Project description:The nature of chromatin as regular succession of nucleosomes has gained iconic status. The average nucleosome repeat length (NRL) determined by classical means serves as index for bulk chromatin of a given specimen. However, this value is dominated by regular heterochromatin since nucleosomal arrays are often not regular at individual single copy sequences. To obtain a measure for nucleosome regularity in euchromatin we subjected nucleosome dyad profiles to autocorrelation and spectral density analyses. This revealed variation in nucleosome regularity and NRL at different types of euchromatin and yielded a comprehensive catalog of regular phased nucleosome arrays (PNA). The absence of the nucleosome sliding factor ACF1 correlated with global loss of regularity in euchromatin and increased NRL and compromised phasing at a novel type of PNA. Our approach is generally applicable to characterize hallmarks of euchromatin organization.

2018-01-01 | GSE85404 | GEO

ACF1 regulates nucleosome spacing in Drosophila euchromatin [MNase-seq BG3]

2018-01-01 | GSE85406 | GEO

ACF1 regulates nucleosome spacing in Drosophila euchromatin [Mnase-seq embryos]

2018-01-01 | GSE85405 | GEO

Quantitative MNase-seq accurately maps nucleosome occupancy levels

Project description:Chromatin mapping using micrococcal nuclease (MNase) has been the standard tool for mapping nucleosomes for >40 years. When coupled with DNA sequencing, MNase-seq can provide base-pair-resolution nucleosome maps. However, determining nucleosome occupancy using MNase-seq has been hampered by its aggressive endo-/exo-nuclease activities, whereby cleavages within linker regions produce oligo- and mono-nucleosomes whereas cleavages within nucleosomes destroy them. Here we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time-course. As expected, DNaseI hypersensitive sites and transcription units are digested by MNase at elevated rates, and the apparent deficiency of nucleosomes at 3’ ends of Drosophila genes is an artifact of MNase preference for AT-rich DNA. Surprisingly, we observed no overall differences between Drosophila euchromatin and heterochromatin, which implies that heterochromatin compaction does not render nucleosomal DNA less accessible than euchromatin.

2019-08-27 | GSE128689 | GEO

Drosophila melanogaster

Project description:modENCODE RNA-Seq of Drosophila BG3 small RNAs

| PRJNA119271 | ENA

Genome-wide chromatin reconstitution with Drosophila embryo extract and nucleosome positioning by CG7372 and su(Hw)

Project description:We have reconstituted chromatin in vitro on a genome-wide level by incubating total genomic Drosophila melanogaster DNA in D. melanogaster preblastoderm embryo extract. We analyzed chromatin reconstituted with 1) extract from wildtype embryos, 2) extract from embryos lacking the chromatin remodeling enzyme subunit Acf , and 3) extract from Acf-mutant embryos supplemented with recombinant ACF (Acf + Iswi). As a comparison, we also reconstituted chromatin on genomic DNA by salt gradient dialysis. We determined nucleosome positions in reconstituted chromatin by MNase-digestion followed by paired-end sequencing of mononucleosomal fragments. We also determined nucleosome positions in D. melanogaster BG3-c2 cells depleted for the proteins su(Hw) and CG7372 by RNA interference.

2018-08-10 | GSE118335 | GEO