Mammalian Genomes Research Articles

Discovery and engineering of ChCas12b for precise genome editing

Many clustered regularly interspaced short palindromic repeat and CRISPR-associated protein 12b (CRISPR-Cas12b) nucleases have been computationally identified, yet their potential for genome editing remains largely unexplored. In this study, we conducted a GFP-activation assay screening 13 Cas12b nucleases for mammalian genome editing, identifying five active candidates. Candidatus hydrogenedentes Cas12b (ChCas12b) was found to recognize a straightforward WTN (W = T or A) proto-spacer adjacent motif (PAM), thereby dramatically expanding the targeting scope. Upon optimization of the single guide RNA (sgRNA) scaffold, ChCas12b exhibited activity comparable to SpCas9 across a panel of nine endogenous loci. Additionally, we identified nine mutations enhancing ChCas12b specificity. More importantly, we demonstrated that both ChCas12b and its high-fidelity variant, ChCas12b-D496A, enabled allele-specific disruption of genes harboring single nucleotide polymorphisms (SNPs). These data position ChCas12b and its high-fidelity counterparts as promising tools for both fundamental research and therapeutic applications.

Human Virus-Like Proteins: Implications for Gene Therapy.

An analysis of mammalian genomes has revealed a significant number of DNA sequences with transposon or viral origin. Some of these elements encode functional proteins, repurposed during evolution to play significant physiological roles in certain tissues. Some human virus-like proteins, such as Peg10 and Arc/Arg3.1, structurally demonstrate significant similarity with Gag retroviral proteins, while others, like syncytins-1 and -2, resemble envelope viral proteins. In recent years, it has become clear that these proteins can be exploited for bioengineering 'humanized' capsid particles aimed at targeted mRNA delivery. Realizing this idea could provide efficient virus-like particles for gene therapy and address the problem of viral vector immunogenicity. This review provides an overview of the most-studied human proteins of viral or transposon origin and highlights their biological functions. Additionally, recent advances in exploiting these proteins for targeted mRNA delivery and prospects for their clinical application are discussed.

Illuminating mitochondrial translation through mouse models.

Mitochondria are hubs of metabolic activity with a major role in ATP conversion by oxidative phosphorylation (OXPHOS). The mammalian mitochondrial genome encodes 11 mRNAs encoding 13 OXPHOS proteins along with 2 rRNAs and 22 tRNAs, that facilitate their translation on mitoribosomes. Maintaining the internal production of core OXPHOS subunits requires modulation of the mitochondrial capacity to match the cellular requirements and correct insertion of particularly hydrophobic proteins into the inner mitochondrial membrane. The mitochondrial translation system is essential for energy production and defects result in severe, phenotypically diverse diseases, including mitochondrial diseases that typically affect postmitotic tissues with high metabolic demands. Understanding the complex mechanisms that underlie the pathologies of diseases involving impaired mitochondrial translation is key to tailoring specific treatments and effectively targeting the affected organs. Disease mutations have provided a fundamental, yet limited, understanding of mitochondrial protein synthesis, since effective modification of the mitochondrial genome has proven challenging. However, advances in next generation sequencing, cryoelectron microscopy, and multi-omic technologies have revealed unexpected and unusual features of the mitochondrial protein synthesis machinery in the last decade. Genome editing tools have generated unique models that have accelerated our mechanistic understanding of mitochondrial translation and its physiological importance. Here we review the most recent mouse models of disease pathogenesis caused by defects in mitochondrial protein synthesis and discuss their value for preclinical research and therapeutic development.

GAGE-seq concurrently profiles multiscale 3D genome organization and gene expression in single cells.

The organization of mammalian genomes features a complex, multiscale three-dimensional (3D) architecture, whose functional significance remains elusive because of limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we introduce genome architecture and gene expression by sequencing (GAGE-seq), a scalable, robust single-cell co-assay measuring 3D genome structure and transcriptome simultaneously within the same cell. Applied to mouse brain cortex and human bone marrow CD34+ cells, GAGE-seq characterized the intricate relationships between 3D genome and gene expression, showing that multiscale 3D genome features inform cell-type-specific gene expression and link regulatory elements to target genes. Integration with spatial transcriptomic data revealed in situ 3D genome variations in mouse cortex. Observations in human hematopoiesis unveiled discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level. GAGE-seq provides a powerful, cost-effective approach for exploring genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

Mammalian and avian species quantification in homogenized foods: real time PCR and digital PCR as tools for label compliance controls

Currently food fraud and authenticity of products composition are topics of great concern; ingredients quantification could allow to identify small amounts of contaminats or voluntary addition of improper components. Many molecular methods are available for species identification in foodstuffs but, for a better application, they should not be affected by the interference of other ingredients. The main purpose of this work was to verify the Real Time PCR and the Digital PCR (dPCR) quantification performances on baby food samples, specifically selected for their high miscibility to limit variability; chicken was selected as target to verify the performance of quantification of methods after having spiked the same quantity in different baby foods. The other aims were: (1) to verify a constant genome copies ratio existence between mammalian and avian species (2) to verify the dPCR performance, set up on housekeeping, to quantify mammalian and avian species in commercial products. Digital PCR showed fewer differences respect to Real Time PCR, at the same 15% w/w chicken spiking level. Despite the constant difference between mammalian and avian genome copies, in samples with the same spiking weight, the confidence intervals increasing towards the extreme values, made impossible to use genome copies ratio as a sort of correction factor between species. Finally, the dPCR system using the myostatin housekeeping gene to determine the chicken content seemed reliable to verify the labelling compliance in meat-based commercial products.

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

The Long Non-Coding RNA Obesity-Related (Obr) Contributes To Lipid Metabolism Through Epigenetic Regulation.

Obesity is a multifactorial disease that is part of today's epidemic and also increases the risk of other metabolic diseases. Long noncoding RNAs (lncRNAs) provide one tier of regulatory mechanisms to maintain metabolic homeostasis. Although lncRNAs are a significant constituent of the mammalian genome, studies aimed at their metabolic significance, including obesity, are only beginning to be addressed. Here, a developmentally regulated lncRNA, termed as obesity related (Obr), whose expression in metabolically relevant tissues such as skeletal muscle, liver, and pancreas is altered in diet-induced obesity, is identified. The Clone 9 cell line and high-fat diet-induced obese Wistar rats are used as a model system to verify the function of Obr. By using stable expression and antisense oligonucleotide-mediated downregulation of the expression of Obr followed by different molecular biology experiments, its role in lipid metabolism is verified. It is shown that Obr associates with the cAMP response element-binding protein (Creb) and activates different transcription factors involved in lipid metabolism. Its association with the Creb histone acetyltransferase complex, which includes the cAMP response element-binding protein (CBP) and p300, positively regulates the transcription of genes involved in lipid metabolism. In addition, Obr is regulated by Pparγ in response to lipid accumulation.

De novo transcriptome assembly and genome annotation of the fat-tailed dunnart (Sminthopsis crassicaudata).

Marsupials exhibit distinctive modes of reproduction and early development that set them apart from their eutherian counterparts and render them invaluable for comparative studies. However, marsupial genomic resources still lag far behind those of eutherian mammals. We present a series of novel genomic resources for the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-like marsupial that, due to its ease of husbandry and ex-utero development, is emerging as a laboratory model. We constructed a highly representative multi-tissue de novo transcriptome assembly of dunnart RNA-seq reads spanning 12 tissues. The transcriptome includes 2,093,982 assembled transcripts and has a mammalian transcriptome BUSCO completeness score of 93.3%, the highest amongst currently published marsupial transcriptomes. This global transcriptome, along with ab initio predictions, supported annotation of the existing dunnart genome, revealing 21,622 protein-coding genes. Altogether, these resources will enable wider use of the dunnart as a model marsupial and deepen our understanding of mammalian genome evolution.

DNA methyltransferase 1 (DNMT1) promotes cyst growth and epigenetic age acceleration in autosomal dominant polycystic kidney disease

Alteration of DNA methylation leads to diverse diseases, and the dynamic changes of DNA methylation (DNAm) on sets of CpG dinucleotides in mammalian genomes are termed “DNAm age” and “epigenetic clocks” that can predict chronological age. However, whether and how dysregulation of DNA methylation promotes cyst progression and epigenetic age acceleration in autosomal dominant polycystic kidney disease (ADPKD) remains elusive. Here, we show that DNA methyltransferase 1 (DNMT1) is upregulated in cystic kidney epithelial cells and tissues and that knockout of Dnmt1 and targeting DNMT1 with hydralazine, a safe demethylating agent, delays cyst growth in Pkd1 mutant kidneys and extends life span of Pkd1 conditional knockout mice. With methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), DNMT1 chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing analysis, we identified two novel DNMT1 targets, PTPRM and PTPN22 (members of the protein tyrosine phosphatase family). PTPRM and PTPN22 function as mediators of DNMT1 and the phosphorylation and activation of PKD associated signaling pathways, including ERK, mTOR and STAT3. With whole genome bisulfide sequencing in kidneys of patients with ADPKD versus normal individuals, we found that the methylation of epigenetic clock associated genes was dysregulated, supporting that epigenetic age is accelerated in the kidneys of patients with ADPKD. Furthermore, five epigenetic clock associated genes, including Hsd17b14, Itpkb, Mbnl1, Rassf5 and Plk2 were identified. Thus, the diverse biological roles of these five genes suggest that their methylation status may not only predict epigenetic age acceleration but also contribute to disease progression in ADPKD.

Uncovering the role of GPR39 in the kidney

G protein-coupled receptors (GPCRs) constitute the largest class of proteins in the mammalian genome. Upon activation by a specific ligand, these receptors initiate downstream cellular responses that regulate various physiological processes. One such receptor under investigation in our lab is the ghrelin family receptor GPR39. Unlike its relatives, GPR39 does not respond to peptide hormones or neuropeptides. Initial studies suggested zinc as the endogenous ligand, but recent research indicates that zinc functions as an allosteric potentiator for another unidentified endogenous ligand. To date, synthetic agonists for GPR39 have revealed functions for GPR39 in the heart, bone, skin, pancreas, and gastrointestinal tract. However, GPR39’s role in renal physiology is currently unknown, despite relatively high kidney expression. To address this gap in knowledge, we first worked to localize GPR39 within the kidney. As a reliable antibody for GPR39 is not available, we utilized a combination of RNAScope (Gpr39) and immunofluorescence (AQP2). We find that GPR39 is expressed in the renal collecting duct, with the highest expression in AQP2-positive cells (principal cells) in the inner medullary collecting duct, and lesser expression in the cortical collecting duct. To determine if GPR39 is expressed apically or basolaterally, we then cloned GPR39 with a C-terminal EGFP tag and observed basolateral targeting of GPR39 in polarized MDCK cells grown on filters. In order to query the function of GPR39 in principal cells, we used murine principal kidney cortical collecting duct cells (mpkCCD). As others have shown, we find that mpkCCD express and traffc AQP2 to the apical plasma membrane only in the presence of the vasopressin analog dDAVP. Furthermore, we observe that treatment of mpkCCD with the GPR39-specific agonist cpd1324 induces internalization of AQP2 into cytoplasmic vesicles, even in the continued presence of dDAVP. Under vehicle conditions (dDAVP + vehicle), 80.9±7.7% (mean±SD) of AQP2 stain colocalized with an apical membrane marker (WGA staining). In contrast, when mpkCCD cells were co-treated ddAVP+cpd1324, only 58.3±6.02% of AQP2 stain colocalized with the apical membrane (p < 0.001, t-test). These results indicate that GPR39 activation may act to antagonize vasopressin-induced AQP2 traffcking in mpkCCD cells. NHLBIT32HL007534 (MK) and American Heart Association Established Investigator Award (JLP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Noncoding RNAs: Emerging regulators of behavioral complexity.

The mammalian genome encodes thousands of non-coding RNAs (ncRNAs), ranging in size from about 20 nucleotides (microRNAs or miRNAs) to kilobases (long non-coding RNAs or lncRNAs). ncRNAs contribute to a layer of gene regulation that could explain the evolution of massive phenotypic complexity even as the number of protein-coding genes remains unaltered. We propose that low conservation, poor expression, and highly restricted spatiotemporal expression patterns-conventionally considered ncRNAs may affect behavior through direct, rapid, and often sustained regulation of gene expression at the transcriptional, post-transcriptional, or translational levels. Besides these direct roles, their effect during neurodevelopment may manifest as behavioral changes later in the organism's life, especially when exposed to environmental cues like stress and seasonal changes. The lncRNAs affect behavior through diverse mechanisms like sponging of miRNAs, recruitment of chromatin modifiers, and regulation of alternative splicing. We highlight the need for synthesis between rigorously designed behavioral paradigms in model organisms and the wide diversity of behaviors documented by ethologists through field studies on organisms exquisitely adapted to their environmental niche. Comparative genomics and the latest advancements in transcriptomics provide an unprecedented scope for merging field and lab studies on model and non-model organisms to shed light on the role of ncRNAs in driving the behavioral responses of individuals and groups. We touch upon the technical challenges and contentious issues that must be resolved to fully understand the role of ncRNAs in regulating complex behavioral traits. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

Genomic Frequencies of Dynamic DNA Sequences and Mammalian Lifespan.

Dynamic DNA sequences (i.e. sequences capable of forming hairpins, G-quadruplexes, i-motifs, and triple helices) can cause replication stress and associated mutations. One example of such a sequence occurs in the RACK7 gene in human DNA. Since this sequence forms i-motif structures at neutral pH that cause replication stress and result in spontaneous deletions in prostate cancer cells, our initial aim was to determine its potential utility as a biomarker of prostate cancer. We cloned and sequenced the region in RACK7 where i-motif deletions often occur in DNA obtained from eight individuals. Expressed prostatic secretions were obtained from three individuals with a positive biopsy for prostate cancer and two with individuals with a negative biopsy for prostate cancer. Peripheral blood specimens were obtained from two control healthy bone marrow donors and a marrow specimen was obtained from a third healthy marrow donor. Follow-up computer searches of the genomes of 74 mammalian species available at the NCBI ftp site or frequencies of 6 dynamic sequences known to produce mutations or replication stress using a program written in Mathematica were subsequently performed. Deletions were found in RACK7 in specimens from both older normal adults, as well as specimens from older patients with cancer, but not in the youngest normal adult. The deletions appeared to show a weak trend to increasing frequency with patient age. This suggested that endogenous mutations associated with dynamic sequences might accumulate during aging and might serve as biomarkers of biological age rather than direct biomarkers of cancer. To test that hypothesis, we asked whether or not the genomic frequencies of several dynamic sequences known to produce replication stress or mutations in human DNA were inversely correlated with maximum lifespan in mammals. Our results confirm this correlation for six dynamic sequences in 74 mammalian genomes studied, thereby suggesting that spontaneously induced replication stress and mutations linked to dynamic sequence frequency may limit lifespan by limiting genome stability.

A Bidirectional Non-Coding RNA Promoter Mediates Long-Range Gene Expression Regulation.

Recent evidence suggests that human gene promoters display gene expression regulatory mechanisms beyond the typical single gene local transcription modulation. In mammalian genomes, genes with an associated bidirectional promoter are abundant; bidirectional promoter architecture serves as a regulatory hub for a gene pair expression. However, it has been suggested that its contribution to transcriptional regulation might exceed local transcription initiation modulation. Despite their abundance, the functional consequences of bidirectional promoter architecture remain largely unexplored. This work studies the long-range gene expression regulatory role of a long non-coding RNA gene promoter using chromosome conformation capture methods. We found that this particular bidirectional promoter contributes to distal gene expression regulation in a target-specific manner by establishing promoter-promoter interactions. In particular, we validated that the promoter-promoter interactions of this regulatory element with the promoter of distal gene BBX contribute to modulating the transcription rate of this gene; removing the bidirectional promoter from its genomic context leads to a rearrangement of BBX promoter-enhancer interactions and to increased gene expression. Moreover, long-range regulatory functionality is not directly dependent on its associated non-coding gene pair expression levels.

Common occurrence of hotspots of single strand DNA breaks at transcriptional start sites

BackgroundWe recently developed two high-resolution methods for genome-wide mapping of two prominent types of DNA damage, single-strand DNA breaks (SSBs) and abasic (AP) sites and found highly complex and non-random patterns of these lesions in mammalian genomes. One salient feature of SSB and AP sites was the existence of single-nucleotide hotspots for both lesions.ResultsIn this work, we show that SSB hotspots are enriched in the immediate vicinity of transcriptional start sites (TSSs) in multiple normal mammalian tissues, however the magnitude of enrichment varies significantly with tissue type and appears to be limited to a subset of genes. SSB hotspots around TSSs are enriched on the template strand and associate with higher expression of the corresponding genes. Interestingly, SSB hotspots appear to be at least in part generated by the base-excision repair (BER) pathway from the AP sites.ConclusionsOur results highlight complex relationship between DNA damage and regulation of gene expression and suggest an exciting possibility that SSBs at TSSs might function as sensors of DNA damage to activate genes important for DNA damage response.

Identification and characterization of endogenous retroviruses upon SARS-CoV-2 infection.

Endogenous retroviruses (ERVs) derived from the long terminal repeat (LTR) family of transposons constitute a significant portion of the mammalian genome, with origins tracing back to ancient viral infections. Despite comprising approximately 8% of the human genome, the specific role of ERVs in the pathogenesis of COVID-19 remains unclear. In this study, we conducted a genome-wide identification of ERVs in human peripheral blood mononuclear cells (hPBMCs) and primary lung epithelial cells from monkeys and mice, both infected and uninfected with SARS-CoV-2. We identified 405, 283, and 206 significantly up-regulated transposable elements (TEs) in hPBMCs, monkeys, and mice, respectively. This included 254, 119, 68, and 28 ERVs found in hPBMCs from severe and mild COVID-19 patients, monkeys, and transgenic mice expressing the human ACE2 receptor (hACE2) and infected with SARS-CoV-2. Furthermore, analysis using the Genomic Regions Enrichment of Annotations Tool (GREAT) revealed certain parental genomic sequences of these up-regulated ERVs in COVID-19 patients may be involved in various biological processes, including histone modification and viral replication. Of particular interest, we identified 210 ERVs specifically up-regulated in the severe COVID-19 group. The genes associated with these differentially expressed ERVs were enriched in processes such as immune response activation and histone modification. HERV1_I-int: ERV1:LTR and LTR7Y: ERV1:LTR were highlighted as potential biomarkers for evaluating the severity of COVID-19. Additionally, validation of our findings using RT-qPCR in Bone Marrow-Derived Macrophages (BMDMs) from mice infected by HSV-1 and VSV provided further support to our results. This study offers insights into the expression patterns and potential roles of ERVs following viral infection, providing a valuable resource for future studies on ERVs and their interaction with SARS-CoV-2.

Coding genomes with gapped pattern graph convolutional network.

Genome sequencing technologies reveal a huge amount of genomic sequences. Neural network-based methods can be prime candidates for retrieving insights from these sequences because of their applicability to large and diverse datasets. However, the highly variable lengths of genome sequences severely impair the presentation of sequences as input to the neural network. Genetic variations further complicate tasks that involve sequence comparison or alignment. Inspired by the theory and applications of "spaced seeds," we propose a graph representation of genome sequences called "gapped pattern graph." These graphs can be transformed through a Graph Convolutional Network to form lower-dimensional embeddings for downstream tasks. On the basis of the gapped pattern graphs, we implemented a neural network model and demonstrated its performance on diverse tasks involving microbe and mammalian genome data. Our method consistently outperformed all the other state-of-the-art methods across various metrics on all tasks, especially for the sequences with limited homology to the training data. In addition, our model was able to identify distinct gapped pattern signatures from the sequences. The framework is available at https://github.com/deepomicslab/GCNFrame.

Multifaceted roles of RNA editing enzyme ADAR1 in innate immunity.

Innate immunity must be tightly regulated to enable sensitive pathogen detection while averting autoimmunity triggered by pathogen-like host molecules. A hallmark of viral infection, double-stranded RNAs (dsRNAs) are also abundantly encoded in mammalian genomes, necessitating surveillance mechanisms to distinguish "self" from "nonself." ADAR1, an RNA editing enzyme, has emerged as an essential safeguard against dsRNA-induced autoimmunity. By converting adenosines to inosines (A-to-I) in long dsRNAs, ADAR1 covalently marks endogenous dsRNAs, thereby blocking the activation of the cytoplasmic dsRNA sensor MDA5. Moreover, beyond its editing function, ADAR1 binding to dsRNA impedes the activation of innate immune sensors PKR and ZBP1. Recent landmark studies underscore the utility of silencing ADAR1 for cancer immunotherapy, by exploiting the ADAR1-dependence developed by certain tumors to unleash an antitumor immune response. In this perspective, we summarize the genetic and mechanistic evidence for ADAR1's multipronged role in suppressing dsRNA-mediated autoimmunity and explore the evolving roles of ADAR1 as an immuno-oncology target.

SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization.

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a-Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.

Abstract 1244: Exploiting the pathogenesis of endogenous retrovirus to tackle squamous cancers

Abstract Retrotransposons are the evolution relic of ancestrally invaded retroviruses interspersed through our genome. Mostly domesticated as host regulome, retrotransposons shape lineage gene expressions or donate coding functions. A few rare evolutionarily young species remain virally active, leading to germline mutations and modulating immune functions. Aberrant retrotransposon activities have been widely observed in adult diseases, although the extent to which they ensue active viral form and function under those contexts is unclear. Their molecular trigger in adult pathophysiology is poorly defined. To address these questions, we use murine skin as our model, given its well-characterized, abundant and highly accessible adult stem cells, not only mediating postnatal remodeling and homeostatic regeneration, but also responding to stress by driving tissue repair and adaptations. By analyzing a genetic model lacking a known retrotransposon suppressor, histone methyltransferase Setdb1, which we found to be essential in the adult skin, we saw hair loss and hair follicle stem cell exhaustion phenotype, accompanied by a robust and selective surge of endogenous retroviruses (ERVs). When combined with squamous cell carcinoma drivers, Setdb1 loss significantly blocked tumor progression in vivo. Interestingly, compared to relatively mild or no induction of commonly observed evolutionarily young ERVs, the most induced species in our model is a class I ERV, murine leukemia virus (MuLV). We detected abundant retroviral peptides originated from its full-length copies through mass spectrometry and viral-like particles through transmission electron microscopy that are immunoreactive to a monoclonal MuLV env antibody. Similar viral-like particles were broadly evident across several epithelial tissues beyond skin, implicating its conserved function across squamous cancers. Epithelial originated ERVs elicit tissue wide inflammatory response, and can be ameliorated by pharmacological or genetic inhibition of retroviral activity. Physiologically, viral-coding ERVs arise as the epigenetic machinery declines in response to age-associated stress, and plummet when host safeguard prevails in UV induced skin hyperplasia. As an evolutionary conundrum, viral-coding ERVs pose a threat to host fitness and yet, they’ve resisted extinction persisting in the mammalian genome. Our findings suggest viral-coding ERVs wrestle with the host surveillance programs to regulate adult tissue physiology and pathology, thus providing a key target in eliciting innate immunity and tackling squamous cancers. Citation Format: Ying Lyu, Yejing Ge. Exploiting the pathogenesis of endogenous retrovirus to tackle squamous cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1244.