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C to T error profile in GCGC (canonical recognition site), ACGC, TCGC, CCGC and GCGT. in R1 reads (orange) and R2 reads (red) for RIMS-seq (upper panel) and DNA-seq(+3H) (lower panel) A. Recombinant HhaI methylase expressed in E. coli B. Native HhaI methylase expressed in Haemophilus parahaemolyticus. Elevation of C to T in the R1 read variant can be observed in the context of GCGC for both the recombinant and native HhaI genomic DNA and in the context of ACGC only for DNA from the recombinant but not the native HhaI.
Source publication
DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylas...
Contexts in source publication
Context 1
... overall sequencing performances were assessed in terms of insert size, GC bias and genome coverage. Similar results were observed between RIMS-seq and the DNA-seq control at all treatment times, indicating that the RIMS-seq heat-alkaline treatment does not affect the quality of the libraries (Supplementary Figure S4). ...
Context 2
... the methylase M.HhaI recognizing GCGC (4), we performed RIMS-seq and a control DNA-seq(+3H) on both the native strain (Haemophilus parahaemolyticus ATCC 10014) and in E. coli K12 expressing the recombinant version of M.HhaI. Interestingly, we found that the de novo RIMS-seq analysis algorithm identifies RCGC (with R being either A or G) for the recombinant strain and GCGC for the native strain ( Figure 4A). Conversely, no notable elevation of C to T read variants are observed at ACGC for the native strain ( Figure 4B), confirming the de novo motif discovery results from the analysis pipeline. ...
Context 3
... we found that the de novo RIMS-seq analysis algorithm identifies RCGC (with R being either A or G) for the recombinant strain and GCGC for the native strain ( Figure 4A). Conversely, no notable elevation of C to T read variants are observed at ACGC for the native strain ( Figure 4B), confirming the de novo motif discovery results from the analysis pipeline. Collectively, these results suggest that the recombinant methylase shows star activity, notably in the context of ACGC, that is not found in the native strain. ...
Citations
... combination with DNA substrates to detect methylation in vitro are feasible approaches, although optimization of experimental conditions may be required. [26,84] In sum, although assignment of prokaryotic enzyme specificity is now largely achieved via high-throughput analysis of genomes and methylomes, [85,86] proper assignment of specificity to novel eukaryotic MTases requires a thorough manual curation of database search outputs, as well as experimental verification of sequence specificity and DNA modification activity. ...
DNA methylation constitutes one of the pillars of epigenetics, relying on covalent bonds for addition and/or removal of chemically distinct marks within the major groove of the double helix. DNA methyltransferases, enzymes which introduce methyl marks, initially evolved in prokaryotes as components of restriction-modification systems protecting host genomes from bacteriophages and other invading foreign DNA. In early eukaryotic evolution, DNA methyltransferases were horizontally transferred from bacteria into eukaryotes several times and independently co-opted into epigenetic regulatory systems, primarily via establishing connections with the chromatin environment. While C5-methylcytosine is the cornerstone of plant and animal epigenetics and has been investigated in much detail, the epigenetic role of other methylated bases is less clear. Recent addition of N4-methylcytosine of bacterial origin as a metazoan DNA modification highlights the prerequisites for foreign gene co-option into the host regulatory networks, and challenges the existing paradigms concerning the origin and evolution of eukaryotic regulatory systems.
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... Genomic DNA was extracted from frozen tissues using the PureLink™ genomic DNA mini kit (K182001; Invitrogen), and the extracted DNA was treated with sodium bisulfite as described above [18,19]. The primers (forward, 5′-GGA TTT GTA TTG AGG TTT TGG AG-3′, reverse, 5′-TAA CCC ATC ACC TCC ACC AC-3′) was designed to amplify the promoter region of OCT4 and sequence the bisulfite genome from − 234 to + 46 in exon 1. ...
There is an ongoing debate regarding whether gliomas originate due to functional or genetic changes in neural stem cells (NSCs). Genetic engineering has made it possible to use NSCs to establish glioma models with the pathological features of human tumors. Here, we found that RAS, TERT, and p53 mutations or abnormal expression were associated with the occurrence of glioma in the mouse tumor transplantation model. Moreover, EZH2 palmitoylation mediated by ZDHHC5 played a significant role in this malignant transformation. EZH2 palmitoylation activates H3K27me3, which in turn decreases miR-1275, increases glial fibrillary acidic protein (GFAP) expression, and weakens the binding of DNA methyltransferase 3A (DNMT3A) to the OCT4 promoter region. Thus, these findings are significant because RAS, TERT, and p53 oncogenes in human neural stem cells are conducive to a fully malignant and rapid transformation, suggesting that gene changes and specific combinations of susceptible cell types are important factors in determining the occurrence of gliomas.
... We first characterized human thymine-DNA glycosylase hTDG repair on G:T and G:hmU mismatch activity on various sequence contexts using a NGS-based assay. For this, we used fully modified XP12 and T4gt phage genomic DNAs that harbor 5mC (16) and 5hmC (17) respectively and were subjected to a limited deamination using heat alkaline treatment following similar conditions as previously published (18) with some minor changes as described in Material and Methods. Using such treatment, the deamination rate of XP12 has been previously shown to be even across all contexts (18). ...
... For this, we used fully modified XP12 and T4gt phage genomic DNAs that harbor 5mC (16) and 5hmC (17) respectively and were subjected to a limited deamination using heat alkaline treatment following similar conditions as previously published (18) with some minor changes as described in Material and Methods. Using such treatment, the deamination rate of XP12 has been previously shown to be even across all contexts (18). We further demonstrate that the deamination rate of 5hmC is proportional to the heat alkaline treatment time (Supplementary Figure S2A) and evenly distributed across all analyzed contexts (Supplementary Figure S2B). ...
Avoiding damage-induced sequencing errors is a critical step for the accurate identification of medium to rare frequency mutations in DNA samples. In the case of FFPE samples, deamination of cytosine moieties represents a major damage resulting in the loss of DNA material and sequencing errors. In this study, we demonstrated that, while damage from deamination of both cytosine and methylated cytosine moieties results in elevated C to T transition, the error profiles and mediation strategies are different and easily distinguishable. While damage-induced sequencing errors from cytosine deamination is driven by the end-repair step commonly used in NGS workflow, DNA damage resulting from deamination of methylated cytosine is another major contributor to sequencing errors at CpG sites. Uracil DNA glycosylase and human thymine DNA glycosylase can respectively eliminate and mitigate both damages in FFPE DNA samples, therefore increasing sequencing accuracy notably for the identification of moderate allelic frequency variants.
Although restriction-modification systems are found in both Eubacterial and Archaeal kingdoms, comparatively less is known about patterns of DNA methylation and genome defense systems in archaea. Here we report the complete closed genome sequence and methylome analysis of Methanococcus aeolicus PL15/H p , a strain of the CO2-reducing methanogenic archaeon and a commercial source for MaeI, MaeII, and MaeIII restriction endonucleases. The M. aeolicus PL15/H p genome consists of a 1.68 megabase circular chromosome predicted to contain 1,615 protein coding genes and 38 tRNAs. A combination of methylome sequencing, homology-based genome annotation, and recombinant gene expression identified five restriction-modification systems encoded by this organism, including the methyltransferase and site-specific endonuclease of MaeIII. The MaeIII restriction endonuclease was recombinantly expressed, purified and shown to have site-specific DNA cleavage activity in vitro.
Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.
Covalent modifications of genomic DNA are crucial for most organisms to survive. Amplicon-based high throughput sequencing technologies erase all DNA modifications to retain only sequence information for the four canonical nucleobases, necessitating specialized technologies for ascertaining epigenetic information. To also capture base modification information, we developed Methyl-SNP-seq, a technology that takes advantage of the complementarity of the double helix to extract the methylation and original sequence information from a single DNA molecule. More specifically, Methyl-SNP-seq uses bisulfite conversion of one of the strands to identify cytosine methylation while retaining the original four-bases sequence information on the other strand. As both strands are locked together to link the dual readouts on a single paired-end read, Methyl-SNP-seq allows detecting methylation status of any DNA even without a reference genome. Because one of the strands retains the original four nucleotide composition, Methyl-SNP-seq can also be used in conjunction with standard sequence-specific probes for targeted enrichment and amplification. We demonstrate the usefulness of this technology in a broad spectrum of applications ranging from allele-specific methylation analysis in humans to identification of methyltransferase specificity in complex bacterial communities.
The development of high-throughput DNA sequencing revolutionized the study of complex bacterial communities called “microbiomes'', in diverse environments, from the central oceans to the human intestine. The research aim of this thesis is to develop new sequencing-based technologies and apply them to provide further insights into changes to the composition and activities of microbiomes. Specifically, Chapter One presents RIMS-seq (Rapid Identification of Methylase Specificity), a method to simultaneously obtain the DNA sequence and 5-methylcytosine (m5C) profile of bacterial genomes. Modification by m5C has been described in the genomes of many bacterial species to modulate gene expression and protect from viral infection. Chapter Two introduces ONT-cappable-seq and Loop-Cappable-seq, two new techniques to reveal operon architecture through full-length transcript sequencing, using Nanopore and LoopSeq sequencing, respectively. In Chapter Three, we applied a multiomics approach using some of the tools developed in the previous chapters tostudy the dynamics of the response of a model human intestinal microbiome after treatment with ciprofloxacin, a widely used broad-spectrum antibiotic. Antibiotics are critical treatments to prevent pathogenic infections, but they also kill commensal species that promote health, enhance the spread of resistant strains, and may degrade the protective effect of microbiota against invasion by pathogens. Therefore, it is crucial to be able to characterize both the composition but also the functional response of a microbial community to antibiotic treatment. We examined both the short and long-term transcriptional and genomic responses of the synthetic community and explored how the immediate transcriptomic response correlates and potentially predicts the later changes of the microbiome composition. The goal is to try to identify a marker appearing a few minutes/hours after the treatment that could be used to potentially predict the outcome of an antibiotic treatment, opening up the path to a more personalized medicine.
