Project description:Positioned nucleosomes limit the access of proteins to DNA and implement regulatory features encoded in eukaryotic genomes. Here we generated the first genome-wide nucleosome positioning map for Schizosaccharomyces pombe and annotated transcription start and termination sites genome-wide. Using this resource we found surprising differences compared to the nucleosome organization in the distantly related yeast Saccharomyces cerevisiae [the cerevisiae data has been published by others (PMID: 17873876) and the raw data is deposited at ArrayExpress(E-MEXP-1172)]. DNA sequence guides nucleosome positioning differently, e.g., poly(dA:dT) elements are not enriched in S. pombe nucleosome-depleted regions (NDRs). Regular nucleosomal arrays emanate more asymmetrically, i.e., mainly co-directionally with transcription, from promoter NDRs, but promoters harbouring the histone variant H2A.Z show regular arrays also upstream. Regular nucleosome phasing in S. pombe has a very short repeat length of 154 base pairs, and requires a remodeler, Mit1, conserved in humans but not found in S. cerevisiae. Nucleosome positioning mechanisms are evidently not universal but evolutionarily plastic.
Project description:Knowing the exact positions of nucleosomes not only advances our understanding of their role in gene regulation, but also the mechanisms that underlie between-species variation in chromatin structure. We have generated a chemical map of nucleosomes in vivo in Schizosaccharomyces pombe at base pair resolution. This new map reveals that S.pombe genome shares a similar periodic linker length distribution with Saccharomyces cerevisiae, but with major distinctions in nucleosomal/linker DNA sequence features. In S.pombe, A/T rich sequences are enriched in the nucleosome core region, particularly +/-20 bp of dyad, while they are disfavored in S.cerevisiae nucleosomes. The poly (dA-dT) tracts only slightly affect the nucleosome occupancy in S.pombe; and they possess preferential rotational positions within the nucleosome core with significant enrichment in the 10-30 bp region from the dyad for longer tracts. S.pombe does not have well-defined nucleosome free region immediately upstream of most transcription start sites (TSS), instead the -1 nucleosome is positioned with regular distance to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. The nucleosomes around TSS show more pronounced bidirectional phasing when the intergenic distance is relatively short, and the downstream nucleosome positioning is strongly correlated with DNA sequence features. We discovered that heterochromatin regions tend to have sparse nucleosome positioning, mixed with both well-positioned and fuzzy nucleosomes. The S.pombe map suggests that some of nucleosome positioning codes, formerly thought to be intrinsic, may largely depend on species-specific extrinsic factors including linker histone, chromatin remodelers and other DNA-binding proteins. 2 samples were analyzed with high throughput paired-end parallel sequencing. Both samples were created using the same chemical mapping protocol
Project description:A key element for defining the centromere identity is the incorporation of a specific histone H3, CENP-A, known as Cnp1p in S. pombe. Previous studies have suggested that functional S. pombe centromeres lack nucleosome arrays and may involve chromatin remodeling as a key step of kinetochore assembly. We used tiling microarrays to show that nucleosomes are in fact positioned in regular intervals in the core of centromere 2, providing the first high resolution map of regional centromere chromatin. Nucleosome locations are not disrupted by mutations in kinetochore proteins cnp1, mis18, mis12, nuf2, mal2, overexpression of Cnp1p, or deletion of ams2. Bioinformatic analysis of the centromere sequence indicates certain enriched motifs in linker regions between nucleosomes and reveals a sequence-bias in nucleosome positioning. We conclude that centromeric nucleosome positions are stable and may be derived from the underlying DNA sequence. In addition, sequence analysis of nucleosome-free regions identifies novel binding sites for the GATA-like protein Ams2p, which participates in CENP-A incorporation. Keywords: Nucleosome Mapping Study
Project description:A key element for defining the centromere identity is the incorporation of a specific histone H3, CENP-A, known as Cnp1p in S. pombe. Previous studies have suggested that functional S. pombe centromeres lack nucleosome arrays and may involve chromatin remodeling as a key step of kinetochore assembly. We used tiling microarrays to show that nucleosomes are in fact positioned in regular intervals in the core of centromere 2, providing the first high resolution map of regional centromere chromatin. Nucleosome locations are not disrupted by mutations in kinetochore proteins cnp1, mis18, mis12, nuf2, mal2, overexpression of Cnp1p, or deletion of ams2. Bioinformatic analysis of the centromere sequence indicates certain enriched motifs in linker regions between nucleosomes and reveals a sequence-bias in nucleosome positioning. We conclude that centromeric nucleosome positions are stable and may be derived from the underlying DNA sequence. In addition, sequence analysis of nucleosome-free regions identifies novel binding sites for the GATA-like protein Ams2p, which participates in CENP-A incorporation. Keywords: Nucleosome Mapping Study Entire cnt regions and histone-related genes were tiled at 1-5 bp spacing using 60-mer probes.
Project description:Hrp3_Purification from Schizosaccharomyces pombe 972h- Eukaryotic genome is composed of repeating units of nucleosomes to form chromatin arrays. A canonical gene is marked by nucleosome free region (NFR) at its 5’ end followed by uniformly spaced arrays of nucleosomes. In fission yeast we show both biochemically and in vivo that both Hrp1 and Hrp3 are key determinants of uniform spacing of genic arrays.
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 pervasive pattern are unclear, but nucleosome remodeling ATPases likely are critical. Now we employ deletion mutants to study the role of nucleosome remodeling ATPases in global nucleosome positioning in S. pombe and the corresponding changes in expression patterns. We find a striking evolutionary shift in remodeling enzyme usage between budding and fission yeast. The S. pombe RSC remodeling complex seems not involved in nucleosome positioning, despite its prominent role in S. cerevisiae. While lacking ISWI-type remodelers, S. pombe has two CHD1-type ATPases, Hrp1 and Hrp3. We demonstrate nucleosome spacing activity for both in vitro, and together they are essential for linking regular genic arrays to most TSSs in vivo. Impaired chromatin may but need not lead to changes in transcription. The absence of both causes changed expression for about 20% and increased antisense transcription for 15% of all annotated elements.
Project description:Knowing the exact positions of nucleosomes not only advances our understanding of their role in gene regulation, but also the mechanisms that underlie between-species variation in chromatin structure. We have generated a chemical map of nucleosomes in vivo in Schizosaccharomyces pombe at base pair resolution. This new map reveals that S.pombe genome shares a similar periodic linker length distribution with Saccharomyces cerevisiae, but with major distinctions in nucleosomal/linker DNA sequence features. In S.pombe, A/T rich sequences are enriched in the nucleosome core region, particularly +/-20 bp of dyad, while they are disfavored in S.cerevisiae nucleosomes. The poly (dA-dT) tracts only slightly affect the nucleosome occupancy in S.pombe; and they possess preferential rotational positions within the nucleosome core with significant enrichment in the 10-30 bp region from the dyad for longer tracts. S.pombe does not have well-defined nucleosome free region immediately upstream of most transcription start sites (TSS), instead the -1 nucleosome is positioned with regular distance to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. The nucleosomes around TSS show more pronounced bidirectional phasing when the intergenic distance is relatively short, and the downstream nucleosome positioning is strongly correlated with DNA sequence features. We discovered that heterochromatin regions tend to have sparse nucleosome positioning, mixed with both well-positioned and fuzzy nucleosomes. The S.pombe map suggests that some of nucleosome positioning codes, formerly thought to be intrinsic, may largely depend on species-specific extrinsic factors including linker histone, chromatin remodelers and other DNA-binding proteins.