Dataset Information

Global histone protein surface accessibility in yeast indicates a uniformly loosely packed genome with canonical nucleosomes.

ABSTRACT:

Background

The vast majority of methods available to characterize genome-wide chromatin structure exploit differences in DNA accessibility to nucleases or chemical crosslinking. We developed a novel method to gauge genome-wide accessibility of histone protein surfaces within nucleosomes by assessing reactivity of engineered cysteine residues with a thiol-specific reagent, biotin-maleimide (BM).

Results

Yeast nuclei were obtained from cells expressing the histone mutant H2B S116C, in which a cysteine resides near the center of the external flat protein surface of the nucleosome. BM modification revealed that nucleosomes are generally equivalently accessible throughout the S. cerevisiae genome, including heterochromatic regions, suggesting limited, higher-order chromatin structures in which this surface is obstructed by tight nucleosome packing. However, we find that nucleosomes within 500 bp of transcription start sites exhibit the greatest range of accessibility, which correlates with the density of chromatin remodelers. Interestingly, accessibility is not well correlated with RNA polymerase density and thus the level of gene expression. We also investigated the accessibility of cysteine mutations designed to detect exposure of histone surfaces internal to the nucleosome thought to be accessible in actively transcribed genes: H3 102, is at the H2A-H2B dimer/H3-H4 tetramer interface, and H3 A110C, resides at the H3-H3 interface. However, in contrast to the external surface site, we find that neither of these internal sites were found to be appreciably exposed.

Conclusions

Overall, our finding that nucleosomes surfaces within S. cerevisiae chromatin are equivalently accessible genome-wide is consistent with a globally uncompacted chromatin structure lacking substantial higher-order organization. However, we find modest differences in accessibility that correlate with chromatin remodelers but not transcription, suggesting chromatin poised for transcription is more accessible than actively transcribed or intergenic regions. In contrast, we find that two internal sites remain inaccessible, suggesting that such non-canonical nucleosome species generated during transcription are rapidly and efficiently converted to canonical nucleosome structure and thus not widely present in native chromatin.

SUBMITTER: Marr LT

PROVIDER: S-EPMC7802155 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Global histone protein surface accessibility in yeast indicates a uniformly loosely packed genome with canonical nucleosomes.

Marr Luke T LT Ocampo Josefina J Clark David J DJ Hayes Jeffrey J JJ

Epigenetics & chromatin 20210111 1

<h4>Background</h4>The vast majority of methods available to characterize genome-wide chromatin structure exploit differences in DNA accessibility to nucleases or chemical crosslinking. We developed a novel method to gauge genome-wide accessibility of histone protein surfaces within nucleosomes by assessing reactivity of engineered cysteine residues with a thiol-specific reagent, biotin-maleimide (BM).<h4>Results</h4>Yeast nuclei were obtained from cells expressing the histone mutant H2B S116C, ...[more]

PMID: 33430969

Similar Datasets

Project description:BACKGROUND: The Streptophyta comprises all land plants and six groups of charophycean green algae. The scaly biflagellate Mesostigma viride (Mesostigmatales) and the sarcinoid Chlorokybus atmophyticus (Chlorokybales) represent the earliest diverging lineages of this phylum. In trees based on chloroplast genome data, these two charophycean green algae are nested in the same clade. To validate this relationship and gain insight into the ancestral state of the mitochondrial genome in the Charophyceae, we sequenced the mitochondrial DNA (mtDNA) of Chlorokybus and compared this genome sequence with those of three other charophycean green algae and the bryophytes Marchantia polymorpha and Physcomitrella patens. RESULTS: The Chlorokybus genome differs radically from its 42,424-bp Mesostigma counterpart in size, gene order, intron content and density of repeated elements. At 201,763-bp, it is the largest mtDNA yet reported for a green alga. The 70 conserved genes represent 41.4% of the genome sequence and include nad10 and trnL(gag), two genes reported for the first time in a streptophyte mtDNA. At the gene order level, the Chlorokybus genome shares with its Chara, Chaetosphaeridium and bryophyte homologues eight to ten gene clusters including about 20 genes. Notably, some of these clusters exhibit gene linkages not previously found outside the Streptophyta, suggesting that they originated early during streptophyte evolution. In addition to six group I and 14 group II introns, short repeated sequences accounting for 7.5% of the genome were identified. Mitochondrial trees were unable to resolve the correct position of Mesostigma, due to analytical problems arising from accelerated sequence evolution in this lineage. CONCLUSION: The Chlorokybus and Mesostigma mtDNAs exemplify the marked fluidity of the mitochondrial genome in charophycean green algae. The notion that the mitochondrial genome was constrained to remain compact during charophycean evolution is no longer tenable. Our data raise the possibility that the emergence of land plants was not associated with a substantial gain of intergenic sequences by the mitochondrial genome.

Project description:Prokaryotic Argonaute proteins (pAgos) widely participate in hosts to defend against the invasion of nucleic acids. Compared with the CRISPR-Cas system, which requires a specific motif on the target and can only use RNA as guide, pAgos exhibit precise endonuclease activity on any arbitrary target sequence and can use both RNA and DNA as guide, thus rendering great potential for genome editing applications. Hitherto, most in-depth studies on the structure-function relationship of pAgos were conducted on thermophilic ones, functioning at ∼60 to 100°C, whose structures were, however, determined experimentally at much lower temperatures (20-33°C). It remains unclear whether these low-temperature structures can represent the true conformations of the thermophilic pAgos under their physiological conditions. The present work studied three pAgos, PfAgo, TtAgo and CbAgo, whose physiological temperatures differ significantly (95, 75 and 37°C). By conducting thorough experimental and simulation studies, we found that thermophilic pAgos (PfAgo and TtAgo) adopt a loosely-packed structure with a partially-melted surface at the physiological temperatures, largely different from the compact crystalline structures determined at moderate temperatures. In contrast, the mesophilic pAgo (CbAgo) assumes a compact crystalline structure at its optimal function temperature. Such a partially-disrupted structure endows thermophilic pAgos with great flexibility both globally and locally at the catalytic sites, which is crucial for them to achieve high DNA-cleavage activity. To further prove this, we incubated thermophilic pAgos with urea to purposely disrupt their structures, and the resulting cleavage activity was significantly enhanced below the physiological temperature, even at human body temperature. Further testing of many thermophilic Agos present in various thermophilic prokaryotes demonstrated that their structures are generally disrupted under physiological conditions. Therefore, our findings suggest that the highly dynamical structure with a partially-melted surface, distinct from the low-temperature crystalline structure, could be a general strategy assumed by thermophilic pAgos to achieve the high DNA-cleavage activity.

Dataset Information

Global histone protein surface accessibility in yeast indicates a uniformly loosely packed genome with canonical nucleosomes.

Background

Results

Conclusions

Publications

Global histone protein surface accessibility in yeast indicates a uniformly loosely packed genome with canonical nucleosomes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets