Project description:Eukaryotic gene expression occurs in the context of structurally distinct chromosomal domains such as the relatively open, gene-rich, and transcriptionally active euchromatin and the condensed and gene-poor heterochromatin where its specific chromatin environment inhibits transcription. To study gene silencing by heterochromatin, we created a minichromosome reporter system where the gene silencer elements were used to repress the URA3 reporter gene. The minichromosome reporters were propagated in yeast Saccharomyces cerevisiae at a stable copy number. Conduction of gene silencing through nucleosome arrays was studied by placing various repeats of clone-601 DNA with high affinity for histones between the silencer and reporter in the yeast minichromosomes. High-resolution chromatin mapping with micrococcal nuclease showed that the clone-601 nucleosome positioning downstream of the HML-E gene silencing element was not significantly altered by chromatin silencing. Using URA3 reporter assays, we observed that gene silencing was conducted through arrays of up to eight nucleosomes. We showed that the shorter nucleosome repeat lengths, typical of yeast (167 and 172 bp), were more efficient in conducting silencing in vivo compared to the longer repeats (207 bp) typical of higher eukaryotes. Both the longer and the shorter repeat lengths were able to conduct silencing in minichromosomes independently of clone-601 nucleosome positioning orientations vs. the silencer element. We suggest that the shorter nucleosome linkers are more suitable for conducting gene silencing than the long repeats in yeast due to their higher propensity to support native-like chromatin higher-order folding.
Project description:<h4>Background</h4>The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA.<h4>Results</h4>We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active). This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent.<h4>Conclusions</h4>These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.
Project description:As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the '601' nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.
Project description:Nucleosome positioning governs access to eukaryotic genomes. Many genes show a stereotypic organisation at their 5'end: a nucleosome free region just upstream of the transcription start site (TSS) followed by a regular nucleosomal array over the coding region. The determinants for this pattern are unclear, but nucleosome remodelers are likely critical. Here we study the role of remodelers in global nucleosome positioning in S. pombe and the corresponding changes in expression. We find a striking evolutionary shift in remodeler usage between budding and fission yeast. The S. pombe RSC complex does not seem to be involved in nucleosome positioning, despite its prominent role in S. cerevisiae. While S. pombe lacks ISWI-type remodelers, it has two CHD1-type ATPases, Hrp1 and Hrp3. We demonstrate nucleosome spacing activity for Hrp1 and Hrp3 in vitro, and that together they are essential for linking regular genic arrays to most TSSs in vivo. Impaired arrays in the absence of either or both remodelers may lead to increased cryptic antisense transcription, but overall gene expression levels are only mildly affected.
Project description:Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.
Project description:Heterochromatic domains are complex structures composed of nucleosome arrays that are bound by silencing factors. This composition raises the possibility that certain configurations of nucleosome arrays facilitate heterochromatic silencing. We tested this possibility in <i>Saccharomyces cerevisiae</i> by systematically altering the distance between heterochromatic nucleosome-depleted regions (NDRs), which is predicted to affect local nucleosome positioning by limiting how nucleosomes can be packed between NDRs. Consistent with this prediction, serial deletions that altered the distance between heterochromatic NDRs revealed a striking oscillatory relationship between inter-NDR distance and defects in nucleosome positioning. Furthermore, conditions that caused poor nucleosome positioning also led to defects in both heterochromatin stability and the ability of cells to generate and inherit epigenetic transcriptional states. These findings strongly suggest that nucleosome positioning can contribute to formation and maintenance of functional heterochromatin and point to previously unappreciated roles of NDR positioning within heterochromatic domains.
Project description:Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal "fingerprint" as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding.
Project description:In the yeast genome, a large proportion of nucleosomes occupy well-defined and stable positions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genome remains controversial. Through quantitative analysis of nucleosome positioning, we show that sequence changes distort the nucleosomal pattern at the level of individual nucleosomes in three species of Schizosaccharomyces and in Saccharomyces cerevisiae This effect is equally detected in transcribed and nontranscribed regions, suggesting the existence of sequence elements that contribute to positioning. To identify such elements, we incorporated information from nucleosomal signatures into artificial synthetic DNA molecules and found that they generated regular nucleosomal arrays indistinguishable from those of endogenous sequences. Strikingly, this information is species-specific and can be combined with coding information through the use of synonymous codons such that genes from one species can be engineered to adopt the nucleosomal organization of another. These findings open the possibility of designing coding and noncoding DNA molecules capable of directing their own nucleosomal organization.
Project description:Nucleosomes form the fundamental building blocks of eukaryotic chromatin, and previous attempts to understand the principles governing their genome-wide distribution have spurred much interest and debate in biology. In particular, the precise role of DNA sequence in shaping local chromatin structure has been controversial. This paper rigorously quantifies the contribution of hitherto-debated sequence features-including G+C content, 10.5?bp periodicity, and poly(dA:dT) tracts-to three distinct aspects of genome-wide nucleosome landscape: occupancy, translational positioning and rotational positioning. Our computational framework simultaneously learns nucleosome number and nucleosome-positioning energy from genome-wide nucleosome maps. In contrast to other previous studies, our model can predict both in vitro and in vivo nucleosome maps in Saccharomyces cerevisiae. We find that although G+C content is the primary determinant of MNase-derived nucleosome occupancy, MNase digestion biases may substantially influence this GC dependence. By contrast, poly(dA:dT) tracts are seen to deter nucleosome formation, regardless of the experimental method used. We further show that the 10.5?bp nucleotide periodicity facilitates rotational but not translational positioning. Applying our method to in vivo nucleosome maps demonstrates that, for a subset of genes, the regularly-spaced nucleosome arrays observed around transcription start sites can be partially recapitulated by DNA sequence alone. Finally, in vivo nucleosome occupancy derived from MNase-seq experiments around transcription termination sites can be mostly explained by the genomic sequence. Implications of these results and potential extensions of the proposed computational framework are discussed.
Project description:Because DNA packaging in nucleosomes modulates its accessibility to transcription factors (TFs), unraveling the causal determinants of nucleosome positioning is of great importance to understanding gene regulation. Although there is evidence that intrinsic sequence specificity contributes to nucleosome positioning, the extent to which other factors contribute to nucleosome positioning is currently highly debated. Here we obtained both in vivo and in vitro reference maps of positions that are either consistently covered or free of nucleosomes across multiple experimental data-sets in Saccharomyces cerevisiae. We then systematically quantified the contribution of TF binding to nucleosome positioning using a rigorous statistical mechanics model in which TFs compete with nucleosomes for binding DNA. Our results reconcile previous seemingly conflicting results on the determinants of nucleosome positioning and provide a quantitative explanation for the difference between in vivo and in vitro positioning. On a genome-wide scale, nucleosome positioning is dominated by the phasing of nucleosome arrays over gene bodies, and their positioning is mainly determined by the intrinsic sequence preferences of nucleosomes. In contrast, larger nucleosome free regions in promoters, which likely have a much more significant impact on gene expression, are determined mainly by TF binding. Interestingly, of the 158 yeast TFs included in our modeling, we find that only 10-20 significantly contribute to inducing nucleosome-free regions, and these TFs are highly enriched for having direct interactions with chromatin remodelers. Together our results imply that nucleosome free regions in yeast promoters results from the binding of a specific class of TFs that recruit chromatin remodelers.