Project description:The positioning of nucleosomes within the coding regions of eukaryotic genes is aligned with respect to transcriptional start sites. This organization is likely to influence many genetic processes, requiring access to the underlying DNA. Here we show that the combined action of Isw1 and Chd1 nucleosome spacing enzymes is required to maintain this organization. In the absence of these enzymes regular positioning of the majority of nucleosomes is lost. Exceptions include the region upstream of the promoter, the +1 nucleosome and a subset of locations distributed throughout coding regions where other factors are likely to be involved. These observations indicated that ATP-dependent remodeling enzymes are responsible for directing the positioning of the majority of nucleosomes within the Saccharomyces cerevisiae genome. Examination of nucleosome positioning in mutants of snf2-related enzymes Other data used in this study are provided in GEO Series GSE31301 and GSE31833.
Project description:Numerous nucleosome remodeling enzymes tightly regulate nucleosome positions in eukaryotic cells. Transcription and statistical positioning of nucleosomes may also contribute to proper nucleosome organization. Individual contributions remain controversial due to strong redundancy of processes acting on the nucleosome landscape. By incisive yeast genome engineering we radically decreased their redundancy. We find the transcriptional machinery to be disruptive of evenly spaced nucleosomes, and proper nucleosome density critical for their biogenesis. INO80 spaces nucleosomes in vivo and positions the first nucleosome covering genes. It requires its Arp8 and Ies2 subunits, but unexpectedly not the Nhp10 module, for spacing. Whereas H2A.Z stimulates INO80 in vitro, its presence is dispensable for INO80 +1 positioning function in vivo. DNA damage, recombination and transposon integration assays suggest that evenly spaced nucleosomes protect cells against genotoxic stress. We derive a unifying model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.
Project description:We addressed the roles of three nucleosome spacing enzymes (ISW1, ISW2 and CHD1) in specifying chromatin organization in S. cerevisiae.
Project description:The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. However, nucleosome number in cells was considered fixed, and no condition was described where nucleosome number was reduced. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker and variant histones, and a correspondingly reduced number of nucleosomes. Yeast nhp6 mutants lacking NHP6A and –B proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform, and our results can be modelled assuming that different nucleosomal sites compete for the available histones: sites with high affinity are almost always packaged into nucleosomes both in wt and nucleosome-depleted cells, whereas sites with low affinity are less frequently packaged in nucleosome-depleted cells. We suggest that by modulating the occupancy of nucleosomes histone availability may constitute a novel layer of epigenetic regulation.