AIRE relies on Z-DNA to flag gene targets for thymic T cell tolerization.

AIRE is an unconventional transcription factor that enhances the expression of thousands of genes in medullary thymic epithelial cells and promotes clonal deletion or phenotypic diversion of self-reactive T cells1-4. The biological logic of AIRE's target specificity remains largely unclear as, in contrast to many transcription factors, it does not bind to a particular DNA sequence motif. Here we implemented two orthogonal approaches to investigate AIRE's cis-regulatory mechanisms: construction of a convolutional neural network and leveraging natural genetic variation through analysis of F1 hybrid mice5. Both approaches nominated Z-DNA and NFE2-MAF as putative positive influences on AIRE's target choices. Genome-wide mapping studies revealed that Z-DNA-forming and NFE2L2-binding motifs were positively associated with the inherent ability of a gene's promoter to generate DNA double-stranded breaks, and promoters showing strong double-stranded break generation were more likely to enter a poised state with accessible chromatin and already-assembled transcriptional machinery. Consequently, AIRE preferentially targets genes with poised promoters. We propose a model in which Z-DNA anchors the AIRE-mediated transcriptional program by enhancing double-stranded break generation and promoter poising. Beyond resolving a long-standing mechanistic conundrum, these findings suggest routes for manipulating T cell tolerance.

African swine fever virus early protein pI73R suppresses the type-I IFN promoter activities.

African swine fever virus is known to suppress type-I interferon (IFN) responses. The main objective of this study was to screen early-expressed viral genes for their ability to suppress IFN production. Out of 16 early genes examined, I73R exhibited robust suppression of cGAS-STING-induced IFN-ß promoter activities, impeding the function of both IRF3 and NF-κB transcription factors. As a result, I73R obstructed IRF3 nuclear translocation following the treatment of cells with poly(dA:dT), a strong inducer of the cGAS-STING signaling pathway. Although the I73R protein exhibits structural homology with the Zα domain binding to the left-handed helical form of DNA known as Z-DNA, its ability to suppress cGAS-STING induction of IFN-ß was independent of Z-DNA binding activity. Instead, the α3 and ß1 domains of I73R played a significant role in suppressing cGAS-STING induction of IFN-ß. These findings offer insights into the protein's functions and support its role as a virulence factor.

Structure-Function Analysis of RBP7910: An Editosome Z-Binding Protein in Trypanosomatids.

RNA editing, a unique post-transcriptional modification, is observed in trypanosomatid parasites as a crucial procedure for the maturation of mitochondrial mRNAs. The editosome protein complex, involving multiple protein components, plays a key role in this process. In Trypanosoma brucei, a putative Z-DNA binding protein known as RBP7910 is associated with the editosome. However, the specific Z-DNA/Z-RNA binding activity and the interacting interface of RBP7910 have yet to be determined. In this study, we conducted a comparative analysis of the binding behavior of RBP7910 with different potential ligands using microscale thermophoresis (MST). Additionally, we generated a 3D model of the protein, revealing potential Z-α and Z-ß nucleic acid-binding domains of RBP7910. RBP7910 belongs to the winged-helix-turn-helix (HTH) superfamily of proteins with an α1α2α3ß1ß2 topology. Finally, using docking techniques, potential interacting surface regions of RBP7910 with notable oligonucleotide ligands were identified. Our findings indicate that RBP7910 exhibits a notable affinity for (CG)n Z-DNA, both in single-stranded and double-stranded forms. Moreover, we observed a broader interacting interface across its Z-α domain when bound to Z-DNA/Z-RNA compared to when bound to non-Z-form nucleic acid ligands.

Flipons and small RNAs accentuate the asymmetries of pervasive transcription by the reset and sequence-specific microcoding of promoter conformation.

The role of alternate DNA conformations such as Z-DNA in the regulation of transcription is currently underappreciated. These structures are encoded by sequences called flipons, many of which are enriched in promoter and enhancer regions. Through a change in their conformation, flipons provide a tunable mechanism to mechanically reset promoters for the next round of transcription. They act as actuators that capture and release energy to ensure that the turnover of the proteins at promoters is optimized to cell state. Likewise, the single-stranded DNA formed as flipons cycle facilitates the docking of RNAs that are able to microcode promoter conformations and canalize the pervasive transcription commonly observed in metazoan genomes. The strand-specific nature of the interaction between RNA and DNA likely accounts for the known asymmetry of epigenetic marks present on the histone tetramers that pair to form nucleosomes. The role of these supercoil-dependent processes in promoter choice and transcriptional interference is reviewed. The evolutionary implications are examined: the resilience and canalization of flipon-dependent gene regulation is contrasted with the rapid adaptation enabled by the spread of flipon repeats throughout the genome. Overall, the current findings underscore the important role of flipons in modulating the readout of genetic information and how little we know about their biology.

The remodeling of Z-DNA in the mammalian germ line.

We recently discovered a novel biological process, the scheduled remodeling of Z-DNA structures in the developing fetal mouse male germ cells [Nat. Cell Biol. 24, 1141-1153]. This process affects purine/pyrimidine dinucleotide repeat (PPR) rich sequences, which can form stable left-handed Z-DNA structures. The protein that carries out this function is identified as ZBTB43, member of a large family of ZBTB proteins. Z-DNA remodeling by ZBTB43 not only coincides with global remodeling of DNA methylation and chromatin events in the male germ line, but it also is a prerequisite for de novo DNA methylation. When ZBTB43 changes DNA structure from the left-handed zigzag shaped Z-DNA to the regular smooth right-handed B-DNA, it also generates a suitable substrate for the de novo DNA methyltransferase, DNMT3A. By instructing de novo DNA methylation at PPRs in prospermatogonia, ZBTB43 safeguards epigenomic integrity of the male gamete. PPRs are fragile sequences, sites of large deletions and rearrangements in mammalian cells, and this fragility is thought to be due to Z-DNA structure formation rather than the sequence itself. This idea is now supported by the in vivo finding that DNA double strand breaks accumulate in mutant prospermatogonia which lack ZBTB43-dependent Z-DNA remodeling. If unrepaired, double stranded DNA breaks can lead to germ line mutations. Therefore, by preventing such breaks ZBTB43 is critical for guarding genome stability between generations. Here, we discuss the significance and implications of these findings in more detail.

Z-DNA is remodelled by ZBTB43 in prospermatogonia to safeguard the germline genome and epigenome.

Mutagenic purine-pyrimidine repeats can adopt the left-handed Z-DNA conformation. DNA breaks at potential Z-DNA sites can lead to somatic mutations in cancer or to germline mutations that are transmitted to the next generation. It is not known whether any mechanism exists in the germ line to control Z-DNA structure and DNA breaks at purine-pyrimidine repeats. Here we provide genetic, epigenomic and biochemical evidence for the existence of a biological process that erases Z-DNA specifically in germ cells of the mouse male foetus. We show that a previously uncharacterized zinc finger protein, ZBTB43, binds to and removes Z-DNA, preventing the formation of DNA double-strand breaks. By removing Z-DNA, ZBTB43 also promotes de novo DNA methylation at CG-containing purine-pyrimidine repeats in prospermatogonia. Therefore, the genomic and epigenomic integrity of the species is safeguarded by remodelling DNA structure in the mammalian germ line during a critical window of germline epigenome reprogramming.

Searching for New Z-DNA/Z-RNA Binding Proteins Based on Structural Similarity to Experimentally Validated Zα Domain.

Z-DNA and Z-RNA are functionally important left-handed structures of nucleic acids, which play a significant role in several molecular and biological processes including DNA replication, gene expression regulation and viral nucleic acid sensing. Most proteins that have been proven to interact with Z-DNA/Z-RNA contain the so-called Zα domain, which is structurally well conserved. To date, only eight proteins with Zα domain have been described within a few organisms (including human, mouse, Danio rerio, Trypanosoma brucei and some viruses). Therefore, this paper aimed to search for new Z-DNA/Z-RNA binding proteins in the complete PDB structures database and from the AlphaFold2 protein models. A structure-based similarity search found 14 proteins with highly similar Zα domain structure in experimentally-defined proteins and 185 proteins with a putative Zα domain using the AlphaFold2 models. Structure-based alignment and molecular docking confirmed high functional conservation of amino acids involved in Z-DNA/Z-RNA, suggesting that Z-DNA/Z-RNA recognition may play an important role in a variety of cellular processes.

Deciphering the Biological Significance of ADAR1-Z-RNA Interactions.

Adenosine deaminase acting on RNA 1 (ADAR1) is an enzyme responsible for double-stranded RNA (dsRNA)-specific adenosine-to-inosine RNA editing, which is estimated to occur at over 100 million sites in humans. ADAR1 is composed of two isoforms transcribed from different promoters: p150 and N-terminal truncated p110. Deletion of ADAR1 p150 in mice activates melanoma differentiation-associated protein 5 (MDA5)-sensing pathway, which recognizes endogenous unedited RNA as non-self. In contrast, we have recently demonstrated that ADAR1 p110-mediated RNA editing does not contribute to this function, implying that a unique Z-DNA/RNA-binding domain α (Zα) in the N terminus of ADAR1 p150 provides specific RNA editing, which is critical for preventing MDA5 activation. In addition, a mutation in the Zα domain is identified in patients with Aicardi-Goutières syndrome (AGS), an inherited encephalopathy characterized by overproduction of type I interferon. Accordingly, we and other groups have recently demonstrated that Adar1 Zα-mutated mice show MDA5-dependent type I interferon responses. Furthermore, one such mutant mouse carrying a W197A point mutation in the Zα domain, which inhibits Z-RNA binding, manifests AGS-like encephalopathy. These findings collectively suggest that Z-RNA binding by ADAR1 p150 is essential for proper RNA editing at certain sites, preventing aberrant MDA5 activation.

Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay.

Methylcytosines in mammalian genomes are the main epigenetic molecular codes that switch off the repertoire of genes in cell-type and cell-stage dependent manners. DNA methyltransferases (DMT) are dedicated to managing the status of cytosine methylation. DNA methylation is not only critical in normal development, but it is also implicated in cancers, degeneration, and senescence. Thus, the chemicals to control DMT have been suggested as anticancer drugs by reprogramming the gene expression profile in malignant cells. Here, we report a new optical technique to characterize the activity of DMT and the effect of inhibitors, utilizing the methylation-sensitive B-Z transition of DNA without bisulfite conversion, methylation-sensing proteins, and polymerase chain reaction amplification. With the high sensitivity of single-molecule FRET, this method detects the event of DNA methylation in a single DNA molecule and circumvents the need for amplification steps, permitting direct interpretation. This method also responds to hemi-methylated DNA. Dispensing with methylation-sensitive nucleases, this method preserves the molecular integrity and methylation state of target molecules. Sparing methylation-sensing nucleases and antibodies helps to avoid errors introduced by the antibody's incomplete specificity or variable activity of nucleases. With this new method, we demonstrated the inhibitory effect of several natural bio-active compounds on DMT. All taken together, our method offers quantitative assays for DMT and DMT-related anticancer drugs.

The Binding of Monoclonal and Polyclonal Anti-Z-DNA Antibodies to DNA of Various Species Origin.

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichiacoli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.

The Simple Biology of Flipons and Condensates Enhances the Evolution of Complexity.

The classical genetic code maps nucleotide triplets to amino acids. The associated sequence composition is complex, representing many elaborations during evolution of form and function. Other genomic elements code for the expression and processing of RNA transcripts. However, over 50% of the human genome consists of widely dispersed repetitive sequences. Among these are simple sequence repeats (SSRs), representing a class of flipons, that under physiological conditions, form alternative nucleic acid conformations such as Z-DNA, G4 quartets, I-motifs, and triplexes. Proteins that bind in a structure-specific manner enable the seeding of condensates with the potential to regulate a wide range of biological processes. SSRs also encode the low complexity peptide repeats to patch condensates together, increasing the number of combinations possible. In situations where SSRs are transcribed, SSR-specific, single-stranded binding proteins may further impact condensate formation. Jointly, flipons and patches speed evolution by enhancing the functionality of condensates. Here, the focus is on the selection of SSR flipons and peptide patches that solve for survival under a wide range of environmental contexts, generating complexity with simple parts.

Special Issue: A, B and Z: The Structure, Function and Genetics of Z-DNA and Z-RNA.

It is now difficult to believe that a biological function for the left-handed Z-DNA and Z-RNA conformations was once controversial. The papers in this Special Issue, "Z-DNA and Z-RNA: from Physical Structure to Biological Function", are based on presentations at the ABZ2021 meeting that was held virtually on 19 May 2021 and provide evidence for several biological functions of these structures. The first of its kind, this international conference gathered over 200 scientists from many disciplines to specifically address progress in research involving Z-DNA and Z-RNA. These high-energy left-handed conformers of B-DNA and A-RNA are associated with biological functions and disease outcomes, as evidenced from both mouse and human genetic studies. These alternative structures, referred to as "flipons", form under physiological conditions, regulate type I interferon responses and induce necroptosis during viral infection. They can also stimulate genetic instability, resulting in adaptive evolution and diseases such as cancer. The meeting featured cutting-edge science that was, for the most part, unpublished. We plan for the ABZ meeting to reconvene in 2022.

Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B-Z Transition of DNA.

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in µs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)-DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1-DNA complex with a slow B-Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s-1, respectively, in agreement with two regimes of residue-dependent chemical shift differences-the "dominant oscillatory regime" and "semi-oscillatory regime". We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.

Sequence dependent influence of an A A mismatch in a DNA duplex: An insight into the recognition by hZαADAR1 protein.

Base pair mismatches can erroneously be incorporated in the DNA. An adenine pairing with another adenine is one of the eight possible mismatches. The atomistic insights about the structure and dynamics of an A…A mismatch in a DNA (unbound form) is not yet accessible to any experimental technique. Earlier molecular dynamics (MD) simulations have shown that A…A mismatch in the midst of 5'CAG/3'GAC, 5'GAC/3'CAG and 5'CAA/3'GAT (underline represents the mismatch) are highly dynamic in nature. By employing MD simulation, the influence of an A…A mismatch in the midst of 5'GAA/3'CAT, 5'GAG/3'CAC, 5'AAC/3'TAG, 5'AAG/3'TAC, 5'TAA/3'AAT, 5'TAT/3'AAA and 5'AAT/3'TAA sequences have been investigated here. The results indicate that irrespective of the flanking sequences, the mismatch samples a variety of transient conformations, including a B-Z junction. Further, circular dichroism studies have been carried out to explore the ability of these sequences to bind with hZαADAR1 which specifically recognizes B-Z junction/Z-DNA. The results indicate that hZαADAR1 could not lead to a complete B to Z transition in the above sequences. Notably, a complete transition to Z-form has been reported earlier for 5'GAC/3'CAG upon titrating with hZαADAR1. Intriguingly, 5'AAC/3'TAG, 5'AAG/3'TAC and 5'GAA/3'CAT exhibit a B-Z junction formation rather than a complete transition to Z-form, similar to the situation of 5'CAA/3'GAT. These indicate that although A…A mismatch could induce a local B-Z junction transiently, hZαADAR1 requires the presence of a G…C/C…G base pair adjacent to the A…A mismatch for the binding. Additionally, the extent of B-Z junction has enhanced upon binding with hZαADAR1 in the presence of the A…A mismatch (specifically when CG, CA, AC, GA and AG steps occur), but not in the presence of the canonical base pairs. These confirm the inclination of A…A mismatch towards the B-Z junction.

Enhanced RIPK3 kinase activity-dependent lytic cell death in M1 but not M2 macrophages.

Macrophages play a crucial role in host innate immune defense against infection and tissue injury. Macrophages are highly plastic cells and their subtypes have been characterized as M1 (also termed classically activated) and M2 (alternatively activated). Although the M1/M2 paradigm has been well documented, less is known regarding the role of macrophage activation/polarization in inflammation-associated necrotic cell death. To address this gap in current knowledge, we prepared bone marrow-derived macrophages, induced them to M1 or M2 subtypes, and then investigated the expression of necroptosis signaling molecules and macrophage subtype-dependent responses to different necroptosis inducers. We found that necroptosis effector mixed lineage kinase domain-like protein (MLKL) and the key necroptosis regulator Z-DNA/RNA binding protein 1 were predominantly induced in M1 but not M2 macrophages. Interestingly, the protein but not mRNA levels of receptor-interacting protein kinase-3 (RIPK3) were also upregulated in M1 macrophages. We further found that macrophage necrotic cell death, the releases of lactate dehydrogenase and dead cell proteases as well as MLKL phosphorylation at Ser345 in response to various necroptosis inducers were greatly augmented in M1 but not M2 macrophages, and the accelerated effects were blocked by two structurally distinct specific RIPK3 inhibitors GSK872 or GSK843. Thus, our findings demonstrate that M1 but not M2 subtypes of macrophages are more susceptible to inflammation-related lytic cell death in an RIPK3 kinase activity-dependent manner.

Remodeling Chromatin Induces Z-DNA Conformation Detected through Fourier Transform Infrared Spectroscopy.

The SWI/SNF complex is a highly conserved chromatin remodeling complex and can hydrolyze ATP by its catalytic subunit BRG1 or BRM to reconstruct the chromatin. To investigate whether this ATP-dependent chromatin remodeling could affect the DNA conformation, we therefore regulated (knocked down or overexpressed) BRG1/BRM in the cells and applied Fourier transform infrared (FTIR) spectroscopy to probe DNA conformational changes. As a result, we found that BRG1/BRM was indeed associated with the DNA conformational changes, in which knockdown of BRG1/BRM reduced Z-DNA conformation, while overexpression of BRG1/BRM enhanced Z-DNA conformation. This Z-DNA conformational transformation was also verified using the Z-DNA-binding proteins. Therefore, this work has provided a direct analytical tool to probe Z-DNA transformation upon ATP-dependent chromatin remodeling.

Protein-induced B-Z transition of DNA duplex containing a 2'-OMe guanosine.

Structural transformation of the canonical right-handed helix, B-DNA, to the non-canonical left-handed helix, Z-DNA, can be induced by the Zα domain of the human RNA editing enzyme ADAR1 (hZαADAR1). To characterize the site-specific preferences of binding and structural changes in DNA containing the 2'-O-methyl guanosine derivative (mG), titration of the imino proton spectra and chemical shift perturbations were performed on hZαADAR1 upon binding to Z-DNA. The structural transition between B-Z conformation as the changing ratio between DNA and protein showed a binding affinity of the modified DNA onto the Z-DNA binding protein similar to wild-type DNA or RNA. The chemical shift perturbation results showed that the overall structure and environment of the modified DNA revealed DNA-like properties rather than RNA-like characteristics. Moreover, we found evidence for two distinct regimes, "Z-DNA Sensing" and "Modification Sensing", based on the site-specific chemical shift perturbation between the DNA (or RNA) binding complex and the modified DNA-hZαADAR1 complex. Thus, we propose that modification of the sugar backbone of DNA with 2'-O-methyl guanosine promotes the changes in the surrounding α3 helical structural segment as well as the non-perturbed feature of the ß-hairpin region.

Dynamic regulation of Z-DNA in the mouse prefrontal cortex by the RNA-editing enzyme Adar1 is required for fear extinction.

DNA forms conformational states beyond the right-handed double helix; however, the functional relevance of these noncanonical structures in the brain remains unknown. Here we show that, in the prefrontal cortex of mice, the formation of one such structure, Z-DNA, is involved in the regulation of extinction memory. Z-DNA is formed during fear learning and reduced during extinction learning, which is mediated, in part, by a direct interaction between Z-DNA and the RNA-editing enzyme Adar1. Adar1 binds to Z-DNA during fear extinction learning, which leads to a reduction in Z-DNA at sites where Adar1 is recruited. Knockdown of Adar1 leads to an inability to modify a previously acquired fear memory and blocks activity-dependent changes in DNA structure and RNA state-effects that are fully rescued by the introduction of full-length Adar1. These findings suggest a new mechanism of learning-induced gene regulation that is dependent on proteins that recognize alternate DNA structure states, which are required for memory flexibility.

Dynamics Studies of DNA with Non-canonical Structure Using NMR Spectroscopy.

The non-canonical structures of nucleic acids are essential for their diverse functions during various biological processes. These non-canonical structures can undergo conformational exchange among multiple structural states. Data on their dynamics can illustrate conformational transitions that play important roles in folding, stability, and biological function. Here, we discuss several examples of the non-canonical structures of DNA focusing on their dynamic characterization by NMR spectroscopy: (1) G-quadruplex structures and their complexes with target proteins; (2) i-motif structures and their complexes with proteins; (3) triplex structures; (4) left-handed Z-DNAs and their complexes with various Z-DNA binding proteins. This review provides insight into how the dynamic features of non-canonical DNA structures contribute to essential biological processes.

Emerging roles for R-loop structures in the management of topological stress.

R-loop structures are a prevalent class of alternative non-B DNA structures that form during transcription upon invasion of the DNA template by the nascent RNA. R-loops form universally in the genomes of organisms ranging from bacteriophages, bacteria, and yeasts to plants and animals, including mammals. A growing body of work has linked these structures to both physiological and pathological processes, in particular to genome instability. The rising interest in R-loops is placing new emphasis on understanding the fundamental physicochemical forces driving their formation and stability. Pioneering work in Escherichia coli revealed that DNA topology, in particular negative DNA superhelicity, plays a key role in driving R-loops. A clear role for DNA sequence was later uncovered. Here, we review and synthesize available evidence on the roles of DNA sequence and DNA topology in controlling R-loop formation and stability. Factoring in recent developments in R-loop modeling and single-molecule profiling, we propose a coherent model accounting for the interplay between DNA sequence and DNA topology in driving R-loop structure formation. This model reveals R-loops in a new light as powerful and reversible topological stress relievers, an insight that significantly expands the repertoire of R-loops' potential biological roles under both normal and aberrant conditions.