Local DNA Research Articles

Anomalies of ATP-dependent chromatin remodeling complexes and human neurodevelopmental genetic disorders

ATP-dependent chromatin remodeling complexes play crucial roles in various biological processes including enhancing local DNA accessibility, regulating gene transcription, and facilitating DNA replication and repair. Based on their functional structural domains, these complexes may be categorized into four families, including SWI/SNF, ISWI, CHD and INO80. Such families are vital factors for regulating gene expression and play pivotal roles in developmental processes. Variants of genes encoding the components of such complexes have been closely associated with human developmental disorders and neurodevelopmental genetic syndromes. This review has summarized the classification, fundamental functions and underlying mechanism of the ATP-dependent chromatin remodeling complexes, as well as common neurological genetic disorders due to variants of genes encoding the subunits of such complexes.

Multispatially Localized DNA Walker Coupling Covalent Organic Framework for Dual-Mode Detection of Nucleocapsid Protein Using the Walking-Recycling-Conversion Strategy.

DNA walkers have emerged as a powerful tool in bioanalysis; however, many existing approaches are still restricted by low reaction kinetics and inaccurate single-mode detection. Herein, a fluorescence (FL) and electrochemical (EC) dual-mode biosensor was proposed based on a multispatially localized DNA walker (m-DNA walker) coupling covalent organic framework (COF) using the walking-recycling-conversion strategy. Specifically, the functionalized COF not only served as a three-dimensional nanocarrier but also acted as an effective quencher of the walking tracks. In the presence of the target, the activated m-DNA walker moved fast along the numerous quenching tracks, leading to the cleavage of Cy3-H1 and the recovery of the FL signal. To further improve the detection sensitivity, the Cy3-H1 fragments' recycling process was implemented with the generation of a large amount of S1 and S2, which caused the assembly of DNA-Fe3+-polydopamine network amplifiers on the electrode. The rapid electrochemical conversion was introduced to convert DNA-Fe3+-polydopamine into electroactive Prussian Blue, providing a significant EC signal output. Using nucleocapsid protein (N-protein) as the model target, the designed biosensing platform produced a FL/EC dual-mode readout with the detection limits of 65.0 fg/mL for FL mode and 2.3 fg/mL for EC mode, which could eliminate the interference from different reactive pathways and improve the detection accuracy, holding potential application in early disease diagnosis and treatment.

Template switching during DNA replication is a prevalent source of adaptive gene amplification.

Copy number variants (CNVs)-gains and losses of genomic sequences-are an important source of genetic variation underlying rapid adaptation and genome evolution. However, despite their central role in evolution little is known about the factors that contribute to the structure, size, formation rate, and fitness effects of adaptive CNVs. Local genomic sequences are likely to be an important determinant of these properties. Whereas it is known that point mutation rates vary with genomic location and local DNA sequence features, the role of genome architecture in the formation, selection, and the resulting evolutionary dynamics of CNVs is poorly understood. Previously, we have found that the GAP1 gene in Saccharomyces cerevisiae undergoes frequent and repeated amplification and selection under long-term experimental evolution in glutamine-limiting conditions. The GAP1 gene has a unique genomic architecture consisting of two flanking long terminal repeats (LTRs) and a proximate origin of DNA replication (autonomously replicating sequence, ARS), which are likely to promote rapid GAP1 CNV formation. To test the role of these genomic elements on CNV-mediated adaptive evolution, we performed experimental evolution in glutamine-limited chemostats using engineered strains lacking either the adjacent LTRs, ARS, or all elements. Using a CNV reporter system and neural network simulation-based inference (nnSBI) we quantified the formation rate and fitness effect of CNVs for each strain. We find that although GAP1 CNVs repeatedly form and sweep to high frequency in strains with modified genome architecture, removal of local DNA elements significantly impacts the rate and fitness effect of CNVs and the rate of adaptation. We performed genome sequence analysis to define the molecular mechanisms of CNV formation for 177 CNV lineages. We find that across all four strain backgrounds, between 26% and 80% of all GAP1 CNVs are mediated by Origin Dependent Inverted Repeat Amplification (ODIRA) which results from template switching between the leading and lagging strand during DNA synthesis. In the absence of the local ARS, a distal ARS can mediate CNV formation via ODIRA. In the absence of local LTRs, homologous recombination mechanisms still mediate gene amplification following de novo insertion of retrotransposon elements at the locus. Our study demonstrates the remarkable plasticity of the genome and reveals that template switching during DNA replication is a frequent source of adaptive CNVs.

Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain

APOBEC3G, part of the AID/APOBEC cytidine deaminase family, is crucial for antiviral immunity. It has two zinc-coordinated cytidine-deaminase domains. The non-catalytic N-terminal domain strongly binds to nucleic acids, whereas the C-terminal domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains is not fully understood. Here, we show that DNA editing function of rhesus macaque APOBEC3G on linear and hairpin loop DNA is enhanced by AA or GA dinucleotide motifs present downstream in the 3’-direction of the target-C editing sites. The effective distance between AA/GA and the target-C sites is contingent on the local DNA secondary structure. We present two co-crystal structures of rhesus macaque APOBEC3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic domain in capturing AA/GA DNA. Our findings elucidate the molecular mechanism of APOBEC3G’s cooperative function, which is critical for its antiviral role and its contribution to mutations in cancer genomes.

Perturbing TET2 condensation promotes aberrant genome-wide DNA methylation and curtails leukaemia cell growth.

The ten-eleven translocation (TET) family of dioxygenases maintain stable local DNA demethylation during cell division and lineage specification. As the major catalytic product of TET enzymes, 5-hydroxymethylcytosine is selectively enriched at specific genomic regions, such as enhancers, in a tissue-dependent manner. However, the mechanisms underlying this selectivity remain unresolved. Here we unveil a low-complexity insert domain within TET2 that facilitates its biomolecular condensation with epigenetic modulators, such as UTX and MLL4. This co-condensation fosters a permissive chromatin environment for precise DNA demethylation. Disrupting low-complexity insert-mediated condensation alters the genomic binding of TET2 to cause promiscuous DNA demethylation and genome reorganization. These changes influence the expression of key genes implicated in leukaemogenesis to curtail leukaemia cell proliferation. Collectively, this study establishes the pivotal role of TET2 condensation in orchestrating precise DNA demethylation and gene transcription to support tumour cell growth.

Optics-free Spatial Genomics for Mapping Mouse Brain Aging.

Spatial transcriptomics has revolutionized our understanding of cellular network dynamics in aging and disease by enabling the mapping of molecular and cellular organization across various anatomical locations. Despite these advances, current methods face significant challenges in throughput and cost, limiting their utility for comprehensive studies. To address these limitations, we introduce IRISeq (Imaging Reconstruction using Indexed Sequencing), a optics-free spatial transcriptomics platform that eliminates the need for predefined capture arrays or extensive imaging, allowing for the rapid and cost-effective processing of multiple tissue sections simultaneously. Its capacity to reconstruct images based solely on sequencing local DNA interactions allows for profiling of tissues without size constraints and across varied resolutions. Applying IRISeq, we examined gene expression and cellular dynamics in thirty brain regions of both adult and aged mice, uncovering region-specific changes in gene expression associated with aging. Further cell type-centric analysis further identified age-related cell subtypes and intricate changes in cell interactions that are distinct to certain spatial niches, emphasizing the unique aspects of aging in different brain regions. The affordability and simplicity of IRISeq position it as a versatile tool for mapping region-specific gene expression and cellular interactions across various biological systems.

Genome-Wide Methylation Analysis Reveals a KCNK3-Prominent Causal Cascade on Hypertension.

Despite advances in understanding hypertension's genetic structure, how noncoding genetic variants influence it remains unclear. Studying their interaction with DNA methylation is crucial to deciphering this complex disease's genetic mechanisms. We investigated the genetic and epigenetic interplay in hypertension using whole-genome bisulfite sequencing. Methylation profiling in 918 males revealed allele-specific methylation and methylation quantitative trait loci. We engineered rs1275988T/C mutant mice using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9), bred them for homozygosity, and subjected them to a high-salt diet. Telemetry captured their cardiovascular metrics. Protein-DNA interactions were elucidated using DNA pull-downs, mass spectrometry, and Western blots. A wire myograph assessed vascular function, and analysis of the Kcnk3 gene methylation highlighted the mutation's role in hypertension. We discovered that DNA methylation-associated genetic effects, especially in non-cytosine-phosphate-guanine (non-CpG) island and noncoding distal regulatory regions, significantly contribute to hypertension predisposition. We identified distinct methylation quantitative trait locus patterns in the hypertensive population and observed that the onset of hypertension is influenced by the transmission of genetic effects through the demethylation process. By evidence-driven prioritization and in vivo experiments, we unearthed rs1275988 in a cell type-specific enhancer as a notable hypertension causal variant, intensifying hypertension through the modulation of local DNA methylation and consequential alterations in Kcnk3 gene expression and vascular remodeling. When exposed to a high-salt diet, mice with the rs1275988C/C genotype exhibited exacerbated hypertension and significant vascular remodeling, underscored by increased aortic wall thickness. The C allele of rs1275988 was associated with elevated DNA methylation levels, driving down the expression of the Kcnk3 gene by attenuating Nr2f2 (nuclear receptor subfamily 2 group F member 2) binding at the enhancer locus. Our research reveals new insights into the complex interplay between genetic variations and DNA methylation in hypertension. We underscore hypomethylation's potential in hypertension onset and identify rs1275988 as a causal variant in vascular remodeling. This work advances our understanding of hypertension's molecular mechanisms and encourages personalized health care strategies.

Spatially Localized Entropy-Driven Evolution of Nucleic Acid-Based Constitutional Dynamic Networks for Intracellular Imaging and Spatiotemporal Programmable Gene Therapy.

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a [2 × 2] CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.

A Dual-Recognition Fluorescence Enzyme-Linked Immunosorbent Assay for Specific Detection of Intact Lipid Nanoparticles via a Localized Scaffolding Autocatalytic DNA Circuit Amplifier.

Lipid nanoparticles (LNPs) are emerging as one of the most promising drug delivery systems. The long-circulating effect of intact LNPs (i-LNPs) is the key to efficacy and toxicity in vivo. However, the significant challenge is specific and sensitive detection of i-LNPs. Herein, a dual-recognition fluorescence enzyme-linked immunosorbent assay (DR-FELISA) was developed to directly isolate and detect i-LNPs by combining dual-recognition separation with a one-step signal amplification strategy. The microplates captured and enriched i-LNPs through antibody-antigen reaction. Dual-chol probes were spontaneously introduced into the lipid bilayer of captured i-LNPs, converting the detection of i-LNPs into the detection of double-cholesterol probes. Finally, the end of the dual-chol probes initiated the localized scaffolding autocatalytic DNA circuits (SADC) system for further signal amplification. The SADC system provides a sensitive and efficient amplifier through localized network structures and self-assembled triggers. Simultaneous recognition of i-LNPs surface PEG-lipid and lipid bilayer structures significantly eliminates interference from biological samples. i-LNPs were detected with high selectivity, ranging from 0.2 to 1.25 mg/mL with a limit of detection of 0.1 mg/mL. Moreover, this method allows the isolation and quantitative analysis of different formulations of i-LNPs in serum samples with a satisfactory recovery rate ranging from 94.8 to 116.3%. Thus, the DR-FELISA method provides an advanced platform for the exclusive and sensitive detection of i-LNPs, providing new insights for the study of the quality and intracorporal process of complex formulations.

Label-free and ultrasensitive detection of environmental lead ions based on spatially localized DNA nanomachines driven by hyperbranched hybridization chain reaction

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring.

The importance of DNA sequence for nucleosome positioning in transcriptional regulation.

Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription machinery at every stage. Influences to steer nucleosome positioning are diverse, and the according importance of the DNA sequence in contrast to active chromatin remodeling has been the subject of long discussion. In this study, we evaluate the functional role of DNA sequence for all major elements along the process of transcription. We developed a random forest classifier based on local DNA structure that assesses the sequence-intrinsic support for nucleosome positioning. On this basis, we created a simple data resource that we applied genome-wide to the human genome. In our comprehensive analysis, we found a special role of DNA in mediating the competition of nucleosomes with cis-regulatory elements, in enabling steady transcription, for positioning of stable nucleosomes in exons, and for repelling nucleosomes during transcription termination. In contrast, we relate these findings to concurrent processes that generate strongly positioned nucleosomes in vivo that are not mediated by sequence, such as energy-dependent remodeling of chromatin.

Long-term prognosis and DNA damage status after oral mucosal epithelial cell sheet transplantation following esophageal endoscopic submucosal dissection for squamous cell carcinoma: A case series

Autologous oral mucosal epithelial cell sheet (AOMECS) transplantation has recently been applied in human patients to prevent postprocedural stenosis following endoscopic submucosal dissection (ESD) for esophageal squamous cell carcinoma. However, the long-term safety of AOMECS transplantation remains unclear. We evaluated the long-term outcomes of 10 patients who participated in a clinical trial of AOMECS transplantation after esophageal ESD. Additionally, we assessed the local DNA damage response in the esophageal epithelium using p53 binding protein 1 (53BP1) immunofluorescence in post-AOMECS biopsy specimens. The median follow-up period was 118.5 months (range: 46–130 months). Two patients developed primary esophageal cancer near the AOMECS site and successfully underwent additional ESD. One patient developed lymph node metastasis and underwent chemotherapy. None of the patients died from the original disease, although one patient died from unrelated causes. The rate of abnormal 53BP1 nuclear foci, indicative of increased genome instability, increased with the progression of neoplasia in patients post AOMECS. Our case series suggests that AOMECS transplantation provides an acceptable long-term prognosis and 53BP1 foci may serve as a useful marker for assessing DNA instability in the post-AOMECS esophageal epithelium.

Dissecting the cis-regulatory syntax of transcription initiation with deep learning.

Despite extensive characterization of mammalian Pol II transcription, the DNA sequence determinants of transcription initiation at a third of human promoters and most enhancers remain poorly understood. Hence, we trained and interpreted a neural network called ProCapNet that accurately models base-resolution initiation profiles from PRO-cap experiments using local DNA sequence. ProCapNet learns sequence motifs with distinct effects on initiation rates and TSS positioning and uncovers context-specific cryptic initiator elements intertwined within other TF motifs. ProCapNet annotates predictive motifs in nearly all actively transcribed regulatory elements across multiple cell-lines, revealing a shared cis-regulatory logic across promoters and enhancers mediated by a highly epistatic sequence syntax of cooperative and competitive motif interactions. ProCapNet models of RAMPAGE profiles measuring steady-state RNA abundance at TSSs distill initiation signals on par with models trained directly on PRO-cap profiles. ProCapNet learns a largely cell-type-agnostic cis-regulatory code of initiation complementing sequence drivers of cell-type-specific chromatin state critical for accurate prediction of cell-type-specific transcription initiation.

A spatially localized DNA linear classifier for cancer diagnosis

Molecular computing is an emerging paradigm that plays an essential role in data storage, bio-computation, and clinical diagnosis with the future trends of more efficient computing scheme, higher modularity with scaled-up circuity and stronger tolerance of corrupted inputs in a complex environment. Towards these goals, we construct a spatially localized, DNA integrated circuits-based classifier (DNA IC-CLA) that can perform neuromorphic architecture-based computation at a molecular level for medical diagnosis. The DNA-based classifier employs a two-dimensional DNA origami as the framework and localized processing modules as the in-frame computing core to execute arithmetic operations (e.g. multiplication, addition, subtraction) for efficient linear classification of complex patterns of miRNA inputs. We demonstrate that the DNA IC-CLA enables accurate cancer diagnosis in a faster (about 3 h) and more effective manner in synthetic and clinical samples compared to those of the traditional freely diffusible DNA circuits. We believe that this all-in-one DNA-based classifier can exhibit more applications in biocomputing in cells and medical diagnostics.

Interpretable deep learning reveals the role of an E-box motif in suppressing somatic hypermutation of AGCT motifs within human immunoglobulin variable regions.

Somatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, long-term humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5' ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context. Here, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions. DeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5'C and 3'G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for E-box transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3'G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation. Our results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions.

H2B oncohistones cause homologous recombination defect and genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe

Canonical oncohistones are histone H3 mutations in the N-terminal tail associated with tumors and affect gene expression by altering H3 post-translational modifications (PTMs) and the epigenetic landscape. Noncanonical oncohistone mutations occur in both tails and globular domains of all 4 core histones and alter gene expression by perturbing chromatin remodeling. However, the effects and mechanisms of noncanonical oncohistones remain largely unknown. Here we characterized 16 noncanonical H2B oncohistones in the fission yeast Schizosaccharomyces pombe. We found that 7 of them exhibited temperature sensitivities and 11 exhibited genotoxic sensitivities. A detailed study of 2 of these onco-mutants H2BG52D and H2BP102L revealed that they were defective in homologous recombination (HR) repair with compromised histone eviction and Rad51 recruitment. Interestingly, their genotoxic sensitivities and HR defects were rescued by inactivation of the H2BK119 deubiquitination function of Ubp8 in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. The levels of H2BK119 monoubiquitination (H2Bub) in the H2BG52D and H2BP102L mutants are reduced in global and local DNA break sites presumably due to enhanced recruitment of Ubp8 onto nucleosomes, and are recovered upon loss of H2B deubiquitination function of the SAGA complex. Moreover, H2BG52D and H2BP102L heterozygotes exhibit genotoxic sensitivities and reduced H2Bub in cis. We therefore conclude that H2BG52D and H2BP102L oncohistones affect HR repair and genome stability via reduction of H2Bub and propose that other noncanonical oncohistones may also affect histone PTMs to cause diseases.

The First International eDNA Workshop in Hong Kong: A beginner's guide for the next‐generation eDNA researcher

AbstractThe field of environmental DNA (eDNA) has rapidly advanced in recent years, providing a non‐invasive and time‐saving method for assessing biodiversity. The First International Environmental DNA (eDNA) Workshop in Hong Kong was held from 16 to 27 October 2023 and provided early career professionals with hands‐on training and collaboration opportunities in eDNA research. With support from The Croucher Foundation Limited (Hong Kong), the workshop covered all stages of an eDNA‐based research project, including study design, field sampling, eDNA extraction, library preparation, bioinformatics, statistical data analysis, and ethics in scientific research. Participants gained insights into the principles and practical applications of eDNA technology, emphasizing the importance of careful experimental design and data interpretation. The workshop also highlighted the need for standardized protocols, comprehensive and local DNA reference databases, and careful selection of primer sets to overcome current issues and limitations. Workshop participants expressed enthusiasm for the potential of eDNA metabarcoding as a valuable tool for ecological monitoring, biodiversity assessment, and conservation decision‐making. The future of eDNA research looks promising overall, with continued advancements in technology, collaboration among researchers, and the integration of eDNA into large‐scale ecological monitoring. Future iterations of the Hong Kong International eDNA workshop will continue to provide hands‐on training and collaboration opportunities for early career professionals interested in eDNA research, focusing on addressing current limitations and challenges in the field.

Imputation of 3D genome structure by genetic–epigenetic interaction modeling in mice

Gene expression is known to be affected by interactions between local genetic variation and DNA accessibility, with the latter organized into three-dimensional chromatin structures. Analyses of these interactions have previously been limited, obscuring their regulatory context, and the extent to which they occur throughout the genome. Here, we undertake a genome-scale analysis of these interactions in a genetically diverse population to systematically identify global genetic–epigenetic interaction, and reveal constraints imposed by chromatin structure. We establish the extent and structure of genotype-by-epigenotype interaction using embryonic stem cells derived from Diversity Outbred mice. This mouse population segregates millions of variants from eight inbred founders, enabling precision genetic mapping with extensive genotypic and phenotypic diversity. With 176 samples profiled for genotype, gene expression, and open chromatin, we used regression modeling to infer genetic–epigenetic interactions on a genome-wide scale. Our results demonstrate that statistical interactions between genetic variants and chromatin accessibility are common throughout the genome. We found that these interactions occur within the local area of the affected gene, and that this locality corresponds to topologically associated domains (TADs). The likelihood of interaction was most strongly defined by the three-dimensional (3D) domain structure rather than linear DNA sequence. We show that stable 3D genome structure is an effective tool to guide searches for regulatory elements and, conversely, that regulatory elements in genetically diverse populations provide a means to infer 3D genome structure. We confirmed this finding with CTCF ChIP-seq that revealed strain-specific binding in the inbred founder mice. In stem cells, open chromatin participating in the most significant regression models demonstrated an enrichment for developmental genes and the TAD-forming CTCF-binding complex, providing an opportunity for statistical inference of shifting TAD boundaries operating during early development. These findings provide evidence that genetic and epigenetic factors operate within the context of 3D chromatin structure.

Imputation of 3D genome structure by genetic-epigenetic interaction modeling in mice.

Gene expression is known to be affected by interactions between local genetic variation and DNA accessibility, with the latter organized into three-dimensional chromatin structures. Analyses of these interactions have previously been limited, obscuring their regulatory context, and the extent to which they occur throughout the genome. Here, we undertake a genome-scale analysis of these interactions in a genetically diverse population to systematically identify global genetic-epigenetic interaction, and reveal constraints imposed by chromatin structure. We establish the extent and structure of genotype-by-epigenotype interaction using embryonic stem cells derived from Diversity Outbred mice. This mouse population segregates millions of variants from eight inbred founders, enabling precision genetic mapping with extensive genotypic and phenotypic diversity. With 176 samples profiled for genotype, gene expression, and open chromatin, we used regression modeling to infer genetic-epigenetic interactions on a genome-wide scale. Our results demonstrate that statistical interactions between genetic variants and chromatin accessibility are common throughout the genome. We found that these interactions occur within the local area of the affected gene, and that this locality corresponds to topologically associated domains (TADs). The likelihood of interaction was most strongly defined by the three-dimensional (3D) domain structure rather than linear DNA sequence. We show that stable 3D genome structure is an effective tool to guide searches for regulatory elements and, conversely, that regulatory elements in genetically diverse populations provide a means to infer 3D genome structure. We confirmed this finding with CTCF ChIP-seq that revealed strain-specific binding in the inbred founder mice. In stem cells, open chromatin participating in the most significant regression models demonstrated an enrichment for developmental genes and the TAD-forming CTCF-binding complex, providing an opportunity for statistical inference of shifting TAD boundaries operating during early development. These findings provide evidence that genetic and epigenetic factors operate within the context of 3D chromatin structure.

Endogenous AND Logic DNA Nanomachine for Highly Specific Cancer Cell Imaging.

Intracellular cancer-related biomarker imaging strategy has been used for specific identification of cancer cells, which was of great importance to accurate cancer clinical diagnosis and prognosis studies. Localized DNA circuits with improved sensitivity showed great potential for intracellular biomarkers imaging. However, the ability of localized DNA circuits to specifically image cancer cells is limited by off-site signal leakage associated with a single-biomarker sensing strategy. Herein, we integrated the endogenous enzyme-powered strategy with logic-responsive and localized signal amplifying capability to construct a self-assembled endogenously AND logic DNA nanomachine (EDN) for highly specific cancer cell imaging. When the EDN encountered a cancer cell, the overexpressed DNA repairing enzyme apurinic/apyrimidinic endonuclease 1 (APE1) and miR-21 could synergistically activate a DNA circuit via cascaded localized toehold-mediated strand displacement (TMSD) reactions, resulting in amplified fluorescence resonance energy transfer (FRET) signal. In this strategy, both endogenous APE1 and miR-21, served as two "keys" to activate the AND logic operation in cancer cells to reduce off-tumor signal leakage. Such a multiplied molecular recognition/activation nanomachine as a powerful toolbox realized specific capture and reliable imaging of biomolecules in living cancer cells.