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Cancer Research Communications Oct 2023African American (AA) women have an excessive risk of developing triple-negative breast cancer (TNBC). We employed Assay for Transposase-Accessible Chromatin using...
UNLABELLED
African American (AA) women have an excessive risk of developing triple-negative breast cancer (TNBC). We employed Assay for Transposase-Accessible Chromatin using sequencing to characterize differences in chromatin accessibility between nine commonly used TNBC cell lines derived from patients of European and African ancestry. Principal component and chromosome mapping analyses of accessibility peaks with the most variance revealed separation of chromatin profiles by patient group. Motif enrichment and footprinting analyses of disparate open chromatin regions revealed differences in transcription factor activity, identifying 79 with ancestry-associated binding patterns (FDR < 0.01). AA TNBC cell lines exhibited increased accessibility for 62 transcription factors associated with epithelial-to-mesenchymal transition, cancer stemness/chemotherapeutic resistance, proliferation, and aberrant p53 regulation, as well as KAISO, which has been previously linked to aggressive tumor characteristics in AA patients with cancer. Differential Assay for Transposase-Accessible Chromatin signal analysis identified 1,596 genes located within promoters of differentially open chromatin regions in AA-derived TNBC, identifying DNA methyltransferase 1 as the top upregulated gene associated with African ancestry. Pathway analyses with these genes revealed enrichment in several pathways, including hypoxia. Culturing cells under hypoxia showed ancestry-specific stress responses that led to the identification of a core set of AA-associated transcription factors, which included members of the Kruppel-like factor and Sp subfamilies, as well as KAISO, and identified ZDHHC1, a gene previously implicated in immunity and STING activation, as the top upregulated AA-specific gene under hypoxia. Together, these data reveal a differential chromatin landscape in TNBC associated with donor ancestry. The open chromatin structure of AA TNBC may contribute to a more lethal disease.
SIGNIFICANCE
We identify an ancestry-associated open chromatin landscape and related transcription factors that may contribute to aggressive TNBC in AA women. Furthermore, this study advocates for the inclusion of diversely sourced cell lines in experimental in vitro studies to advance health equity at all levels of scientific research.
PubMed: 37732899
DOI: 10.1158/2767-9764.CRC-23-0236 -
Chemistry (Weinheim An Der Bergstrasse,... Dec 2020Bulges are essential structural elements in nucleic acids. The detection and targeting of bulged DNA sequences are highly important. Small molecules capable of targeting...
Bulges are essential structural elements in nucleic acids. The detection and targeting of bulged DNA sequences are highly important. Small molecules capable of targeting DNA bulges have attracted considerable attention because they cannot only be used as reagents for bulge recognition, but also as potential therapeutic drugs. Herein, the interactions of DNA duplexes, containing bulges of various sizes and base compositions, with a series of Fe triplex-forming metallohelices are reported. The results obtained, with the aid of molecular biophysics methods, show that the investigated metallohelices prefer to bind to bulged DNA, rather than double-stranded DNA, and that their binding affinities towards bulges differ among individual metallohelices. Moreover, their binding affinities towards bulges strongly depend on the bulge size and the base composition of the bulge loop. The investigated metallohelices can enter eukaryotic cells and accumulate in the cell nucleus, allowing them to interact with nucleic acids. Hence, it is reasonable to suggest that the interaction of metallohelices with nucleic acid bulges might contribute to the mechanism of their biological activity.
Topics: Antineoplastic Agents; Base Sequence; DNA; Ferrous Compounds
PubMed: 33026666
DOI: 10.1002/chem.202004060 -
Methods in Molecular Biology (Clifton,... 2020R-loops are three-stranded structures that form during transcription when the nascent RNA hybridizes with the template DNA resulting in a DNA:RNA hybrid and a looped-out...
R-loops are three-stranded structures that form during transcription when the nascent RNA hybridizes with the template DNA resulting in a DNA:RNA hybrid and a looped-out single-stranded DNA (ssDNA) strand. These structures are important for normal cellular processes and aberrant R-loop formation has been implicated in a number of pathological outcomes, including certain cancers and neurodegenerative diseases. Mapping R-loops has primarily been performed using DRIP (DNA:RNA immunoprecipitation) based methods that are dependent on the anti-DNA:RNA hybrid S9.6 antibody and short-read sequencing. While DRIP-based methods are robust and report R-loop formation genome-wide, they only do so at the population average level; interrogating R-loop formation at the single molecule level is not feasible with such approaches. Here we present single molecule R-loop footprinting (SMRF-seq), a method that relies on the chemical reactivity of the displaced ssDNA strand to non-denaturing sodium bisulfite and single molecule long-read sequencing as a readout, to characterize R-loops. SMRF-seq can be used independently of S9.6 to generate high resolution, strand-specific, maps of individual R-loops at ultra-deep coverage on kilobases-length DNA fragments.
Topics: HeLa Cells; Humans; R-Loop Structures; Sequence Analysis, RNA
PubMed: 32681515
DOI: 10.1007/978-1-0716-0680-3_15 -
BMC Microbiology Jan 2023The control of cellular zinc (Zn) concentrations by dedicated import and export systems is essential for the survival and virulence of Pseudomonas aeruginosa. The...
The control of cellular zinc (Zn) concentrations by dedicated import and export systems is essential for the survival and virulence of Pseudomonas aeruginosa. The transcription of its many Zn transporters is therefore tightly regulated by a known set of transcription factors involved in either the import or the export of Zn. In this work, we show that the Zur protein, a well-known repressor of Zn import, plays a dual role and functions in both import and export processes. In a situation of Zn excess, Zur represses Zn entry, but also activates the transcription of czcR, a positive regulator of the Zn export system. To achieve this, Zur binds at two sites, located by DNA footprinting in the region downstream the czcR transcription start site. In agreement with this regulation, a delay in induction of the efflux system is observed in the absence of Zur and Zn resistance is reduced. The discovery of this regulation highlights a new role of Zur as global regulator of Zn homeostasis in P. aeruginosa disclosing an important link between Zur and zinc export.
Topics: Zinc; Pseudomonas aeruginosa; Bacterial Proteins; Gene Expression Regulation, Bacterial; Transcription Factors
PubMed: 36617571
DOI: 10.1186/s12866-022-02750-4 -
Frontiers in Microbiology 2021After several decades of use, trimethoprim (TMP) remains one of the key access antimicrobial drugs listed by the World Health Organization. To circumvent the problem of...
After several decades of use, trimethoprim (TMP) remains one of the key access antimicrobial drugs listed by the World Health Organization. To circumvent the problem of trimethoprim resistance worldwide, a better understanding of drug-resistance mechanisms is required. In this study, we screened the single-gene knockout library of , and identified and other several genes involved in trimethoprim resistance. Subsequent comparative transcriptional analysis between Δ and the wild-type strain showed that expression levels of , , and were significantly upregulated in Δ. Further deleting or could partially restore trimethoprim sensitivity to Δ, and co-overexpression of caused TMP resistance, suggesting the involvement of PhoP/Q in trimethoprim resistance. Correspondingly, MgrB and PhoP were shown to be able to modulated expression . After that, efforts were made to test if PhoP could directly modulate the expression of . Though phosphorylated PhoP could bind to the promotor region of , the former only provided a weak protection on the latter as shown by the DNA footprinting assay. In addition, deleting the deduced PhoP box in Δ could only slightly reverse the TMP resistance phenotype, suggesting that it is less likely for PhoP to directly modulate the transcription of . Taken together, our data suggested that, in , MgrB affects susceptibility to trimethoprim by modulating the expression of with the involvement of PhoP/Q. This work broadens our understanding of the regulation of folate metabolism and the mechanisms of TMP resistance in bacteria.
PubMed: 34394028
DOI: 10.3389/fmicb.2021.682205 -
Methods in Enzymology 2024DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to...
DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to G-quadruplexes, cruciforms, Z-DNA or H-DNA structures, amongst others (collectively called 'non-B DNA'), which DNA molecules can adopt beyond the B conformation. DNA secondary structures have significant biological roles, and their landscape is dynamic and can rearrange due to various factors, including changes in cellular conditions, temperature, and DNA-binding proteins. Understanding this dynamic nature is crucial for unraveling their functions in cellular processes. Detecting DNA secondary structures remains a challenge. Conventional methods, such as gel electrophoresis and chemical probing, have limitations in terms of sensitivity and specificity. Emerging techniques, including next-generation sequencing and single-molecule approaches, offer promise but face challenges since these techniques are mostly limited to only one type of secondary structure. Here we describe an updated version of a technique permanganate/S1 nuclease footprinting, which uses potassium permanganate to trap single-stranded DNA regions as found in many non-B structures, in combination with S1 nuclease digest and adapter ligation to detect genome-wide non-B formation. To overcome technical hurdles, we combined this method with direct adapter ligation and sequencing (PDAL-Seq). Furthermore, we established a user-friendly pipeline available on Galaxy to standardize PDAL-Seq data analysis. This optimized method allows the analysis of many types of DNA secondary structures that form in a living cell and will advance our knowledge of their roles in health and disease.
Topics: DNA; G-Quadruplexes; Oxides; Manganese Compounds; Oligonucleotides
PubMed: 38521584
DOI: 10.1016/bs.mie.2023.12.003 -
International Journal of Biological... Mar 2020As a member of the SWI/SNF family, ARID1A plays an essential role in modulating chromatin structure and gene expression. The tumor suppressive function of ARID1A has...
As a member of the SWI/SNF family, ARID1A plays an essential role in modulating chromatin structure and gene expression. The tumor suppressive function of ARID1A has been well-defined and its downregulation in cancers is attributed to genomic deletion, DNA methylation and microRNA-mediated inhibition. In this study, we demonstrated that the negative strand of a C-rich region in the upstream vicinity of the human ARID1A transcription start site could form G-quadruplexes. Synthesized oligonucleotides based on the sequence of this region exhibited molar ellipticity at specific wavelengths characteristic of G-quadruplex structures in circular dichroism analyses. The formation of G-quadruplexes by these oligonucleotides were also proved by native polyacrylamide gel electrophoresis, DNA synthesis block assays, immunofluorescent staining and dimethyl sulfate footprinting studies. In reporter assays, mutations of the G-quadruplex forming sequence reduced ARID1A promoter-mediated transcription. Transfection of the oligonucleotide with the full length of G-quadruplex motif region, but not its partial sequences or the mutants, could both promote endogenous ARID1A expression and reduce cell proliferation.
Topics: Cell Line, Tumor; Cell Proliferation; Chromatin; Circular Dichroism; DNA; DNA-Binding Proteins; G-Quadruplexes; Gene Expression Regulation; Humans; Neoplasms; Oligonucleotides; Promoter Regions, Genetic; Transcription Factors; Transcription Initiation Site; Transfection
PubMed: 31982538
DOI: 10.1016/j.ijbiomac.2020.01.210 -
Genes Oct 2022Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral...
Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked (eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to . To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in , and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the -refined critical interval. We conclude that the refined critical region at chrX: 56,145,000-58,385,000 contains multiple genetic variants that may be linked to altered regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in , but further work is required to determine the precise causative allele and its genetic mechanism.
Topics: Mice; Male; Animals; Anophthalmos; Regulatory Sequences, Nucleic Acid; Transcription Factors; Chromatin; DNA; Homeodomain Proteins
PubMed: 36292683
DOI: 10.3390/genes13101797 -
Microorganisms Apr 2021Type II bacterial toxin-antitoxin (TA) systems are found in most bacteria, archaea, and mobile genetic elements. TAs are usually found as a bi-cistronic operon composed...
Type II bacterial toxin-antitoxin (TA) systems are found in most bacteria, archaea, and mobile genetic elements. TAs are usually found as a bi-cistronic operon composed of an unstable antitoxin and a stable toxin that targets crucial cellular functions like DNA supercoiling, cell-wall synthesis or mRNA translation. The type II RelBE system encoded by the pathogen is highly conserved among different strains and participates in biofilm formation and response to oxidative stress. Here, we have analyzed the participation of the RelB antitoxin and the RelB:RelE protein complex in the self-regulation of the pneumococcal operon. RelB acted as a weak repressor, whereas RelE performed the role of a co-repressor. By DNA footprinting experiments, we show that the proteins bind to a region that encompasses two palindromic sequences that are located around the -10 sequences of the single promoter that directs the synthesis of the mRNA. High-resolution footprinting assays showed the distribution of bases whose deoxyriboses are protected by the bound proteins, demonstrating that RelB and RelB:RelE contacted the DNA backbone on one face of the DNA helix and that these interactions extended beyond the palindromic sequences. Our findings suggest that the binding of the RelBE proteins to its DNA target would lead to direct inhibition of the binding of the host RNA polymerase to the relBE promoter.
PubMed: 33921033
DOI: 10.3390/microorganisms9040851 -
Journal of Molecular Biology Mar 2020R-loops are a prevalent class of non-B DNA structures that have been associated with both positive and negative cellular outcomes. DNA:RNA immunoprecipitation (DRIP)...
R-loops are a prevalent class of non-B DNA structures that have been associated with both positive and negative cellular outcomes. DNA:RNA immunoprecipitation (DRIP) approaches based on the anti-DNA:RNA hybrid S9.6 antibody revealed that R-loops form dynamically over conserved genic hotspots. We have developed an orthogonal approach that queries R-loops via the presence of long stretches of single-stranded DNA on their looped-out strand. Nondenaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long-read, single-molecule PacBio sequencing allows the identification of R-loop 'footprints' at near nucleotide resolution in a strand-specific manner on long single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting coupled with PacBio sequencing (SMRF-seq) revealed a strong agreement between S9.6-based and bisulfite-based R-loop mapping and confirmed that R-loops form over genic hotspots, including gene bodies and terminal gene regions. Based on the largest single-molecule R-loop dataset to date, we show that individual R-loops form nonrandomly, defining discrete sets of overlapping molecular clusters that pileup through larger R-loop zones. R-loops most often map to intronic regions and their individual start and stop positions do not match with intron-exon boundaries, reinforcing the model that they form cotranscriptionally from unspliced transcripts. SMRF-seq further established that R-loop distribution patterns are not simply driven by intrinsic DNA sequence features but most likely also reflect DNA topological constraints. Overall, DRIP-based and SMRF-based approaches independently provide a complementary and congruent view of R-loop distribution, consolidating our understanding of the principles underlying R-loop formation.
Topics: DNA; Embryonal Carcinoma Stem Cells; Humans; R-Loop Structures; RNA; Single-Cell Analysis; Transcription, Genetic
PubMed: 32105733
DOI: 10.1016/j.jmb.2020.02.014