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ELife Dec 2020Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved...
Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.
Topics: Acetylation; Binding Sites; Chromatin; DNA; Epigenesis, Genetic; High-Throughput Nucleotide Sequencing; Histones; Humans; K562 Cells; Nucleic Acid Conformation; Nucleosomes; Proof of Concept Study; Protein Conformation; Protein Processing, Post-Translational; Single Molecule Imaging; Site-Specific DNA-Methyltransferase (Adenine-Specific); Transcription Factors
PubMed: 33263279
DOI: 10.7554/eLife.59404 -
Wiley Interdisciplinary Reviews.... Jan 2018Enhancers serve as critical regulatory elements in higher eukaryotic cells. The characterization of enhancer function has evolved primarily from genome-wide... (Review)
Review
Enhancers serve as critical regulatory elements in higher eukaryotic cells. The characterization of enhancer function has evolved primarily from genome-wide methodologies, including chromatin immunoprecipitation (ChIP-seq), DNase-I hypersensitivity (DNase-seq), digital genomic footprinting (DGF), and the chromosome conformation capture techniques (3C, 4C, and Hi-C). These population-based assays average signals across millions of cells and lead to enhancer models characterized by static and sequential binding. More recently, fluorescent microscopy techniques, including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single molecule tracking (SMT), reveal a highly dynamic binding behavior for these factors in live cells. Furthermore, a refined analysis of genomic footprinting suggests that many transcription factors leave minimal or no footprints in chromatin, even when present and active in a given cell type. In this study, we review the implications of these new approaches for an accurate understanding of enhancer function in real time. In vivo SMT, in particular, has recently evolved as a promising methodology to probe enhancer function in live cells. Integration of findings from the many approaches now employed in the study of enhancer function suggest a highly dynamic view for the action of enhancer activating factors, viewed on a time scale of milliseconds to seconds, rather than minutes to hours. WIREs Syst Biol Med 2018, 10:e1390. doi: 10.1002/wsbm.1390 This article is categorized under: Analytical and Computational Methods > Computational Methods Laboratory Methods and Technologies > Genetic/Genomic Methods Laboratory Methods and Technologies > Imaging.
Topics: Chromatin; DNA; Enhancer Elements, Genetic; Genome; Humans; Protein Binding; Transcription Factors
PubMed: 28544514
DOI: 10.1002/wsbm.1390 -
Acta Chimica Slovenica Dec 2016The synthesis and biological activity of a variety of analogues to the naturally occurring antibacterial and antifungal Distamycin A were explored by a number of... (Review)
Review
The synthesis and biological activity of a variety of analogues to the naturally occurring antibacterial and antifungal Distamycin A were explored by a number of authors. These compounds were subject to a large array of assays. Some of these compounds showed high activity against a range of Gram-positive, Gram-negative bacteria as well as fungi. To explore the anti-parasitic activity of this class of compounds, specific modifications had to be made. A number of these compounds proved to be active against Trypanosoma brucei. The binding of a number of these compounds to short sequences of DNA were also examined using footprinting assays as well as NMR spectroscopy. Computer modelling was employed on selected compounds to understand the way these compounds bind to specific DNA sequences. A large number of variations were made to the standard structure of Distamycin. These changes involved the replacement of the pyrrole moieties as well as the head and tail groups with a number of heterocyclic compounds. Some of these minor groove binders (MGBs) were also investigated for their capability for the treatment of cancer and in particular lung cancer.
Topics: Animals; Anti-Bacterial Agents; Computer Simulation; DNA; DNA Footprinting; Distamycins; Humans; Magnetic Resonance Spectroscopy; Trypanocidal Agents
PubMed: 28004090
DOI: 10.17344/acsi.2016.2775 -
BioRxiv : the Preprint Server For... Mar 2023-regulatory elements control gene expression and are dynamic in their structure, reflecting changes to the composition of diverse effector proteins over time. Here we...
-regulatory elements control gene expression and are dynamic in their structure, reflecting changes to the composition of diverse effector proteins over time. Here we sought to connect the structural changes at regulatory elements to alterations in cellular fate and function. To do this we developed PRINT, a computational method that uses deep learning to correct sequence bias in chromatin accessibility data and identifies multi-scale footprints of DNA-protein interactions. We find that multi-scale footprints enable more accurate inference of TF and nucleosome binding. Using PRINT with single-cell multi-omics, we discover wide-spread changes to the structure and function of candidate -regulatory elements (cCREs) across hematopoiesis, wherein nucleosomes slide, expose DNA for TF binding, and promote gene expression. Activity segmentation using the co-variance across cell states identifies "sub-cCREs" as modular cCRE subunits of regulatory DNA. We apply this single-cell and PRINT approach to characterize the age-associated alterations to cCREs within hematopoietic stem cells (HSCs). Remarkably, we find a spectrum of aging alterations among HSCs corresponding to a global gain of sub-cCRE activity while preserving cCRE accessibility. Collectively, we reveal the functional importance of cCRE structure across cell states, highlighting changes to gene regulation at single-cell and single-base-pair resolution.
PubMed: 37034577
DOI: 10.1101/2023.03.28.533945 -
Biochemistry Mar 2019Paranemic crossover DNA (PX-DNA) is a four-stranded multicrossover structure that has been implicated in recombination-independent recognition of homology. Although...
Paranemic crossover DNA (PX-DNA) is a four-stranded multicrossover structure that has been implicated in recombination-independent recognition of homology. Although existing evidence has suggested that PX is the DNA motif in homologous pairing (HP), this conclusion remains ambiguous. Further investigation is needed but will require development of new tools. Here, we report characterization of the complex between PX-DNA and T7 endonuclease I (T7endoI), a junction-resolving protein that could serve as the prototype of an anti-PX ligand (a critical prerequisite for the future development of such tools). Specifically, nuclease-inactive T7endoI was produced and its ability to bind to PX-DNA was analyzed using a gel retardation assay. The molar ratio of PX to T7endoI was determined using gel electrophoresis and confirmed by the Hill equation. Hydroxyl radical footprinting of T7endoI on PX-DNA is used to verify the positive interaction between PX and T7endoI and to provide insight into the binding region. Cleavage of PX-DNA by wild-type T7endoI produces DNA fragments, which were used to identify the interacting sites on PX for T7endoI and led to a computational model of their interaction. Altogether, this study has identified a stable complex of PX-DNA and T7endoI and lays the foundation for engineering an anti-PX ligand, which can potentially assist in the study of molecular mechanisms for HP at an advanced level.
Topics: Bacteriophage T7; DNA; Deoxyribonuclease I; Electrophoretic Mobility Shift Assay; Gene Rearrangement; Models, Molecular; Nanostructures; Nucleic Acid Conformation; Nucleotide Motifs; Oligonucleotides; Protein Conformation; Sequence Homology
PubMed: 30794750
DOI: 10.1021/acs.biochem.9b00057 -
Journal of Molecular Biology Jul 2017G-quadruplexes (G4s) are extremely stable DNA or RNA secondary structures formed by sequences rich in guanine. These structures are implicated in many essential cellular... (Review)
Review
G-quadruplexes (G4s) are extremely stable DNA or RNA secondary structures formed by sequences rich in guanine. These structures are implicated in many essential cellular processes, and the number of biological functions attributed to them continues to grow. While DNA G4s are well understood on structural and, to some extent, functional levels, RNA G4s and their functions have received less attention. The presence of bona fide RNA G4s in cells has long been a matter of debate. The development of G4-specific antibodies and ligands hinted on their presence in vivo, but recent advances in RNA sequencing coupled with chemical footprinting suggested the opposite. In this review, we will critically discuss the biology of RNA G4s focusing on the molecular mechanisms underlying their proposed functions.
Topics: G-Quadruplexes; Gene Expression Regulation; Protein Biosynthesis; RNA, Messenger; Transcription, Genetic
PubMed: 28554731
DOI: 10.1016/j.jmb.2017.05.017 -
Nature Genetics Mar 2021The advent of single-cell chromatin accessibility profiling has accelerated the ability to map gene regulatory landscapes but has outpaced the development of scalable...
The advent of single-cell chromatin accessibility profiling has accelerated the ability to map gene regulatory landscapes but has outpaced the development of scalable software to rapidly extract biological meaning from these data. Here we present a software suite for single-cell analysis of regulatory chromatin in R (ArchR; https://www.archrproject.com/ ) that enables fast and comprehensive analysis of single-cell chromatin accessibility data. ArchR provides an intuitive, user-focused interface for complex single-cell analyses, including doublet removal, single-cell clustering and cell type identification, unified peak set generation, cellular trajectory identification, DNA element-to-gene linkage, transcription factor footprinting, mRNA expression level prediction from chromatin accessibility and multi-omic integration with single-cell RNA sequencing (scRNA-seq). Enabling the analysis of over 1.2 million single cells within 8 h on a standard Unix laptop, ArchR is a comprehensive software suite for end-to-end analysis of single-cell chromatin accessibility that will accelerate the understanding of gene regulation at the resolution of individual cells.
Topics: Animals; Chromatin; Cluster Analysis; Gene Expression Regulation; Genome; Humans; Mice; Sequence Analysis, RNA; Single-Cell Analysis; Software; Transcription Factors; User-Computer Interface; Web Browser
PubMed: 33633365
DOI: 10.1038/s41588-021-00790-6 -
Frontiers in Cell and Developmental... 2022ATR is a PI3K-like kinase protein, regulating checkpoint responses to DNA damage and replication stress. Apart from its checkpoint function in the nucleus, ATR actively...
ATR is a PI3K-like kinase protein, regulating checkpoint responses to DNA damage and replication stress. Apart from its checkpoint function in the nucleus, ATR actively engages in an antiapoptotic role at mitochondria following DNA damage. The different functions of ATR in the nucleus and cytoplasm are carried out by two prolyl isomeric forms of ATR: - and -ATR, respectively. The isomerization occurs at the Pin1 Ser428-Pro429 motif of ATR. Here, we investigated the structural basis of the subcellular location-specific functions of human ATR. Using a mass spectrometry-based footprinting approach, the surface accessibility of ATR lysine residues to sulfo-NHS-LC-biotin modification was monitored and compared between the - and the -isomers. We have identified two biotin-modified lysine residues, K459 and K469, within the BH3-like domain of -ATR that were not accessible in -ATR, indicating a conformational change around the BH3 domain between and -ATR. The conformational alteration also involved the N-terminal domain and the middle HEAT domain. Moreover, experimental results from an array of complementary assays show that -ATR with the accessible BH3 domain was able to bind to tBid while -ATR could not. In addition, both - and -ATR can directly form homodimers their C-terminal domains without ATRIP, while nuclear (-ATR) in the presence of ATRIP forms dimer-dimer complexes involving both N- and C-termini of ATR and ATRIP after UV. Structural characteristics around the Ser428-Pro429 motif and the BH3 domain region are also analyzed by molecular modeling and dynamics simulation. In support, conformation was found to be significantly more energetically favorable than at the Ser428-Pro429 bond in a 20-aa wild-type ATR peptide. Taken together, our results suggest that the isomerization-induced structural changes of ATR define both its subcellular location and compartment-specific functions and play an essential role in promoting cell survival and DNA damage responses.
PubMed: 35721505
DOI: 10.3389/fcell.2022.826576 -
Frontiers in Plant Science 2017is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome...
is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of is unknown. Sequencing of strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete spp. genomes and across the , as well as a methylation motif associated to a phage-borne methylase unique to UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in evolution and its adaptation to different plants.
PubMed: 28450872
DOI: 10.3389/fpls.2017.00504 -
Bio-protocol Dec 2020DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to...
DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use traditional footprinting methods to determine protein-DNA contacts.
PubMed: 33659492
DOI: 10.21769/BioProtoc.3843