Flanking DNA Research Articles

Mobile genetic elements contributing to horizontal gene transfer of blaNDM among Escherichia coli in the community setting

ObjectiveTo investigate the distribution of carbapenem-resistant Enterobacterales (CRE) in the community and to describe the genomic characteristics. MethodsCRE screened from fecal samples in healthy people at the health examination center of a tertiary hospital in China underwent Whole genome sequencing (WGS) to analyze genotypic characteristics of CRE. The flanking DNA sequence of blaNDM-5 and mcr1.1 genes were analyzed by Gcluster software. ResultsA total of 7187 fecal samples were screened, and CRE carriage was detected in 0.4 % of the sampled population. In total, 30 Escherichia coli, one Citrobacter freundii and one Klebsiella aerogene were screened. The 30 carbapenem-resistant Escherichia coli (CREC) isolates displayed slight resistance to amikacin (13.3 %) and aztreonam (20.0 %). All the CRE isolates contained blaNDM, and blaNDM-5 (84.4 %) was the most common one. B1 (n = 11) and A (n = 7) were predominant phylogroups. Furthermore, 34 distinct plasmid replicons, 67 different VFs, 22 distinct STs, 17 different FimH types, 26 O:H serotypes as well as 74 MGEs including 61 insertion sequences and 13 transposons were identified. The flanking DNA sequence analysis of blaNDM-5 and mcr1.1 genes indicates the key role of horizontal transfer of blaNDM-5 in the CRE development evidenced by diverse STs and phylogenetic tree. ConclusionE. coli was the most predominant CRE isolates in community setting, and blaNDM (blaNDM-5) was the main CHβL encoding genes. The high prevalence of ARGs was associated with high resistance to commonly used antimicrobials. Besides, the genetic diversity of these isolates suggested the key role of blaNDM horizontal transfer in the CRE development. Thus, active screening of blaNDM in communities is particularly important for the prevention and control of CRE.

Quantitative trait loci for bearing habit in a ‘Sparrow’ × ‘Schessler’ Juglans nigra mapping population

Eastern black walnut, Juglans nigra L., is an economically important tree species valued for its high-quality timber and edible nuts. A regional industry for the species’ nut and kernel products resides in Missouri, where over 9 million kg of hulled in-shell nuts are purchased in masting years. The crop is primarily based upon nuts harvested from wild trees, placing a ceiling on nut volume and quality (e.g. small nut size, dark pellicle color, and 10–14% kernel by weight). Orchards of named cultivars, like ‘Kwik Krop’ and ‘Sparrow’, supply up to 22,000 kg of nuts with a higher kernel percentage (> 26%) and improved quality. Such cultivars often represent chance wild or on-farm seedlings, clonally propagated since the late 1800’s by enthusiasts. Today, continued improvement of eastern black walnut as an orchard crop is limited by a long generation time, delayed expression of important traits, and space requirements – creating a strong need for marker-trait association studies that inform progeny selection. The first linkage map for J. nigra was recently created using the ‘Sparrow’ × ‘Schessler’ F1 population and loci for phenology traits discovered. The objective of this study is to utilize these genetic resources to detect quantitative trait loci (QTL) and report associated DNA markers for the spur-bearing habit, which promotes precocity and high yield. Using single-year data from the 11-year-old population, we observe that segregation for the spur-bearing habit appears to be recessive and multigenic. Three QTLs (p > 0.99) were identified on linkage group (LG) 8, LG11, and LG16 that explain 7.2%, 8.7%, and 10% of trait variation, respectively. Regions between flanking DNA markers were 3.16 cM, 4.32 cM, and 9.69 cM, respectively. This study is the first to examine the genetic control of bearing habit and yield in eastern black walnut and informs breeders’ approach for their future genetic improvement.

Colorimetric detection of furin based on enhanced catalytic activity of G-quadruplex/hemin DNAzyme

BackgroundRapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative. ResultsHerein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS2− for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM. SignificanceThe DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers.

Variation of the 3'RR1 HS1.2 Enhancer and Its Genomic Context.

In humans, the HS1.2 enhancer in the Ig heavy-chain locus is modular, with length polymorphism. Previous studies have shown the following features for this variation: (i) strong population structuring; (ii) association with autoimmune diseases; and (iii) association with developmental changes in Ig expression. The HS1.2 region could then be considered as a contributor to inter-individual diversity in humoral response in adaptive immunity. We experimentally determined the HS1.2-length class genotype in 72 of the 1000 Genomes CEU cell lines and assigned the HS1.2 alleles to haplotypes defined by 18 landmark SNPs. We also sequenced the variable portion and ~200 bp of the flanking DNA of 34 HS1.2 alleles. Furthermore, we computationally explored the ability of different allelic arrangements to bind transcription factors. Non-random association between HS1.2 and Gm allotypes in the European population clearly emerged. We show a wealth of variation in the modular composition of HS1.2, with five SNPs further contributing to diversity. Longer alleles offer more potential sites for binding but, for same-length alleles, SNP variation creates/destroys potential binding sites. Altogether, the arrangements of modules and SNP alleles both inside and outside HS1.2 denote an organization of diversity far from randomness. In the context of the strong divergence of human populations for this genomic region and the reported disease associations, our results suggest that selective forces shaped the pattern of its diversity.

Microsatellite break-induced replication generates highly mutagenized extrachromosomal circular DNAs.

Extrachromosomal circular DNAs (eccDNAs) are produced from all regions of the eucaryotic genome. We used inverse PCR of non-B microsatellites capable of forming hairpin, triplex, quadruplex and AT-rich structures integrated at a common ectopic chromosomal site to show that these non-B DNAs generate highly mutagenized eccDNAs by replication-dependent mechanisms. Mutagenesis occurs within the non-B DNAs and extends several kilobases bidirectionally into flanking and nonallelic DNA. Each non-B DNA exhibits a different pattern of mutagenesis, while sister clones containing the same non-B DNA also display distinct patterns of recombination, microhomology-mediated template switching and base substitutions. Mutations include mismatches, short duplications, long nontemplated insertions, large deletions and template switches to sister chromatids and nonallelic chromosomes. Drug-induced replication stress or the depletion of DNA repair factors Rad51, the COPS2 signalosome subunit or POLη change the pattern of template switching and alter the eccDNA mutagenic profiles. We propose an asynchronous capture model based on break-induced replication from microsatellite-induced DNA double strand breaks to account for the generation and circularization of mutagenized eccDNAs and the appearance of genomic homologous recombination deficiency (HRD) scars. These results may help to explain the appearance of tumor eccDNAS and their roles in neoantigen production, oncogenesis and resistance to chemotherapy.

Analysis of the properties of Komagataella phaffii CF-st401, a genetically modified producer of the sweet protein brazzein by using in silico methods

Currently, in order to reduce the consumption of mono- and disaccharides in diets, sweeteners are widely used. At the same time, none of the sweeteners approved for food industry usage matches the organoleptic properties of natural sugars. This circumstance was the basis for the development of technology for producing a new type of food ingredient with a sweet taste - brazzein using the producer strain Komagataella phaffii CF-st401. The purpose of the study was the application of in silico methods to assess the safety of K. phaffii CF-st401 genetically modified (GM) microorganism, which is the producer of the "Sweet protein Brazzein". Material and methods. The research object was the map of K. phaffii CF-st401 plasmid obtained by Biryuch LLC as a result of sequencing the plasmid of GM strain K. phaffii CF-st401, including: a synthetic nucleotide sequence similar to the sequence encoding the brazzein protein in the plant (Pentadiplandra brazzeana) and optimized for expression in the DNA of the recipient strain; the nucleotide sequence of plasmid M4794 from Saccharomyces cerevisiae and a linear fragment of the flanking DNA regions from K. phaffii used for integration into the genome of the recipient strain K. phaffii YIB Δleu2 VKPM Y-476, as well as amino acid sequence data of recombinant brazzein. By using bioinformatics methods (in silico), we investigated the DNA structure of the vector sequence of K. phaffii CF-st401, including the presence of operons responsible for toxin production, antibiotic resistance, and allergenicity. Results. As a result of the studies of K. phaffii CF-st401 vector plasmid, introduced into K. phaffii YIB Δleu2 VKPM Y-4761, it was shown that its regions responsible for the structure of the "Sweet protein Brazzein" coincide by more than 70% with elements of the brazzein P56552 reference protein from the plant P. brazzeana. The absence of selective markers and allergenicity in the vector plasmid was confirmed. Conclusion. The analysis of the structure of the DNA vector sequence of the K. phaffii CF-st401 GM strain confirmed the feasibility of using bioinformatics methods to predict the properties of technological microorganisms when assessing their safety for consumers.

Interpretable predictive models of genome-wide aryl hydrocarbon receptor-DNA binding reveal tissue-specific binding determinants.

The aryl hydrocarbon receptor (AhR) is an inducible transcription factor whose ligands include the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ligand-activated AhR binds to DNA at dioxin response elements (DREs) containing the core motif 5'-GCGTG-3'. However, AhR binding is highly tissue specific. Most DREs in accessible chromatin are not bound by TCDD-activated AhR, and DREs accessible in multiple tissues can be bound in some and unbound in others. As such, AhR functions similarly to many nuclear receptors. Given that AhR possesses a strong core motif, it is suited for a motif-centered analysis of its binding. We developed interpretable machine learning models predicting the AhR binding status of DREs in MCF-7, GM17212, and HepG2 cells, as well as primary human hepatocytes. Cross-tissue models predicting transcription factor (TF)-DNA binding generally perform poorly. However, reasons for the low performance remain unexplored. By interpreting the results of individual within-tissue models and by examining the features leading to low cross-tissue performance, we identified sequence and chromatin context patterns correlated with AhR binding. We conclude that AhR binding is driven by a complex interplay of tissue-agnostic DRE flanking DNA sequence and tissue-specific local chromatin context. Additionally, we demonstrate that interpretable machine learning models can provide novel and experimentally testable mechanistic insights into DNA binding by inducible TFs.

Regulation of eosinophils by the transcription factor Ikaros

Abstract Immune cell development is a tightly controlled process that enables a homeostatic level of innate and adaptive immune cells of the myeloid and lymphoid lineages, respectively. Imbalance in immune cells can lead to diseases due to immune defects or over-activation. such as immunodeficiency, allergies or autoimmunity. Ikaros is a transcription factor known to be critically important for lymphoid development, with recurrent mutations in different human diseases, but less is known about the role of Ikaros in myeloid development. Ikaros is encoded by the Ikzf1gene, and contains four DNA-binding zinc fingers (ZnF) and two ZnFs mediating protein dimerization. The four DNA-binding ZnFs (ZnF1 through ZnF4) are encoded by three exons, with the two central ZnF2 and ZnF3 encoded by exon 5 being required for binding to the core DNA recognition site. The two flanking ZnFs 1 and 4 are encoded by exon 4 and exon 6, and can modulate DNA binding by contacting the flanking DNA. Interestingly, Ikaros is highly alternatively spliced, creating multiple isoforms with different combinations of the DNA-binding ZnFs. We study the role of Ikaros in immune cell development using Ikzf1-mutant mouse models, and analysis of mice with deletions of exon 4 or exon 6 (encoding ZnF1 and ZnF4, respectively) has revealed differential phenotypes in the two mutant mouse models. Here, we describe a new role of Ikaros in regulation of eosinophils. We have found that Ikaros is required for limiting eosinophil production in bone marrow (BM), as both mutants show increased levels of BM eosinophils. Interestingly, the levels of peripheral tissue eosinophils differ between the two mutants, suggesting a second role of Ikaros in regulating migration, tissue recruitment or retention.

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Chinese Journal of ChemistryVolume 41, Issue 10 p. 1288-1288 Back CoverFree Access Back Cover First published: 15 April 2023 https://doi.org/10.1002/cjoc.202390105AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract The cover picture shows TET3 CXXC domain searching CpG motifs with preferences on their flanking DNA sequences, which is profiled at the single-molecule limit. The selective binding dynamics of a CXXC on a gene facilitates our understanding to epigenetic information reading mechanisms. More details are discussed in the article by Yu et al. on page 1177—1184. Volume41, Issue1015 May, 2023Pages 1288-1288 RelatedInformation

Non-specific vs. specific DNA binding free energetics of a transcription factor domain protein between search and recognition.

Transcription factor (TF) proteins are key in genetic regulation by locating specific sites on the genome. It has been proposed that an optimized search can be achieved by protein alternating between 3D diffusion in the bulk and 1D diffusion along DNA in the facilitated diffusion model. While undergoing 1D diffusion, the protein can switch from a “search” mode for fast diffusion along non-specific DNA, to a “recognition” mode for protein stable binding to specific DNA. Though it was regarded that protein conformation transitions enable the search to recognition transitions, it was recently noticed that for a small TF domain protein, re-orientation of the protein on the DNA, without protein conformational changes, happens between the non-specific and specific DNA binding. We further conducted all-atom molecular dynamics simulations on the TF domain protein bound to a nonspecific or specific DNA site, to energetically confirm that the protein-DNA binding free energetics between the two systems is consistent with the “search” and “recognition”. Using both the Jarzynski equality and umbrella sampling methods pulling protein away from DNA, we obtained protein binding free energetics and a free energy difference of about 10 kBT between the two systems. However, the binding free energy difference estimated from experimental measurements was about 4 kBT on 15-bp DNA. We suggest that the discrepancy between the experimental and computational measurements can be due to DNA sequences flanking the 6-bp central specific and nonspecific binding sites. Such a proposal was also investigated through a highly simplified spherical protein model along with stochastic simulations, which well supported that flanking DNA sequences impact on overall protein dissociation kinetics and therefore on measuring binding affinity variations with specific or non-specific DNA in the central binding site.

PLP1-lacZ transgenic mice reveal that splice variants containing "human-specific" exons are relatively minor in comparison to the archetypal transcript and that an upstream regulatory element bolsters expression during early postnatal brain development.

Much of what is known about the mechanisms that control the developmental expression of the myelin proteolipid protein gene (PLP1) has been attained through use of transgenic animal models. In this study, we analyzed expression of related transgenes which utilize PLP1 genomic DNA from either human or mouse to drive expression of a lacZ reporter. Human PLP1 (hPLP1) sequence span either the proximal 6.2 or 2.7 kb of 5'-flanking DNA to an internal site in Exon 2, while those from mouse comprise the proximal 2.3 kb of 5'-flanking DNA to an analogous site in Exon 2. Transgenes with hPLP1 sequence were named, in part, to the amount of upstream sequence they have [6.2hPLP(+)Z/FL and 2.7hPLP(+)Z]. The transgene containing mouse sequence is referred to here as mPLP(+)Z, to denote the species origin of PLP1 DNA. Mice which harbor the 6.2hPLP(+)Z/FL transgene were used as a model system to investigate the developmental expression of splice variants that incorporate supplementary exons from what is classically defined as PLP1 intron 1. While expression of the splice variants were detected in brain through RT-PCR analysis, they are present at much lower levels relative to the archetypal (classic) transcript. Additionally, we show that mice which harbor the 6.2hPLP(+)Z/FL transgene demonstrate wide-ranging expression throughout brain at P2, whereas expression of mPLP(+)Z is quite limited at this age. Therefore, we generated new transgenic mouse lines with the 2.7hPLP(+)Z transgene, which contains hPLP1 sequence orthologous to just that in mPLP(+)Z. Of the seven lines analyzed, six showed higher levels of 2.7hPLP(+)Z expression in brain at P21 compared to P2; the other line expressed the transgene, only weakly, at either age. This trend, coupled with the robust expression observed for 6.2hPLP(+)Z/FL at P2, suggests that the distal 3.5 kb of 5'-flanking PLP1 DNA specific to 6.2hPLP(+)Z/FL contains regulatory element(s) important for promoting early postnatal expression in brain.

Chromatin Sensing by the Auxiliary Domains of KDM5C Regulates Its Demethylase Activity and Is Disrupted by X-linked Intellectual Disability Mutations

The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie between the segments of the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. Through in vitro binding and kinetic studies using nucleosomes, we find that while the ARID domain is required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. In addition, the unstructured linker region between the ARID and PHD1 domains interacts with PHD1 and is necessary for nucleosome binding. Our data suggests a model in which the PHD1 domain inhibits DNA recognition by KDM5C. This inhibitory effect is relieved by the H3 tail, enabling recognition of flanking DNA on the nucleosome. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains break this regulation by enhancing DNA binding, resulting in the loss of specificity of substrate chromatin recognition and rendering demethylase activity lower in the presence of flanking DNA. Our findings suggest a model by which specific XLID mutations could alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.

Exploring novel QTLs among backcross lines for salinity tolerance in rice

Wild progenitor species of rice (Oryza rufipogon Griff./ Oryza nivara Sharma et. Shastry) are rich source of genes for both the biotic as well as abiotic stress tolerance. Wild rice accession NKSWR 173 has been identified as highly tolerant to salinity stress at seedling stage and moderately tolerant at reproductive stage after evaluation of more than two hundred wild rice accessions from across India. In a bid to introgress the salt tolerance trait from NKSWR 173 to a high-yielding mega variety of rice IR 64, we screened a segregating BC1 population for identification of suitable lines for making the second backcross using both controlled phenotyping and QTL flanking DNA markers. Four lines, namely SN 32, SN 33, SN 39 and SN 45 were found highly tolerant to salinity at both seedling and reproductive stage and were backcrossed to IR 64 to generate BC2F1 seeds for development of advance introgressed lines. Introgression of novel salinity tolerance genes for both the seedling and reproductive stages in mega variety of rice will be useful in achieving high productivity in salt affected rice areas.

Fusion primer driven racket PCR: A novel tool for genome walking.

The limitations of the current genome-walking strategies include strong background and cumbersome experimental processes. Herein, we report a genome-walking method, fusion primer-driven racket PCR (FPR-PCR), for the reliable retrieval of unknown flanking DNA sequences. Four sequence-specific primers (SSP1, SSP2, SSP3, and SSP4) were sequentially selected from known DNA (5'→3') to perform FPR-PCR. SSP3 is the fragment that mediates intra-strand annealing (FISA). The FISA fragment is attached to the 5' end of SSP1, generating a fusion primer. FPR-PCR comprises two rounds of amplification reactions. The single-fusion primary FPR-PCR begins with the selective synthesis of the target first strand, then allows the primer to partially anneal to some place(s) on the unknown region of this strand, producing the target second strand. Afterward, a new first strand is synthesized using the second strand as the template. The 3' end of this new first strand undergoes intra-strand annealing to the FISA site, followed by the formation of a racket-like DNA by a loop-back extension. This racket-like DNA is exponentially amplified in the secondary FPR-PCR performed using SSP2 and SSP4. We validated this FPR-PCR method by identifying the unknown flanks of Lactobacillus brevis CD0817 glutamic acid decarboxylase genes and the rice hygromycin gene.

DAR-PCR: a new tool for efficient retrieval of unknown flanking genomic DNA

Various PCR-based genome-walking methods have been developed to acquire unknown flanking DNA sequences. However, the specificity and efficacy levels, and the operational processes, of the available methods are unsatisfactory. This work proposes a novel walking approach, termed differential annealing-mediated racket PCR (DAR-PCR). The key to DAR-PCR is the use of primer-mediated intra-strand annealing (ISA). An ISA primer consists of a 5’ root homologous to the known sequence and a heterologous 3’ bud. In the single low-stringency cycle, the ISA primer anneals to a site on an unknown region and extends towards the sequence-specific primer (SSP) 1 site, thereby forming a target single-stranded DNA bound by the SSP1 complement and the ISA primer. In the subsequent more stringent cycles, its complementary strand is accumulated, owing to the differential annealing between the moderate-stringency ISA primer and the high-stringency SSP1. The accumulation of this strand provides an opportunity for ISA mediated by the ISA primer root. A loop-back extension subsequent to ISA occurs, creating a racket-like DNA with the known region positioned at both ends of the unknown sequence. This DNA is exponentially amplified during the secondary PCR driven by an SSP pair inner to SSP1. DAR-PCR was validated as an efficient walking method by determining unknown flanking sequences in Lactobacillus brevis and Oryza sativa.

Individual-specific levels of CTG•CAG somatic instability are shared across multiple tissues in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a complex disease caused by a genetically unstable CTG repeat expansion in the 3'-untranslated region of the DMPK gene. Age-dependent, tissue-specific somatic instability has confounded genotype-phenotype associations, but growing evidence suggests that it also contributes directly toward disease progression. Using a well-characterized clinical cohort of DM1 patients from Costa Rica, we quantified somatic instability in blood, buccal cells, skin and skeletal muscle. Whilst skeletal muscle showed the largest expansions, modal allele lengths in skin were also very large and frequently exceeded 2000 CTG repeats. Similarly, the degree of somatic expansion in blood, muscle and skin were associated with each other. Notably, we found that the degree of somatic expansion in skin was highly predictive of that in skeletal muscle. More importantly, we established that individuals whose repeat expanded more rapidly than expected in one tissue (after correction for progenitor allele length and age) also expanded more rapidly than expected in other tissues. We also provide evidence suggesting that individuals in whom the repeat expanded more rapidly than expected in skeletal muscle have an earlier age at onset than expected (after correction for the progenitor allele length). Pyrosequencing analyses of the genomic DNA flanking the CTG repeat revealed that the degree of methylation in muscle was well predicted by the muscle modal allele length and age, but that neither methylation of the flanking DNA nor levels of DMPK sense and anti-sense transcripts could obviously explain individual- or tissue-specific patterns of somatic instability.

H2A-H2B Histone Dimer Plasticity and Its Functional Implications.

The protein core of the nucleosome is composed of an H3-H4 histone tetramer and two H2A-H2B histone dimers. The tetramer organizes the central 60 DNA bp, while H2A-H2B dimers lock the flanking DNA segments. Being positioned at the sides of the nucleosome, H2A-H2B dimers stabilize the overall structure of the nucleosome and modulate its dynamics, such as DNA unwrapping, sliding, etc. Such modulation at the epigenetic level is achieved through post-translational modifications and the incorporation of histone variants. However, the detailed connection between the sequence of H2A-H2B histones and their structure, dynamics and implications for nucleosome functioning remains elusive. In this work, we present a detailed study of H2A-H2B dimer dynamics in the free form and in the context of nucleosomes via atomistic molecular dynamics simulations (based on X. laevis histones). We supplement simulation results by comparative analysis of information in the structural databases. Particularly, we describe a major dynamical mode corresponding to the bending movement of the longest H2A and H2B α-helices. This overall bending dynamics of the H2A-H2B dimer were found to be modulated by its interactions with DNA, H3-H4 tetramer, the presence of DNA twist-defects with nucleosomal DNA and the amino acid sequence of histones. Taken together, our results shed new light on the dynamical mechanisms of nucleosome functioning, such as nucleosome sliding, DNA-unwrapping and their epigenetic modulation.

Flexibility of flanking DNA is a key determinant of transcription factor affinity for the core motif

Selective gene regulation is mediated by recognition of specific DNA sequences by transcription factors (TFs). The extremely challenging task of searching out specific cognate DNA binding sites among several million putative sites within the eukaryotic genome is achieved by complex molecular recognition mechanisms. Elements of this recognition code include the core binding sequence, the flanking sequence context, and the shape and conformational flexibility of the composite binding site. To unravel the extent to which DNA flexibility modulates TF binding, in this study, we employed experimentally guided molecular dynamics simulations of ternary complex of closely related Hox heterodimers Exd-Ubx and Exd-Scr with DNA. Results demonstrate that flexibility signatures embedded in the flanking sequences impact TF binding at the cognate binding site. A DNA sequence has intrinsic shape and flexibility features. While shape features are localized, our analyses reveal that flexibility features of the flanking sequences percolate several basepairs and allosterically modulate TF binding at the core. We also show that lack of flexibility in the motif context can render the cognate site resistant to protein-induced shape changes and subsequently lower TF binding affinity. Overall, this study suggests that flexibility-guided DNA shape, and not merely the static shape, is a key unexplored component of the complex DNA-TF recognition code.

CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.

A stable XPG protein is required for proper ribosome biogenesis: Insights on the phenotype of combinate Xeroderma Pigmentosum/Cockayne Syndrome patients.

Nucleotide Excision Repair is one of the five DNA repair systems. More than 30 proteins are involved in this process, including the seven XP proteins. When mutated, the genes coding for these proteins are provoking the rare disease Xeroderma Pigmentosum, which causes cutaneous defects and a high prevalence of skin cancers in patients. The CSA and CSB proteins are also involved in Nucleotide Excision Repair, and their mutation leads to Cockayne Syndrome, another rare disease, causing dwarfism, neurodegeneration, and ultimately early death, but without high skin cancer incidence. Some mutations of ERCC5, the gene coding for XPG, may give rise to a combined Xeroderma Pigmentosum and Cockayne Syndrome. A defect in Nucleotide Excision Repair alone cannot explain all these phenotypes. XPG has been located in the nucleolus, where ribosome biogenesis happens. This energy-consuming process starts with the transcription of the ribosomal DNA in a long ribosomal RNA, the pre-rRNA 47S, by RNA Polymerase 1. 47S pre-rRNA undergoes several cleavages and modifications to form three mature products: the ribosomal RNAs 18S, 5.8S and 28S. In the cytoplasm, these three products will enter the ribosomes’ composition, the producers of protein in our cells. Our work aimed to observe ribosome biogenesis in presence of an unstable XPG protein. By working on Xeroderma Pigmentosum/Cockayne Syndrome cell lines, meaning in the absence of XPG, we uncovered that the binding of UBF, as well as the number of unresolved R-loops, is increased along the ribosomal DNA gene body and flanking regions. Furthermore, ribosomal RNA maturation is impaired, with increased use of alternative pathways of maturation as well as an increase of immature precursors. These defective processes may explain the neurodegeneration observed when the XPG protein is heavily truncated, as ribosomal homeostasis and R-loops resolution are critical for proper neuronal development.