Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.
- MeSH
- acetylace MeSH
- chromatin * metabolismus MeSH
- histony metabolismus MeSH
- lidé MeSH
- myši MeSH
- nukleozomy * metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- protaminy metabolismus MeSH
- restrukturace chromatinu MeSH
- sperma metabolismus MeSH
- spermatidy metabolismus MeSH
- spermatogeneze fyziologie MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.
- MeSH
- centromera MeSH
- histonový kód MeSH
- homologní rekombinace * MeSH
- nukleozomy metabolismus MeSH
- proteiny buněčného cyklu antagonisté a inhibitory metabolismus MeSH
- regulace genové exprese u hub * MeSH
- represorové proteiny fyziologie MeSH
- Schizosaccharomyces pombe - proteiny antagonisté a inhibitory metabolismus fyziologie MeSH
- Schizosaccharomyces genetika MeSH
- transkripční faktory antagonisté a inhibitory metabolismus MeSH
- umlčování genů * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The three-dimensional (3D) organization of chromatin plays a crucial role in the regulation of gene expression. Chromatin conformation is strongly affected by the composition, structural features and dynamic properties of the nucleosome, which in turn determine the nature and geometry of interactions that can occur between neighboring nucleosomes. Understanding how chromatin is spatially organized above the nucleosome level is thus essential for understanding how gene regulation is achieved. Towards this end, great effort has been made to understand how an array of nucleosomes folds into a regular chromatin fiber. This review summarizes new insights into the 3D structure of the chromatin fiber that were made possible by recent advances in cryo-electron microscopy.
The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.
OBJECTIVE: While circulating nucleosome levels are high in obese mouse models, it is unknown where these nucleosomes originate from and whether they are a marker of cardio-metabolic health in humans. Here, we aimed to determine whether an association exists between circulating nucleosomes and the risk of developing obesity, metabolic syndrome (MetS) and/or a dysfunctional cardiovascular performance. METHODS: We randomly selected 120 participants of the Kardiovize Brno 2030 study across three BMI strata: BMI 18-25, 25-30, and > 30. We assessed the association between circulating nucleosome levels and the risk of obesity, MetS, and poor cardiovascular health. We then cultured human neutrophils, adipocytes, and hepatoma cells to study nucleosome origins in a fat-rich environment. RESULTS: Circulating nucleosome levels positively correlated with BMI (R = 0.602, p < 0.05), fatty liver index (R = 0.622, p < 0.05), left ventricular mass (R = 0.457, p < 0.05), and associated with MetS (p < 0.001) and poor cardiovascular health (p < 0.001). Incubating neutrophils with 1-10 μM free fatty acids triggered nucleosome production without concomitant cell death. Nucleosomes were not produced during pre-adipocyte differentiation or upon incubation of hepatic cells with palmitic acid. CONCLUSIONS: Neutrophils are a bona fide source of circulating nucleosomes in an obesogenic environment and in overweight/obese patients. High nucleosome levels are associated with MetS and cardiovascular performance, and might represent novel candidate biomarkers for cardio-metabolic health.
- MeSH
- buňky Hep G2 MeSH
- dospělí MeSH
- index tělesné hmotnosti MeSH
- kardiovaskulární nemoci etiologie MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- metabolický syndrom krev komplikace MeSH
- nadváha krev komplikace metabolismus MeSH
- neutrofily cytologie metabolismus MeSH
- nukleozomy metabolismus MeSH
- obezita krev komplikace metabolismus MeSH
- senioři MeSH
- tukové buňky cytologie metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.
- MeSH
- adenosintrifosfát metabolismus farmakologie MeSH
- bezbuněčný systém MeSH
- chromozomální proteiny, nehistonové fyziologie MeSH
- DNA bakterií metabolismus MeSH
- DNA footprinting MeSH
- fungální proteiny fyziologie MeSH
- histony genetika metabolismus MeSH
- mikroskopie atomárních sil MeSH
- multiproteinové komplexy fyziologie MeSH
- nukleozomy fyziologie ultrastruktura MeSH
- plazmidy chemie MeSH
- proteiny vázající RNA fyziologie MeSH
- rekombinantní proteiny metabolismus MeSH
- restrikční endonukleasy typu II MeSH
- restrukturace chromatinu genetika fyziologie MeSH
- Saccharomyces cerevisiae metabolismus ultrastruktura MeSH
- Xenopus laevis genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.
- MeSH
- DNA chemie genetika metabolismus MeSH
- elektronová kryomikroskopie MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- genetické vektory chemie metabolismus MeSH
- histony chemie genetika metabolismus MeSH
- hydroxylový radikál chemie MeSH
- interakční proteinové domény a motivy MeSH
- klonování DNA MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- nukleozomy chemie metabolismus ultrastruktura MeSH
- osmolární koncentrace MeSH
- protein 1 vytvářející nukleozómy chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Xenopus laevis MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Psoriasis is a multifactorial chronic inflammatory disease. We aimed to examine blood levels of nucleosomes derived from apoptotic cells, nucleosomal cell-free DNA (cfDNA) and immune-inflammatory biomarkers tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), and interleukin 6 (IL-6) in psoriatic subjects. The study included 28 patients with exacerbated psoriasis vulgaris and 22 controls. The clinical and laboratory investigations included the determination of PASI score, BMI, cfDNA (by real-time PCR), nucleosomes, TNF-α, CRP, and IL-6. The range of PASI score in psoriatic patients was 10-34 (median 19). In the patients, we found significantly elevated levels (p < 0.001) of cfDNA, nucleosomes, TNF-α, CRP, and IL-6. We did not find any significant relationship between the analyzed parameters in either group (i.e., experimental or control). Elevated levels of the biomarkers of inflammation (TNF-α, CRP, and IL-6) and the indicators of apoptosis (cfDNA, circulating nucleosomes) proved that exacerbated psoriasis vulgaris is associated with a high degree of systemic inflammatory responses and dysregulated apoptotic pathways.
- MeSH
- apoptóza MeSH
- biologické markery krev MeSH
- C-reaktivní protein analýza MeSH
- dospělí MeSH
- interleukin-6 krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- nukleozomy genetika metabolismus MeSH
- progrese nemoci MeSH
- psoriáza krev patologie MeSH
- senioři MeSH
- stupeň závažnosti nemoci MeSH
- TNF-alfa krev MeSH
- volné cirkulující nukleové kyseliny krev MeSH
- zánět MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Chromatin Assembly Factor 1 (CAF-1) is a major nucleosome assembly complex which functions particularly during DNA replication and repair. Here we studied how the nucleosome landscape changes in a CAF-1 mutant in the model plant Arabidopsis thaliana. Globally, most nucleosomes were not affected by loss of CAF-1, indicating the presence of efficient alternative nucleosome assemblers. Nucleosomes that we found depleted in the CAF-1 mutant were enriched in non-transcribed regions, consistent with the notion that CAF-1-independent nucleosome assembly can compensate for loss of CAF-1 mainly in transcribed regions. Depleted nucleosomes were particularly enriched in proximal promoters, suggesting that CAF-1-independent nucleosome assembly mechanisms are often not efficient upstream of transcription start sites. Genes related to plant defense were particularly prone to lose nucleosomes in their promoters upon CAF-1 depletion. Reduced nucleosome occupancy at promoters of many defense-related genes is associated with a primed gene expression state that may considerably increase plant fitness by facilitating plant defense. Together, our results establish that the nucleosome landscape in Arabidopsis is surprisingly robust even in the absence of the dedicated nucleosome assembly machinery CAF-1 and that CAF-1-independent nucleosome assembly mechanisms are less efficient in particular genome regions.
- MeSH
- Arabidopsis genetika imunologie metabolismus MeSH
- chromatin genetika MeSH
- faktor 1 pro uspořádání chromatinu genetika metabolismus MeSH
- imunita rostlin genetika MeSH
- mutace MeSH
- nukleozomy genetika metabolismus MeSH
- oprava DNA genetika MeSH
- počátek transkripce MeSH
- promotorové oblasti (genetika) genetika MeSH
- replikace DNA genetika MeSH
- restrukturace chromatinu genetika MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication.
- MeSH
- buněčný cyklus genetika fyziologie MeSH
- chromatin metabolismus MeSH
- G2 fáze genetika MeSH
- histony metabolismus MeSH
- HL-60 buňky MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- nukleozomy metabolismus MeSH
- replikace DNA genetika fyziologie MeSH
- S fáze genetika MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
