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Cancers Jul 2023Liquid biopsies are revolutionary tools used to detect tumor-specific genetic alterations in body fluids, including the use of cell-free DNA (cfDNA) for molecular...
BACKGROUND
Liquid biopsies are revolutionary tools used to detect tumor-specific genetic alterations in body fluids, including the use of cell-free DNA (cfDNA) for molecular diagnosis in cancer patients. In brain tumors, cerebrospinal fluid (CSF) cfDNA might be more informative than plasma cfDNA. Here, we assess the use of CSF cfDNA in pediatric embryonal brain tumors (EBT) for molecular diagnosis.
METHODS
The CSF cfDNA of pediatric patients with medulloblastoma ( = 18), ATRT ( = 3), ETMR ( = 1), CNS NB FOXR2 ( = 2) and pediatric EBT NOS ( = 1) (mean cfDNA concentration 48 ng/mL; range 4-442 ng/mL) and matched tumor genomic DNA were sequenced by WES and/or a targeted sequencing approach to determine single-nucleotide variations (SNVs) and copy number alterations (CNA). A specific capture covering transcription start sites (TSS) of genes of interest was also used for nucleosome footprinting in CSF cfDNA.
RESULTS
15/25 CSF cfDNA samples yielded informative results, with informative CNA and SNVs in 11 and 15 cases, respectively. For cases with paired tumor and CSF cfDNA WES ( = 15), a mean of 83 (range 1-160) shared SNVs were observed, including SNVs in classical medulloblastoma genes such as SMO and KMT2D. Interestingly, tumor-specific SNVs (mean 18; range 1-62) or CSF-specific SNVs (mean 5; range 0-25) were also observed, suggesting clonal heterogeneity. The TSS panel resulted in differential coverage profiles across all 112 studied genes in 7 cases, indicating distinct promoter accessibility.
CONCLUSION
CSF cfDNA sequencing yielded informative results in 60% (15/25) of all cases, with informative results in 83% (15/18) of all cases analyzed by WES. These results pave the way for the implementation of these novel approaches for molecular diagnosis and minimal residual disease monitoring.
PubMed: 37444642
DOI: 10.3390/cancers15133532 -
Biomolecules Feb 2023The CRISPR-Cas system is an adaptive immune system for many bacteria and archaea to defend against foreign nucleic acid invasion, and this system is conserved in the...
The CRISPR-Cas system is an adaptive immune system for many bacteria and archaea to defend against foreign nucleic acid invasion, and this system is conserved in the genome of (). Although the CRISPR-Cas system-mediated immune defense mechanism has been revealed in , the regulation of gene expression is poorly understood. In this study, we identified a transcription factor, CasR (CRISPR-associated protein repressor, encoded by ), and it could bind to the upstream DNA sequence of the CRISPR-Cas gene cluster and regulate the expression of genes. EMSA and ChIP assays confirmed that CasR could interact with the upstream sequence of the promoter, both in vivo and in vitro. Furthermore, DNA footprinting assay revealed that CasR recognized a 20 bp palindromic sequence motif and negatively regulated the expression of In conclusion, our research elucidates the regulatory effect of CasR on the expression of CRISPR-associated genes in mycobacteria, thus providing insight into gene expression regulation of the CRISPR-Cas system.
Topics: Mycobacterium tuberculosis; Archaea; CRISPR-Cas Systems; Transcription Factors
PubMed: 36830769
DOI: 10.3390/biom13020400 -
Molecular Cell Jan 2021Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short...
Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we present a DNA footprinting method that detects individual molecular interactions of transcription factors and nucleosomes with DNA in vivo. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules at mouse CREs. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that high DNA co-occupancy occurs for most types of transcription factors, in the absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy in binding cooperativity. Our results reveal the interactions regulating CREs at molecular resolution and identify DNA co-occupancy as a widespread cooperativity mechanism used by transcription factors to remodel chromatin.
Topics: Animals; Binding Sites; DNA; DNA Footprinting; Male; Mice; Mouse Embryonic Stem Cells; Nucleosomes; Protein Binding; Regulatory Elements, Transcriptional; Transcription Factors; Transcription, Genetic
PubMed: 33290745
DOI: 10.1016/j.molcel.2020.11.015 -
Nucleic Acids Research Mar 2015DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these...
DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines.
Topics: Algorithms; DNA; DNA Footprinting; Depsipeptides; Elasticity; Intercalating Agents; Kinetics; Ligands; Models, Molecular; Nucleic Acid Conformation; Optical Tweezers; Protein Binding; Thermodynamics; Time Factors
PubMed: 25690887
DOI: 10.1093/nar/gkv087 -
The Journal of Biological Chemistry Jul 2021Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc...
Binding of antibodies to their receptors is a core component of the innate immune system. Understanding the precise interactions between antibodies and their Fc receptors has led to the engineering of novel mAb biotherapeutics with tailored biological activities. One of the most significant findings is that afucosylated monoclonal antibodies demonstrate increased affinity toward the receptor FcγRIIIa, with a commensurate increase in antibody-dependent cellular cytotoxicity. Crystal structure analysis has led to the hypothesis that afucosylation in the Fc region results in reduced steric hindrance between antibody-receptor intermolecular glycan interactions, enhancing receptor affinity; however, solution-phase data have yet to corroborate this hypothesis. In addition, recent work has shown that the fragment antigen-binding (Fab) region may directly interact with Fc receptors; however, the biological consequences of these interactions remain unclear. By probing differences in solvent accessibility between native and afucosylated immunoglobulin G1 (IgG1) using hydroxyl radical footprinting-MS, we provide the first solution-phase evidence that an IgG1 bearing an afucosylated Fc region appears to require fewer conformational changes for FcγRIIIa binding. In addition, we performed extensive molecular dynamics (MD) simulations to understand the molecular mechanism behind the effects of afucosylation. The combination of these techniques provides molecular insight into the steric hindrance from the core Fc fucose in IgG1 and corroborates previously proposed Fab-receptor interactions. Furthermore, MD-guided rational mutagenesis enabled us to demonstrate that Fab-receptor interactions directly contribute to the modulation of antibody-dependent cellular cytotoxicity activity. This work demonstrates that in addition to Fc-polypeptide and glycan-mediated interactions, the Fab provides a third component that influences IgG-Fc receptor biology.
Topics: Animals; Antibody-Dependent Cell Cytotoxicity; CHO Cells; Cricetulus; DNA Mutational Analysis; Fucose; Glycosylation; Hydroxyl Radical; Immunoglobulin Fab Fragments; Immunoglobulin G; Molecular Dynamics Simulation; Mutation; Protein Binding; Protein Conformation; Receptors, Fc
PubMed: 34044019
DOI: 10.1016/j.jbc.2021.100826 -
Nature Communications Sep 2021Chromatin remodeling and genomic alterations impact spatio-temporal regulation of gene expression, which is central to embryonic development. The analysis of mouse and...
Chromatin remodeling and genomic alterations impact spatio-temporal regulation of gene expression, which is central to embryonic development. The analysis of mouse and chicken limb development provides important insights into the morphoregulatory mechanisms, however little is known about the regulatory differences underlying their morphological divergence. Here, we identify the underlying shared and species-specific epigenomic and genomic variations. In mouse forelimb buds, we observe striking synchrony between the temporal dynamics of chromatin accessibility and gene expression, while their divergence in chicken wing buds uncovers species-specific regulatory heterochrony. In silico mapping of transcription factor binding sites and computational footprinting establishes the developmental time-restricted transcription factor-DNA interactions. Finally, the construction of target gene networks for HAND2 and GLI3 transcriptional regulators reveals both conserved and species-specific interactions. Our analysis reveals the impact of genome evolution on the regulatory interactions orchestrating vertebrate limb bud morphogenesis and provides a molecular framework for comparative Evo-Devo studies.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Body Patterning; Chick Embryo; Chickens; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation Sequencing; Computer Simulation; Embryo, Mammalian; Embryonic Development; Gene Expression Regulation, Developmental; Gene Regulatory Networks; Limb Buds; Mice; Nerve Tissue Proteins; RNA-Seq; Species Specificity; Zinc Finger Protein Gli3
PubMed: 34584102
DOI: 10.1038/s41467-021-25935-3 -
Computational and Structural... 2022Cooperativity between transcription factors is important to regulate target gene expression. In particular, the binding grammar of TFs in relation to each other, as well...
Cooperativity between transcription factors is important to regulate target gene expression. In particular, the binding grammar of TFs in relation to each other, as well as in the context of other genomic elements, is crucial for TF functionality. However, tools to easily uncover co-occurrence between DNA-binding proteins, and investigate the regulatory modules of TFs, are limited. Here we present TF-COMB (Transcription Factor Co-Occurrence using Market Basket analysis) - a tool to investigate co-occurring TFs and binding grammar within regulatory regions. We found that TF-COMB can accurately identify known co-occurring TFs from ChIP-seq data, as well as uncover preferential localization to other genomic elements. With the use of ATAC-seq footprinting and TF motif locations, we found that TFs exhibit both preferred orientation and distance in relation to each other, and that these are biologically significant. Finally, we extended the analysis to not only investigate individual TF pairs, but also TF pairs in the context of networks, which enabled the investigation of TF complexes and TF hubs. In conclusion, TF-COMB is a flexible tool to investigate various aspects of TF binding grammar.
PubMed: 35983231
DOI: 10.1016/j.csbj.2022.07.025 -
Methods (San Diego, Calif.) Apr 2019Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is...
Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins. However, in cells, the transcription of genes is influenced by several factors, including DNA structure, chromatin, co-transcriptional processes, and DNA binding proteins, which impede the smooth progression of RNAPII down the template. Many transcription elongation proteins have evolved to mitigate the complications and barriers encountered by polymerase during transcription. Many of these elongation factors physically interact with components of the RNAPII elongation complex, including the growing RNA transcript and the DNA template entering and exiting RNAPII. To better understand how transcription elongation factors (EFs) regulate RNAPII, elegant methods are required to probe the structure of the elongation complex. Here, we describe a collection of biochemical assays to interrogate the structure of the RNAPII elongation complex of Saccharomyces cerevisiae that are capable of providing insights into the function of EFs and the elongation process.
Topics: Humans; Optical Imaging; RNA Polymerase II; Single Molecule Imaging; Transcription Elongation, Genetic; Transcriptional Elongation Factors
PubMed: 30684536
DOI: 10.1016/j.ymeth.2019.01.011 -
Photochemistry and Photobiology Jan 2019While is it well known that human telomeric DNA sequences can adopt G-quadruplex structures, some promoters sequences have also been found to form G-quadruplexes, and...
While is it well known that human telomeric DNA sequences can adopt G-quadruplex structures, some promoters sequences have also been found to form G-quadruplexes, and over 40% of promoters contain putative G-quadruplex-forming sequences. Because UV light has been shown to crosslink human telomeric G-quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G-quadruplexes in promoters. To investigate this possibility, 15 potentially UV-crosslinkable G-quadruplex-forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G-quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non-B DNA structures, such as G-quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.
Topics: G-Quadruplexes; Humans; Mass Spectrometry; Photochemical Processes; Promoter Regions, Genetic; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 30084501
DOI: 10.1111/php.12991 -
Genome Biology Nov 2023Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing...
BACKGROUND
Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing their origin to evolutionary innovations first made in their archaeal ancestors. One of the most notable such features is nucleosomal chromatin, although archaeal histones and chromatin differ significantly from those of eukaryotes, not all archaea possess histones and it is not clear if histones are a main packaging component for all that do. Despite increased interest in archaeal chromatin in recent years, its properties have been little studied using genomic tools.
RESULTS
Here, we adapt the ATAC-seq assay to archaea and use it to map the accessible landscape of the genome of the euryarchaeote Haloferax volcanii. We integrate the resulting datasets with genome-wide maps of active transcription and single-stranded DNA (ssDNA) and find that while H. volcanii promoters exist in a preferentially accessible state, unlike most eukaryotes, modulation of transcriptional activity is not associated with changes in promoter accessibility. Applying orthogonal single-molecule footprinting methods, we quantify the absolute levels of physical protection of H. volcanii and find that Haloferax chromatin is similarly or only slightly more accessible, in aggregate, than that of eukaryotes. We also evaluate the degree of coordination of transcription within archaeal operons and make the unexpected observation that some CRISPR arrays are associated with highly prevalent ssDNA structures.
CONCLUSIONS
Our results provide the first comprehensive maps of chromatin accessibility and active transcription in Haloferax across conditions and thus a foundation for future functional studies of archaeal chromatin.
Topics: Chromatin; Histones; Haloferax volcanii; Nucleosomes; Biological Evolution; Eukaryota; Archaeal Proteins
PubMed: 37932847
DOI: 10.1186/s13059-023-03095-5