Rapid and inexpensive preparation of genome-wide nucleosome footprints from model and non-model organisms.

MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste.

Basis of specificity for a conserved and promiscuous chromatin remodeling protein.

Eukaryotic genomes are organized dynamically through the repositioning of nucleosomes. Isw2 is an enzyme that has been previously defined as a genome-wide, nonspecific nucleosome spacing factor. Here, we show that Isw2 instead acts as an obligately targeted nucleosome remodeler in vivo through physical interactions with sequence-specific factors. We demonstrate that Isw2-recruiting factors use small and previously uncharacterized epitopes, which direct Isw2 activity through highly conserved acidic residues in the Isw2 accessory protein Itc1. This interaction orients Isw2 on target nucleosomes, allowing for precise nucleosome positioning at targeted loci. Finally, we show that these critical acidic residues have been lost in the Drosophila lineage, potentially explaining the inconsistently characterized function of Isw2-like proteins. Altogether, these data suggest an 'interacting barrier model,' where Isw2 interacts with a sequence-specific factor to accurately and reproducibly position a single, targeted nucleosome to define the precise border of phased chromatin arrays.

DNA encodes the genetic instructions for life in a long, flexible molecular chain that is packaged up neatly to fit inside cells. Short sections of DNA are wound around proteins to form bundles called nucleosomes, and then spun into chromatin fibres, a more compact form of DNA. While nucleosomes are a fundamental part of this space-saving packaging process, they also play a key regulatory role in gene expression, which is where genes are decoded into working proteins. Placing nucleosomes at regular intervals along DNA invariably controls which parts of the DNA ­ and which genes ­ the cell's machinery can access and 'read' to make proteins. But the nucleosomes' positions are not fixed, and gene expression is a dynamic process. The cell often uncoils and repackages its DNA while molecular motors called chromatin remodelling proteins move nucleosomes up and down the DNA, exposing some genes and obstructing others. One group of chromatin remodelling proteins are called Imitation Switch (ISWI) complexes. It has long been thought that these complexes position nucleosomes with little regard to the underlying DNA sequence or the genes encoded, that is to say in a non-specific way. However, this theory has not been thoroughly tested. It is possible that ISWI complexes actually place nucleosomes on certain parts of DNA at particular times in an organism's development, or in response to other environmental factors. Except how such precision is achieved remains unknown. To test this alternative theory of nucleosome positioning, Donovan et al. studied ISWI proteins and nucleosomes in common baker's yeast. This involved systematically removing sections of ISWI proteins to see whether the complexes could still position nucleosomes, and which parts of the proteins where essential for the job. By doing so, Donovan et al. identified multiple 'targeting' proteins that bind to ISWI proteins and deliver the complexes to specific target sequences of DNA. From there, the complex remodels the nucleosome, positioning it at a specific distance from its landing site on DNA, as further experiments showed. This research provides a new model for explaining how nucleosomes are positioned to package DNA and control gene expression. Donovan et al. have identified a new mechanism of interaction between nucleosomes and chromatin remodelling proteins of the ISWI variety. It is possible that more interactions of this kind will be discovered with further research.

Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning.

Regulation of chromatin structure is essential for controlling access of DNA to factors that require association with specific DNA sequences. Here we describe the development and validation of engineered chromatin remodeling proteins (E-ChRPs) for inducing programmable changes in nucleosome positioning by design. We demonstrate that E-ChRPs function both in vitro and in vivo to specifically reposition target nucleosomes and entire nucleosomal arrays. We show that induced, systematic positioning of nucleosomes over yeast Ume6 binding sites leads to Ume6 exclusion, hyperacetylation, and transcriptional induction at target genes. We also show that programmed global loss of nucleosome-free regions at Reb1 targets is generally inhibitory with mildly repressive transcriptional effects. E-ChRPs are compatible with multiple targeting modalities, including the SpyCatcher and dCas9 moieties, resulting in high versatility and enabling diverse future applications. Thus, engineered chromatin remodeling proteins represent a simple and robust means to probe and disrupt DNA-dependent processes in different chromatin contexts.