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PLoS Pathogens Jun 2024Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction....
Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction. Typically, the P22 DNA packaging machinery initiates packaging when a short sequence of DNA, known as the pac site, is recognized on the P22 genome. However, sequences similar to the pac site in the host genome, called pseudo-pac sites, lead to erroneous packaging and subsequent generalized transduction of Salmonella DNA. While the general genomic locations of the Salmonella pseudo-pac sites are known, the sequences themselves have not been determined. We used visualization of P22 sequencing reads mapped to host Salmonella genomes to define regions of generalized transduction initiation and the likely locations of pseudo-pac sites. We searched each genome region for the sequence with the highest similarity to the P22 pac site and aligned the resulting sequences. We built a regular expression (sequence match pattern) from the alignment and used it to search the genomes of two P22-susceptible Salmonella strains-LT2 and 14028S-for sequence matches. The final regular expression successfully identified pseudo-pac sites in both LT2 and 14028S that correspond with generalized transduction initiation sites in mapped read coverages. The pseudo-pac site sequences identified in this study can be used to predict locations of generalized transduction in other P22-susceptible hosts or to initiate generalized transduction at specific locations in P22-susceptible hosts with genetic engineering. Furthermore, the bioinformatics approach used to identify the Salmonella pseudo-pac sites in this study could be applied to other phage-host systems.
PubMed: 38913753
DOI: 10.1371/journal.ppat.1012301 -
Molecular Cell Jun 2024The construction of synthetic gene circuits requires the rational combination of multiple regulatory components, but predicting their behavior can be challenging due to...
The construction of synthetic gene circuits requires the rational combination of multiple regulatory components, but predicting their behavior can be challenging due to poorly understood component interactions and unexpected emergent behaviors. In eukaryotes, chromatin regulators (CRs) are essential regulatory components that orchestrate gene expression. Here, we develop a screening platform to investigate the impact of CR pairs on transcriptional activity in yeast. We construct a combinatorial library consisting of over 1,900 CR pairs and use a high-throughput workflow to characterize the impact of CR co-recruitment on gene expression. We recapitulate known interactions and discover several instances of CR pairs with emergent behaviors. We also demonstrate that supervised machine learning models trained with low-dimensional amino acid embeddings accurately predict the impact of CR co-recruitment on transcriptional activity. This work introduces a scalable platform and machine learning approach that can be used to study how networks of regulatory components impact gene expression.
Topics: Chromatin; Synthetic Biology; Saccharomyces cerevisiae; Gene Expression Regulation, Fungal; Transcription, Genetic; Gene Regulatory Networks; High-Throughput Screening Assays; Saccharomyces cerevisiae Proteins; Supervised Machine Learning; Chromatin Assembly and Disassembly; Transcription Factors
PubMed: 38906116
DOI: 10.1016/j.molcel.2024.05.025 -
Nature Reviews. Genetics Jun 2024Dense packaging of genomic DNA is crucial for organismal survival, as DNA length always far exceeds the dimensions of the cells that contain it. Organisms, therefore,... (Review)
Review
Dense packaging of genomic DNA is crucial for organismal survival, as DNA length always far exceeds the dimensions of the cells that contain it. Organisms, therefore, use sophisticated machineries to package their genomes. These systems range across kingdoms from a single ultra-powerful rotary motor that spools the DNA into a bacteriophage head, to hundreds of thousands of relatively weak molecular motors that coordinate the compaction of mitotic chromosomes in eukaryotic cells. Recent technological advances, such as DNA proximity-based sequencing approaches, polymer modelling and in vitro reconstitution of DNA loop extrusion, have shed light on the biological mechanisms driving DNA organization in different systems. Here, we discuss DNA packaging in bacteriophage, bacteria and eukaryotic cells, which, despite their extreme variation in size, structure and genomic content, all rely on the action of molecular motors to package their genomes.
PubMed: 38886215
DOI: 10.1038/s41576-024-00740-y -
Communications Biology Jun 2024Before each cell division, eukaryotic cells must replicate their chromosomes to ensure the accurate transmission of genetic information. Chromosome replication involves... (Review)
Review
Before each cell division, eukaryotic cells must replicate their chromosomes to ensure the accurate transmission of genetic information. Chromosome replication involves more than just DNA duplication; it also includes chromatin assembly, inheritance of epigenetic marks, and faithful resumption of all genomic functions after replication. Recent progress in quantitative technologies has revolutionized our understanding of the complexity and dynamics of DNA replication forks at both molecular and genomic scales. Here, we highlight the pivotal role of these novel methods in uncovering the principles and mechanisms of chromosome replication. These technologies have illuminated the regulation of genome replication programs, quantified the impact of DNA replication on genomic mutations and evolutionary processes, and elucidated the mechanisms of replication-coupled chromatin assembly and epigenome maintenance.
Topics: DNA Replication; Humans; Epigenesis, Genetic; Animals; Chromosomes; High-Throughput Screening Assays; High-Throughput Nucleotide Sequencing; Chromatin Assembly and Disassembly
PubMed: 38877080
DOI: 10.1038/s42003-024-06412-1 -
PloS One 2024The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on...
The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on the condition of the genetic material. All potential damage induced by oxidative stress may affect the final results of the analysis of genetic material obtained using commonly used techniques such as polymerase chain reaction or sequencing. The aim of this work was to evaluate the effects of high temperature and pH on DNA structure in the context of the occurrence of oxidative damage, using square-wave voltammetry and two independent research protocols. We resulted in visible oxidation damage registered in acidic conditions after the thermal denaturation process (pH 4.7) with changes in the intensity of guanine and adenine signals. However, using phosphate buffer (pH 7.0) for DNA denaturation negatively affected the DNA structure, but without any oxidized derivatives present. This leads to the conclusion that oxidation occurring in the DNA melting process results in the formation of various derivatives of nucleobases, both electrochemically active and inactive. These derivatives may distort the results of molecular tests due to the possibility of forming complementary bonds with various nucleobases. For example, 8-oxoguanine can form pairs with both cytosine and adenine.
Topics: DNA; Oxidative Stress; Nucleic Acid Denaturation; Temperature; Oxidation-Reduction; DNA Damage; Hydrogen-Ion Concentration; Guanine; Electrochemical Techniques; Adenine
PubMed: 38875261
DOI: 10.1371/journal.pone.0305590 -
Nature Cell Biology Jun 2024The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes...
The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes young and old bone marrow progenitor (pro-) B cells. These changes result in increased interactions at the compartment level and reduced interactions within topologically associated domains (TADs). The gene encoding Ebf1, a key B cell regulator, switches from compartment A to B with age. Genetically reducing Ebf1 recapitulates some features of old pro-B cells. TADs that are most reduced with age contain genes important for B cell development, including the immunoglobulin heavy chain (Igh) locus. Weaker intra-TAD interactions at Igh correlate with altered variable (V), diversity (D) and joining (J) gene recombination. Our observations implicate three-dimensional chromatin reorganization as a major driver of pro-B cell phenotypes that impair B lymphopoiesis with age.
Topics: Animals; Aging; B-Lymphocytes; Chromatin Assembly and Disassembly; Lymphopoiesis; Immunoglobulin Heavy Chains; Trans-Activators; Chromatin; Precursor Cells, B-Lymphoid; Mice, Inbred C57BL; Mice; Cell Differentiation; Mice, Knockout
PubMed: 38866970
DOI: 10.1038/s41556-024-01424-9 -
Genomics, Proteomics & Bioinformatics May 2024Ribonuclease P (RNase P) was first described in the 1970's as an endoribonuclease acting in the maturation of precursor transfer RNAs (tRNAs). More recent studies,... (Review)
Review
Ribonuclease P (RNase P) was first described in the 1970's as an endoribonuclease acting in the maturation of precursor transfer RNAs (tRNAs). More recent studies, however, have uncovered non-canonical roles for RNase P and its components. Here, we review the recent progress of its involvement in chromatin assembly, DNA damage response, and maintenance of genome stability with implications in tumorigenesis. The possibility of RNase P as a therapeutic target in cancer is also discussed.
Topics: Ribonuclease P; Humans; RNA, Transfer; Neoplasms; RNA Precursors; Genomic Instability; Animals; DNA Damage; RNA Processing, Post-Transcriptional; Chromatin Assembly and Disassembly
PubMed: 38862431
DOI: 10.1093/gpbjnl/qzae016 -
BioRxiv : the Preprint Server For... May 2024There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we...
There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the sperm, the short one (Mw 36 kDa) is mislocalized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/B2/B3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 protein or SUN4 expression in testes. Furthermore, the sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone- protamine 1/2 ratio and modified levels of cytoskeletal proteins. We detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions, which was not true for healthy normozoospermic men. In conclusion, our findings expand the current knowledge regarding the connection between Protamine 2 and Septin 12 expression and localization, resulting in low sperm motility and morphological abnormalities.
PubMed: 38854089
DOI: 10.1101/2024.05.28.596175 -
Biochemical and Biophysical Research... Sep 2024SWI/SNF chromatin remodeling complexes play a key role in gene transcription as epigenetic regulators and are typically considered to act as tumor suppressors in...
SWI/SNF chromatin remodeling complexes play a key role in gene transcription as epigenetic regulators and are typically considered to act as tumor suppressors in cancers. Compared to other cancer-related components of the SWI/SNF complex, research on SMARCC2, a component of the initial BAF core, has been relatively limited. This study aimed to elucidate the role of SMARCC2 in breast cancer by employing various in vitro and in vivo methods including cell proliferation assays, mammosphere formation, and xenograft models, complemented by RNA-seq, ATAC-seq, and ChIP analyses. The results showed that SMARCC2 silencing surprisingly led to the suppression of breast tumorigenesis, indicating a pro-tumorigenic function for SMARCC2 in breast cancer, which contrasts with the roles of other SWI/SNF subunits. In addition, SMARCC2 depletion reduces cancer stem cell features of breast cancer cells. Mechanistic study showed that SMARCC2 silencing downregulated the oncogenic Ras-PI3K signaling pathway, likely by directly regulating the chromatin accessibility of the enhancers of the key genes such as PIK3CB. Together, these results expand our understanding of the SWI/SNF complex's role in cancer development and identify SMARCC2 as a promising new target for breast cancer therapies.
Topics: Humans; Breast Neoplasms; Female; Chromatin; Animals; Gene Silencing; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Mice; Transcription Factors; Cell Proliferation; Carcinogenesis; Chromosomal Proteins, Non-Histone; Signal Transduction; Mice, Nude; Chromatin Assembly and Disassembly
PubMed: 38852505
DOI: 10.1016/j.bbrc.2024.150223 -
Advanced Drug Delivery Reviews Jun 2024Gene editing technologies have the potential to correct genetic disorders by modifying, inserting, or deleting specific DNA sequences or genes, paving the way for a new... (Review)
Review
Gene editing technologies have the potential to correct genetic disorders by modifying, inserting, or deleting specific DNA sequences or genes, paving the way for a new class of genetic therapies. While gene editing tools continue to be improved to increase their precision and efficiency, the limited efficacy of in vivo delivery remains a major hurdle for clinical use. An ideal delivery vehicle should be able to target a sufficient number of diseased cells in a transient time window to maximize on-target editing and mitigate off-target events and immunogenicity. Here, we review major advances in novel delivery platforms based on cell-derived vesicles - extracellular vesicles and virus-like particles - for transient delivery of gene editing payloads. We discuss major findings regarding packaging, in vivo biodistribution, therapeutic efficacy, and safety concerns of cell-derived vesicles delivery of gene-editing cargos and their potential for clinical translation.
PubMed: 38849005
DOI: 10.1016/j.addr.2024.115346