Linker DNA Research Articles

Temperature switchable linkers suitable for triggered drug release in cancer thermo-chemotherapy

In drug delivery systems, a stimuli-responsive linker that attaches a targeting carrier and a cytotoxic payload can be dissociated to release the payload on the target over the action of a stimuli, thereby it would harden the selectivity, specificity and potency of the cytotoxic agent against targeted tissues whilst sparing the drug-induced toxicity on normal cells. Oligonucleotide duplexes can unwind and be separated into single-stranded random coils under a defined temperature, and this property makes the oligonucleotide an appealing thermo-responsive linker. In this work, we studied the melting temperatures of different DNA linkers with various lengths and mismatches inserted in the double helix with either different numbers or positions. We further chose the DNA linkers that can unwind at the hyperthermia temperature and used them in the construction of four different drug delivery systems both in vitro and in vivo. Results showed that the chosen DNA linkers in all of the constructed delivery systems can successfully unwind and release cargos or drugs after application of heat compared to control groups. This research demonstrated the potential applications of DNA duplexes as temperature-sensitive linkers of drug delivery systems for cancer therapy.

Filter Membrane-Based Colorimetric Approach for Point-of-Care Detection of Biomarkers Using CRISPR-Cas12a.

CRISPR-Cas-based point-of-care testing (POCT) strategies have been widely explored for the detection of diverse biomarkers. However, these methods often require complicated operations, such as careful solution transfer steps, to achieve high sensitivity and accuracy. In this study, we combine a filter membrane-based POCT method with CRISPR-Cas12a for colorimetric detection of biomarkers. For the nucleic acid target, the trans-cleavage activity of CRISPR-Cas12a is directly triggered, cutting the single-stranded DNA linkers on glucose oxidase (GOx)-modified polymer nanoparticles. Due to the size difference between GOx and the polymer nanoparticles, GOx can be separated using a filter membrane. The filtrate containing GOx reacts with the substrate to generate a colorimetric signal. For the non-nucleic acid target, the non-nucleic acid signal is converted into a nucleic acid signal that activates CRISPR-Cas12a, resulting in a colorimetric signal. The entire operation is easy to perform, and the signal can be directly observed via the naked eye, which circumvents the use of costly instruments. The developed strategy holds great promise for accurate and accessible POCT detection of disease biomarkers in resource-limited settings.

Dimeric-molecular beacon based intramolecular strand displacement amplification enables robust analysis of miRNA

Given the critical role of miRNAs in regulating gene expression and their potential as biomarkers for various diseases, accurate and sensitive miRNA detection is essential for early diagnosis and monitoring of conditions such as cancer. In this study, we introduce a dimeric molecular beacon (Di-MB) based isothermal strand displacement amplification (ISDA) system (Di-MB-ISDA) for enhanced miRNA detection. The Di-MB system is composed of two monomeric MBs (Mono-MBs) connected by a double-stranded DNA linker with single-stranded sequences in the middle, facilitating binding with the flexible arms of the Mono-MBs. This design forms a compact, high-density structure, significantly improving biostability against nuclease degradation. In the absence of target miRNA, the Di-MB maintains its stable structure. When target miRNA is present, it binds to the stem-loop regions, causing the hairpin structure to unfold and expose the stem sequences. These sequences serve as templates for the built-in primers, triggering DNA replication through an intramolecular recognition mechanism. This spatial confinement effect accelerates the strand displacement reaction, allowing the target miRNA to initiate additional reaction cycles and amplify the detection signal. The Di-MB-ISDA system addresses key challenges such as poor biostability and limited sensitivity seen in traditional methods. By enhancing biostability and optimizing reaction conditions, this system demonstrates robust performance for miRNA detection with a detection limit of 100 pM. The findings highlight the potential of Di-MB-ISDA for sensitive and accurate miRNA analysis, paving the way for its application in biomedical study and disease diagnosis in complex biological samples.

Ligation initiated self-priming isothermal polymerization enabled nano-signal amplification for APE1 sensing and logical unlocking

In this work, we developed a label-free and one-tube method for apurinic/apyrimidinic endonuclease 1 (APE1) sensing based on ligation initiated self-priming isothermal polymerization (SPIP) and signal amplification of fluorescent copper nanoparticles (CuNPs). This method was rationally proposed with modular design. In target recognition module, APE1 could specifically cleave substrate to release a linker DNA that served the ligation of an adenine/thymine (AT)-rich hairpin and a phosphorothioate-modified hairpin with the assistance of ligase. Then in SPIP amplification and signal transduction modules, once driven by polymerase, enzymatic extension and self-folding alternately occurred on the primary DNA originated from the ligation reaction, which resulted in accumulation of numerous AT-rich templates repeatedly included in the elongated dsDNA products, and ultimately enabled rapid formation of fluorescent CuNPs to output amplified signal. The accurate target identification and efficient signal amplification facilitated the highly selective and sensitive detection of APE1 with limit of detection of 8.6 × 10−4 U/mL. Benefiting from the robustness of this method, the normal cells and tumor cells could be distinguished unambiguously with this method according to the test results of cellular APE1. Moreover, the sensing strategy was employed in the concatenated AND logical operation and molecular keypad unlocking, which further displayed its extensibility and versatility.

Hmo1: A versatile member of the high mobility group box family of chromosomal architecture proteins.

Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures, which is facilitated by linker histone H1. Formation of chromatin compacts and protects the genome, but also hinders DNA transactions. Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions. The high mobility group box (HMGB) proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion. They play a major role in chromatin dynamics. The Saccharomyces cerevisiae (yeast hereafter) HMGB protein Hmo1 contains two HMGB motifs. However, unlike a canonical HMGB protein that has an acidic C-terminus, Hmo1 ends with a lysine rich, basic, C-terminus, resembling linker histone H1. Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions. For instance, Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones. Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome. This minireview reviews the functions of Hmo1 and the underlying mechanisms, highlighting recent discoveries.

Structural insights into the cooperative nucleosome recognition and chromatin opening by FOXA1 and GATA4

Mouse FOXA1 and GATA4 are prototypes of pioneer factors, initiating liver cell development by binding to the N1 nucleosome in the enhancer of the ALB1 gene. Using cryoelectron microscopy (cryo-EM), we determined the structures of the free N1 nucleosome and its complexes with FOXA1 and GATA4, both individually and in combination. We found that the DNA-binding domains of FOXA1 and GATA4 mainly recognize the linker DNA and an internal site in the nucleosome, respectively, whereas their intrinsically disordered regions interact with the acidic patch on histone H2A-H2B. FOXA1 efficiently enhances GATA4 binding by repositioning the N1 nucleosome. Invivo DNA editing and bioinformatics analyses suggest that the co-binding mode of FOXA1 and GATA4 plays important roles in regulating genes involved in liver cell functions. Our results reveal the mechanism whereby FOXA1 and GATA4 cooperatively bind to the nucleosome through nucleosome repositioning, opening chromatin by bending linker DNA and obstructing nucleosome packing.

A DNA condensation code for linker histones

Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.

Alternative splicing of BAZ1A in colorectal cancer disrupts the DNA damage response and increases chemosensitization

Bromodomain Adjacent to Zinc Finger Domain 1A (BAZ1A) is a critical regulator of chromatin remodeling. We sought to clarify the roles of BAZ1A in the etiology of colorectal cancer, including the mechanisms of its alternatively spliced variants. Public databases were examined and revealed high BAZ1A expression in the majority of colorectal cancer patients, which was corroborated in a panel of human colon cancer cell lines. BAZ1A silencing reduced cell viability and increased markers of DNA damage, apoptosis, and senescence, along with the downregulation of Wnt/β-catenin signaling. The corresponding molecular changes resulted in tumor growth inhibition when BAZ1A-knockout cells were implanted into nude mice. In rescue experiments, a short isoform of BAZ1A that was associated with alternative splicing by the DBIRD complex failed to restore DNA repair activity in colon cancer cells and maintained chemosensitivity to phleomycin treatment, unlike the full-length BAZ1A. A working model proposes that a buried domain in the N-terminus of the BAZ1A short isoform lacks the ability to access linker DNA, thereby disrupting the activity of the associated chromatin remodeling complexes. Given the current interest in RNA splicing deregulation and cancer etiology, additional mechanistic studies are warranted with new lead compounds targeting BAZ1A, and other members of the BAZ family, with a view to improved therapeutic interventions.

DNA nanosensor based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly for sensitive and fast TK1 mRNA detection.

Hyperproliferative diseases are the first step for tumor formation; thymidine kinase 1 (TK1) mRNA is closely related to cell proliferation. Therefore, the risk of malignant proliferation can be identified by sensitively detecting the variance in TK1 mRNA concentration, which can be used for tumor auxiliary diagnosis and monitoring tumor treatment. Owing to the low abundance and instability of TK1 mRNA in real samples, the development of a sensitive and fast mRNA detection method is necessary. A DNA nanosensor that can be used for detecting TK1 mRNA based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly (P-CHA) was developed. P-CHA hairpins were hybridized to a linker DNA strand coupled with magnetic nanoparticles to increase their local concentrations. The bipedal DNA walking on the surface of NPs accelerates reaction kinetics using the proximity effect. Taking advantage of the signal amplification of P-CHA as well as the rapid reaction rate of the DNA walker in 80min, the proposed sensor detects TK1 mRNA with a low detection limit of 14pM and may then be applied toclinical diagnosis.

Interpretable deep residual network uncovers nucleosome positioning and associated features.

Nucleosomes represent elementary building units of eukaryotic chromosomes and consist of DNA wrapped around a histone octamer flanked by linker DNA segments. Nucleosomes are central in epigenetic pathways and their genomic positioning is associated with regulation of gene expression, DNA replication, DNA methylation and DNA repair, among other functions. Building on prior discoveries that DNA sequences noticeably affect nucleosome positioning, our objective is to identify nucleosome positions and related features across entire genome. Here, we introduce an interpretable framework based on the concepts of deep residual networks (NuPoSe). Trained on high-coverage human experimental MNase-seq data, NuPoSe is able to learn sequence and structural patterns associated with nucleosome organization in human genome. NuPoSe can bealsoapplied to unseen data from different organisms and cell types. Our findings point to 43 informative features, most of them constitute tri-nucleotides, di-nucleotides and one tetra-nucleotide. Most features are significantly associated with the nucleosomal structuralcharacteristics, namely, periodicity of nucleosomal DNA and its location with respect to a histone octamer. Importantly, we show that features derived from the 27 bp linker DNAflanking nucleosomescontribute up to 10% to the quality of the prediction model. This,alongwith the comprehensive training sets, deep-learning architecture, and feature selection method,may contribute totheNuPoSe's 80-89% classificationaccuracy on different independentdatasets.

Three-Dimensional DNA Hydrogel Mediated Dual-Mode Sensing Method for Quantification of Epithelial Cell Adhesion Molecule in Biological Fluid Samples.

Monitoring changes in the expression of marker proteins in biological fluids is essential for biomarker-based disease diagnosis. Epithelial cell adhesion molecule (EpCAM) has been identified as a broad-spectrum biomarker for various chronic diseases and as a therapeutic target. However, the development of simple and reliable methods for quantifying EpCAM changes in biological fluids faces challenges due to the variability of its expression across different diseases, the presence of soluble forms, and matrix effects. In this paper, a surface-enhanced Raman scattering (SERS)-fluorescence (FL) dual-mode sensing method was established for quantification of trace EpCAM in biological fluids based on bimetallic Au@Ag nanoparticles and nitrogen-doped quantum dots encapsulated DNA hydrogel hybrid with graphene oxide (Au@Ag-NQDs/GO). The DNA hydrogel was constructed based on three-dimensional (3D) structure DNA-mediated strategy using an aptamer DNA (AptDNA) linker. The interaction of the AptDNA with EpCAM triggered the disassembly of the DNA hydrogel. Consequently, the release of Au@Ag nanoparticles induced an "on-off" switch in the SERS signal while the weakened FL quenching effect in Au@Ag-NQDs/GO system achieved "off-on" switch of FL signal, enabling the simultaneous SERS-FL quantification of EpCAM. The established dual-mode method exhibited outstanding sensitivity and stability in quantifying EpCAM in the range of 0.5-60.0 pg/mL, with the limits of detection (LODs) of SERS and FL as 0.17 and 0.35 pg/mL, respectively. When applied for real sample analysis, the method showed satisfactory specificity and recoveries in cancer cells lysate, serum, and urine samples with RSDs of 2.8-6.3%, 4.0-6.3%, and 2.8-5.7%, respectively. The developed SERS-FL sensing method offered a sensitive, reliable, and practical quantification strategy for trace EpCAM in diverse biological fluid samples, which would benefit the early diagnosis of disease and further health management.

Near-Infrared Light-Activatable DNA Tentacles for Efficient Inhibition of Tumor Metastasis by Bio-Orthogonal Cell Assembly.

Tumor metastasis remains a major challenge in cancer management. Among various treatment strategies, immune cell-based cancer therapy holds a great potential for inhibiting metastasis. However, its wide application in cancer therapy is restricted by complex preparations, as well as inadequate homing and controllability. Herein, we present a groundbreaking approach for bioorthogonally manipulating tumor-NK (natural killer) cell assembly to inhibit tumor metastasis. Multiple dibenzocyclootyne (DBCO) groups decorated long single-stranded DNA were tail-modified on core-shell upconversion nanoparticles (CSUCNPs) and condensed by photosensitive chemical linker (PC-Linker) DNA to shield most of the DBCO groups. On the one hand, the light-triggered DNA scaffolds formed a cross-linked network by click chemistry, effectively impeding tumor cell migration. On the other hand, the efficient cellular assembly facilitated the effective communication between tumor cells and NK-92 cells, leading to enhanced immune response against tumors and further suppression of tumor metastasis. These features make our strategy highly applicable to a wide range of metastatic cancers.

Enhanced Circular Dichroism for Achiral Sensing Based on a DNA-Origami-Empowered Anapole Metasurface.

Circular dichroism (CD) spectroscopy has been extensively utilized for detecting and distinguishing the chirality of diverse substances and structures. However, CD spectroscopy is inherently weak and conventionally associated with chiral sensing, thus constraining its range of applications. Here, we report a DNA-origami-empowered metasurface sensing platform through the collaborative effect of metasurfaces and DNA origami, enabling achiral/slightly chiral sensing with high sensitivity via the enhanced ΔCD. An anapole metasurface, boasting over 60 times the average optical chirality enhancement, was elaborately designed to synergize with reconfigurable DNA origami. We experimentally demonstrated the detection of achiral/slightly chiral DNA linker strands via the enhanced ΔCD of the proposed platform, whose sensitivity was a 10-fold enhancement compared with the platform without metasurfaces. Our work presents a high-sensitivity platform for achiral/slightly chiral sensing through chiral spectroscopy, expanding the capabilities of chiral spectroscopy and inspiring the integration of multifunctional artificial nanostructures across diverse domains.

Physical modeling of nucleosome clustering in euchromatin resulting from interactions between epigenetic reader proteins

Euchromatin is an accessible phase of genetic material containing genes that encode proteins with increased expression levels. The structure of euchromatin in vitro has been described as a 30-nm fiber formed from ordered nucleosome arrays. However, recent advances in microscopy have revealed an in vivo euchromatin architecture that is much more disordered, characterized by variable-length linker DNA and sporadic nucleosome clusters. In this work, we develop a theoretical model to elucidate factors contributing to the disordered in vivo architecture of euchromatin. We begin by developing a 1D model of nucleosome positioning that captures the interactions between bound epigenetic reader proteins to predict the distribution of DNA linker lengths between adjacent nucleosomes. We then use the predicted linker lengths to construct 3D chromatin configurations consistent with the physical properties of DNA within the nucleosome array, and we evaluate the distribution of nucleosome cluster sizes in those configurations. Our model reproduces experimental cluster-size distributions, which are dramatically influenced by the local pattern of epigenetic marks and the concentration of reader proteins. Based on our model, we attribute the disordered arrangement of euchromatin to the heterogeneous binding of reader proteins and subsequent short-range interactions between bound reader proteins on adjacent nucleosomes. By replicating experimental results with our physics-based model, we propose a mechanism for euchromatin organization in the nucleus that impacts gene regulation and the maintenance of epigenetic marks.

The nucleosome reference frame and standard geometries for octasomes.

There are over 533 nucleosome structures in the Research Collaboratory for Structural Bioinformatics (RCSB). Collectively, numerous variants and species are present, as are sub-nucleosomal and super-nucleosomal assemblies within the nucleosome family. The organization of the histones and DNA is highly conserved in all standard octasomes containing 145, 146, or 147 base pairs. This observation is used to establish a nucleosome reference frame that enables us to describe and compare the gross structure and organization of all nucleosomes. We observe that cumulative sums of Rise, Twist, and DNA arc length are linear functions of the base pair index with values exceeding 0.999 for almost all octasome structures. These relationships enable us to readily compare the location and orientation of DNA director frames extracted from the crystal structures to ideal superhelix values. Such comparisons reveal that the DNA superhelix extracted from X-ray structures exhibits a sinusoidal variation with an amplitude of approximately 5Å about a constant superhelix radius of Å, in agreement with early descriptions of nucleosome organization as tripartite. There is also a distinct straightening of the nucleosomal DNA over the outermost turn of DNA's double helix. The straightening of the DNA superhelix marks the transition to linker DNA and is easily recognized as a rapid increase in superhelix radius and is concomitant with a change in pitch. This provides a rigorous means of separating nucleosomal DNA from linker DNA. For all X-ray structures, we find that near the dyad, there exists a set of DNA director frames for which the spatial location and orientation are highly conserved. Away from the dyad, the DNA superhelix exhibits "singletrack" and "multipath" regions. In the singletrack region, all structures exhibit a single highly conserved pathway along which all base pairs must track, but at varying rates. In the multipath regions, the base pairs are allowed to map out a limited number of different pathways along the surface of the histone octamer. To demonstrate the utility of the proposed reference geometries, standard and distorted octasome structures, super-nucleosomal structures, nucleosomes with linker DNA, and nucleosomes in closed circular DNA are analyzed. The online version contains supplementary material available at 10.1007/s12551-024-01206-5.

Cryo-EM structure and functional analysis of the chromatin remodeler RSF.

The RSF complex belongs to the ISWI chromatin-remodeling family and is composed of two subunits: RSF1 (remodeling and spacing factor 1) and SNF2h (sucrose nonfermenting protein 2 homolog). The RSF complex participates in nucleosome spacing and assembly, and subsequently promotes nucleosome maturation. Although SNF2h has been extensively studied in the last few years, the structural and functional properties of the remodeler RSF1 still remain vague. Here, a cryo-EM structure of the RSF-nucleosome complex is reported. The 3D model shows a two-lobe architecture of RSF, and the structure of the RSF-nucleosome (flanked with linker DNA) complex shows that the RSF complex moves the DNA away from the histone octamer surface at the DNA-entry point. Additionally, a nucleosome-sliding assay and a restriction-enzyme accessibility assay show that the RSF1 subunit may cause changes in the chromatin-remodeling properties of SNF2h. As a `nucleosome ruler', the results of an RSF-dinucleosome binding affinity test led to the proposal that the critical distance that RSF `measures' between two nucleosomes is about 24 base pairs.

Angle between DNA linker and nucleosome core particle regulates array compaction revealed by individual-particle cryo-electron tomography

The conformational dynamics of nucleosome arrays generate a diverse spectrum of microscopic states, posing challenges to their structural determination. Leveraging cryogenic electron tomography (cryo-ET), we determine the three-dimensional (3D) structures of individual mononucleosomes and arrays comprising di-, tri-, and tetranucleosomes. By slowing the rate of condensation through a reduction in ionic strength, we probe the intra-array structural transitions that precede inter-array interactions and liquid droplet formation. Under these conditions, the arrays exhibite irregular zig-zag conformations with loose packing. Increasing the ionic strength promoted intra-array compaction, yet we do not observe the previously reported regular 30-nanometer fibers. Interestingly, the presence of H1 do not induce array compaction; instead, one-third of the arrays display nucleosomes invaded by foreign DNA, suggesting an alternative role for H1 in chromatin network construction. We also find that the crucial parameter determining the structure adopted by chromatin arrays is the angle between the entry and exit of the DNA and the corresponding tangents to the nucleosomal disc. Our results provide insights into the initial stages of intra-array compaction, a critical precursor to condensation in the regulation of chromatin organization.

An antifouling electrochemical sensor integrating ‘‘3-in-1″ dual-targeted aptamer for the detection of the SARS-CoV-2 spike protein

Electrochemical biosensors have shown promise in bioanalysis and diagnosis. However, they suffer from the accumulation and adhesion of biomolecules in biofluids on sensing surfaces, while the hiring of potential antifouling is limited by the cost and complexity of the construction methods. Here, an economical three-dimensional (3D) conductive antifouling coating was prepared. This coating was composed of bovine serum albumin (BSA) crosslinked glutaraldehyde (GA) and copper-gold nanowires (CuAu NWs) with excellent electrical conductivity. Furthermore, a dual-targeted aptamer (D-Apt), with a “3-in-1″ structure of two specific binding sequences and a DNA linker, was designed to specifically recognize BSA and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, affording low nanomolar binding affinity. Importantly, the introduction of the D-Apt greatly simplified the functionalization procedure on the electrode surface and the immobilization of the biorecognition element. Then, a simple electrochemical biosensor based on the CuAu/BSA/GA and D-Apt was constructed for the detection of S protein. This sensor exhibited merits of cost-effectiveness, sensitivity and practicality for detection of S protein. The work could provide a facile avenue to construct electrochemical biosensors with low-cost antifouling for the accurate diagnosis of epidemic antigenic disease markers.

Semiconservative transmission of DNA N 6-adenine methylation in a unicellular eukaryote.

Although DNA N 6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.

Structures and dynamics of Rpd3S complex bound to nucleosome.

The Rpd3S complex plays a pivotal role in facilitating local histone deacetylation in the transcribed regions to suppress intragenic transcription initiation. Here, we present the cryo-electron microscopy structures of the budding yeast Rpd3S complex in both its apo and three nucleosome-bound states at atomic resolutions, revealing the exquisite architecture of Rpd3S to well accommodate a mononucleosome without linker DNA. The Rpd3S core, containing a Sin3 Lobe and two NB modules, is a rigid complex and provides three positive-charged anchors (Sin3_HCR and two Rco1_NIDs) to connect nucleosomal DNA. In three nucleosome-bound states, the Rpd3S core exhibits three distinct orientations relative to the nucleosome, assisting the sector-shaped deacetylase Rpd3 to locate above the SHL5-6, SHL0-1, or SHL2-3, respectively. Our work provides a structural framework that reveals a dynamic working model for the Rpd3S complex to engage diverse deacetylation sites.