Genome Balance: Repeats, Immunity, and Cancer

Cancer is usually described as a disease of mutations. Genes break, pathways fail, and cells escape control. That framing has been powerful, but it misses a deeper layer that may reveal how it begins.

The human genome is not primarily a coding genome. It is a repeat genome. More than half of our DNA consists of repetitive elements, with Alu retroelements alone numbering over a million copies. These sequences are a defining feature of primate genomes and they create a unique biological problem that human cells must continuously manage. Recent work suggests that cancer may emerge, in part, when this management system loses balance.

Alu elements are short retrotransposons that readily form double‑stranded RNA stem‑loop structures when transcribed, particularly in antisense orientation within introns and untranslated regions. To the innate immune system, these structures resemble viral RNA. This means that normal gene expression in human cells constantly risks triggering antiviral immune responses against self‑derived RNA.

A striking recent study shows that human cells rely on active suppression to avoid this outcome. In Ku suppresses RNA‑mediated innate immune responses in human cells to accommodate primate‑specific Alu expansion, the authors demonstrate that the DNA repair protein Ku (Ku70/Ku80) plays an essential second role: binding Alu‑derived dsRNA stem‑loops and preventing activation of innate immune sensors such as MDA5, RIG‑I, PKR, and OAS/RNase L.

When Ku is depleted interferon and NF‑κB signaling are strongly activated, translation is suppressed, and cells undergo growth arrest or death. Notably, Ku levels scale tightly with Alu expansion across primates, and Ku is essential in human cells but not in mice. The implication is clear:

Human cell viability depends on continuous suppression of Alu‑derived innate immune activation.

Alu expression is not harmless noise, it is actively tolerated! Ku functions as a finite buffer that allows primate cells to tolerate structurally immunogenic RNA produced by repeat‑rich genomes. When structured RNA load increases simultaneously from endogenous repeat transcription and exogenous viral RNA infection, Ku becomes functionally saturated and redistributed, weakening nuclear retention and cytoplasmic buffering. This pressurizes the cell’s capacity to contain dsRNA stress, promoting escape of repeat‑derived RNA, activation of innate sensors, and eventual selection for immune‑tolerant states.

A second line of evidence connects this tolerance to cancer evolution. A 2025 bioRxiv preprint, p53 loss promotes chronic viral mimicry and immune tolerance, shows that loss of p53 permits transcription of immunogenic repetitive elements, generating signals that resemble viral infection. Rather than leading to effective immune clearance, this state becomes chronic. Tumor cells adapt by dampening innate immune responses and tolerating persistent repeat‑derived nucleic acids.

In this view, “viral mimicry” is not a one‑time immune alarm. It is a conditioning process repeat RNAs accumulate, immune pathways are activated, and progressively suppressed or rewired to allow survival. Cancer cells do not simply evade immunity, they learn to live with endogenous viral‑like signals.

These immune findings align with earlier evidence that repeat control begins at the level of genome structure itself. A 2022 Nature Communications study demonstrated that retroelements embedded within the first intron of TP53 act as cis‑repressive genomic architecture. Removing this intron increases TP53 expression, indicating that long‑embedded repeats contribute directly to regulating a core tumor suppressor gene.

Importantly, this repression is architectural rather than motif‑driven. The repeats do not act through a single conserved sequence, but through repeat‑dense structure.

Together, these findings suggest a layered system of control:

1. Structural repression of repeats within introns.

2. Immune suppression of repeat‑derived dsRNA.

3. p53‑dependent governance of both genome stability and immune signaling. 

One long‑standing challenge in repeat biology is inconsistency. Different tumors show different repeat fragments. Even different regions of the same tumor can look unrelated at the sequence level.

From a traditional biomarker perspective, this appears discouraging. From a structural perspective, it is expected. Codondex analyses of repeat‑dense introns, including TP53 intron 1, show that cancer does not preserve specific Alu sequences. Instead, it perturbs repeat topology:

• dominance and skew within intronic scaffolds,

• stem‑loop‑prone architectures,

• context‑specific fragmentation patterns.

The sequences vary. The instability regime does not. This is characteristic of a state change, not a discrete genetic event. Repeat‑dense introns behave like stress recorders. They integrate replication stress, chromatin relaxation, repair pathway bias, and immune tolerance history.

Unlike coding mutations, these signals are heterogeneous, region‑specific, and reflective of ongoing cellular state.

They are difficult to interpret with gene‑centric tools, but powerful when viewed architecturally. 

Most cancer diagnostics ask:

What mutation is present? A repeat‑aware framework asks:

Has this tissue entered a stable state of repeat derepression coupled with immune tolerance?

That state may precede aggressive behavior, accompany treatment resistance, or mark transitions in disease evolution. Future prognostic approaches may therefore combine repeat‑topology instability metrics, repeat RNA burden, and evidence of immune decoupling from dsRNA load. Not to identify a single driver, but to detect loss of containment.

Alu repeats do not cause cancer on their own, but human cells must continuously restrain them, structurally and immunologically. Cancer appears, at least in part, when that restraint erodes and tolerance replaces control. Introns, long treated as background, may be one of the clearest places to see this shift, not because they encode instructions, but because they actively record genomic history and project it into a measure of present state.

Repeats as Signatures of Regulatory Potential

In the vast landscape of AI genomics, emerging analyses reveals non-coding DNA (ncDNA) as a treasure trove of regulatory information. At Codondex, our innovative k-mer-based approach uncovers how repetitive subsequences—short DNA fragments known as k-mers—serve as powerful signatures of regulatory potential. By viewing these repeats through a topological lens, we transform linear sequences into dynamic networks that highlight subtle distinctions in gene transcripts, offering new insights into gene regulation, isoform diversity, and disease mechanisms.

The Codondex Method: From Sequences to Topology

Codondex begins by "amplifying" ncDNA sequences associated with gene transcripts, generating all contiguous k-mers of length 8 or greater. For a gene like TP53, with its multiple isoforms (variants), we associate these k-mers with transcript-specific signatures derived from cDNA, mRNA or protein constants. The result? A rich dataset of subsequences, where repeats—identical k-mers appearing multiple times—emerge as key players.

Rather than treating DNA as a flat string, we interpret it topologically: k-mers as nodes in a graph, with repeats forming edges that indicate connections, clusters, and symmetries. Metrics like i-Score (normalizing contained k-mers by length) and inclusiveness (repeat frequency) rank these patterns, while cDNA or protein vectors capture fine distinctions. In our analyses of genes such as MEN1 and TP53, symmetries in repeat length and frequency stand out, unrelated to obvious features like reverse complements. These non-random patterns suggest repeats are not artifacts but deliberate signatures encoded for regulation.

Repeats as Regulatory Hotspots

How do these repeats signal regulatory potential? First, they often manifest as binding sites for proteins. Repetitive motifs can amplify affinity for transcription factors or splicing regulators. In TP53 introns, high-frequency k-mers align with p53-binding elements, potentially modulating tumor-suppressive isoforms. Variants with asymmetric repeats might weaken these interactions, leading to dysregulation in cancer.

Second, repeats influence secondary structures. Topologically, frequent repeats create "hubs" in the network, fostering DNA/RNA folds like hairpins that affect chromatin accessibility or mRNA stability. Our MEN1 intron1 study, analyzing 15 variants, revealed length-biased repeat clusters in scatter-graphs—despite length-agnostic algorithms—indicating structured motifs that differentiate stable from unstable transcripts. Disruptions from low-length repeats, as seen in TP53 vectors, act like regulatory "switches," fine-tuning expression in response to cellular stress.

Third, symmetries in repeats point to evolutionary conservation. Equal-length k-mers recurring with balanced frequencies form symmetric graphs, preserving robust modules across species. In MEN1, linked to endocrine tumors, these patterns suggest intron-driven adaptations for hormone regulation. Disruptions in variants could flag pathogenicity, enabling predictive modeling without coding-sequence reliance.

Real-World Implications and Validation

Our deep k-mer analysis, first detailed in a 2018 blog post, showcased MEN1 intron symmetries predicting protein outcomes, later validated through lab tests at Tel Aviv University. For TP53, stable vector positions disrupted by specific repeats correlated with isoform-specific roles, highlighting ncDNA's influence on cancer hallmarks.

This topological view empowers genomics: identifying regulatory elements for drug targeting, differentiating disease variants, and advancing precision medicine. At Codondex, we're excited to explore how these repeat signatures unlock ncDNA's secrets—join us in redefining genomic potential.

P53 - Stability and Life Or Disorder and Death!

There is something ancient about the struggle between order and disorder in biology. A cell does not merely live by dividing, signaling, and repairing itself. It lives by maintaining interpretability. Its genome must remain legible enough to be copied, restrained enough not to erupt into instability, and coherent enough that surrounding systems — especially the immune system — can still distinguish function from failure. In that sense, p53 is not simply a tumor suppressor in the narrow modern meaning of the phrase. It is closer to a molecular governor of biological intelligibility, one of the factors that helps determine whether stress remains containable or tips into forms of disorder.

The broader role has appeared repeatedly across Codondex discussions, from Expanding Treatment Horizons to Does SARS-CoV2 Strangle P53 to kill Natural Killer Immunity?, where p53 was already being read less as an isolated tumor suppressor and more as part of a wider immune and genomic control system.

That older and deeper role becomes clearer once p53 is viewed not only through apoptosis or cell-cycle arrest, but through its relationship with the repetitive genome. Work over the last decade has shown that p53 does not merely respond to genetic insult after the fact. It can directly repress human LINE1 retrotransposons by binding the 5′UTR and promoting local repressive chromatin, and more recent work has extended that picture by showing p53-dependent restraint of LINE1-associated RNA-DNA hybrid states as well. One of the great guardians of cellular integrity is therefore also engaged in policing one of the genome’s recurrent internal threats: mobile and semi-mobile repetitive sequence that, when released from restraint, can destabilize chromosomal order and provoke inflammatory consequences. p53 directly represses human LINE1 transposons and p53-mediated regulation of LINE1 retrotransposon-derived R-loops both push that picture into sharper focus.

But the relationship runs in both directions. Transposable elements are not only targets of p53; they have also helped shape the p53 regulatory landscape itself. A substantial body of work has shown that human retrotransposons contain p53 responsive elements, meaning that the repetitive genome has donated part of the sequence architecture through which p53 now reads and regulates stress. This is one of those places where the older division between “functional genome” and “junk” becomes difficult to maintain. Repetitive sequence has not only threatened order. It has also contributed to the grammar by which order is defended.

Once that is appreciated, the immune side of the problem begins to look less like a separate field and more like a continuation of the same one. If p53 helps determine whether genomic instability remains suppressed, then it also helps determine whether such instability becomes visible to immune surveillance. Emerging views now frame p53 as a major regulator of NK-cell tumor immunosurveillance, not because NK cells are somehow subordinate to p53, but because p53 influences so many of the target-cell properties that NK cells are built to read: stress ligands, metabolic distress, microenvironmental signals, and the broader state of cellular legitimacy.

One of the clearest examples is the p53-dependent induction of ligands visible to NK activation pathways. When wild-type p53 is induced in tumor cells, NK cells can be alerted through upregulation of the NKG2D ligands ULBP1 and ULBP2. That is an important bridge. It means p53 is not only preserving internal order; it can also help convert intracellular stress into a surface-readable signal that tells NK cells something has gone wrong. The target is no longer merely unstable. It becomes interpretable to innate immune surveillance.

A parallel bridge exists through antigen processing. p53 has also been shown to increase MHC class I expression by upregulating ERAP1, a trimming enzyme involved in preparing peptides for class I presentation. That does not collapse NK and T-cell biology into one another, but it does reinforce the larger point: p53 influences whether a distressed cell remains hidden, partially legible, or fully exposed to immune scrutiny. It affects not just whether the cell survives, but how clearly that cell can be judged by the systems around it.

The recombination thread can also be preserved, though it needs to be understood in the right register. Mature NK cells do not generate their recognition receptors through classical V(D)J recombination in the way T and B cells do. Their receptors are fundamentally germline encoded. Yet that is not the end of the recombination story. Work on NK ontogeny has shown that a history of RAG expression in progenitors and NK precursors marks functionally distinct NK subsets later in the periphery, with consequences for fitness, survival, and responsiveness. Recombination machinery therefore leaves a developmental trace on NK biology, even if it does not build the mature receptor repertoire in the adaptive sense.

That developmental nuance matters because it prevents the argument from becoming either too weak or too strong. Too weak, and the relationship between recombination-linked stress and NK function disappears. Too strong, and NK cells are mistakenly described as if they were just another rearranging lymphocyte lineage. The better reading is that recombination biology, DNA damage response history, and developmental programming can shape the later functional competence of NK cells without making their mature surveillance logic identical to that of T cells.

A similar caution, and opportunity, appears in the KIR story. One of the most interesting findings in NK regulation is that KIR expression can be governed by bidirectional promoter logic and antisense transcription. In particular, KIR antisense transcripts processed into a 28-base PIWI-like small RNA have been linked to transcriptional silencing, while related work on KIR antisense lncRNAs and probabilistic promoter switching suggests that inhibitory receptor expression is shaped by a layered interaction between transcription, antisense regulation, and epigenetic commitment. This does not establish a direct p53→piRNA→KIR3DL1 pathway. But it does show that the NK lineage is not insulated from the wider world of small RNA restraint and genome-governed silencing.

That is where the Codondex theme begins to re-emerge. If p53 sits in one part of the cell as a governor of repetitive-element restraint, and if NK inhibitory receptor choice is itself touched by small-RNA and antisense-mediated silencing logic, then the two systems may not be identical, but they may still rhyme. Both are concerned with the management of unstable potential. Both are concerned with whether latent disorder is allowed to become active. Both are concerned with which signals are permitted to surface and which are held in reserve. This is not yet a single proven pathway. It is a systems-level parallel supported by a growing amount of molecular detail.

There is another reason the p53–NK connection deserves attention. In some settings, p53 activation appears able to convert repetitive-element biology into something resembling a warning flare. Pharmacologic activation of p53 has been linked to antiviral-like and immune-stimulatory states, and the broader literature now places p53 within a network that can enhance NK recognition and tumor destruction through multiple channels rather than one single canonical mechanism. The significance of that shift should not be underestimated. It means p53 is no longer best understood only as the decider of cell fate from within. It is also a participant in the communication of cellular fate to the outside world.

So the central question remains a fruitful one. Is p53 merely a brake on instability, or is it also part of the language by which instability becomes visible to elimination? The literature increasingly favors the second possibility. p53 restrains transposable elements. p53 shapes stress-ligand display. p53 influences antigen processing and MHC-I expression. p53 intersects with developmental and regulatory processes that matter to NK-cell competence and target recognition. The picture that emerges is not of a single linear circuit, but of a pressure point where genome integrity, immune legibility, and cellular fate begin to converge.

In that light, p53 can still be read as this article’s central character without overstatement. When p53 function is preserved, a cell under strain is more likely to remain ordered, to arrest, to die cleanly, or to become visible enough for immune removal. When p53 function is lost, not only does instability grow, but the cell’s interpretability may degrade with it. Disorder then becomes doubly dangerous: more abundant internally, and more ambiguous externally. That may be one of the deeper meanings of p53 in cancer and perhaps in biology more generally. It is not only a guardian against mutation. It is one of the means by which life keeps disorder readable.

Electrons Rule Your Biology!

The mitochondrial Electron Transport Chain (ETC) is responsible for almost all cellular energy - ATP. One protein, GPD2 was adopted into the inner mitochondrial membrane, perhaps because it enabled ETC production to move to its electron processing limit. To do this, lipids are metabolized when cytoplasmic GPD1-DHAP convert Glycerol Kinase to G3P, which passes two additional electrons from the cytoplasm, through GPD2, to the internalized ETC complexes. 

When Mitochondrial Membrane Potential "Δψm" is within normal range, the GPD2 electrons enhance ATP energy production. When damage to lipids, fatty chains, cholesterols or other elements, constituting the inner mitochondrial membrane, disrupt Δψm the anchored ETC proteins can move fractionally apart causing electrons passing along the chain of ETC complexes to leak.

During disrupted Δψm the additional flow of GPD2 electrons can burden the ETC complexes, resulting in unstable molecules that contain oxygen and are highly reactive known as reactive oxygen species (ROS). Prolific ROS can increase CA+ levels, damage lipids in mitochondrial membranes, which can cause dysfunction and disease. In  a normal cellular environment this process can lead to ferroptosis, an iron-dependent form of cell death, induced by lipid peroxidation. 

A key bidirectional regulator of ferroptosis, p53 can adjust metabolism of iron, lipids, glutathione peroxidase 4, reactive oxygen species, and amino acids via a canonical pathway. GPD2 is transcribed by multiple factors that interact with p53 including Nrf2 and others during stress, but findings with E2F suggest a critical function controls a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation. 

P53 can also induce apoptosis through the mitochondrial pathway, contribute to necrosis by accumulating in the mitochondrial matrix and regulating autophagy. Mitochondrial p53 accumulation is an early event  not merely a consequence of apoptosis or a consequence of binding to damaged organelles in dying cells. Now, emerging evidence shows that ferroptosis plays a crucial role in tumor suppression via p53. 

Immune cells require massive energy boosts during synapse formation and lysis of a target cell when mitochondrial fitness is essential. However, tumor micro environments (TME's) alter lipid metabolism disrupting Δψm causing immune cells to function sub-optimally. Stimulation of T cells triggers a spike in cellular ATP production that doubles intracellular levels in <30 s and causes prolonged ATP release into the extracellular space. ATP release and autocrine feedback, via purinergic receptors collectively contribute to the influx of extracellular Ca2+ that is required for IL-2 production. The process has also been described for Natural Killer (NK) cells.

In the TME innate NK cells are dysfunctional due to lipid peroxidation inhibiting glucose metabolism. If innate immune cells are initially successful, adaptive immune responses may still fail because mitochondria reposition to the immune synapse where they transfer, including to immune cells, which can assist the target to evade immune response. Rapidly proliferating cancer cells may overwhelm initial immune responses and modify immune signaling promoting cancer and vascular remodeling.

ΔΨm as a measure of functional integrity maybe the flawed alert, a blind spot for of a cells' ADP-ATP pipeline. Likewise the status of TP53, from transcription through p53 isoform, may signal wide ranging affects of ΔΨm changes that incorporate fragmentation, accumulating damaged mitochondria, mitophagy, apoptosis or normal immune signaling and response through mitochondrial biogenesis, differentiation and angiogenesis. This modal duality aligns known functions of NK cells that under physiological conditions promote angiogenesis growth (as in Blastocyst implantation and placental vascularization) or NK's classic, cytolytic role in the innate immune response. 

Mitochondrial Phospholipid (MitoPLD), is anchored to the mitochondrial surface. It regulates mitochondrial shape, facilitating fusion and in the electron-dense nuage, of adjacent mitochondria, performs a critical piRNA generating function that is known to generate a spermatocyte-specific piRNA required for meiosis. piRNA are known to be aberrantly expressed in cancer cells.

Changes in mitochondrial membrane potential and ETC complexes can also influence piRNA-mediated control of transposable elements (TE's) through energy availability, ROS generation, and direct or indirect effects on piRNA biogenesis and function. piRNA restrain TE's that disrupt genes, chromosomal stability, damage DNA, cause inflammation, disease and/or cell death. For example, increased levels of endogenous retroviruses (ERV's), a TE subclass, trigger fibro inflammation and play a role in kidney disease development.

In mammals, the transcription of TEs is important for maintaining early embryonic development and related vital aspects of NK cell immune development. Intriguingly, regardless of the cell type, p53 sites are highly enriched in the endogenous retroviral elements of the ERV1 family. This highlights the importance of this repeat family in shaping the transcriptional network of p53 and its transcriptional role in interferon-mediated antiviral immunity. 

 

 

All Roads Lead to (Ch)Romosome 19!

A hepatocellular carcinoma (HCC) co-regulatory network exists between chromosome 19 microRNA cluster (C19MC) at 19q13.42, melanoma-A antigens, IFN-γ and p53, promoting an oncogenic role of C19MC that is disrupted by metal ions zinc and nickel. IFN-γ plays a co-operative role whereas IL-6 is antagonistic, each have a major bearing on the expression of HLA molecules on cancer cells. Analysis of Mesenchymal stem cells and cancer cells predicted C19MC modulation of apoptosis in induced pluripotency and tumorigenesis.

Key, differentially expressed genes in HCC included cancer-related transcription factors (TF) EGR1, FOS, and FOSB. From mRNA and miRNA expression profiles these were most enriched in the p53 signaling pathway where mRNA levels of each decreased in HCC tissues. In addition, mRNA levels of CCNB1, CCNB2, and CHEK1, key markers of the p53 signaling pathway, were all increased. miR-181a-5p regulated FOS and EGR1 to promote the invasion and progression of HCC by p53 signaling pathway and it plays an important role in maturation or impairment of natural killer (NK) cells.

A pan-cancer analysis, on microRNA-associated gene activation, produced the top 57 miRNAs that positively correlated with at least 100 genes. miR-150, at 19q13.33 was the most active, it positively correlated with 1009 different genes each covering at least 10 cancers. It is an important hematopoietic, especially B, T, and NK, cell specific miRNA.

Rapid functional impairment of NK cells following tumor entry limits anti-tumor immunity. Gene regulatory network analysis revealed downregulation of TF regulons, over pseudo-time, as NK cells transition to their impaired end state. These included AP-1 complex TF's, Fos, Fosb (19q13.32), Jun, Junb (19p13.13), which are activated during NK cell cytolytic programs and down regulated by interactions with inhibitory ligands. Other down-regulated TF's included Irf8, Klf2 (19p13.11), Myc, which support NK cell activation and proliferation. There were no significantly upregulated TF's suggesting that the tumor-retained NK state arises from the reduced activity of core transcription factors associated with promoting mature NK cell development and expansion.

Innate immune, intra-tumoral, stimulatory dendritic cells (SDCs) and NK cells cluster together and are necessary for enhanced T cell tumor responses. In human melanoma, SDC abundance is associated with intra-tumoral expression of the cytokine producing gene FLT3LG (19q13.33) that is predominantly produced by NK cells in tumors. Computed tomography exposes patients to ionizing X-irradiation. Determined trends in the expression of 24 radiation-responsive genes linked to cancer, in vivo, found that TP53 and FLT3LG expression increased linearly with CT dose. 

Undifferentiated embryonal sarcoma of the liver displays high aneuploidy with recurrent alterations of 19q13.4 that are uniformly associated with aberrantly high levels of transcriptional activity of C19MC microRNA. Further, TP53 mutation or loss was present with all samples that also display C19MC changes. The 19q13.4 locus is gene-poor with highly repetitive sequences. Given the noncoding nature and lack of an obvious oncogene, disruption of the nearby C19MC regulatory region became a target for tumorigenesis. 

The endogenous retroviral, hot-spot deletion rate at 19p13.11-19p13.12 and 19q33-19q42 occurs at double the background deletion rate. Clustered in and around these regions are many gene families including KIR, Siglec, Leukocyte immunoglobulin-like receptors and cytokines that associate important NK gene features to proximal NK genes that were overrepresented in a meta analysis of blood pressure. 

Endogenous retroviruses that invite p53 and its transcriptional network, at retroviral hot-spots, suggest that lymphocyte progenitors, such as ILC's and expanded, NK cells are synergistically responsive to transcription from this busy region including by the top differentially expressed blood pressure genes MYADM, GZMB, CD97, NKG7, CLC, PPP1R13L , GRAMD1A as well as (RAS-KKS) Kallikrein related peptidases to educate early and expanded NK cells that shape immune responses.  

Can Ancient Pathways Defeat Cancer?

It has been widely acknowledged that non-coding RNAs are master-regulators of genomic function. The association between human introns and ncRNAs has a pronounced synergistic effect with important implications for fine-tuning gene expression patterns across the entire genome. There is also strong preference of ncRNA from intronic regions particularly associated with the transcribed strand. 

Accumulating evidence demonstrates that, analogous to other small ncRNAs (e.g. miRNAs, siRNA's etc.) piRNAs have both oncogenic and tumor suppressive roles in cancer development. Functionally, piRNAs maintain genomic integrity and cell age by silencing repetitive, transposable elements, and are capable of regulating the expression of specific downstream target genes in a post-transcriptional manner. 

Unlike miRNAs and siRNAs, the precursors of piRNAs are single stranded transcripts without any prominent secondary hairpin structures. These precursors are usually generated from specific genomic locations containing repetitive elements, a process that is typically orchestrated via a Dicer-independent pathway. 

Without restraint, the ancient, L1 class of transposable elements can interrupt the genome through insertions, deletions, rearrangements, and copy number variations. L1 activity has contributed to instability and evolution of genomes, and is tightly regulated by DNA methylation, histone modifications, and piRNA. They can impact genome variation by mispairing and unequal crossing-over during meiosis due to repetitive DNA sequences. Indeed meiotic double-strand breaks are the proximal trigger for retrotransposon eruptions as highlighted in animals lacking p53.

Through a novel 28-base small piRNA of the KIR3DL1 gene, antisense transcripts mediate Killer Ig-like receptor (KIR) transcriptional silencing in immune somatic, Natural Killer (NK) cell lineage, a mechanism that may be broadly used in orchestrating immune development. Expressed on NK cells, KIR's are important determinants of NK cell function. Silencing  individual KIR genes is strongly correlated with the presence of CpG dinucleotide methylation within the promoter. 

Structural research exposed the enormous binding complexity behind KIR haplotypes and HLA allotypes. Not only via protein structures, but also plasticity and selective binding behavior's as influenced by extrinsic factors. One study links a specific recognition of HLA-C*05:01 by KIR2DS4 receptor through a peptide highly conserved among bacteria pathogenic in humans. Another demonstrated a hierarchy of functional peptide selectivity by KIR–HLA-C interactions, including cross-reactive binding, with relevance to NK cell biology and human disease associations. Additionally a p53 peptide most overlapped other high performance peptides for a HLA-C allotype C*02:02 that shares identical contact residues with C*05:01.

Ancient pathways linking p53 to attenuation of aberrant stem cell proliferation may predate the divergence between vertebrates and invertebrates. Human stem cell proliferation, as determined by p53 transposable element silencing, may also serve a NK progenitor to promote the repertoire of more than 30,000 NK cell subsets. 

A recent study showed that wild type p53 can restrain transposon mobility through interaction with PIWI-piRNA complex. Also, cellular metabolism regulates sensitivity to NK cells depending on P53 status and P53 pathway is coupled to NK cell maturation leaving open the possibility that a direct relationship exists. Further, functional interactions between KIR and HLA modify risks of basal cell carcinoma (BCC) and squamous cell carcinomas (SCC) and KIR B haplotypes provide selective pressure for altered P53 in BCC tumors. 

Anticipating p53's broader influences or responses, cells, extracted from 48 different sections of 7 tumor biopsies were sequenced and TP53 DNA computed using Codondex algorithm. Each section produced a TP53 Consensus Variant (CV), represented by its intron1, ncDNA Key Sequence's (KS). Bioinformatic correlations between each KS and cytotoxicity resulting from NK coculture with the section may predict KIR-HLA and extrinsic factor plasticity to reliably determine from KS's, optimal cell/tissue selections for NK cell education and licensing. 

Tolerating Your Non-self!

Immune cells get comfortable with cancer
Courtesy https://deepai.org

A hallmark of cancer, autoimmunity and disease is the aberrant transcription of typically silenced, repetitive genetic elements that mimic Pathogen-Associated Molecular Patterns (PAMP's) that bind Pattern Recognition Receptors (PPR's) triggering the innate immune system and inflammation. Unrestrained, this 'viral mimicry' activates a generally conserved mechanism that, under restraint, supports homeostasis. These repetitive viral DNA sequences normally act as a quality control over genomic dysregulation responding in ways that preferentially promote immune conditions for stability. If aberrantly unrestrained and the 'viral mimicry' is transcribed it may result in undesirable immune reactions that disrupt the homeostasis of cells.

Mitochondrial DNA (mtDNA) are one source of cytosolic double stranded RNA (dsRNA) that is commonly present in cells. Trp53 Mutant Embryonic Fibroblasts (MEF's) contain innate immune stimulating endogenous dsRNA, from mtDNA that mimic PAMP's. The immune response, via RIG-1 like PRR, leads to expression of type 1 interferon (IFN) and proinflammatory cytokine genes. Further, Natural Killer cells also produce a multitude of cytokines that can promote or dampen an immune response. Wild-type p53 suppresses viral repeats and contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its function as a proapoptotic and tumor suppressor gene. 

Post-translationally modified P53, located in the cytoplasm, enhances the permeability of the mitochondrial outer membrane thus stimulating apoptosis. However, treating Trp53 mutant MEF's with DNA demethylating agent caused a huge increase in the level of transcripts encoding short interspersed nuclear elements and other species of noncoding RNAs that generated a strong type 1 IFN response. This did not occur in p53 wild-type MEF's. Thus it appears that another function of p53 is to silence repeats that can accidentally induce an immune response.

This has several implications for how we understand self versus non-self discrimination. When pathogen-associated features were quantified, specific repeats in the genome not only display PAMP's capable of stimulating PRRs but, in some instances, have seemingly maintained such features under selection. For organisms with a high degree of epigenetic regulation and chromosomal organization immuno-stimulatory repeats release a danger signal, such as repeats released after p53 mutations. Here, immune stimulation may act as back-up for the failure of other p53 functions such as apoptosis or senescence due to mutation. This supports the hypothesis that specific repeats gained favor by maintaining non-self PAMPs to act as sensors for loss of heterochromatin as an epigenetic checkpoint of quality control that avoids genome instability generally. 

When P53 mutates it begins to fail its restraint of viral suppression, this enables a 'viral mimicry' and aberrant immune reactions. These may promote survival of cells that can leverage immunity, promote angiogenesis and heightened proliferation of cancers, or other diseases under modified conditions for non-self tolerance. 

Toward Customized Natural Killer Cells

An important role of Natural Killer (NK) cells is to eliminate other cells that extinguish or diminish expression of self-MHC class I molecules or Human Leukocyte Antigen (HLA), which commonly occurs as a result of viral infection or cellular transformation. This capacity arises because NK cells express stimulatory and inhibitory receptors that engage ligands on normal cells. The majority of inhibitory receptors belong to the Killer-cell immunoglobulin-like receptors (KIR) and CD94/NKG2A  families and are specific for MHC I molecules. When an NK cell encounters a normal cell, engagement of the inhibitory receptors conveys signals that counteract stimulatory signaling. Lysis occurs when inhibition is lost because the target cell lacks one or more self-MHC molecules or when target cells express high levels of stimulatory ligands that counter inhibition.

Mitochondrial DNA (MtDNA) embedded in the genomes of 66,000 humans was associated with adverse consequences including cancer. Overall tumor specific nuclear embedded MtDNA was more common on Chromosome (Chr)19, less common on Chr6 and tended to involve non-coding, repetitive elements or satellite repeats. 

The dimorphic relationship between genes on Chr6, encoding HLA and  Chr19, encoding KIRs  may elucidate how, why and when NK cells determine self restraint or attack cells infected by pathogens and disease. Chr19 has also been linked to blood pressure mechanics, immunity and checkpoints associated with P53. Cancer mutation burden is shaped by G4 DNA, cell cycle replication stress, DNA repair pathway and mitochondrial dysfunction. G4 DNA overrepresentation generally occurs in tumors with mutations in tumor suppressor gene's such as TP53. 

Whether KIR-HLA relationships are associated with p53 status of NK cells and of its target is unknown. However, it has been reported that cellular metabolism regulates a cells sensitivity to NK cells depending on its P53 status and that P53 pathway is coupled to NK cell maturation leaving open the possibility that a relationship exists. 

KIR and HLA genes are polymorphic and display significant variations, The independent segregation of these unlinked gene families produces extraordinary diversity in the number and type of KIR-HLA pairs inherited in individuals. Variation affects the KIR repertoire of NK cell clones, NK cell maturation, the capability to deliver signals, and consequently the NK cell response to human diseases.

One study suggests that functional interactions between KIR and HLA modify risks of basal cell carcinoma (BCC) and squamous cell carcinomas (SCC) and that KIR B haplotypes provide selective pressure for altered P53 in BCC tumors.

MtDNA and other insertions into nuclear DNA may have altered Chr19-Chr6 linkage relationships and KIR-HLA validity, affecting the integrity of NK missing-self surveillance. Therefore, P53 dependent metabolism and P53 coupled NK cell education may point to a required synchronicity, obtained through NK education, licensing KIR-HLA and other receptor-ligand combinations for a global NK symbiosis.

The altered landscape of cancer is often characterized by a heterogeneous mix of immunosuppressive metabolites, glucose and amino acid deprivation, hypoxia and acidity, which, in concert, prevent effective anti-tumor immunity, here NK therapies herald great potential.

NK cell co-culture with patient cells selected using precise P53 rankings for a distinct P53-coupled-NK cell education may realize a mature NK subset with P53-paired characteristics. Trojan therapy using autologous or combined allogeneic NK cells may promote licensing, through a broad synchronization including at least KIR-HLA. This ex-vivo approach may resist re-education in vivo and activate against P53-decoupled-KIR-HLA affected cells. The objective is an NK subset that, in vivo will initiate and progress a limited innate immune response and disrupt near-neighbor targets that will contribute to a broader immune response.  

Retroviral Defense And Mitochondrial Offense

Chromosomal DNA has played host to the long game of viral insertions that repeat and continue as a genetic and epigenetic symbiosis along its phosphate and pentose sugar backbone. But, the bacterial origin of mitochondria and its hosted DNA also promotes its offense. 

Research suggests that retrovirus insertions evolved from a type of transposon called a retrotransposon. The evolutionary time scales of inherited, endogenous retroviruses (ERV) and the appearance of the zinc finger gene that binds its unique sequences occur over same time scales of primate evolution. Additionaly the zinc-finger genes that inactivate transposable elements are commonly located on chromosome 19. The recurrence of independent ERV invasions can be countered by a reservoir of zinc-finger repressors that are continuously generated on copy number variant (CNV) formation hotspots.

One of the more intiguing aspects of prevalent CNV hotspots on chromosome 19 are their proximity to killer immunoglobulin receptor gene's (KIR's) and other critical gene's of the innate immune system.

Frequently occuring DNA breaks can cause genomic instability, which is a hallmark of cancer. These breaks are over represented at G4 DNA quadruplexes within, hominid-specific, SVA retrotransposons and generally occur in tumors with mutations in tumor suppressor genes, such as TP53. Cancer mutational burden is shaped by G4 DNA, replication stress and mitochondrial dysfunction, that in lung adenocarcinoma downlregulates SPATA18, a mitochondrial eating protein (MIEAP) that contributes to mitophagy. 

Genetic variations, in non-coding regions can control the activity of conserved protein-coding genes resulting in the establishment of species-specific transcriptional networks. A chromosome 19 zinc finger, ZNF558 evolved as a suppressor of LINE-1 transposons, but has since been co-opted to singly regulate SPATA18. These variations are evident from a panel of 409 human lymphoblastoid cell lines where the lengths of the ZNF558 variable number tandem repeats (VNTR) negatively correlated with its expression. 

Colon cancer cells with p53 deletion were used to analyze deregulated p53 target genes in HCT116 p53 null cells compared to HCT116-p53 +/+ cells. SPATA18 was the most upregulted gene in the differential expression providing further insight to p53 and mitophagy via SPATA18-MIEAP.

p53 response elements (p53RE) can be shaped by long terminal repeats from endogenous retroviruses, long interspersed nuclear repeats, and ALU repeats in humans and fuzzy tandem repeats in mice. Further, p53 pervasively binds to p53REs derived from retrotransposons or other mobile genetic elements and can suppress transcription of retroelements. The p53- mediated mechanisms conferring protection from retroelements is also conserved through evolution. Certainly, p53 has been shown to have other roles in DNA  context, such as playing an important role in replication restart and replication fork progression. The absence of these p53-dependent processes can lead to further genomic instability. 

The frequency of variable length, long or short nucleotide repeats and their locations within a gene may be key to the repression of DNA sequences that would otherwise cause genomic instability or protein expressions that would eat bacterial mitochondria or destroy its cell host. 

The complexity of variable length insertions is made evident when exhaustively analyzing a simple length 12 sequence for the potential frequency of each of its variable length repeats starting from a minumum variable length of 8.

Then, for TGTGGGCCCACA(12)

All possible internal variable length combinations from and including length 8:

TGTGGGCC(8)|GTGGGCCC(8)|TGTGGGCCC(9)|TGGGCCCA(8)|GTGGGCCCA(9)|TGTGGGCCCA(10|GGGCCCAC(8)|TGGGCCCAC(9)|GTGGGCCCAC(10)|TGTGGGCCCAC(11)|GGCCCACA(8)|GGGCCCACA(9)|TGGGCCCACA(10)|GTGGGCCCACA(11)|TGTGGGCCCACA(12)

For example, reviewing length (8) only:

TGTGGGCC (8) occurs 5 times

GTGGGCCC (8) occurs 8 times

TGGGCCCA (8) occurs 9 times

GGGCCCAC (8) occurs 8 times

GGCCCACA (8) occurs 5 times

Any repeat can be ranked based on its ocurrence within all possible combinations of a given sequence, known as the repeats' iScore rank. This illustrates a potential useful statistical ranking that, subject to biology may describe a repeats inherency to be more or less effective, in increments of the gene sequence. 

Repression of the most active sequences, especially in context of repeats may result in genetic variation. 

First Intron DNA - Site for a Genetic Brain?

DNA Methylation

The first intron of a gene, regardless of tissue or species is conserved as a site of downstream methylation with an inverse relationship to transcription and gene expression. Therefore, it is an informative gene feature regarding the relationship between DNA methylation and gene expression. But, expression in induced pluripotent stem cells (iPSC's) has been a major challenge to the stem cell industry, because by comparison these cells have not yet reached the state of natural pluripotent or embryonic stem cells (ESC's).

In mice two X chromosomes (XC) are active in the epiblasts of blastocysts as well as in pluripotent stem cells. One XC is inactivated triggered by Xist (non coding) RNA transcripts coating it to become silent. Designer transcription factor (dTF) repressors, binding the Xist intron 1 enhancer region caused higher H3K9me3 methylation and led to XC's opening and X-linked gene repression in MEFs. This substantially improved iPSC production and somatic cell nuclear transfer (SCNT) preimplantation embryonic development. This also correlated with much fewer abnormally expressed genes frequently associated with SCNT, even though it did not affect Xist expression. In stark contrast, the dTF activator targeting the same enhancer region drastically decreased both iPSC generation and SCNT efficiencies and induced ESC differentiation. 

A genome-wide, tissue-independent quasi-linear, inverse relationship exists between DNA methylation of the first intron and gene expression. More tissue-specific, differentially methylated regions exist in the first intron than in any other gene feature. These have positive or negative correlation with gene expression, indicative of distinct mechanisms of tissue-specific regulation. CpGs in transcription factor binding motifs are enriched in the first intron and methylation tends to increase with distance from the first exon–first intron boundary, with a concomitant decrease in gene expression.

Since the relationship between sequence, methylation, repression and transcription is determinative in ESC differentiation it may also suggest a broader link to differential translation. Translation is required for miRNA-dependent transcript destabilization that alters levels of coding and noncoding transcripts. But, steady-state abundance and decay rates of cytosolic long non-coding RNA's (lncRNAs) are insensitive to miRNA loss. Instead lncRNAs fused to protein-coding reporter sequences become susceptible to miRNA-mediated decay. 

In this model, first intron DNA sequences that are differentially methylated, bind transcription factors that effect transcription, impact splicing, expressions of coding or non-coding transcripts and transcript destabilizations resulting in differential rates and possible variations in translation. This bottom-up, dynamic view of the classical process may elevate the first intron from 'junk' to a DNA 'brain' because it plays a more extensive role, heading the process toward translation of any gene or switching it off entirely.  

For this reason, among others Codondex uses first intron k-mers relative to the transcripts mRNA as the basis for comparing same gene transcripts in diseased cells or tissue samples. Further, p53 and BRCA1 miRNA key sequences, discovered using Codondex iScore algorithm, when transfected into HeLa cells resulted in significantly reduced proliferation that may result from this accelerated, transfected miRNA dependent decay.

 

Short Sequences of Proximally Disordered DNA

Oxford Nanopore Device Reducing Sequencing Cost

Relationships exist between short sequences of proximal DNA (SSPD) of a gene that when transcribed into RNA present stronger or weaker binding attractions to RNA binding proteins (RBP'S) that settle, edit, splice and resolve messenger RNA (mRNA). Responsive to epigenetic stimuli on Histones and DNA, mRNA are constantly transcribed in different quantity, at different times such that different mRNA strands are transported from the nucleus to cytoplasm where they are translated into and produce any of more than 30,000 different proteins.

Single nucleotide polymorphisms and DNA mutations can alter SSPD combinations in different diseased cells thus altering sequence proximity, ordering that affects transcribed RNA's attraction and optimal binding of RBP's. This may result in modified splicing of RNA, assembly of mRNA and slight or major variations in some or all translated protein derived from that gene. 

The specific effects of these DNA variations, on the multitude of proteins produced are generally unknown. However, it remains important to understand their effects in disease, diagnosis and therapy. Typically these have historically been researched by large scale analysis of RBP on RNA as opposed to the more fundamental, yet underrepresented massive array of diseased variant DNA to mRNA transitions.

Most pharmaceutical research is directed to a molecular interference targeting an aberrant protein to cure widely represented or highly impactful disease conditions of society. Economic assessments generally influence government decisions to support research based on loss of GDP contribution by a specific disease in a  patient cohort. However, in the modern multi-omics era top down research into protein-RNA activity is descending deeper into the cell to include RNA-mRNA and mRNA-DNA customizable therapies that will eventually resolve individually assessed diseases at a price that addresses much larger array of patient needs.  

SNP's and other mutations can vary considerably in cells. These variations can cause instability during division and lead to translated differences that can ultimately drive cancerous cell growth to escape patient immunity. Like a 'whack-a-mole' game, pattern variation and mechanistic persistence eventually beat the player. Without effective immune clearance these cells can replicate into tumors and contribute to microenvironments that support their existence.

Link to video on tumor microenvironment https://youtu.be/Z9H2utcnBic

We thought to analyze DNA and mRNA transcripts from cells in tumors and their microenvironments to see if we could expose the SSPD disordered combinations that may have promoted sub-optimal RBP attractions and led to sustained immune escape. Given the complexity of DNA to mRNA transcription, for any given gene many distortions in gene data sets have to be filtered. To do that we focused on p53, the most mutated gene in cancer. We designed a method to compare sequences arrays of DNA and mRNA Ensembl transcripts, from the consensus of healthy patients to multiple cell samples extracted from different sections of a patients tumor and tumor microenvironment.     

We previously identified and measured different levels of Natural Killer (NK) cell cytotoxicity, produced from cocultures with the extracted samples of each of the multiple sites of a biopsy. We will measure the different p53 transcript SSPD combinations associated with each sample and determine whether disordered SSPD's corelate with NK cytotoxicity from each coculture. We expect to identify whether biopsied tumor cells, ranked by SSPD's predict the cytotoxicity resulting from NK cell cocultures. We will narrow our research to identify the varied expressions of receptor combinations associated with degrees of cytotoxicity. We will test immune efficacy to lyse and destroy tumor cells. Finally we will test for adaptive immune response. 

Our vision is for per-patient, predictable cell co-culture pairings, for innate immune cell education based on ranking DNA-mRNA combinations to lead to multiple effective therapies. The falling cost of sequencing and sophistication of GMP laboratories presently servicing oncologists may support a successful use of this analytical approach to laboratory assisted disease management.

   

 

Non-Coding DNA Key Sequences

DNA Structural Inherency

Wind two strands of elastic, eventually it will knot, ultimately it will double up on itself. Separate the strands. From the point of unwinding, forces will be directed to different regions and the separation will approximately return to the wound state of the band. Do the same with each of 10 different bands or strings of any type, they will all behave in much the same way. For a given section of DNA being transcribed, the effect of separation will be much the same. For a given gene, there will be sequences that can tolerate force to greater or lesser degrees. For different transcripts, of a gene variation at those sequences may be crucial to the integrity of transcription machinery that separates DNA strands to initiate replication to RNA and for the outcome.

Cellular biology is enormously complex in all regards. The physics of molecular interaction, fluid dynamics, and chemistry combine in a system where cause and effect is near impossible to predict. At the most elementary level we hypothesize some non-coding DNA (ncDNA) possess structural inherencies that can be deployed to direct gene proteins and cell function for diagnosis or therapy.

Coding DNA and its regulatory, non-coding gene compliment is transcribed and spliced from a transcribed gene. Transcription to RNA, edited mRNA, spliced non-coding RNA and ultimately mRNA translation to protein can produce wide ranging, variable outcomes that may not be re-captured experimentally. 

A single nucleotide polymorphism (SNP) or SNP combinations within a gene may affect the finely tuned balance that results. Under different environmental conditions this could be material to the protein produced. Additionally other mutations of the gene could add complexity to the environment and/or the  resulting protein translation. 

At this level of cellular biology, genetic DNA stores instruction for protein assemblies to produce new protein required for the fully functional cell. However, DNA's stored mutations can lead to different functional or non-functional versions of protein depending on many different factors. Relationships between ncDNA, including mutations and the transcripts' edited, protein coding mRNA may represent unexplored inherencies that can regulate the gene's mRNA or translated protein.

We built an algorithm to elaborately compare ncDNA sequences of multiple protein coding transcripts of the same gene. For each transcript it steps through every variable length ncDNA sequence (kmer) (specifically intron1), computes a signature for each and indexes it to the constant of the transcripts' mRNA signature. For each step these signatures order the kmers for each of the transcript's. The order is represented in a vector of all the transcripts being compared.  

At millions of successive steps (depending on total intron 1 length's) transcripts mostly retain their vector ordering except, as expected at a kmer length change. Mostly transcript order in the vector does not change, occasionally a few positions change, vary rarely do all positions change. Position changes that cause another, like a domino effect are filtered out. For the rarest positions changes at a step, we look to the root causes in the kmer (sequence). We call this a Key Sequence because it is identified by the significance of changes to transcript positions in the vector compared to the vector at the next step. 

Therefore, Key Sequences cause the most position changes between transcripts being compared by the algorithm. This relative measure is step dependent and Key Sequences are discovered by comparing transcript positions in the vector at the next step location. Logically, this infers a genes structural inherency discovered through ncDNA Key Sequence relationships to mRNA, to other transcripts, error in gene alignments, sequenced reads or the algorithm. 

In assay testing we were able to predict and synthesize non-coding RNA Key Sequences that significantly reduced proliferation of HeLa cells. In our pre-clinical work, based on comparisons to transcripts of the TP53 we will be predicting the efficacy of cell and tissue selections that educate and activate Natural Killer cells.

If Key Sequences are inherent they could open a new frontier for diagnosis and therapy.