SINE Elements Research Articles

Abstract A076: LINE-1 ORF1p mimics viral innate immune evasion mechanisms in pancreatic cancer

Abstract Aberrant expression of repeat elements displaying viral mimicry is a common feature found in both mouse models and human pancreatic ductal adenocarcinoma (PDAC). However, cancer cells must employ mechanisms to manage this "viral" repeat element load to avoid triggering innate immune responses. Here, we demonstrate that the long interspersed nuclear element 1 (LINE-1) ORF1 protein (ORF1p) in PDAC cells plays a role in shielding repeat RNAs from activating pathogen recognition receptor (PRR)-mediated antiviral responses, independent of retrotransposition. Suppression of ORF1p using lentiviral shRNA induces innate immune responses through the PRRs, RIG-I and MAVS (double-stranded RNA sensors). Interestingly, PDAC cell lines with low ORF1p lack these two receptors, suggesting convergent mechanisms to suppress PRR signaling. Furthermore, RNA immunoprecipitation sequencing (RIP-seq) demonstrates ORF1p association with repeat RNA, including LINE and SINE elements that typically trigger antiviral responses. Subcellular localization of ORF1p in processing bodies (p-bodies) reveals specific interaction with MOV10, a dsRNA helicase crucial for antiviral responses. Loss of ORF1p results in significant growth reduction in vitro and in vivo, which supports ORF1p as a potential immune oncology therapeutic target. Altogether, our results demonstrate a novel function of ORF1p in shielding cytoplasmic repeat RNA from PRR sensing, shedding light on how PDAC cells evade host immune responses triggered by viral-like repeat RNA. Citation Format: Eunae You, Bidish Patel, Alexandra Rojas, Siyu Sun, Natalie Ho, Ildiko Phillips, Michael Raabe, Yuhui Song, Katherine Xu, Joshua Kocher, Peter Richieri, Martin Taylor, Linda Nieman, Benjamin Greenbaum, David Ting. LINE-1 ORF1p mimics viral innate immune evasion mechanisms in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A076.

The Detection of a Functional 168 bp Deletion of the HOXB13 Gene Determining Short Tail and Its Association with Senior Growth Traits in Sheep Breeds Worldwide.

In recent years, genome-wide association studies (GWAS) have uncovered that the HOXB13 gene is a key regulatory factor for the tail length trait of sheep. Further research has found that there is a functional 168 bp SINE element insertion upstream of the HOXB13 gene, which leads to the occurrence of long tails in sheep. However, the frequency of mutations in the 168 bp SINE element of the HOXB13 gene among different sheep breeds around the world and its relationship with growth traits are still unclear. This study used whole-genome sequencing (WGS) data, including 588 samples from 33 different sheep breeds around the world, to evaluate the frequency of HOXB13 gene mutations in different sheep breeds globally. At the same time, this study also selected 3392 sheep samples from six breeds. The genetic variation in the 168 bp InDel locus in the HOXB13 gene was determined through genotyping, and its association with the growth traits of Luxi black-headed sheep was analyzed. The research results indicate that the polymorphism of the 168 bp InDel locus is significantly correlated with the hip width of adult ewes in the Luxi black-headed sheep breed (p < 0.05) and that the hip width of adult ewes with the DD genotype is significantly larger than that of adult ewes with the ID genotype (p < 0.05). This study indicates that there is consistency between the research results on the sheep tail length trait and growth traits, which may contribute to the promotion of sheep breed improvement.

Mapping and functional characterization of structural variation in 1060 pig genomes

BackgroundStructural variations (SVs) have significant impacts on complex phenotypes by rearranging large amounts of DNA sequence.ResultsWe present a comprehensive SV catalog based on the whole-genome sequence of 1060 pigs (Sus scrofa) representing 101 breeds, covering 9.6% of the pig genome. This catalog includes 42,487 deletions, 37,913 mobile element insertions, 3308 duplications, 1664 inversions, and 45,184 break ends. Estimates of breed ancestry and hybridization using genotyped SVs align well with those from single nucleotide polymorphisms. Geographically stratified deletions are observed, along with known duplications of the KIT gene, responsible for white coat color in European pigs. Additionally, we identify a recent SINE element insertion in MYO5A transcripts of European pigs, potentially influencing alternative splicing patterns and coat color alterations. Furthermore, a Yorkshire-specific copy number gain within ABCG2 is found, impacting chromatin interactions and gene expression across multiple tissues over a stretch of genomic region of ~200 kb. Preliminary investigations into SV’s impact on gene expression and traits using the Pig Genotype-Tissue Expression (PigGTEx) data reveal SV associations with regulatory variants and gene-trait pairs. For instance, a 51-bp deletion is linked to the lead eQTL of the lipid metabolism regulating gene FADS3, whose expression in embryo may affect loin muscle area, as revealed by our transcriptome-wide association studies.ConclusionsThis SV catalog serves as a valuable resource for studying diversity, evolutionary history, and functional shaping of the pig genome by processes like domestication, trait-based breeding, and adaptive evolution.

Broadband four-shoulder sine antenna of radio monitoring and direction finding equipment with a note system of two orthogonally combined printed resistance transformers

Problem statement. The article presents material on the development of antennas used in radio monitoring and radio direction finding equipment of electronic warfare equipment. The basic requirements for the design and characteristics of antennas of radio monitoring and direction finding equipment are presented. Aspects of the fundamentals of the development of a four-arm broadband antenna powered by two orthogonally combined printed resistance transformers, which has not been used before, for the possibility of controlling polarization, are considered, the results of modeling the developed antenna are presented and recommendations for its use in the antenna array are given. Purpose. To develop a small-sized antenna that allows to increase the efficiency of the operation of radio direction finding and radio monitoring complexes in an ultra-wide frequency band, with a special power supply system of two orthogonally combined printed resistance transformers for the possibility of implementing polarization control. Results. Based on the methods of electrodynamic modeling, computational methods of technical electrodynamics, methods of full–scale experimental measurements of antenna characteristics and methods of computer processing of data of full-scale experimental measurements, an antenna has been developed that allows the control of amplitude, phase and polarization parameters in an ultra-wide frequency band. The developed antenna is based on a flat four-arm sine element with a power supply system of two orthogonally arranged printed resistance transformers. The resistance transformer significantly reduces the level of the reflection coefficient at the input and radiation losses. The use of this development in radio direction finding and radio monitoring complexes will reduce the number of sublattices of the antenna system due to the functioning of its elements in an ultra-wide frequency band. The practical significance lies in reducing the number of letters of radio direction finding antenna systems consisting of the developed antenna elements, minimizing the degree of distortion of ultra-wideband signals and ultrashort pulses of arbitrary (varying) polarization received by antenna devices and systems from the antenna presented in the article. The developed antenna and antenna devices based on it can be used for electronic warfare, as well as improving communication and remote control systems.

A germline chimeric KANK1-DMRT1 transcript derived from a complex structural variant is associated with a congenital heart defect segregating across five generations.

Structural variants (SVs) pose a challenge to detect and interpret, but their study provides novel biological insights and molecular diagnosis underlying rare diseases. The aim of this study was to resolve a 9p24 rearrangement segregating in a family through five generations with a congenital heart defect (congenital pulmonary and aortic valvular stenosis and pulmonary artery stenosis), by applying a combined genomic analysis. The analysis involved multiple techniques, including karyotype, chromosomal microarray analysis (CMA), FISH, genome sequencing (GS), RNA-seq, and optical genome mapping (OGM). A complex 9p24 SV was hinted at by CMA results, showing three interspersed duplicated segments. Combined GS and OGM analyses revealed that the 9p24 duplications constitute a complex SV, on which a set of breakpoints matches the boundaries of the CMA duplicated sequences. The proposed structure for this complex rearrangement implies three duplications associated with an inversion of ~ 2Mb region on chromosome 9 and a SINE element insertion at the more distal breakpoint. Interestingly, this genomic structure of rearrangement forms a chimeric transcript of the KANK1/DMRT1 loci, which was confirmed by both RNA-seq and Sanger sequencing on blood samples from 9p24 rearrangement carriers. Altogether with breakpoint amplification and FISH analysis, this combined approach allowed a deep characterization of this complex rearrangement. Although the genotype-phenotype correlation remains elusive from the molecular mechanism point of view, this study identified a large genomic rearrangement at 9p24 segregating with a familial congenital heart defect, revealing a genetic biomarker that was successfully applied for embryo selection, changing the reproductive perspective of affected individuals.

Multi-Omics Factor Analysis (MOFA) Identifies Transposable Element Expression As a Risk Factor and Inflammaging As a Protective Factor in Myelodysplastic Syndromes

Introduction Myelodysplastic syndromes (MDS) are haematological diseases characterised by clonal proliferation due to genetic and epigenetic alterations within haematopoietic stem and progenitor cells. Thus far, the prediction of a patient's overall survival (OS) and event-free survival (EFS) has been dependent predominantly on the degree of peripheral blood cytopenias, bone marrow blast percentage, cytogenetics, and genetic features. However, this may overlook other biological phenotypes, including, for example, the expression of transposable elements (TEs), ageing, and immunology profile. In this study, we present a comprehensive analysis of three data modalities (clinical, genotypic, and transcriptomic) and eight different views derived from these modalities to identify the hidden factors that significantly impact MDS prognosis. Methods Three (immunology, ageing, and cellular composition) out of eight views of the data were derived from RNA-seq by applying singscore, a gene set scoring tool developed by Foroutan et al., 2018, on RNA-seq gene expression from bone marrow samples of 94 MDS patients obtained from Shiozawa et al., 2017 (Fig 1.A). Each of these three views was carried forward in the workflow by a number of gene sets. The other five views were clinical numeric data, clinical categorical data, TE expression, genotype data, and known AML/MDS antigens (obtained from TANTIGEN 2.0), expressed in at least 10% of MDS samples in the study. We then employed Multi-Omics Factor Analysis (MOFA), a computational framework developed by Argelaguet et al., 2018, to uncover hidden phenotypic states within a multi-omic dataset. Through MOFA, we generated factors representing specific biological data modalities, establishing connections between features across these different categories. The total variance ( R2) that can be attributed to specific factors is also exhibited by MOFA. Results Factors were ranked based on their explained variance. We present the 10 factors with a threshold of variance explained of 2% from the 15 default factors generated by MOFA (Fig 1.A). Factor 1 was one of the factors that linked the most categories of data: immunology, cellular composition, ageing, and TEs. Most notably, Factor 1 was aligned with features of less differentiated cells (more HSC-like and less GMP-like). We observed that samples represented by high Factor 1 scores also had a higher expression of SINE:Alu elements. (Fig 1.A). Moreover, Factor 1 aligns the following immunology features: increased T-helper 1 (Th1) cells, macrophages, and T regulatory cells. The relationship between Th1 and SINE elements is particularly interesting, given the role of Th1 cell activation in host defence against infection that may be modulated by SINE activation. Factor 2 comprised of ageing and clinical categorical data, which showed that less inflammaging may lead to a poorer EFS ( P=0.00023) (Fig 1.B). Factor 4 linked immunology, cellular decomposition, ageing and clinical categorical data. The following features mainly influenced Factor 4: increased HSC-like cells, decreased CD8+ T cells, decreased immunosenescence and increased inflammatory chemokines. Using Kaplan-Meier plots to predict OS, we observed that patients with more HSC-like cells, fewer leukocytes and T cells, and less immunosenescence and inflammaging were linked to poorer EFS ( P=0.0001) and OS ( P=0.00017, data not shown). Furthermore, Factor 9 comprised solely of the TEs category and exhibited a correlation between increased TE expression and poorer EFS ( P=0.0074) and OS ( P=0.0065, data not shown) (Fig 1.B). Conclusions Our analysis using MOFA on a diverse set of biological data modalities uncovered hidden factors in MDS, providing insights into the complex interplay between various biological layers. Notably, we identified TE expression as a risk factor and inflammaging as a protective factor in MDS. HSC-like cells and specific immunological profiles were also associated with significantly poor OS and EFS. This study contributes to a deeper understanding of MDS pathogenesis and identifies potential prognostic markers for this disease. It also elucidates the importance of considering the relationships between different pathways, markers, and mutations in predicting patient outcomes, highlighting the efficacy of a comprehensive approach that goes beyond all the scoring systems described thus far for MDS.

SINE-Associated LncRNA SAWPA Regulates Porcine Zygotic Genome Activation.

In mice, retrotransposon-associated long noncoding RNAs (lncRNA) play important regulatory roles in pre-implantation development; however, it is largely unknown whether they function in the pre-implantation development in pigs. The current study aims to screen for retrotransposon-associated lncRNA in porcine early embryos and identifies a porcine 8-cell embryo-specific SINE-associated nuclear long noncoding RNA named SAWPA. SAWPA is essential for porcine embryonic development as depletion of SAWPA results in a developmental arrest at the 8-cell stage, accompanied by the inhibition of the JNK-MAPK signaling pathway. Mechanistically, SAWPA works in trans as a transcription factor for JNK through the formation of an RNA-protein complex with HNRNPA1 and MED8 binding the SINE elements upstream of JNK. Therefore, as the first functional SINE-associated long noncoding RNAs in pigs, SAWPA provides novel insights for the mechanism research on retrotransposons in mammalian pre-implantation development.

The transposable element-derived transcript of LIN28B has a placental origin and is not specific to tumours

Transposable elements (TEs) are genetic elements that have evolved as crucial regulators of human development and cancer, functioning as both genes and regulatory elements. When TEs become dysregulated in cancer cells, they can serve as alternate promoters to activate oncogenes, a process known as onco-exaptation. This study aimed to explore the expression and epigenetic regulation of onco-exaptation events in early human developmental tissues. We discovered co-expression of some TEs and oncogenes in human embryonic stem cells and first trimester and term placental tissues. Previous studies identified onco-exaptation events in various cancer types, including an AluJb SINE element–LIN28B interaction in lung cancer cells, and showed that the TE-derived LIN28B transcript is associated with poor patient prognosis in hepatocellular carcinoma. This study further characterized the AluJb–LIN28B transcript and confirmed that its expression is restricted to the placenta. Targeted DNA methylation analysis revealed differential methylation of the two LIN28B promoters between placenta and healthy somatic tissues, indicating that some TE–oncogene interactions are not cancer-specific but arise from the epigenetic reactivation of developmental TE-derived regulatory events. In conclusion, our findings provide evidence that some TE–oncogene interactions are not limited to cancer and may originate from the epigenetic reactivation of TE-derived regulatory events that are involved in early development. These insights broaden our understanding of the role of TEs in gene regulation and suggest the potential importance of targeting TEs in cancer therapy beyond their conventional use as cancer-specific markers.

Mouse B2 SINE elements function as IFN-inducible enhancers.

Regulatory networks underlying innate immunity continually face selective pressures to adapt to new and evolving pathogens. Transposable elements (TEs) can affect immune gene expression as a source of inducible regulatory elements, but the significance of these elements in facilitating evolutionary diversification of innate immunity remains largely unexplored. Here, we investigated the mouse epigenomic response to type II interferon (IFN) signaling and discovered that elements from a subfamily of B2 SINE (B2_Mm2) contain STAT1 binding sites and function as IFN-inducible enhancers. CRISPR deletion experiments in mouse cells demonstrated that a B2_Mm2 element has been co-opted as an enhancer driving IFN-inducible expression of Dicer1. The rodent-specific B2 SINE family is highly abundant in the mouse genome and elements have been previously characterized to exhibit promoter, insulator, and non-coding RNA activity. Our work establishes a new role for B2 elements as inducible enhancer elements that influence mouse immunity, and exemplifies how lineage-specific TEs can facilitate evolutionary turnover and divergence of innate immune regulatory networks.

ZFP92, a KRAB domain zinc finger protein enriched in pancreatic islets, binds to B1/Alu SINE transposable elements and regulates retroelements and genes.

Repressive KRAB domain-containing zinc-finger proteins (KRAB-ZFPs) are abundant in mammalian genomes and contribute both to the silencing of transposable elements (TEs) and to the regulation of developmental stage- and cell type-specific gene expression. Here we describe studies of zinc finger protein 92 (Zfp92), an X-linked KRAB-ZFP that is highly expressed in pancreatic islets of adult mice, by analyzing global Zfp92 knockout (KO) mice. Physiological, transcriptomic and genome-wide chromatin binding studies indicate that the principal function of ZFP92 in mice is to bind to and suppress the activity of B1/Alu type of SINE elements and modulate the activity of surrounding genomic entities. Deletion of Zfp92 leads to changes in expression of select LINE and LTR retroelements and genes located in the vicinity of ZFP92-bound chromatin. The absence of Zfp92 leads to altered expression of specific genes in islets, adipose and muscle that result in modest sex-specific alterations in blood glucose homeostasis, body mass and fat accumulation. In islets, Zfp92 influences blood glucose concentration in postnatal mice via transcriptional effects on Mafb, whereas in adipose and muscle, it regulates Acacb, a rate-limiting enzyme in fatty acid metabolism. In the absence of Zfp92, a novel TE-Capn11 fusion transcript is overexpressed in islets and several other tissues due to de-repression of an IAPez TE adjacent to ZFP92-bound SINE elements in intron 3 of the Capn11 gene. Together, these studies show that ZFP92 functions both to repress specific TEs and to regulate the transcription of specific genes in discrete tissues.

Antiretroviral APOBEC3 cytidine deaminases alter HIV-1 provirus integration site profiles

APOBEC3 (A3) proteins are host-encoded deoxycytidine deaminases that provide an innate immune barrier to retroviral infection, notably against HIV-1. Low levels of deamination are believed to contribute to the genetic evolution of HIV-1, while intense catalytic activity of these proteins can induce catastrophic hypermutation in proviral DNA leading to near-total HIV-1 restriction. So far, little is known about how A3 cytosine deaminases might impact HIV-1 proviral DNA integration sites in human chromosomal DNA. Using a deep sequencing approach, we analyze the influence of catalytic active and inactive APOBEC3F and APOBEC3G on HIV-1 integration site selections. Here we show that DNA editing is detected at the extremities of the long terminal repeat regions of the virus. Both catalytic active and non-catalytic A3 mutants decrease insertions into gene coding sequences and increase integration sites into SINE elements, oncogenes and transcription-silencing non-B DNA features. Our data implicates A3 as a host factor influencing HIV-1 integration site selection and also promotes what appears to be a more latent expression profile.

Novel selectively amplified DNA sequences in the germline genome of the Japanese hagfish, Eptatretus burgeri

In the Japanese hagfish Eptatretus burgeri, 16 chromosomes (eliminated [E]-chromosomes) have been lost in somatic cells (2n = 36), which is equivalent to approx. 21% of the genomic DNA in germ cells (2n = 52). At least seven of the 12 eliminated repetitive DNA families isolated in eight hagfish species were selectively amplified in the germline genome of this species. One of them, EEEb1 (eliminated element of E.burgeri 1) is exclusively localized on all E-chromosomes. Herein, we identified four novel eliminated repetitive DNA families (named EEEb3-6) through PCR amplification and suppressive subtractive hybridization (SSH) combined with Southern-blot hybridization. EEEb3 was mosaic for 5S rDNA and SINE elements. EEEb4 was GC-rich repeats and has one pair of direct and inverted repeats, whereas EEEb5 and EEEb6 were AT-rich repeats with one pair and two pairs of sub-repeats, respectively. Interestingly, all repeat classes except EEEb3 were transcribed in the testes, although no open reading frames (ORF) were identified. We conducted fluorescence in situ hybridization (FISH) to examine the chromosomal localizations of EEEb3-6 and EEEb2, which was previously isolated from the germline genome of E. burgeri. All sequences were only found on all EEEb1-positive E-chromosomes. Copy number estimation of the repeated elements by slot-blot hybridization revealed that (i) the EEEb1-6 family members occupied 39.9% of the total eliminated DNA, and (ii) a small number of repeats were retained in somatic cells, suggesting that there is incomplete elimination of the repeated elements. These results provide new insights into the mechanisms involved in the chromosome elimination and the evolution of E-chromosomes.

Wood feeding and social living: Draft genome of the subterranean termite Reticulitermes lucifugus (Blattodea; Termitoidae).

Termites (Insecta, Blattodea, Termitoidae) are a widespread and diverse group of eusocial insects known for their ability to digest wood matter. Herein, we report the draft genome of the subterranean termite Reticulitermes lucifugus, an economically important species and among the most studied taxa with respect to eusocial organization and mating system. The final assembly (~813 Mb) covered up to 88% of the estimated genome size and, in agreement with the Asexual Queen Succession Mating System, it was found completely homozygous. We predicted 16,349 highly supported gene models and 42% of repetitive DNA content. Transposable elements of R. lucifugus show similar evolutionary dynamics compared to that of other termites, with two main peaks of activity localized at 25% and 8% of Kimura divergence driven by DNA, LINE and SINE elements. Gene family turnover analyses identified multiple instances of gene duplication associated with R. lucifugus diversification, with significant lineage-specific gene family expansions related to development, perception and nutrient metabolism pathways. Finally, we analysed P450 and odourant receptor gene repertoires in detail, highlighting the large diversity and dynamical evolutionary history of these proteins in the R. lucifugus genome. This newly assembled genome will provide a valuable resource for further understanding the molecular basis of termites biology as well as for pest control.

Abstract A004: Repetitive elements and viral mimicry co-evolve in pancreatic cancer

Abstract Overexpression of repetitive DNA is an emerging hallmark of human cancers and several repeats, such as SINE elements and satellite, transcribe immunostimulatory RNA that mimic features associated with viral infection. However, the extent to which specific repeat families affect tumor evolution and the immune microenvironment in a pro-or anti-tumorigenic manner remains poorly understood. We present an integrated, evolutionary multi-omic analysis pf repeat evolution in multiregion-sampled human metastatic and primary pancreatic cancers for the first time. We found that LINE-1 (L1) somatic insertion location coincides with tumor evolution. Moreover, we found L1 activity is negatively correlated with immunogenic SINE element expression. Two orthogonal metrics were used in quantifying immunogenicity of SINE elements: IFN signature and capability of forming dsRNA, where both methods revealed that evolutionary young SINE elements have stronger correlation with IFN signatures and are more likely to form dsRNAs. Moreover, the anti-correlation between L1 activity and SINE expression is validated through both immunohistological analysis of human samples and pancreatic cancer cell lines. In the end, we proposed that the model of hypoactive L1 is beneficial for tumors by suppression of immunogenic SINE elements through either their direct reverse transcription or accelerating their degradation. Our analysis therefore sheds light on the evolutionary role of dark matter repeats in a tumor’s adaptation to immunogenic vulnerabilities. Citation Format: Siyu Sun. Repetitive elements and viral mimicry co-evolve in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A004.

Fine-mapping and identification of candidate causal genes for tail length in the Merinolandschaf breed

Docking the tails of lambs in long-tailed sheep breeds is a common practice worldwide. But this practice is associated with pain. Breeding for a shorter tail could offer an alternative. Therefore, this study aimed to analyze the natural tail length variation in the Merinolandschaf and to identify causal alleles for the short tail phenotype segregating within long-tailed breeds. We used SNP-based association analysis and haplotype-based mapping in 362 genotyped (Illumina OvineSNP50) and phenotyped Merinolandschaf lambs. Genome-wide significant regions were capture sequenced in 48 lambs and comparatively analyzed in various long and short-tailed sheep breeds and wild sheep subspecies. Here we show a SNP located in the first exon of HOXB13 and a SINE element located in the promotor of HOXB13 as promising candidates. These results enable more precise breeding towards shorter tails, improve animal welfare by amplification of ancestral alleles and contribute to a better understanding of differential embryonic development.

Abstract 6196: Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma

Abstract Fanconi anemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink (ICL) repair resulting in chromosome breakage. The FA repair pathway comprises at least 22 FANC proteins including BRCA1 and BRCA2 and protects against carcinogenic endogenous and exogenous aldehydes. Individuals with FA are hundreds to thousands-fold more likely to develop head and neck (HNSCC), esophageal and anogenital squamous cell carcinomas (SCCs) with a median onset age of 31 years. The aggressive nature of these tumors and poor patient tolerance of platinum and radiation-based therapy have been associated with short survival in FA. Molecular studies of SCCs from individuals with FA (FA SCCs) have been limited, and it is unclear how they relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or human papillomavirus (HPV) infection. Here, by sequencing FA SCCs, we demonstrate that the primary genomic signature of FA-deficiency is the presence of a high number of structural variants (SVs). SVs are enriched for small deletions, unbalanced translocations, and fold-back inversions that arise in the context of TP53 loss. The SV breakpoints preferentially localize to early replicating regions, common fragile sites, tandem repeats, and SINE elements. SVs are often connected forming complex rearrangements. Resultant genomic instability underlies elevated copy number alteration (CNA) rates of key HNSCC-associated genes, including PIK3CA, MYC, CSMD1, PTPRD, YAP1, MXD4, and EGFR. In contrast to sporadic HNSCC, we find no evidence of HPV infection in FA HNSCC, although positive cases were identified in gynecologic tumors. A murine allograft model of FA pathway-deficient SCC was enriched in SVs, exhibited dramatic tumor growth advantage, more rapid epithelial-to-mesenchymal transition, and enhanced autonomous inflammatory signaling when compared to an FA pathway-proficient model. In light of the protective role of the FA pathway against SV formation uncovered here, and recent findings of FA pathway insufficiency in the setting of increased formaldehyde load resulting in hematopoietic stem cell failure and carcinogenesis, we propose that high copy-number instability in sporadic HNSCC may result from functional overload of the FA pathway by endogenous and exogenous DNA crosslinking agents. Our work lays the foundation for improved FA patient treatment and demonstrates that FA SCC is a powerful model to study tumorigenesis resulting from DNA crosslinking damage. Citation Format: Andrew L. Webster, Mathijs A. Sanders, Krupa Patel, Ralf Dietrich, Raymond J. Noonan, Francis P. Lach, Ryan R. White, Audrey M. Goldfarb, Kevin Hadi, Matthew M. Edwards, Frank X. Donovan, Moonjung Jung, Sunandini Sridhar, Olivier Fedrigo, Huasong Tian, Joel Rosiene, Thomas Heineman, Jennifer Kennedy, Lorenzo Bean, Rasim O. Rosti, Rebecca Tryon, Ashlyn-Maree Gonzalez, Allana Rosenberg, Ji-Dung Luo, Thomas Carrol, Eunike Velleuer, Jeff C. Rastatter, Susanne I. Wells, Jordi Surrallés, Grover Bagby, Margaret L. MacMillan, John E. Wagner, Maria Cancio, Farid Boulad, Theresa Scognamiglio, Roger Vaughan, Amnon Koren, Marcin Imielinski, Settara Chandrasekharappa, Arleen D. Auerbach, Bhuvanesh Singh, David Kutler, Peter J. Campbell, Agata Smogorzewska. Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6196.

The Emergence of a New Isoform of POU2F1 in Primates through the Use of Egoistic Mobile Genetic Elements

The emergence of new genes and functions is of paramount importance in the emergence of new animal species. For example, the insertion of the mobile element Tigger 2 into the sequence of the functional gene POU2F1 in primates led to the formation of a new chimeric primate-specific isoform POU2F1Z, the translation of which is activated under cellular stress. Its mRNA was found in all species of monkeys, starting with macaques. Analysis of the fragments of the Tigger2 copy corresponding to the human exon Z showed that the splicing sites of exon Z are homologous in humans and in most monkeys, with the exception of lemurs and galagos. The stop codon introduced into the mRNA by the Tigger2 sequence is present in all primates, starting with macaques. The internal ATG codon is also present in all primates, with the exception of lemurs and galagos. In the course of evolution, other MGEs, mainly of the SINE type, were inserted into the Tigger2 copy. In the course of evolution, both the location and the number of mobile SINE elements within the POU2F1 gene changed. Starting with macaques, the pattern of the arrangement of SINE elements within the Tigger2 copy in the studied region of the POU2F1 gene was fixed and then remained unchanged in other primates and humans, which may indicate its functional significance.

The PMEL gene and merle (dapple) in the dachshund: cryptic, hidden, and mosaic variants demonstrate the need for genetic testing prior to breeding.

One of the most unique coat color patterns in the domestic dog is merle (also known as dapple in the dachshund breed), characterized by patches of normal pigmentation surrounded by diluted eumelanin pigment. In dogs, this striking variegated pattern is caused by an insertion of a SINE element into the PMEL gene. Differences in the length of the SINE insertion [due to a variable-length poly(A)-tail] has been associated with variation in the merle coat color and patterning. We previously performed a systematic evaluation of merle in 175 Australian shepherds and related breeds and correlated the length of the merle insertion variants with four broad phenotypic clusters designated as "cryptic", "atypical", "classic", and "harlequin" merle. In this study, we evaluated the SINE insertions in 140 dachshunds and identified the same major merle phenotypic clusters with only slight variation between breeds. Specifically, we identified numerous cases of true "hidden" merle in dachshunds with light/red (pheomelanin) coats with little to no black/brown pigment (eumelanin) and thus minimal or no observable merle phenotype. In addition, we identified somatic and gonadal mosaicism, with one dog having a large insertion in the harlequin size range of M281 that had no merle phenotype and unintentionally produced a double merle puppy with anophthalmia. The frequent identification of cryptic, hidden, and mosaic merle variants, which can be undetectable by phenotypic inspection, should be of particular concern to breeders and illustrates the critical need for genetic testing for merle prior to breeding to avoid producing dogs with serious health problems.

Amplified Fragments of an Autosome-Borne Gene Constitute a Significant Component of the W Sex Chromosome of Eremias velox (Reptilia, Lacertidae).

Heteromorphic W and Y sex chromosomes often experience gene loss and heterochromatinization, which is frequently viewed as their “degeneration”. However, the evolutionary trajectories of the heterochromosomes are in fact more complex since they may not only lose but also acquire new sequences. Previously, we found that the heterochromatic W chromosome of a lizard Eremias velox (Lacertidae) is decondensed and thus transcriptionally active during the lampbrush stage. To determine possible sources of this transcription, we sequenced DNA from a microdissected W chromosome sample and a total female DNA sample and analyzed the results of reference-based and de novo assembly. We found a new repetitive sequence, consisting of fragments of an autosomal protein-coding gene ATF7IP2, several SINE elements, and sequences of unknown origin. This repetitive element is distributed across the whole length of the W chromosome, except the centromeric region. Since it retained only 3 out of 10 original ATF7IP2 exons, it remains unclear whether it is able to produce a protein product. Subsequent studies are required to test the presence of this element in other species of Lacertidae and possible functionality. Our results provide further evidence for the view of W and Y chromosomes as not just “degraded” copies of Z and X chromosomes but independent genomic segments in which novel genetic elements may arise.

Gene and Transposable Element Expression Evolution Following Recent and Past Polyploidy Events in Spartina (Poaceae).

Gene expression dynamics is a key component of polyploid evolution, varying in nature, intensity, and temporal scales, most particularly in allopolyploids, where two or more sub-genomes from differentiated parental species and different repeat contents are merged. Here, we investigated transcriptome evolution at different evolutionary time scales among tetraploid, hexaploid, and neododecaploid Spartina species (Poaceae, Chloridoideae) that successively diverged in the last 6–10 my, at the origin of differential phenotypic and ecological traits. Of particular interest are the recent (19th century) hybridizations between the two hexaploids Spartina alterniflora (2n = 6x = 62) and S. maritima (2n = 6x = 60) that resulted in two sterile F1 hybrids: Spartina × townsendii (2n = 6x = 62) in England and Spartina × neyrautii (2n = 6x = 62) in France. Whole genome duplication of S. × townsendii gave rise to the invasive neo-allododecaploid species Spartina anglica (2n = 12x = 124). New transcriptome assemblies and annotations for tetraploids and the enrichment of previously published reference transcriptomes for hexaploids and the allododecaploid allowed identifying 42,423 clusters of orthologs and distinguishing 21 transcribed transposable element (TE) lineages across the seven investigated Spartina species. In 4x and 6x mesopolyploids, gene and TE expression changes were consistent with phylogenetic relationships and divergence, revealing weak expression differences in the tetraploid sister species Spartina bakeri and Spartina versicolor (<2 my divergence time) compared to marked transcriptome divergence between the hexaploids S. alterniflora and S. maritima that diverged 2–4 mya. Differentially expressed genes were involved in glycolysis, post-transcriptional protein modifications, epidermis development, biosynthesis of carotenoids. Most detected TE lineages (except SINE elements) were found more expressed in hexaploids than in tetraploids, in line with their abundance in the corresponding genomes. Comparatively, an astonishing (52%) expression repatterning and deviation from parental additivity were observed following recent reticulate evolution (involving the F1 hybrids and the neo-allododecaploid S. anglica), with various patterns of biased homoeologous gene expression, including genes involved in epigenetic regulation. Downregulation of TEs was observed in both hybrids and accentuated in the neo-allopolyploid. Our results reinforce the view that allopolyploidy represents springboards to new regulatory patterns, offering to worldwide invasive species, such as S. anglica, the opportunity to colonize stressful and fluctuating environments on saltmarshes.