Saccharomyces telomeres assume a non-nucleosomal chromatin structure.

The chromatin structures of the telomeric and subtelomeric regions on chromosomal DNA molecules in Saccharomyces cerevisiae were analyzed using micrococcal nuclease and DNAse I. The subtelomeric repeats X and Y' were assembled in nucleosomes. However, the terminal tracts of C1-3A repeats were protein protected in a particle larger than a nucleosome herein called a telosome. The proximal boundary of the telosome was a DNase I hypersensitive site. This boundary between the telosome and adjacent nucleosomes was completely accessible to Escherichia coli dam methylase when this enzyme was expressed in yeast, whereas a site 250 bp internal to the telomeric repeats was relatively inaccessible. Telosomes could be cleaved from chromosome ends with nuclease and solubilized as protein-DNA complexes. Immunoprecipitation of chromosomal telosomes with antiserum to the RAP1 protein indicated that RAP1 was one component of isolated telosomes. Thus, the termini of chromosomal DNA molecules in yeast are assembled in a non-nucleosomal structure encompassing the entire terminal C1-3A tract. This structure is separated from adjacent nucleosomes by a region of DNA that is highly accessible to enzymes.     

A high-resolution map of nucleosome positioning on a fission yeast centromere

A key element for defining the centromere identity is the incorporation of a specific histone H3, CENPA, known as Cnp1p in Schizosaccharomyces pombe. Previous studies have suggested that functional S. pombe centromeres lack regularly positioned nucleosomes 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 protein genes cnp1, mis18, mis12, nuf2, mal2; overexpression of cnp1; or the deletion of ams2, which encodes a GATA-like factor participating in CENPA incorporation. Bioinformatics analysis of the centromere sequence indicates certain enriched motifs in linker regions between nucleosomes and reveals a sequence bias in nucleosome positioning. In addition, sequence analysis of nucleosome-free regions identifies novel binding sites of Ams2p. We conclude that centromeric nucleosome positions are stable and may be derived from the underlying DNA sequence.     

Molecular organization of terminal repetitive DNA in Beta species

We have isolated families of subtelomeric satellite DNA sequences from species of four sections of the genus Beta and from spinach, a related Chenopodiaceae. Twenty-five clones were sequenced and representative repeats of each family were characterized by Southern blotting and FISH. The families of ApaI restriction satellite repeats were designated pAv34, pAc34, the families of RsaI repeats pRp34, pRn34 and pRs34. The repeating units are 344–362 bp long and 45.7–98.8% homologous with a clear species-specific divergence. Each satellite monomer consists of two subrepeats SR1 and SR2 of 165–184 bp, respectively. The repeats of each subrepeat group are highly identical across species, but share only a homology of 40.8–54.8% with members of the other subrepeat group. Two evolutionary steps could be supposed in the phylogeny of the subtelomeric satellite family: the diversification of an ancestor satellite into groups representing SR1 and SR2 in the progenitor of Beta and Spinacea species, followed by the dimerization and diversification of the resulting 360 bp repeats into section-specific satellite DNA families during species radiation. The chromosomal localization of telomeric, subtelomeric and rDNA tandem repeats was investigated by multi-colour FISH. High-resolution analysis by fibre FISH revealed a unique physical organization of B. vulgaris chromosome ends with telomeric DNA and subtelomeric satellites extending over a maximum of 63 kb and 125 kb, respectively.     

Widespread signatures of recent selection linked to nucleosome positioning in the human lineage

In this study we investigated the strengths and modes of selection associated with nucleosome positioning in the human lineage through the comparison of interspecies and intraspecies rates of divergence. We identify significant evidence for both positive and negative selection linked to human nucleosome positioning for the first time, implicating a widespread and important role for DNA sequence in the location of well-positioned nucleosomes. Selection appears to be acting on particular base substitutions to maintain optimum GC compositions in core and linker regions, with, e.g., unexpectedly elevated rates of C→T substitutions during recent human evolution at linker regions 60-90 bp from the nucleosome dyad but significant depletion of the same substitutions within nucleosome core regions. These patterns are strikingly consistent with the known relationships between genomic sequence composition and nucleosome assembly. By stratifying nucleosomes according to the GC content of their genomic neighborhood, we also show that the strength and direction of selection detected is dictated by local GC content. Intriguingly these signatures of selection are not restricted to nucleosomes in close proximity to exons, suggesting the correct positioning of nucleosomes is not only important in and around coding regions. This analysis provides strong evidence that the genomic sequences associated with nucleosomes are not evolving neutrally, and suggests that underlying DNA sequence is an important factor in nucleosome positioning. Recent signatures of selection linked to genomic features as ubiquitous as the nucleosome have important implications for human genome evolution and disease.     

Asymmetric nucleosomes flank promoters in the budding yeast genome

Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that 5% of budding yeast (Saccharomyces cerevisiae) nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling.Nucleosomes, the fundamental units of chromatin, are dynamic structures characterized by spontaneous conformational fluctuations that lead to reversible loss of histone-DNA and histone-histone contacts. Every nucleosome in the genome has to disassemble at least once during the cell cycle to allow for passage of the DNA replication machinery (Annunziato 2005), and nucleosomes at active regions might turn over several times during each cell cycle (Dion et al. 2007; Deal et al. 2010). Intrinsic to nucleosome dynamics is the formation of nucleosomal intermediates with alternative structures. However, the nature of such intermediate nucleosome structures formed in vivo is not known.Intermediate nucleosome structures can potentially be identified in vivo using base-pair resolution methods that interrogate histone-DNA contacts genome-wide. The traditional method for high-resolution mapping of nucleosomes is to use micrococcal nuclease (MNase) digestion, which digests away linker regions between nucleosomes (Reeves and Jones 1976). Subjecting MNase-digested DNA fragments to paired-end sequencing (MNase-seq) results in a high-resolution map of nucleosome positions (Hughes and Rando 2014). An alternative method for mapping nucleosomes is H4S47C-anchored cleavage mapping (Brogaard et al. 2012b), which has been used to determine the precise position of nucleosomes in yeast genomes (Brogaard et al. 2012a; Moyle-Heyrman et al. 2013). In this method, histone H4 mutant S47C is derivatized ex vivo with a phenanthroline ligand, converting H4 into a site-specific DNA cleavage agent. Using a modified library preparation and a structural model for H4S47C-anchored cleavage, we extended this method to determine the precise position and orientation of half-nucleosomes (hemisomes) at centromeres (Henikoff et al. 2014), thus showing that alternative nucleosome structures can be probed using this method. In this study, we ask whether alternative nucleosome structures can be found in regions of the yeast genome other than at the centromeres.     

Concerted interaction between origin recognition complex (ORC), nucleosomes and replication origin DNA ensures stable ORC–origin binding

Chromosomal replication origins, where DNA replication is initiated, are determined in eukaryotic cells by specific binding of a six‐subunit origin recognition complex (ORC). Many biochemical analyses have showed the detailed properties of the ORC–DNA interaction. However, because of the lack of in vitro analysis, the molecular architecture of the ORC–chromatin interaction is unclear. Recently, mainly from in vivo analyses, a role of chromatin in the ORC–origin interaction has been reported, including the existence of a specific pattern of nucleosome positioning around origins and of a specific interaction between chromatin—or core histones—and Orc1, a subunit of ORC. Therefore, to understand how ORC establishes its interaction with origin in vivo, it is essential to know the molecular mechanisms of the ORC–chromatin interaction. Here, we show that ORC purified from yeast binds more stably to origin‐containing reconstituted chromatin than to naked DNA and forms a nucleosome‐free region at origins. Molecular imaging using atomic force microscopy (AFM) shows that ORC associates with the adjacent nucleosomes and forms a larger complex. Moreover, stable binding of ORC to chromatin requires linker DNA. Thus, ORC establishes its interaction with origin by binding to both nucleosome‐free origin DNA and neighboring nucleosomes.     

Independent and complementary methods for large-scale structural analysis of mammalian chromatin

The fundamental building block of chromatin, the nucleosome, occupies 150 bp of DNA in a spaced arrangement that is a primary determinant in regulation of the genome. The nucleosomal organization of some regions of the human genome has been described, but mapping of these regions has been limited to a few kilobases. We have explored two independent and complementary methods for the high-throughput analysis of mammalian chromatin structure. Through adaptations to a protocol used to map yeast chromatin structure, we determined sites of nucleosomal protection over large regions of the mammalian genome using a tiling microarray. By modifying classical primer extension methods, we localized specific internucleosomally cleaved mammalian genomic sequences using a capillary electrophoresis sequencer in a manner that allows high-throughput nucleotide-resolution characterization of nucleosome protection patterns. We developed algorithms for the automated and unbiased analysis of the resulting data, a necessary step toward large-scale analysis. We validated these assays using the known positions of nucleosomes on the mouse mammary tumor virus LTR, and additionally, we characterized the previously unreported chromatin structure of the LCMT2 gene. These results demonstrate the effectiveness of the combined methods for reliable analysis of mammalian chromatin structure in a high-throughput manner.     

Telomeric DNA-protein interactions of Oxytricha macronuclear DNA

Telomeres of Oxytricha macronuclear chromatin exist as discrete nonnucleosomal DNA-protein complexes, each of which encompasses the terminal 100-150 bp of a macronuclear DNA molecule. We have used chemical and nuclease footprinting to examine the internal structure of these telomeric complexes. Remarkably salt-stable DNA-protein interactions result in methylation-protection of specific guanine residues in the 3'-terminal T4G4T4G4 tail. The methylation pattern seen in vivo and in isolated macronuclei is reconstituted in vitro when purified 55-kD and 43-kD telomere proteins are added to purified macronuclear DNA. Very different interactions are observed between protein and DNA within the region approximately 45-135 bp from the 5' terminus. The DNase I cleavage pattern indicates that this DNA lies on the outside surface of protein but is not part of a nucleosome. Our data suggest that the telomeric complexes have two structural domains characterized by their dissimilar DNA-protein interactions. We propose that functionally equivalent telomeres from other organisms could be accommodated in a similar telomeric chromatin structure.     

A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning

Using the massively parallel technique of sequencing by oligonucleotide ligation and detection (SOLiD; Applied Biosystems), we have assessed the in vivo positions of more than 44 million putative nucleosome cores in the multicellular genetic model organism Caenorhabditis elegans. These analyses provide a global view of the chromatin architecture of a multicellular animal at extremely high density and resolution. While we observe some degree of reproducible positioning throughout the genome in our mixed stage population of animals, we note that the major chromatin feature in the worm is a diversity of allowed nucleosome positions at the vast majority of individual loci. While absolute positioning of nucleosomes can vary substantially, relative positioning of nucleosomes (in a repeated array structure likely to be maintained at least in part by steric constraints) appears to be a significant property of chromatin structure. The high density of nucleosomal reads enabled a substantial extension of previous analysis describing the usage of individual oligonucleotide sequences along the span of the nucleosome core and linker. We release this data set, via the UCSC Genome Browser, as a resource for the high-resolution analysis of chromatin conformation and DNA accessibility at individual loci within the C. elegans genome.     

Nucleosome positioning signals in genomic DNA

Although histones can form nucleosomes on virtually any genomic sequence, DNA sequences show considerable variability in their binding affinity. We have used DNA sequences of Saccharomyces cerevisiae whose nucleosome binding affinities have been experimentally determined (Yuan et al. 2005) to train a support vector machine to identify the nucleosome formation potential of any given sequence of DNA. The DNA sequences whose nucleosome formation potential are most accurately predicted are those that contain strong nucleosome forming or inhibiting signals and are found within nucleosome length stretches of genomic DNA with continuous nucleosome formation or inhibition signals. We have accurately predicted the experimentally determined nucleosome positions across a well-characterized promoter region of S. cerevisiae and identified strong periodicity within 199 center-aligned mononucleosomes studied recently (Segal et al. 2006) despite there being no periodicity information used to train the support vector machine. Our analysis suggests that only a subset of nucleosomes are likely to be positioned by intrinsic sequence signals. This observation is consistent with the available experimental data and is inconsistent with the proposal of a nucleosome positioning code. Finally, we show that intrinsic nucleosome positioning signals are both more inhibitory and more variable in promoter regions than in open reading frames in S. cerevisiae.     

Nucleosomal occupancy and CGG repeat expansion: a comparative analysis of triplet repeat region from mouse and human fragile X mental retardation gene 1

The expansion of CGG repeats in the 5′-untranslated region (5′UTR) of FMR1 gene is the molecular basis of fragile X syndrome in most of the patients. The nature of the flanking sequences in addition to the length and interruption pattern of repeats is predicted to influence CGG repeat instability in the FMR1 gene. We investigated nucleosome occupancy as a contributor to CGG repeat instability in a transgenic mouse model containing unstable (CGG)_26, from human FMR1 cloned downstream of nucleosome-excluding sequence. We observe that the transgene has an open chromatin structure compared to the stable endogenous mouse Fmr1 within the same nucleus. CGG repeats in mouse Fmr1 are flanked by nucleosomes unlike the repeats in the transgene in all the tissues examined. Further in vitro chromatin reconstitution experiments show that DNA fragment without the SV40ori/EPR (nucleosome-excluding sequence) forms more stable chromatin than the one containing it, despite having the same number of CGG repeats. The correlation between nucleosomal organisation of the FMR1 gene and CGG repeat instability was supported by significantly lower frequency of repeat expansion in mice containing an identical transgene without the SV40ori/EPR. Our studies demonstrate that flanking DNA sequences can influence repeat instability through modulation of nucleosome occupancy in the region.     

The prenucleosome,a stable conformational isomer of the nucleosome

Chromatin comprises nucleosomes as well as nonnucleosomal histone–DNA particles. Prenucleosomes are rapidly formed histone–DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome. It consists of a histone octamer associated with ∼80 base pair (bp) of DNA, which is located at a position that corresponds to the central 80 bp of a nucleosome core particle. Monomeric prenucleosomes with free flanking DNA do not spontaneously fold into nucleosomes but can be converted into canonical nucleosomes by an ATP-driven motor protein such as ACF or Chd1. In addition, histone H3K56, which is located at the DNA entry and exit points of a canonical nucleosome, is specifically acetylated by p300 in prenucleosomes relative to nucleosomes. Prenucleosomes assembled in vitro exhibit properties that are strikingly similar to those of nonnucleosomal histone–DNA particles in the upstream region of active promoters in vivo. These findings suggest that the prenucleosome, the only known stable conformational isomer of the nucleosome, is related to nonnucleosomal histone–DNA species in the cell.     

Two breakpoint clusters at fragile site FRA3B form phased nucleosomes

Fragile sites are gaps and breaks in metaphase chromosomes generated by specific culture conditions. Fragile site FRA3B is the most unstable site and is directly involved in the breakpoints of deletion and translocation in a wide spectrum of cancers. To learn about the general characteristics of common fragile sites, we investigated the chromatin structure of the FRA3B site. Because FRA3B spans several hundred kilobases, we focused our study on two breakpoint clusters found in FRA3B. Using various nucleases, we demonstrated that these two regions contain phased nucleosomes, regardless of treatment with aphidicolin. Because these regions are located in intron 4 of the FHIT gene, it is very interesting to observe phased nucleosomes over these regions, which are several hundred kilobases downstream from the promoter. Further, by using nucleosome assembly assays, we demonstrate that these two regions do not contain strong nucleosome positioning elements. These results suggest that other factors appear to cooperate with the DNA sequence of these regions to impart nucleosome phasing. This study provides the first information on the chromatin structure of breakpoint regions in a common fragile site. The observation of phased nucleosomes over these breakpoint regions could offer a foundation to understand the expression of fragile sites.