LADs were only mapped to the moment, they are neither H3K9me2-enriched, nor overlap with the domains of Heterochromatin Protein 1a (HP1a). Here, using a cell type-specific DamID approach we mapped genome-wide LADs, HP1a and Pc domains from the central brain, Repo-positive glia, Elav-positive neurons and the fat body of Drosophila third instar larvae. Silent or weakly-expressed genes occupy LADs, HP1a and Pc domains, with genes residing in the HP1a-bound LADs expressed at the lowest level. However, a fraction of HP1a domains contain actively expressed genes. The inter-LAD regions that are shared by all cell types are populated by ubiquitously expressed genes, whereas the conserved LADs are less abundant than in mammals. Importantly, in the central brain and in neurons we found strong overlap of HP1a domains with LADs both in the chromosome arms and in pericentromeric regions. In the glia and fat bodies, this overlap was less frequent. Consistent with these results, centromeres appear to reside closer to nuclear lamina in neurons than in Kc167 cells. The discovery of Drosophila peripheral chromatin bound by both HP1a and B-type lamin implies that mechanisms of heterochromatin compaction and attachment to nuclear lamina may be similar in Drosophila and mammals.
Project description:Genome-wide identification of the binding sites of the Drosophila transcription factors Achaete, Asense, E(spl)m3-HLH and Senseless in wing imaginal cells using DamID profiling.
Project description:Heterochromatin protein 1 (HP1) proteins are important regulators of heterochromatin mediated gene silencing and chromosome structure and it is well known as the reader of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me). In Drosophila three different histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9; Su(var)3-9, Setdb1 and G9a. To gain insights on the dependence of HP1a on the three different HKMTs, the division of labor between these methyl transferases and the dependence of HP1a on H3K9me we have studied HP1a binding in relation to H3K9me in mutants of these HKMTs. We show that Su(var)3-9 is responsible for the HP1a H3K9me-dependent binding in pericentromeric regions while Setdb1 controls the HP1a H3K9me-dependent binding to cytological region 2L:31 and together with POF chromosome 4. HP1a binds to the promoters and within gene bodies of active genes in these three regions. More importantly, HP1a bound at promoters of active genes are independent of H3K9me and POF and is associated to heterochromatin protein 2 (HP2) and open chromatin. Our results supports a model where HP1a nucleates with high affinity independent of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.
Project description:Histone modifications represent one of the key factors contributing to proper genome regulation. One of the histone modifications involved in gene silencing is H3K9 methylation, which is found in the chromosomes across different eukaryotes and controlled by SU(VAR)3-9 and its orthologs. Although SU(VAR)3-9 was discovered over two decades ago, little is known about the details of its chromosomal distribution pattern. To fill in this gap, we used DamID-seq approach and obtained high-resolution genome-wide profiles for SU(VAR)3-9 in two somatic and two germline tissues of fruitfly.
Project description:DamID is a powerful technique for identifying regions of the genome bound by a DNA-binding (or DNA-associated) protein. Currently no method exists for automatically processing next-generation sequencing DamID (DamID-seq) data, and the use of DamID-seq datasets with normalisation based on read-counts alone can lead to high background and the loss of bound signal. DamID-seq thus presents novel challenges in terms of normalisation and background minimisation. We describe here damidseq_pipeline, a software pipeline that performs automatic normalisation and background reduction on multiple DamID-seq FASTQ or BAM datasets. Single replicate profiling of pol II occupancy in 3rd instar larval neuroblasts of Drosophila