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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.

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.

Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZβDAI) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZβDAI/Z-DNA complex reveals that hZβDAI has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first β-strand rather than residues in the β-loop contributes to DNA binding, and part of the (α3) recognition helix adopts a 3₁₀ helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation.

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.

B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)n sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. \"Bubble\" templates containing 50–100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand–strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context.

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.

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.

Background Z-DNA is a left-handed DNA conformation with a zigzag backbone whose formation depends on base composition, modifications, and environmental factors. Although energetically unfavorable, Z-DNA has been implicated in both normal physiology and disease. The Z-Hunt algorithm predicts Z-DNA potential from thermodynamic principles, but its command-line interface and plain-text outputs limit adoption by users without coding expertise. Results We introduce Z-GENIE, an R/Shiny GUI that automates Z-Hunt execution, parses its output, and presents interactive visualizations. Z-GENIE accepts FASTA files, NCBI accession IDs, or manual sequences and produces CSV and BED summaries compatible with genomic browsers. In benchmarks on small to medium genomes (< 20 Mb), Z-Hunt completes in minutes and the full Z-GENIE pipeline (data retrieval, parsing, visualization) finishes in under five minutes. For large genomes (> 50 Mb), Z-Hunt may require up to two hours, whereas Z-GENIE’s parsing and BED-file export take < 2 min. In a human ADAM12 case study, Z-GENIE reproduced a published Z-score (3.0 × 10^7) and uncovered orientation-dependent Z-DNA clusters. Another case study compared predictions for Z-DNA in the rice genome ( Oryza sativa ) with experimental ZIP-Seq and CUT&Tag data; this study highlights the complementarity between in silico and in vivo approaches. Conclusions By encapsulating Z-Hunt within an intuitive GUI and offering flexible inputs and downstream-ready outputs, Z-GENIE democratizes genome-wide Z-DNA analysis. Its rapid performance and advanced visualization features should broaden exploration of Z-DNA’s roles in health and disease.