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Briefings in Bioinformatics Sep 2019Deoxyribonuclease I (DNase I)-hypersensitive site sequencing (DNase-seq) has been widely used to determine chromatin accessibility and its underlying regulatory lexicon.... (Review)
Review
Deoxyribonuclease I (DNase I)-hypersensitive site sequencing (DNase-seq) has been widely used to determine chromatin accessibility and its underlying regulatory lexicon. However, exploring DNase-seq data requires sophisticated downstream bioinformatics analyses. In this study, we first review computational methods for all of the major steps in DNase-seq data analysis, including experimental design, quality control, read alignment, peak calling, annotation of cis-regulatory elements, genomic footprinting and visualization. The challenges associated with each step are highlighted. Next, we provide a practical guideline and a computational pipeline for DNase-seq data analysis by integrating some of these tools. We also discuss the competing techniques and the potential applications of this pipeline for the analysis of analogous experimental data. Finally, we discuss the integration of DNase-seq with other functional genomics techniques.
Topics: Computational Biology; DNA Footprinting; Data Management; Deoxyribonuclease I; Quality Control; Sequence Analysis, DNA
PubMed: 30010713
DOI: 10.1093/bib/bby057 -
Molecules (Basel, Switzerland) Aug 2020Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this...
Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.
Topics: Amino Acid Sequence; Basic-Leucine Zipper Transcription Factors; Binding Sites; Calorimetry, Differential Scanning; Catalysis; Chromatography, High Pressure Liquid; Click Chemistry; Copper; DNA; DNA Damage; DNA, Single-Stranded; Molecular Dynamics Simulation; Nucleic Acid Conformation; Peptides; Protein Domains; Saccharomyces cerevisiae Proteins; Spectrometry, Mass, Electrospray Ionization; Transition Temperature
PubMed: 32784992
DOI: 10.3390/molecules25163630 -
PNAS Nexus Sep 2022The tumor suppressor p53 functions as a pioneer transcription factor that binds a nucleosomal target DNA sequence. However, the mechanism by which p53 binds to its...
The tumor suppressor p53 functions as a pioneer transcription factor that binds a nucleosomal target DNA sequence. However, the mechanism by which p53 binds to its target DNA in the nucleosome remains elusive. Here we report the cryo-electron microscopy structures of the p53 DNA-binding domain and the full-length p53 protein complexed with a nucleosome containing the 20 base-pair target DNA sequence of p53 (p53BS). In the p53-nucleosome structures, the p53 DNA-binding domain forms a tetramer and specifically binds to the p53BS DNA, located near the entry/exit region of the nucleosome. The nucleosomal position of the p53BS DNA is within the genomic p21 promoter region. The p53 binding peels the DNA from the histone surface, and drastically changes the DNA path around the p53BS on the nucleosome. The C-terminal domain of p53 also binds to the DNA around the center and linker DNA regions of the nucleosome, as revealed by hydroxyl radical footprinting. These results provide important structural information for understanding the mechanism by which p53 binds the nucleosome and changes the chromatin structure for gene activation.
PubMed: 36714865
DOI: 10.1093/pnasnexus/pgac177 -
Molecular Cell Mar 2023Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA...
Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA demethylation. However, whether this epigenetic remodeling is necessary for enhancer activation is unknown. Here, we adapted single-molecule footprinting to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their chromatin accessibility in multiple cell lineages. Although reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where DNA methylation antagonizes the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding require active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at active enhancers.
Topics: Animals; DNA Methylation; Enhancer Elements, Genetic; Chromatin; Transcription Factors; Gene Expression Regulation; Mammals
PubMed: 36758546
DOI: 10.1016/j.molcel.2023.01.017 -
Journal of Inorganic Biochemistry Jan 2022The investigation of compounds capable of strongly and selectively interacting with DNA comprises a field of research in constant development. In this work, we...
The investigation of compounds capable of strongly and selectively interacting with DNA comprises a field of research in constant development. In this work, we demonstrate that a trinuclear coordination complex based on a dinuclear Fe(III)Zn(II) core designed for biomimicry of the hydrolytic enzyme kidney bean purple acid phosphatase, containing an additional pendant arm coordinating a Pd(II) ion, has the ability to interact with DNA and to promote its hydrolytic cleavage. These results were found through analysis of plasmid DNA interaction and cleavage by the trinuclear complex 1 and its derivatives 2 and 3, in addition to the analysis of alteration in the DNA structure in the presence of the complexes through circular dichroism and DNA footprinting techniques. The suggested covalent interaction of the palladium-containing complex with DNA was analysed using an electrophoretic mobility assay, circular dichroism, high resolution gel separation techniques and kinetic analysis. This is a new and promising metal complex targeted to nucleic acids and acting in two separate ways: strong DNA interaction and hydrolytic cleavage.
Topics: Coordination Complexes; DNA Cleavage; Deoxyribonucleases; Metals; Plasmids
PubMed: 34717251
DOI: 10.1016/j.jinorgbio.2021.111631 -
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 -
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