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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 cells 1 – 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 mice 5 . 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. Z-DNA anchors the AIRE-mediated transcriptional program by enhancing the generation of double-stranded breaks and promoter poising.

DNA topology is critical for regulating transcription and maintaining cellular homeostasis. Z-DNA is a left-handed DNA helix in regions with high transcriptional activity. Its physiological function remains poorly understood. Here, we demonstrate that oncoprotein MYC induces the formation of Z-DNA by recruiting the chromatin remodeler FACT, independent of RNA Polymerase II activity. FACT facilitates Z-DNA formation by remodeling H2A/H2B dimers within intact nucleosomes. Additionally, the phosphorylation of FACT regulates its liquid-liquid phase separation, promoting its efficient recruitment to chromatin by MYC. Through a genome-wide analysis and characterization of engineered Z-DNA promoters, we found that Z-DNA directly facilitates the loading of RNA Polymerase II, thereby promoting transcriptional activity. This study elucidates the molecular mechanisms of Z-DNA dynamics and emphasizes its functional importance in transcriptional regulation, providing insights into the role of left-handed DNA structures in chromatin biology and MYC-driven cancer. Z-DNA forms at highly active genes, but its function is unclear. Here, the authors show that MYC recruits FACT to promote Z-DNA formation within nucleosomes, enabling RNA Polymerase II loading and enhancing transcription, revealing a mechanistic role for Z-DNA in MYC-driven regulation.

The role of alternative nucleic acid structures (ANS) in biology is an area of increasing interest. These non-canonical structures include the Z-DNA and Z-RNA duplexes (ZNA), the three-stranded triplex, the four-stranded G-quadruplex (GQ), and i-motifs. Previously, the biological relevance of ANS was dismissed. Their formation in vitro often required non-physiological conditions, and there was no genetic evidence for their function. Further, structural studies confirmed that sequence-specific transcription factors (TFs) bound B-DNA. In contrast, ANS are formed dynamically by a subset of repeat sequences, called flipons. The flip requires energy, but not strand cleavage. Flipons are enriched in promoters where they modulate transcription. Here, computational modeling based on AlphaFold V3 (AF3), under optimized conditions, reveals that known B-DNA-binding TFs also dock to ANS, such as ZNA and GQ. The binding of HLH and bZIP homodimers to Z-DNA is promoted by methylarginine modifications. Heterodimers only bind preformed Z-DNA. The interactions of TFs with ANS likely enhance genome scanning to identify cognate B-DNA-binding sites in active genes. Docking of TF homodimers to Z-DNA potentially facilitates the assembly of heterodimers that dissociate and are stabilized by binding to a cognate B-DNA motif. The process enables rapid discovery of the optimal heterodimer combinations required to regulate a nearby promoter.

Left-handed Z-DNA/Z-RNA is bound with high affinity by the Zα domain protein family that includes ADAR (a double-stranded RNA editing enzyme), ZBP1 and viral orthologs regulating innate immunity. Loss-of-function mutations in ADAR p150 allow persistent activation of the interferon system by Alu dsRNAs and are causal for Aicardi-Goutières Syndrome. Heterodimers of ADAR and DICER1 regulate the switch from RNA- to protein-centric immunity. Loss of DICER1 function produces age-related macular degeneration, a different type of Alu-mediated disease. The overlap of Z-forming sites with those for the signal recognition particle likely limits invasion of primate genomes by Alu retrotransposons. Alan Herbert discusses the properties of Z-DNA and Z-RNA, interactions with ADAR and other Z-binding proteins, and the role these elements play in disease. He also discusses the implication of Z-forming sites in genome evolution.

Z-DNA binding protein 1 (ZBP1) is a crucial player in the intracellular recognition of Z-form nucleic acids (Z-NAs) through its Zαβ domain, initiating downstream interactions with RIPK1 and RIPK3 via RHIM domains. This engagement leads to the assembly of PANoptosomes, ultimately inducing programmed cell death to curb pathogen dissemination. How Zαβ and RHIM domain cooperate to trigger Z-NAs recognition and signal transduction remains unclear. Here, we show that ZBP1 condensate formation facilitates Z-NAs binding and antiviral signal transduction. The ZBP1 Zαβ dimerizes in a concentration-dependent manner, forming characteristic condensates in solutions evidenced by DLS and SAXS methods. ZBP1 exhibits a binding preference for 10-bp length CG (10CG) DNA and Z-RNA ligand, which in turn enhanced Zαβ dimerization, expediting the formation of droplet condensates in vitro and amyloid-like puncta in cells. Subsequent investigations reveal that Zαβ could form condensates with liquid-liquid phase separation property upon HSV and IAV infections, while full-length ZBP1 forms amyloid-like puncta with or without infections. Furthermore, ZBP1 RHIM domains show typical amyloidal fibril characterizations and cross-polymerize with RIPK1 depending on the core motif of 206 IQIG 209 , while mutated ZBP1 could impede necroptosis and antiviral immunity in HT-29 cells. Thus, ZBP1 condensate formation facilitates the recognition of viral Z-NAs and activation of downstream signal transduction via synergic action of different domains, revealing its elaborated mechanism in innate immunity.

Summary Z‐DNA is a left‐handed double helix form of DNA that is believed to be involved in various DNA transactions. However, comprehensive investigations aimed at global profiling of Z‐DNA landscapes are still missing in both humans and plants. We here report the development of two techniques: anti‐Z‐DNA antibody‐based immunoprecipitation followed by sequencing (ZIP‐seq), and cleavage under targets and tagmentation (CUT&TAG) for characterizing Z‐DNA in nipponbare rice (Oryza sativa L., Japonica). We found that Z‐DNA‐IP+ (Z‐DNA recognized by the antibody) exhibits distinct genomic features as compared to Z‐DNA‐IP− (Z‐DNA not recognized by the antibody). The concomitant presence of G‐quadruplexes (G4s) and i‐motifs (iMs) may promote Z‐DNA formation. DNA modifications such as DNA‐6mA/‐4acC generally disfavours Z‐DNA formation, while modifications like DNA‐5mC (CHH) and 8‐oxodG promote it, highlighting the distinct roles of DNA base modifications in modulating Z‐DNA formation. Importantly, Z‐DNA located at transcription start sites (TSSs) enhances gene expression, whereas Z‐DNA in genic regions represses it, underscoring its dual roles in regulating the expression of genes involved in fundamental biological functions and responses to salt stress. Furthermore, Z‐DNA may play a role in transcriptional initiation and termination rather than in transcriptional elongation. Finally, the presence of Z‐DNA in promoters is correlated with the coevolution of overlapping genes, thereby regulating gene domestication. Consequently, our study represents as a pivotal point and a solid foundation for reliably launching genome‐wide investigations of Z‐DNA, thereby advancing the understanding of Z‐DNA biology in both plants and non‐plant systems.

Variants in the human double-stranded RNA editing enzyme ADAR produce three well-characterized rare Mendelian Diseases: Dyschromatosis Symmetrica Hereditaria (OMIM: 127400), Aicardi-Goutières syndrome (OMIM: 615010) and Bilateral Striatal Necrosis/Dystonia. ADAR encodes p150 and p110 protein isoforms. p150 incorporates the Zα domain that binds left-handed Z-DNA and Z-RNA with high affinity through contact of highly conserved residues with the DNA and RNA double helix. In certain individuals, frameshift variants on one parental chromosome in the second exon of ADAR produce haploinsufficiency of p150 while maintaining normal expression of p110. In other individuals, loss of p150 expression from one chromosome allows mapping of Zα p150 variants from the other parental chromosome directly to phenotype. The analysis reveals that loss of function Zα variants cause dysregulation of innate interferon responses to double-stranded RNA. This approach confirms a biological role for the left-handed conformation in human disease, further validating the power of Mendelian genetics to deliver unambiguous answers to difficult questions. The findings reveal that the human genome encodes genetic information using both shape and sequence.

Sequences called flipons can adopt discrete, alternative nucleic acid conformations, such as the left-handed Z-DNA and Z-RNA double helices (referred to collectively as ZNA), and the four-stranded RNA and DNA G-quadruplexes. Each flipon conformation encodes different information. For example, the base-specific interactions of proteins with B-DNA enable sequence-specific recognition. In contrast, the higher energy Z-DNA and G-quadruplexes facilitate the speedy scanning of chromosomes to locate active regions of the genome. Results synthesized from small-scale benchside and large-scale computational experimental approaches provide compelling evidence that zinc-finger protein domains (ZFDs) not only engage in base-specific recognition of B-DNA, but also bind directly to Z-DNA and G-quadruplexes. The findings address the long-standing speed–stability paradox of how high-affinity ZFPs with multiple zinc fingers can rapidly localize to a specific binding site. The energy gap between different DNA interaction modes enables fast off-rates during the scanning of Z-DNA for cognate binding sites, and a slow off-rate following engagement of the B-DNA conformer. ZFPs represent the most prominent human transcription factor family with 804 annotated members. The coevolution of flipons and ZFP enhances suppression of retroelements and enables rapid, context-specific responses. ZNA and GQ binding proteins are consequently more frequent in the proteome than currently conceded.

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.