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.

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.

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.

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.

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.

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.

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.

The ancient Z-DNA and Z-RNA specific Zα fold has evolved modern roles in immunity and transcription through the natural selection of flipons.

The Zα fold specifically binds to both Z-DNA and Z-RNA, left-handed nucleic acid structures that form under physiological conditions and are encoded by flipons. I trace the Zα fold back to unicellular organisms representing all three domains of life and to the realm of giant nucleocytoplasmic DNA viruses (NCVs). The canonical Zα fold is present in the earliest known holozoan unicellular symbiont Capsaspora owczarzaki and persists in vertebrates and some invertebrates, but not in plants or fungi. In metazoans, starting with porifera, Zα is incorporated into the double-stranded RNA editing enzyme ADAR and reflects an early symbiont relationship with NCV. In vertebrates, Zα is also present in ZBP1 and PKZ proteins that recognize host-derived Z-RNAs to defend against modern-day viruses. A related Zα fold, also likely to bind Z-DNA, is present in proteins thought to modulate gene expression, including a subset of prokaryote arsR proteins and the p15 (PC4) family present in algae. Other Zα variants that probably play a more general role in the reinitiation of transcription include the archaeal and human transcription factor E and the human RNA polymerase 3 subunit C proteins. The roles in immunity and transcription underlie the natural selection of flipons.

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.