Mobile DNA

Multicellularity has evolved multiple times across the eukaryotic tree of life, including among protist lineages. Because transposable elements (TEs) strongly influence genome architecture and gene regulation, understanding their potential impact on genome structure and their relationship with gene expression may provide insight into the evolution of multicellularity. Here, we generated a new genome assembly for the facultatively multicellular amoeba Acrasis kona and performed comparative analyses of TE composition, TE diversity, and TE-density organization across diverse protist lineages. Comparative analyses included unicellular and multicellular representatives from across the tree of eukaryotes, (Heterolobosea, Filasterea, Cristidiscoidea, and Chlorophyceae), including Naegleria spp., Tetramitus jugosus, Capsaspora owczarzaki, Pigoraptor spp., Fonticula alba, Parvularia atlantis, Volvox carteri, and Chlamydomonas reinhardtii. To examine relationships between TEs and gene regulation, we integrated transcriptomic datasets from A. kona, Capsaspora owczarzaki, and Volvox carteri with genome-wide TE-density analyses of differentially expressed genes. TE abundance and composition varied substantially among lineages, with species that exhibit more complex developmental or cellular organization generally containing higher TE proportions than closely related unicellular taxa. Patterns of TE-density organization near up-regulated, down-regulated, and non-differentially expressed genes also differed among systems, ranging from strong TE depletion in A. kona to weaker or cell-type-specific patterns in Capsaspora and Volvox. Together, these findings suggest that transposable elements are associated with multicellularity across diverse protist lineages, although the specific roles they play appear to be complex, lineage-specific, and not yet fully understood.

Transposable elements (TE) jump from one genomic locus to another. Since increase in their copy number is a metabolic burden for the host, TE are considered as genomic parasites. Although host-TE co-existence is regarded as an evolutionary arms race, the hypothesis is not extensively tested especially using evolutionary genomics. We provide a hypothesis testing framework to understand the distribution of TE in genic regions of the host genome, variation in the regulation of TE by host, and effect of these two factors on host-TE co-evolutionary dynamics. We test our hypothesis by understanding the distributions of potentially active TEs in the genome of 78 teleost fishes, representing major families and orders within the clade. Our analysis reveals coevolutionary arms race predicted by the Red Queen dynamics.

The Extensive de-novo TE Annotator (EDTA) automates transposable element annotation in plant genomes but lacks direct LINE/SINE detection, limiting its applicability to animal genomes. We present EDTA v2, which integrates LINE and SINE detection, completely rewrites TIR-Learner for deployability and scalability, and accelerates structural detectors by up to two orders of magnitude. Tested in 30 animal genomes from the Vertebrate Genomes Project Phase I, EDTA v2 bridges the non-LTR detection gap that has prevented automated TE annotation in animals.

Transcribed repeats represent a major challenge in the de novo assembly of transcriptomes from short RNA-seq reads. Young transposable elements (TEs) and other inexact repeats create dense and ambiguous regions in the assembly graph, preventing the correct assembly of transcripts. In this paper, we introduce a fully de novo method based on the discovery of dense regions in the compacted De Bruijn graph (DBG) to identify such repeats directly from short reads RNA-seq data, without requiring a reference genome or repeat database. Our approach defines the extended t-cores, subgraphs of the DBG that capture the complex topology induced by highly expressed inexact repeats appearing in RNA-seq reads. Independently of its interest for transcriptome assembly, the proposed method appears to be effective for the de novo identification of repeats in transcriptomes. After classifying cores using sequence-based motifs to distinguish simple repeats from potential TEs, we demonstrate its potential for the de novo discovery of transposable elements. We validate the approach on a Mus musculus dataset using expressed TE consensus sequences, showing that extended t-cores correspond to known expressed TE families. We also illustrate its de novo discovery potential on a non-model species, Canis lupus familiaris, where the method was also able to recover known transposable elements.

Drosophila RNA viruses often persist in wild and lab populations, yet their tissue and cellular tropism is poorly understood. In the Fly Cell Atlas (a comprehensive Drosophila single-nucleus transcriptome) data, we detected four RNA virus infections: Nora virus, Drosophila A virus, Drosophila C virus, and Newfield virus. Nora and Drosophila A virus were the most abundant and widespread across tissues and cell types, while Drosophila C virus and Newfield virus RNA transcript were only found in oenocyte and fat body tissues. We found transcriptional changes associated with viral infection in canonical viral immunity genes (e.g. Vago, vir-1). Additionally, we observed that during persistent viral infections, transposable element (TE) transcripts were upregulated in somatic cells. TEs are traditionally associated with the germline, but recent studies and our data suggest they are also expressed in somatic cells. Using the Fly Cell Atlas data, we found that distinct somatic cell types express specific TE subtypes, indicating regulated and cell-type specific TE activity often overlooked in transcriptomic studies. We present Fly Viral Atlas (https://flyviralatlas.shinyapps.io/home/), a single-nucleus level atlas of RNA viruses and TE expressions in Drosophila, providing new insights into viral tropism and TE dynamics across cell types and tissues.

Endogenous retroviruses (ERVs) constitute a significant fraction of vertebrate genomes and serve as genomic records of past retroviral infections, while also influencing host biology through regulatory co-option and, in some cases, ongoing retrotransposition. Despite extensive examination of ERVs in haplorrhine primates, equivalent analyses in strepsirrhines remain absent, leaving a substantial gap in our understanding of ERV diversity and evolutionary dynamics across the primate order. Here, we present the first comprehensive characterisation of ERVs in a strepsirrhine primate, identifying 15 Loris Endogenous Retrovirus (LERV) families encompassing 34 subfamilies and over 6,000 insertions in the Nycticebus coucang reference genome. Phylogenetic analyses resolved LERVs into three retroviral genera: betaretroviruses (LERV1-4), type-D betaretroviruses (LERV5-9), and gammaretroviruses (LERV10-15). LERV2a shows multiple hallmarks of recent or potentially ongoing retrotransposition, including a median insertion age of zero, a high proportion of identical LTR pairs, dN/dS ratios comparable to the active retrovirus HTLV, and insertional polymorphism between two conspecific genomes. Comparative genomic screening across Lorisidae revealed that LERV subfamily distribution broadly mirrors estimated insertion ages, with progressively fewer subfamilies detected in more distantly related species. These findings establish a detailed foundation for understanding retroviral evolution in Strepsirrhini and reveal that ongoing retroviral activity is not restricted to haplorrhine primates.

Highly repetitive sequences pose problems for genome assembly and analysis. While advances in long-read sequencing technologies have helped reveal the organization of repetitive genomic sequences at unprecedented resolution, their functional characterization remains difficult because molecular assays that probe protein-DNA interactions and characterize expression often rely on short read sequencing. The repetitive nature of these regions poses major challenges for methods relying on sequence mapping, which is exacerbated for short reads. Repetitive genome regions often have low mappability, leading to substantial information loss during downstream filtering. To address this challenge, we developed a bioinformatic tool--kmerRRR--that leverages k-mer frequency analyses to enhance the mappability of repetitive regions. KmerRRR compares k-mer frequencies within user-defined loci to their frequencies across the genome to identify repetitive sequences that are overrepresented locally relative to the global background. This approach quantifies locus uniqueness, allowing users to distinguish sequences that are globally repetitive from those that are repetitive, but restricted to specific genomic loci. We demonstrated the utility of this method by reanalyzing chromatin profiling data from human, Drosophila, and Arabidopsis centromeres and small RNA sequencing data. Our results show that incorporating local k-mer ratio information enhances read retention and signal interpretation within repetitive regions, thereby recovering biologically meaningful information that is typically lost in conventional analyses. The tool is freely available under MIT license in github: (https://github.com/LarracuenteLab/kmerRRR).

The continuous transfer of organellar DNA into the nuclear genome during eukaryotic evolution has resulted in the widespread occurrence of nuclear plastid DNA insertions (NUPTs) and nuclear mitochondrial DNA insertions (NUMTs). However, their functional significance in nuclear gene expression and genome evolution remains largely unresolved. In this study, we employed Oxford Nanopore Direct RNA Sequencing (DRS) to investigate the transcription of NUPTs and NUMTs in the Populus nuclear genome and compared their transcriptional characteristics with their genome-wide insertion patterns. Our analyses revealed that the majority of transcribed NUPTs and NUMTs are enriched within introns and are co-transcribed with their host or adjacent genes in polycistronic-like transcriptional units. In addition, NUPTs and NUMTs frequently generate intronless transcripts, features reminiscent of their prokaryotic ancestry. We further identified a putatively functional NUPT-derived psbH gene that is unique to P. trichocarpa, providing new insights into the evolution of nuclear-encoded organelle-targeted genes. In addition, we identified transcribed NUPT and NUMT insertion polymorphisms among alleles, suggesting that organellar DNA insertions contribute to allelic variation and may participate in environmental adaptation. Collectively, our findings reveal previously unrecognized roles of NUPT and NUMT transcription in gene regulation, allelic variation, genome evolution, and the emergence of novel genes.

Transposable elements (TEs) are powerful drivers of genome evolution, yet how they persist under selection and become incorporated into host regulatory networks remains poorly understood. In the Drosophila male germline, TEs are highly expressed during the spermatocyte stage, coinciding with activation of giant fertility genes on the Y chromosome. These genes contain megabase-scale introns enriched for repetitive DNA, and three of these genes form prominent nuclear structures known as Y-loops, providing a unique system to investigate gene regulation. Here, we show that multiple TEs expressed in spermatocytes are transcribed from the introns of Y-linked fertility genes. RNA fluorescence in situ hybridization (FISH) targeting several TEs, including accord2, Juan, and HMS Beagle, illuminates distinct nuclear regions corresponding to kl-2, kl-3, and kl-5, respectively. Genetic perturbation of these fertility gene loci or disruption of RNA-processing factors eliminates these TE transcripts, demonstrating that these TE sequences are embedded within Y chromosome-associated nascent transcripts rather than being independently transcribed. The identity and expression patterns of Y-loop-associated TEs vary extensively among closely related Drosophila species, consistent with the previously documented rapid evolution of Y-linked loci and suggesting that TEs may contribute to the genetic diversification of these giant fertility genes. We propose that continual turnover of repetitive elements within Y-linked introns provides a mechanism by which rapidly evolving repetitive DNA influences germline gene regulation, male fertility, and speciation.

BackgroundHeterochromatic long arm of mouse Y chromosome harbors the multicopy species-specific sequences Ssty, Sly, Asty and Orly that are transcribed in testis and have known functions in male fertility. Of these Ssty and Sly encode proteins - yet all the transcripts are not translated. To investigate the roles of these Y-heterochromatic transcripts further, we analyzed them. MethodsMice with 2/3rd deletion of the Y-chromosome (XYRIIIqdel) and its wild type (XYRIII) were used in this study. Bioinformatic approaches, small RNA northern blots, Electrophoretic Mobility Shift Assays, Luciferase reporter assays, dPCR analysis, RT-qPCR assays and western blotting techniques were used to identify piRNAs that regulate autosomal genes. ResultsWe demonstrate that the multicopy gene families from mouse Y-long arm generate piRNAs predominantly in testis. We observed sequences homologous to these piRNAs in the UTRs of a few autosomal genes, which are differentially expressed in the sperms of XYRIIIqdel mice. Furthermore, the Endogenous Retrovirus Element (ERV) LTR, found in the Orly1 transcript identified piRNAs in the database, showed homology to UTRs and associated genomic regions of a few autosomal genes. Orly1 showed a reduction in genomic copy number by digital PCR in XYRIIIqdel mice. One of the four autosomal genes containing the ERV segment in their UTRs, showed a differential testicular protein expression in the mutant mice. ConclusionsThus, we further elucidate that different classes of repeats from Y-chromosome regulate autosomal gene expression via piRNAs. Besides, this study also identified novel roles for a Y-derived ERV in autosomal gene regulation in testis.

Human-specific long interspersed nuclear element-1 (L1HS) is an active and autonomous retrotransposon in the human genome. Changes in its transcription and transposition are known to affect cellular processes involved in development and aging, and diseases such as neurological disorders and cancer. To better understand natural variability in epigenetic patterns that affect L1HS regulation, we developed a targeted long-read method to simultaneously profile individual haplotypes for DNA methylation and chromatin accessibility across L1HS loci in a healthy human cell line trio. We show that the intronic L1HS in the ZNF638 gene consistently displays high chromatin accessibility and DNA hypomethylation with bidirectional transcription. Our approach also reveals additional intronic and intergenic L1HS copies with allele-specific chromatin accessibility and methylation, and instances of reduced promoter DNA methylation that does not correspond with increased chromatin accessibility. We also identify potential cases of non-Mendelian inheritance of DNA methylation patterns over a subset of L1HS promoters. Our methods high coverage over L1HS loci enables detection and profiling of loci that are missed even by long-read-based assemblies and enables more accurate inheritance tracing of L1HS insertions. Overall, our results offer new insights into the locus-specific regulation of both reference and non-reference L1HS within the human genome.

BackgroundStructural variants (SVs) are large insertions or deletions of DNA sequences. While less numerous than single nucleotide polymorphisms, SVs often account for a greater proportion of nucleotide differences between genomes. Their size and frequent association with repetitive sequences has historically hindered their detection, which has limited the ability to associate this variation with molecular and phenotypic trait variation. While some SVs have been linked to observable traits, it remains unclear whether such effects are rare or broadly distributed across the genome. ResultsTo test for genome-wide relationships between SVs and gene expression, we analyzed genome assemblies and transcriptomic data from 10 tissues across 26 diverse maize inbred lines. We identified SVs amongst these lines and examined variants located within the 1kb promoter region upstream of genes. Thousands of genes showed expression differences associated with promoter SVs, often in a tissue-specific manner. One common feature of these SVs was the presence of transposable element sequences. LTR retrotransposons were enriched amongst promoter SVs associated with differential expression and often reduced expression of the nearby gene. Despite widespread expression changes, we found no enrichment for specific biological functions or pathways among affected genes. ConclusionsOur findings indicate that extant TE-mediated promoter SVs play a significant role in shaping gene expression patterns across the maize genome. However, their phenotypic effects appear limited or context-dependent, suggesting that many variants may have minimal impact outside specific developmental stages or environmental conditions.

BackgroundThe black soldier fly (Hermetia illucens) is an emerging model for bioconversion and industrial rearing. Its genome is highly repetitive, yet the contribution of transposable elements (TEs) to population divergence and demographic processes. The sampled populations represent a gradient of demographic histories, including wild and near-wild North American populations, and domesticated European strains with shared industrial origins. Difference in TE composition may influence genome structure, regulatory variation, and evolutionary responses to captive environments. ResultsA comparative analysis of the repetitive landscape was done for four H. illucens genomes, one of which is a wild-caught specimen. Total repeat content was high across all assemblies (67.6% to 70.8%) and dominated by LINE elements. Class-level TE diversity was nearly identical among genomes, but multiple DNA transposon families showed distinct lineage-specific differences. Large families including Maverick and Academ were generally depleted relative to the wild sample. Divergence profiles revealed patterns consistent with recent turnover in several families. Family level turnover, rather than class level change, accounted for the most difference among the genomes. TE-associated structural variants (TESVs) were also not uniformly distributed. Most chromosomes showed mid-chromosome enrichment, and a pronounced TESV peak on chromosome 5 overlapped a histone rich region containing many unclassified repeats. Use of a repeat library derived from multiple genomes increased the number of detected TESVs and improved classification within complex regions, demonstrating that multi-genome libraries enhance annotation accuracy compared to single reference-based models. ConclusionsMultiple DNA transposon families show evidence of recent or lineage-specific amplification in H. illucens, suggesting that TE amplification contributes to genome variation during demography-associated TE turnover. The multi-genome-based library improved TE detection and classification, providing a proof of concept that even a small lineage-inclusive repeat library enhances annotation accuracy and capture TE diversity missed by single-reference approaches. Together, these findings demonstrate that TE family turnover plays a significant role in shaping genome architecture and adaptation in this species.

Nuclear organization is dynamic and originates from the fundamental subunit of chromatin, the nucleosome. Post-translational modification of nucleosomal histones, particularly within intrinsically disordered histone tail regions, provides a dynamic regulatory mechanism of accessibility for chromatin-templated processes. While the epigenomic impacts of lysine acetylation and serine phosphorylation in the histone H3 tail are well-known, how these charge-altering post-translational modifications (PTMs) alter nucleosomal tail conformational dynamics remains incompletely characterized. Given that the functional implications of these PTMs are, at least in part, a consequence of modified nucleosome conformation, systematically cataloging the impact of histone PTMs on nucleosome dynamics provides crucial insight into both baseline cellular activity and epigenetic dysregulation that occurs in disease. Previously, our lab demonstrated that arginine citrullination mimetics lead to regional increases in H3 tail dynamics within nucleosome core particles. Here, we performed nuclear magnetic resonance spin relaxation experiments to investigate the effects of lysine acetylation and serine phosphorylation on H3 tail picosecond-nanosecond (ps-ns) dynamics. Using lysine-to-glutamine and serine-to-glutamate mutations as acetyllysine and phosphoserine mimetics, respectively, we found that these PTMs increase ps-ns conformational dynamics regionally around the PTM site, with a position-dependent effect. Additionally, we show that the type of PTM influences the extent of these increases: in general, the effect of mimetics trends in the order of phosphorylation [&le;] acetylation < citrullination, suggesting a tunable method for altering histone tail dynamics. Taken together, these results illustrate the role of nucleosome conformational dynamics in conveying the effects of epigenomic PTMs, elucidating a mechanism of the histone language.

Abstract The Duroc-derived reference genome Sscrofa11.1 has provided a critical foundation for pig genomics, providing a high-quality reference genome for accurate variant detection and comparative genomics but does not capture breed-specific variation. Here, we present a near-complete, gap-free genome assembly for the Landrace pig (Landrace_v1, GCA_963921485.1), spanning all 20 chromosomes and totaling 2.6 Gb, including 176 Mb of sequence absent from Sscrofa11.1. Comparative analyses with recently published high-quality pig genomes reveal a conserved centromere organization across breeds, accompanied by substantial variation in repeat composition and length, and identify a pig specific pattern of telomere variant repeats across eight pig breeds. The improved resolution of repetitive regions in Landrace_v1 enables more complete reconstruction of complex gene families, including olfactory receptors, and uncovers structural variation at the KIT proto-oncogene receptor tyrosine kinase locus not represented in the Duroc reference. Together, these findings highlight the limitations of single-reference genomes and demonstrate the value of breed-specific assemblies for capturing genomic diversity and improving downstream analyses.

Transposable element-derived long intergenic noncoding RNAs are increasingly recognized as context-dependent regulators of gene expression, but the functional consequences of their ectopic activation in somatic cells remain poorly understood. We previously showed that U7 snRNA represses a subset of LTR12-associated lincRNAs, including lnc-ARRDC4-1 and lnc-ADCYAP1-2, two testis-enriched lincRNAs with minimal expression in somatic cells. Here, we examined the consequences of their increased expression in somatic cells. We showed that overexpression of either lincRNA led to overlapping transcriptomic and proteomic changes, impaired migration, altered adhesion and proliferation, and a [~]50% reduction in protein synthesis. Furthermore, we identified lnc-ARRDC4-1 as an upstream regulator of lnc-ADCYAP1-2 transcription. Downstream of this event, lnc-ADCYAP1-2 interacts with the RNA helicase DHX36, a regulator of G-quadruplex-containing mRNAs. lnc-ADCYAP1-2 activation reduces DHX36 protein levels which is accompanied by decreased protein output from a subset of DHX36 mRNA targets. At the cellular level, these effects correlate with altered cell proliferation, migration, adhesion, and global translation. Our results suggest a lnc-ARRDC4-1: lnc-ADCYAP1-2 : DHX36 regulatory cascade linking de-repression of LTR12-containing lincRNAs to reduced protein synthesis and altered cellular processes in somatic cells.

Homing endonucleases (HEs) are selfish genetic elements that drive the mobilization of their own coding sequences, often in concert with surrounding introns. Homing endonuclease genes (HEGs) usually display life cycles in which they accumulate inactivating mutations after invading a host genomic target site, leading to eventual removal from the genome. We identified several hundred novel HEGs and determined the distribution of their proteins behaviors and activities. Approximately 10% are expressed as properly folded functional proteins that cleave predictable DNA target sites. Another [~]20% display significant expression but little to no cleavage activity; the remainder display severely reduced expression. Despite the presence of debilitating mutations throughout most HEGs, ancestral reconstructions yielded endonucleases with improved expression and stability. One such reconstruction, at a hypothetical node preceding highly diverged HEs that cleave unique target sites, binds (but does not cleave) their individual targets. It instead cleaves a DNA sequence that represents a hybrid of those modern-day DNA targets, while displaying a specificity profile that resembled those of previously characterized HEs. Its DNA-bound crystal structure adds detail to our understanding of how homing endonuclease DNA contacting surfaces and residues shift and rearrange during evolution, ultimately leading to their action at new target sites.

Background: Accurate identification of differentially methylated regions (DMRs) is fundamental to epigenomic research but remains challenging due to biological variability among replicates, heterogeneous effect sizes, and the tendency of adjacent cytosines to share similar methylation states. Many existing methods aggregate methylation measurements before statistical testing or do not explicitly account for replicate-level variability, contributing to elevated false-positive rates. Results: We developed glmmDMR, a DMR detection framework that combines generalized linear mixed models with a seed-based strategy for reconstructing DMRs from locally high-confidence signals while explicitly modeling replicate-level variability. Using simulated datasets with known ground-truth DMRs, we demonstrate that false-positive detections are more strongly associated with methylation variance among biological replicates than with the magnitude of methylation differences between groups. glmmDMR achieved higher precision than existing approaches while maintaining competitive recall, particularly for subtle methylation differences. Site-level modeling with beta regression provided the strongest overall performance, and seed-based region construction reduced artificial DMR fragmentation, improving recovery of true DMR boundaries and producing more contiguous, biologically interpretable DMRs. Applied to Arabidopsis thaliana ddm1 methylomes and a rice DEMETER-LIKE DNA demethylase mutant (Osdml3a-1), glmmDMR identified biologically meaningful DMRs, revealing widespread TE-associated hypomethylation and subtle TE-family-specific hypermethylation. Conclusions: Replicate-level methylation variance is an important determinant of DMR detection performance, and explicitly modeling this variance improves discrimination of biologically meaningful methylation changes from high-variance signals. By combining variance-aware statistical modeling with seed-based region construction, glmmDMR provides a robust framework for identifying contiguous, biologically interpretable DMRs across diverse methylome datasets.

Some key inflammatory genes controlled by the RELA transcription factor are thought to be highly expressed in fibroblasts induced into senescence by ionizing radiation (IR) as part of the Senescent-Associated Secretory Phenotype (SASP). However, this view is based largely on studies of a limited number of fibroblast cell lines derived from fetal lung or neonatal foreskin. Here, we show that more than half of the primary adult fibroblast strains examined exhibit only weak induction of RELA-dependent inflammatory genes following IR-induced senescence. We define these fibroblasts as "low-responding" to distinguish them from fibroblasts that express high levels of inflammatory gene expression in response to IR. RNA-seq analysis indicated particularly weak IL1A and IL1B expression in low-responding fibroblasts. IL1-alpha and IL1-beta participate in a positive amplification loop for inflammatory gene expression in senescence. Addition of recombinant IL1-alpha or IL1-beta to these fibroblasts sufficed to induce high expression of inflammatory genes. Low-responding fibroblasts thus exhibit cell-autonomous defects in IL1A and IL1B gene activation in response to IR that explains their overall low expression of RELA-targeted inflammatory genes. This defect was correlated with reduced chromatin accessibility and H3-K27-acetylation at 2 putative enhancers in the intergenic region separating IL1A and IL1B, and deletion of either of these enhancers inhibited inflammatory gene expression in IR-induced senescence. Fibroblasts express distinct transcriptomes and we found that differential expression of the FOXF1 transcription factor gene in high-responding WI38 fetal lung fibroblasts contributes to inflammatory gene expression after IR. Our observations indicate that fibroblasts can be distinguished by their ability to manifest cell-autonomous induction of inflammatory genes under conditions of IR-induced senescence.

Merkel cell carcinoma (MCC) is a neuroendocrine skin tumor that is frequently driven by integration of Merkel cell polyomavirus (MCPyV). In MCC, the MCPyV genome is truncated, but expression of the viral tumor antigens, truncated large tumor antigen (LTT) and small tumor antigen (sT), is maintained and drives uncontrolled proliferation. We introduced constitutive expression of the MCPyV T antigens (TAs) into primary mouse dermal fibroblasts (MDFs) to determine whether these cells are susceptible to MCPyV-driven transformation. TA expression alone in MDFs induced key MCC markers, cytokeratin-20 (CK20) and Sry-box transcription factor 2 (SOX2), and promoted anchorage-independent growth indicative of cellular transformation. Subcutaneous implantation of TA-transformed fibroblasts produced high-grade MCC-like tumors that grew persistently in immunodeficient NSG mice but not in immunocompetent C57BL/6 mice. Serial in vivo passaging of the tumor cell line enhanced tumor growth, reduced expression of p53-target genes and MHC-1, and was accompanied by a shift in T antigen isoform expression, with decreased LTT and increased sT expression. Our data demonstrate that MCPyV-driven tumors acquire immune-evasive adaptions during tumor progression in vivo and suggest that the anti-tumor immune response exerts selective pressure in MCC that favors expression of sT rather than LTT. The model established in this study provides a unique platform for studying evolution of MCPyV-driven tumors under immune pressure and identifying mechanisms of immune evasion in MCC that could be used to develop new therapeutic strategies. Significance StatementMCPyV tumor antigen expression transforms mouse dermal fibroblasts to generate MCC-like tumors. Serial in vivo passaging reveals tumor evolution under immune pressure, providing a model to study immune evasion mechanisms in MCC.