ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis.

Only a small proportion of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy and prevents ICB responsiveness by repressing immunogenic double-stranded RNAs (dsRNAs), such as those arising from the dysregulated expression of endogenous retroviral elements (EREs)1-4. These dsRNAs trigger an interferon-dependent antitumour response by activating A-form dsRNA (A-RNA)-sensing proteins such as MDA-5 and PKR5. Here we show that ADAR1 also prevents the accrual of endogenous Z-form dsRNA elements (Z-RNAs), which were enriched in the 3' untranslated regions of interferon-stimulated mRNAs. Depletion or mutation of ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, which culminated in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumour immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily translatable avenue for rekindling the immune responsiveness of ICB-resistant human cancers.

Osteogenesis imperfecta type 10 and the cellular scaffolds underlying common immunological diseases.

Osteogenesis imperfecta type 10 (OI10) is caused by loss of function codon variants in the gene SERPINH1 that encodes heat shock protein 47 (HSP47), rather than in a gene specifying bone formation. The HSP47 variants disrupt the folding of both collagen and the endonuclease IRE1α (inositol-requiring enzyme 1α) that splices X-Box Binding Protein 1 (XBP1) mRNA. Besides impairing bone development, variants likely affect osteoclast differentiation. Three distinct biochemical scaffold play key roles in the differentiation and regulated cell death of osteoclasts. These scaffolds consist of non-templated protein modifications, ordered lipid arrays, and protein filaments. The scaffold components are specified genetically, but assemble in response to extracellular perturbagens, pathogens, and left-handed Z-RNA helices encoded genomically by flipons. The outcomes depend on interactions between RIPK1, RIPK3, TRIF, and ZBP1 through short interaction motifs called RHIMs. The causal HSP47 nonsynonymous substitutions occur in a novel variant leucine repeat region (vLRR) that are distantly related to RHIMs. Other vLRR protein variants are causal for a variety of different mendelian diseases. The same scaffolds that drive mendelian pathology are associated with many other complex disease outcomes. Their assembly is triggered dynamically by flipons and other context-specific switches rather than by causal, mendelian, codon variants.

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 role of DNA in the pathogenesis of SLE: DNA as a molecular chameleon.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterised by antibodies to DNA (anti-DNA) and other nuclear macromolecules. Anti-DNA antibodies are markers for classification and disease activity and promote pathogenesis by forming immune complexes that deposit in the tissue or stimulate cytokine production. Studies on the antibody response to DNA have focused primarily on a conformation of DNA known as B-DNA, the classic right-handed double helix. Among other conformations of DNA, Z-DNA is a left-handed helix with a zig-zag backbone; hence, the term Z-DNA. Z-DNA formation is favoured by certain base sequences, with the energetically unfavourable flip from B-DNA to Z-DNA dependent on conditions. Z-DNA differs from B-DNA in its immunogenicity in animal models. Furthermore, anti-Z-DNA antibodies, but not anti-B-DNA antibodies, can be present in otherwise healthy individuals. In SLE, antibodies to Z-DNA can occur in association with antibodies to B-DNA as a cross-reactive response, rising and falling together. While formed transiently in chromosomal DNA, Z-DNA is stably present in bacterial biofilms; biofilms can provide protection against antibiotics and other challenges including elements of host defence. The high GC content of certain bacterial DNA also favours Z-DNA formation as do DNA-binding proteins of bacterial or host origin. Together, these findings suggest that sources of Z-DNA can enhance the immunogenicity of DNA and, in SLE, stimulate the production of cross-reactive antibodies that bind both B-DNA and Z-DNA. As such, DNA can act as a molecular chameleon that, when stabilised in the Z-DNA conformation, can drive autoimmunity.

Nucleosomes and flipons exchange energy to alter chromatin conformation, the readout of genomic information, and cell fate.

Alternative non-B-DNA conformations formed under physiological conditions by sequences called flipons include left-handed Z-DNA, three-stranded triplexes, and four-stranded i-motifs and quadruplexes. These conformations accumulate and release energy to enable the local assembly of cellular machines in a context specific manner. In these transactions, nucleosomes store power, serving like rechargeable batteries, while flipons smooth energy flows from source to sink by acting as capacitors or resistors. Here, I review the known biological roles for flipons. I present recent and unequivocal findings showing how innate immune responses are regulated by Z-flipons that identify endogenous RNAs as self. Evidence is also presented supporting important roles for other flipon classes. In these examples, the dynamic exchange of energy between flipons and nucleosomes enables rapid switching of genetic programs without altering flipon sequence. The increased phenotypic diversity enabled by flipons drives their natural selection, with adaptations evolving faster than is possible by codon mutation alone.

To "Z" or not to "Z": Z-RNA, self-recognition, and the MDA5 helicase.

Double-stranded RNA (dsRNA) is produced both by virus and host. Its recognition by the melanoma differentiation-associated gene 5 (MDA5) initiates type I interferon responses. How can a host distinguish self-transcripts from nonself to ensure that responses are targeted correctly? Here, I discuss a role for MDA5 helicase in inducing Z-RNA formation by Alu inverted repeat (AIR) elements. These retroelements have highly conserved sequences that favor Z-formation, creating a site for the dsRNA-specific deaminase enzyme ADAR1 to dock. The subsequent editing destabilizes the dsRNA, ending further interaction with MDA5 and terminating innate immune responses directed against self. By enabling self-recognition, Alu retrotransposons, once invaders, now are genetic elements that keep immune responses in check. I also discuss the possible but less characterized roles of the other helicases in modulating innate immune responses, focusing on DExH-box helicase 9 (DHX9) and Mov10 RISC complex RNA helicase (MOV10). DHX9 and MOV10 function differently from MDA5, but still use nucleic acid structure, rather than nucleotide sequence, to define self. Those genetic elements encoding the alternative conformations involved, referred to as flipons, enable helicases to dynamically shape a cell's repertoire of responses. In the case of MDA5, Alu flipons switch off the dsRNA-dependent responses against self. I suggest a number of genetic systems in which to study interactions between flipons and helicases further.

Simple Repeats as Building Blocks for Genetic Computers.

Processing of RNA involves heterogeneous nuclear ribonucleoproteins. The simple sequence repeats (SSRs) they bind can also adopt alternative DNA structures, like Z DNA, triplexes, G quadruplexes, and I motifs. Those SSRs capable of switching conformation under physiological conditions (called flipons) are genetic elements that can encode alternative RNA processing by their effects on RNA processivity, most likely as DNA:RNA hybrids. Flipons are elements of a binary, instructive genetic code directing how genomic sequences are compiled into transcripts. The combinatorial nature of this code provides a rich set of options for creating genetic computers able to reproduce themselves and use a heritable and evolvable code to optimize survival. The underlying computational logic potentiates a diverse set of genetic programs that modify cis-mediated heritability and disease risk.

The Intransitive Logic of Directed Cycles and Flipons Enhances the Evolution of Molecular Computers by Augmenting the Kolmogorov Complexity of Genomes.

Cell responses are usually viewed as transitive events with fixed inputs and outputs that are regulated by feedback loops. In contrast, directed cycles (DCs) have all nodes connected, and the flow is in a single direction. Consequently, DCs can regenerate themselves and implement intransitive logic. DCs are able to couple unrelated chemical reactions to each edge. The output depends upon which node is used as input. DCs can also undergo selection to minimize the loss of thermodynamic entropy while maximizing the gain of information entropy. The intransitive logic underlying DCs enhances their programmability and impacts their evolution. The natural selection of DCs favors the persistence, adaptability, and self-awareness of living organisms and does not depend solely on changes to coding sequences. Rather, the process can be RNA-directed. I use flipons, nucleic acid sequences that change conformation under physiological conditions, as a simple example and then describe more complex DCs. Flipons are often encoded by repeats and greatly increase the Kolmogorov complexity of genomes by adopting alternative structures. Other DCs allow cells to regenerate, recalibrate, reset, repair, and rewrite themselves, going far beyond the capabilities of current computational devices. Unlike Turing machines, cells are not designed to halt but rather to regenerate.

Conserved microRNAs and Flipons Shape Gene Expression during Development by Altering Promoter Conformations.

The classical view of gene regulation draws from prokaryotic models, where responses to environmental changes involve operons regulated by sequence-specific protein interactions with DNA, although it is now known that operons are also modulated by small RNAs. In eukaryotes, pathways based on microRNAs (miR) regulate the readout of genomic information from transcripts, while alternative nucleic acid structures encoded by flipons influence the readout of genetic programs from DNA. Here, we provide evidence that miR- and flipon-based mechanisms are deeply connected. We analyze the connection between flipon conformation and the 211 highly conserved human miR that are shared with other placental and other bilateral species. The direct interaction between conserved miR (c-miR) and flipons is supported by sequence alignments and the engagement of argonaute proteins by experimentally validated flipons as well as their enrichment in promoters of coding transcripts important in multicellular development, cell surface glycosylation and glutamatergic synapse specification with significant enrichments at false discovery rates as low as 10-116. We also identify a second subset of c-miR that targets flipons essential for retrotransposon replication, exploiting that vulnerability to limit their spread. We propose that miR can act in a combinatorial manner to regulate the readout of genetic information by specifying when and where flipons form non-B DNA (NoB) conformations, providing the interactions of the conserved hsa-miR-324-3p with RELA and the conserved hsa-miR-744 with ARHGAP5 genes as examples.

A Genetic Instruction Code Based on DNA Conformation.

Flipons are sequences capable of forming either right- or left-handed DNA under physiological conditions, forming a class of dissipative structures that trade metabolic energy for information by cycling DNA between different chromatin states. Flipons enhance the diversity of transcriptomes, increasing entropy while enabling the evolution of features both new and unexpected.

Mono a Mano: ZBP1's Love-Hate Relationship with the Kissing Virus.

Z-DNA binding protein (ZBP1) very much represents the nuclear option. By initiating inflammatory cell death (ICD), ZBP1 activates host defenses to destroy infectious threats. ZBP1 is also able to induce noninflammatory regulated cell death via apoptosis (RCD). ZBP1 senses the presence of left-handed Z-DNA and Z-RNA (ZNA), including that formed by expression of endogenous retroelements. Viruses such as the Epstein-Barr "kissing virus" inhibit ICD, RCD and other cell death signaling pathways to produce persistent infection. EBV undergoes lytic replication in plasma cells, which maintain detectable levels of basal ZBP1 expression, leading us to suggest a new role for ZBP1 in maintaining EBV latency, one of benefit for both host and virus. We provide an overview of the pathways that are involved in establishing latent infection, including those regulated by MYC and NF-κB. We describe and provide a synthesis of the evidence supporting a role for ZNA in these pathways, highlighting the positive and negative selection of ZNA forming sequences in the EBV genome that underscores the coadaptation of host and virus. Instead of a fight to the death, a state of détente now exists where persistent infection by the virus is tolerated by the host, while disease outcomes such as death, autoimmunity and cancer are minimized. Based on these new insights, we propose actionable therapeutic approaches to unhost EBV.

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.

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.

Genome-wide association study identifies novel loci association with fasting insulin and insulin resistance in African Americans.

Insulin resistance (IR) is a key determinant of type 2 diabetes (T2D) and other metabolic disorders. This genome-wide association study (GWAS) was designed to shed light on the genetic basis of fasting insulin (FI) and IR in 927 non-diabetic African Americans. 5 396 838 single-nucleotide polymorphisms (SNPs) were tested for associations with FI or IR with adjustments for age, sex, body mass index, hypertension status and first two principal components. Genotyped SNPs (n = 12) with P < 5 × 10(-6) in African Americans were carried forward for de novo genotyping in 570 non-diabetic West Africans. We replicated SNPs in or near SC4MOL and TCERG1L in West Africans. The meta-analysis of 1497 African Americans and West Africans yielded genome-wide significant associations for SNPs in the SC4MOL gene: rs17046216 (P = 1.7 × 10(-8) and 2.9 × 10(-8) for FI and IR, respectively); and near the TCERG1L gene with rs7077836 as the top scoring (P = 7.5 × 10(-9) and 4.9 × 10(-10) for FI and IR, respectively). In silico replication in the MAGIC study (n = 37 037) showed weak but significant association (adjusted P-value of 0.0097) for rs34602777 in the MYO5A gene. In addition, we replicated previous GWAS findings for IR and FI in Europeans for GCKR, and for variants in four T2D loci (FTO, IRS1, KLF14 and PPARG) which exert their action via IR. In summary, variants in/near SC4MOL, and TCERG1L were associated with FI and IR in this cohort of African Americans and were replicated in West Africans. SC4MOL is under-expressed in an animal model of T2D and plays a key role in lipid biosynthesis, with implications for the regulation of energy metabolism, obesity and dyslipidemia. TCERG1L is associated with plasma adiponectin, a key modulator of obesity, inflammation, IR and diabetes.

Genomic screening and replication using the same data set in family-based association testing.

The Human Genome Project and its spin-offs are making it increasingly feasible to determine the genetic basis of complex traits using genome-wide association studies. The statistical challenge of analyzing such studies stems from the severe multiple-comparison problem resulting from the analysis of thousands of SNPs. Our methodology for genome-wide family-based association studies, using single SNPs or haplotypes, can identify associations that achieve genome-wide significance. In relation to developing guidelines for our screening tools, we determined lower bounds for the estimated power to detect the gene underlying the disease-susceptibility locus, which hold regardless of the linkage disequilibrium structure present in the data. We also assessed the power of our approach in the presence of multiple disease-susceptibility loci. Our screening tools accommodate genomic control and use the concept of haplotype-tagging SNPs. Our methods use the entire sample and do not require separate screening and validation samples to establish genome-wide significance, as population-based designs do.

The four Rs of RNA-directed evolution.

The way we quantify the human genome has changed markedly. The estimated percentage of the genome derived from retrotransposition has increased (now 45%; refs. 1,2), as have the estimates for alternative splicing (now 41-60% of multiexon genes), antisense transcription (now 10-20% of genes) and non-protein coding RNA (now approximately 7% of full-length cDNAs). Concomitantly, the estimated number of protein-coding genes (now approximately 24,500) has decreased. These numbers support an RNA-centric view of evolution in which phenotypic diversity arises through extensive RNA processing and widespread RNA-directed rewriting of DNA enables dissemination of 'selfish' RNAs associated with successful outcomes. The numbers also indicate important roles for sense-antisense transcription units (SATs) and coregulatory RNAs (coRNAs) in directing the read-out of genetic information, in reconciling different regulatory inputs and in transmitting epigenetic information to progeny. Together, the actions of reading, 'riting, 'rithmetic and replication constitute the four Rs of RNA-directed evolution.

Z-DNA and Z-RNA: Methods-Past and Future.

A quote attributed to Yogi Berra makes the observation that "It's tough to make predictions, especially about the future," highlighting the difficulties posed to an author writing a manuscript like the present. The history of Z-DNA shows that earlier postulates about its biology have failed the test of time, both those from proponents who were wildly enthusiastic in enunciating roles that till this day still remain elusive to experimental validation and those from skeptics within the larger community who considered the field a folly, presumably because of the limitations in the methods available at that time. If anything, the biological roles we now know for Z-DNA and Z-RNA were not anticipated by anyone, even when those early predictions are interpreted in the most favorable way possible. The breakthroughs in the field were made using a combination of methods, especially those based on human and mouse genetic approaches informed by the biochemical and biophysical characterization of the Zα family of proteins. The first success was with the p150 Zα isoform of ADAR1 (adenosine deaminase RNA specific), with insights into the functions of ZBP1 (Z-DNA-binding protein 1) following soon after from the cell death community. Just as the replacement of mechanical clocks by more accurate designs changed expectations about navigation, the discovery of the roles assigned by nature to alternative conformations like Z-DNA has forever altered our view of how the genome operates. These recent advances have been driven by better methodology and by better analytical approaches. This article will briefly describe the methods that were key to these discoveries and highlight areas where new method development is likely to further advance our knowledge.

Z-form nucleic acid-binding protein 1 (ZBP1) as a sensor of viral and cellular Z-RNAs: walking the razor's edge.

Z-form nucleic acid-binding protein 1 (ZBP1) detects viral Z-form RNAs (Z-RNAs), activates receptor-interacting protein kinase 3, and triggers cell death during both RNA and DNA virus infections. Such cell death promotes virus clearance by eliminating infected cells and galvanizing antiviral immunity, and is thus often targeted for evasion by virus-encoded suppressors. Recent evidence demonstrates that ZBP1 can also be activated by cellular Z-RNAs transcribed from endogenous retroelements within mammalian genomes. These cellular Z-RNAs, if not edited and neutralized by adenosine deaminase RNA-specific 1, trigger ZBP1-dependent cell death and inflammation, which may drive disease in Aicardi-Goutière's syndrome and related interferonopathies. Thus, while well-controlled activation of ZBP1 by viral Z-RNAs during infections is beneficial, the same pathway can have harmful consequences when inappropriately triggered by cellular Z-RNAs in other disease settings.

Unsupervised domain adaptation methods for cross-species transfer of regulatory code signals.

Due to advances in NGS technologies whole-genome maps of various functional genomic elements were generated for a dozen of species, however experiments are still expensive and are not available for many species of interest. Deep learning methods became the state-of-the-art computational methods to analyze the available data, but the focus is often only on the species studied. Here we take advantage of the progresses in Transfer Learning in the area of Unsupervised Domain Adaption (UDA) and tested nine UDA methods for prediction of regulatory code signals for genomes of other species. We tested each deep learning implementation by training the model on experimental data from one species, then refined the model using the genome sequence of the target species for which we wanted to make predictions. Among nine tested domain adaptation architectures non-adversarial methods Minimum Class Confusion (MCC) and Deep Adaptation Network (DAN) significantly outperformed others. Conditional Domain Adversarial Network (CDAN) appeared as the third best architecture. Here we provide an empirical assessment of each approach using real world data. The different approaches were tested on ChIP-seq data for transcription factor binding sites and histone marks on human and mouse genomes, but is generalizable to any cross-species transfer of interest. We tested the efficiency of each method using species where experimental data was available for both. The results allows us to assess how well each implementation will work for species for which only limited experimental data is available and will inform the design of future experiments in these understudied organisms. Overall, our results proved the validity of UDA methods for generation of missing experimental data for histone marks and transcription factor binding sites in various genomes and highlights how robust the various approaches are to data that is incomplete, noisy and susceptible to analytic bias.