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Science Advances Jun 2024The hierarchical chromatin organization begins with formation of nucleosomes, which fold into chromatin domains punctuated by boundaries and ultimately chromosomes. In a...
The hierarchical chromatin organization begins with formation of nucleosomes, which fold into chromatin domains punctuated by boundaries and ultimately chromosomes. In a hierarchal organization, lower levels shape higher levels. However, the dependence of higher-order 3D chromatin organization on the nucleosome-level organization has not been studied in cells. We investigated the relationship between nucleosome-level organization and higher-order chromatin organization by perturbing nucleosomes across the genome by deleting () and () chromatin remodeling factors in budding yeast. We find that changes in nucleosome-level properties are accompanied by changes in 3D chromatin organization. Short-range chromatin contacts up to a few kilo-base pairs decrease, chromatin domains weaken, and boundary strength decreases. Boundary strength scales with accessibility and moderately with width of nucleosome-depleted region. Change in nucleosome positioning seems to alter the stiffness of chromatin, which can affect formation of chromatin contacts. Our results suggest a biomechanical "bottom-up" mechanism by which nucleosome distribution across genome shapes 3D chromatin organization.
Topics: Nucleosomes; Chromatin Assembly and Disassembly; Chromatin; Genome, Fungal; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; DNA-Binding Proteins; Transcription Factors; Adenosine Triphosphatases
PubMed: 38848364
DOI: 10.1126/sciadv.adn2955 -
AAPS PharmSciTech Jun 2024In this paper, we report two Accelerated Stability Assessment Program (ASAP) studies for a pediatric drug product. Whereas the first study using a generic design failed...
In this paper, we report two Accelerated Stability Assessment Program (ASAP) studies for a pediatric drug product. Whereas the first study using a generic design failed to establish a predictive model, the second one was successful after troubleshooting the first study and customizing the study conditions. This work highlighted important lessons learned from designing an ASAP study for formulations containing excipients that could undergo phase change at high humidity levels. The stability predictions by the second ASAP model were consistent with available long-term stability data of the drug product under various storage conditions in two different packaging configurations. The ASAP model was part of the justifications accepted by the health authority to submit a stability package with reduced long-term stability data from the primary stability batches for a Supplemental New Drug Application (sNDA).
Topics: Drug Stability; Excipients; Chemistry, Pharmaceutical; Humidity; Drug Storage; Drug Packaging; Drug Compounding; Humans; Child; Pharmaceutical Preparations; Pediatrics
PubMed: 38844721
DOI: 10.1208/s12249-024-02848-0 -
Horticulture Research Jun 2024Tomato fruit ripening is triggered by the demethylation of key genes, which alters their transcriptional levels thereby initiating and propagating a cascade of...
Tomato fruit ripening is triggered by the demethylation of key genes, which alters their transcriptional levels thereby initiating and propagating a cascade of physiological events. What is unknown is how these processes are altered when fruit are ripened using postharvest practices to extend shelf-life, as these practices often reduce fruit quality. To address this, postharvest handling-induced changes in the fruit DNA methylome and transcriptome, and how they correlate with ripening speed, and ripening indicators such as ethylene, abscisic acid, and carotenoids, were assessed. This study comprehensively connected changes in physiological events with dynamic molecular changes. Ripening fruit that reached 'Turning' (T) after dark storage at 20°C, 12.5°C, or 5°C chilling (followed by 20°C rewarming) were compared to fresh-harvest fruit 'FHT'. Fruit stored at 12.5°C had the biggest epigenetic marks and alterations in gene expression, exceeding changes induced by postharvest chilling. Fruit physiological and chronological age were uncoupled at 12.5°C, as the time-to-ripening was the longest. Fruit ripening to Turning at 12.5°C was not climacteric; there was no respiratory or ethylene burst, rather, fruit were high in abscisic acid. Clear differentiation between postharvest-ripened and 'FHT' was evident in the methylome and transcriptome. Higher expression of photosynthetic genes and chlorophyll levels in 'FHT' fruit pointed to light as influencing the molecular changes in fruit ripening. Finally, correlative analyses of the -omics data putatively identified genes regulated by DNA methylation. Collectively, these data improve our interpretation of how tomato fruit ripening patterns are altered by postharvest practices, and long-term are expected to help improve fruit quality.
PubMed: 38840937
DOI: 10.1093/hr/uhae095 -
Nature Communications Jun 2024SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of...
SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the "simultaneous inhibition of a paralog pair" concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.
Topics: SMARCB1 Protein; Humans; Animals; Cell Line, Tumor; Mice; Gene Expression Regulation, Neoplastic; p300-CBP Transcription Factors; E1A-Associated p300 Protein; Enhancer of Zeste Homolog 2 Protein; Chromatin Assembly and Disassembly; Mice, Nude; RNA, Small Interfering; Xenograft Model Antitumor Assays; Promoter Regions, Genetic; Cell Proliferation; Rhabdoid Tumor
PubMed: 38839769
DOI: 10.1038/s41467-024-49063-w -
The Journal of General Virology Jun 2024Relatively few phages that infect plant pathogens have been isolated and investigated. The species complex is present in various environments, including plants. It can...
Relatively few phages that infect plant pathogens have been isolated and investigated. The species complex is present in various environments, including plants. It can cause major crop diseases, such as bacterial canker on apricot trees. This study presents a collection of 25 unique phage genomes that infect . These phages were isolated from apricot orchards with bacterial canker symptoms after enrichment with 21 strains of . This collection comprises mostly virulent phages, with only three being temperate. They belong to 14 genera, 11 of which are newly discovered, and 18 new species, revealing great genetic diversity within this collection. Novel DNA packaging systems have been identified bioinformatically in one of the new phage species, but experimental confirmation is required to define the precise mechanism. Additionally, many phage genomes contain numerous potential auxiliary metabolic genes with diversified putative functions. At least three phages encode genes involved in bacterial tellurite resistance, a toxic metalloid. This suggests that viruses could play a role in bacterial stress tolerance. This research emphasizes the significance of continuing the search for new phages in the agricultural ecosystem to unravel novel ecological diversity and new gene functions. This work contributes to the foundation for future fundamental and applied research on phages infecting phytopathogenic bacteria.
Topics: Pseudomonas syringae; Genome, Viral; Plant Diseases; Pseudomonas Phages; Phylogeny; Genetic Variation
PubMed: 38833289
DOI: 10.1099/jgv.0.001990 -
MBio Jun 2024The phylum consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining...
UNLABELLED
The phylum consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining followed by extensive phylogenomic analysis and protein structure comparison, we delineate a distinct group of viruses with double-stranded (ds) DNA genomes in the range of 35-45 kb that appear to be related to the . In phylogenetic trees of the conserved double jelly-roll major capsid proteins (MCPs) and DNA packaging ATPases, these viruses do not show affinity to any particular branch of the and accordingly would comprise a class which we propose to name "" (after Ukrainian "mriya," dream). Structural comparison of the MCP suggests that, among the extant virus lineages, mriyaviruses are the closest one to the ancestor of the . In the phylogenetic trees, mriyaviruses split into two well-separated branches, the family and proposed new family "" The previously characterized members of these families, yaravirus and sp. endemic viruses, infect amoeba and haptophytes, respectively. The genomes of the rest of the mriyaviruses were assembled from metagenomes from diverse environments, suggesting that mriyaviruses infect various unicellular eukaryotes. Mriyaviruses lack DNA polymerase, which is encoded by all other members of the , and RNA polymerase subunits encoded by all cytoplasmic viruses among the , suggesting that they replicate in the host cell nuclei. All mriyaviruses encode a HUH superfamily endonuclease that is likely to be essential for the initiation of virus DNA replication via the rolling circle mechanism.
IMPORTANCE
The origin of giant viruses of eukaryotes that belong to the phylum is not thoroughly understood and remains a matter of major interest and debate. Here, we combine metagenome database searches with extensive protein sequence and structure analysis to describe a distinct group of viruses with comparatively small genomes of 35-45 kilobases that appear to comprise a distinct class within the phylum that we provisionally named "." Mriyaviruses appear to be the closest identified relatives of the ancestors of the . Analysis of proteins encoded in mriyavirus genomes suggests that they replicate their genome via the rolling circle mechanism that is unusual among viruses with double-stranded DNA genomes and so far not described for members of .
PubMed: 38832788
DOI: 10.1128/mbio.01035-24 -
Life Science Alliance Aug 2024Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription...
Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription machinery at every stage. Influences to steer nucleosome positioning are diverse, and the according importance of the DNA sequence in contrast to active chromatin remodeling has been the subject of long discussion. In this study, we evaluate the functional role of DNA sequence for all major elements along the process of transcription. We developed a random forest classifier based on local DNA structure that assesses the sequence-intrinsic support for nucleosome positioning. On this basis, we created a simple data resource that we applied genome-wide to the human genome. In our comprehensive analysis, we found a special role of DNA in mediating the competition of nucleosomes with cis-regulatory elements, in enabling steady transcription, for positioning of stable nucleosomes in exons, and for repelling nucleosomes during transcription termination. In contrast, we relate these findings to concurrent processes that generate strongly positioned nucleosomes in vivo that are not mediated by sequence, such as energy-dependent remodeling of chromatin.
Topics: Nucleosomes; Humans; Chromatin Assembly and Disassembly; Transcription, Genetic; DNA; Gene Expression Regulation; Chromatin; Genome, Human; Base Sequence
PubMed: 38830772
DOI: 10.26508/lsa.202302380 -
Journal of Virology Jun 2024We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The satellite podovirus Aci01-2-Phanie (short name Phanie)...
We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The satellite podovirus Aci01-2-Phanie (short name Phanie) uses a phage phi29-like DNA replication and packaging mode. Its linear 11,885 bp dsDNA genome bears 171 bp inverted terminal repeats (ITR). Phanie is related to phage DU-PP-III from and to members of the from . Together, they form a new clade of phages with 27% to 30% identity over the whole genome. Detailed 3D protein structure prediction and mass spectrometry analyses demonstrate that Phanie encodes its capsid structural genes and genes necessary to form a short tail. However, our study reveals that Phanie virions are non-infectious unless they associate with the contractile tail of an unrelated phage, Aci01-1, to produce chimeric myoviruses. Following the coinfection of Phanie with myovirus Aci01-1, hybrid viral particles composed of Phanie capsids and Aci01-1 contractile tails are assembled together with Phanie and Aci01-1 particles.IMPORTANCEThere are few reported cases of satellite-helper phage interactions but many more may be yet undiscovered. Here we describe a new mode of satellite phage dependence on a helper phage. Phanie, like phage phi29, replicates its linear dsDNA by a protein primed-mechanism and protects it inside podovirus-like particles. However, these particles are defective, requiring the acquisition of the tail from a myovirus helper for production of infectious virions. The formation of chimeras between a phi29-like podovirus and a helper contractile tail reveals an unexpected association between very different bacterial viruses.
PubMed: 38829140
DOI: 10.1128/jvi.00667-24 -
Acta Crystallographica. Section F,... Jun 2024The RSF complex belongs to the ISWI chromatin-remodeling family and is composed of two subunits: RSF1 (remodeling and spacing factor 1) and SNF2h (sucrose nonfermenting...
The RSF complex belongs to the ISWI chromatin-remodeling family and is composed of two subunits: RSF1 (remodeling and spacing factor 1) and SNF2h (sucrose nonfermenting protein 2 homolog). The RSF complex participates in nucleosome spacing and assembly, and subsequently promotes nucleosome maturation. Although SNF2h has been extensively studied in the last few years, the structural and functional properties of the remodeler RSF1 still remain vague. Here, a cryo-EM structure of the RSF-nucleosome complex is reported. The 3D model shows a two-lobe architecture of RSF, and the structure of the RSF-nucleosome (flanked with linker DNA) complex shows that the RSF complex moves the DNA away from the histone octamer surface at the DNA-entry point. Additionally, a nucleosome-sliding assay and a restriction-enzyme accessibility assay show that the RSF1 subunit may cause changes in the chromatin-remodeling properties of SNF2h. As a `nucleosome ruler', the results of an RSF-dinucleosome binding affinity test led to the proposal that the critical distance that RSF `measures' between two nucleosomes is about 24 base pairs.
Topics: Cryoelectron Microscopy; Nucleosomes; Chromatin Assembly and Disassembly; DNA-Binding Proteins; Models, Molecular; Protein Binding; Nuclear Proteins; DNA; Histones; Humans; Transcription Factors; Adenosine Triphosphatases; Chromosomal Proteins, Non-Histone; Trans-Activators
PubMed: 38818823
DOI: 10.1107/S2053230X24004655 -
The Journal of Physical Chemistry. B Jun 2024The integration host factor (IHF) in is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination....
The integration host factor (IHF) in is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.
Topics: DNA, Cruciform; Integration Host Factors; Escherichia coli; Nucleic Acid Conformation; Escherichia coli Proteins; Protein Binding
PubMed: 38812070
DOI: 10.1021/acs.jpcb.4c02997