Evolution of Virus-like Features and Intrinsically Disordered Regions in Retrotransposon-derived Mammalian Genes.

Several mammalian genes have originated from the domestication of retrotransposons, selfish mobile elements related to retroviruses. Some of the proteins encoded by these genes have maintained virus-like features; including self-processing, capsid structure formation, and the generation of different isoforms through -1 programmed ribosomal frameshifting. Using quantitative approaches in molecular evolution and biophysical analyses, we studied 28 retrotransposon-derived genes, with a focus on the evolution of virus-like features. By analyzing the rate of synonymous substitutions, we show that the -1 programmed ribosomal frameshifting mechanism in three of these genes (PEG10, PNMA3, and PNMA5) is conserved across mammals and originates alternative proteins. These genes were targets of positive selection in primates, and one of the positively selected sites affects a B-cell epitope on the spike domain of the PNMA5 capsid, a finding reminiscent of observations in infectious viruses. More generally, we found that retrotransposon-derived proteins vary in their intrinsically disordered region content and this is directly associated with their evolutionary rates. Most positively selected sites in these proteins are located in intrinsically disordered regions and some of them impact protein posttranslational modifications, such as autocleavage and phosphorylation. Detailed analyses of the biophysical properties of intrinsically disordered regions showed that positive selection preferentially targeted regions with lower conformational entropy. Furthermore, positive selection introduces variation in binary sequence patterns across orthologues, as well as in chain compaction. Our results shed light on the evolutionary trajectories of a unique class of mammalian genes and suggest a novel approach to study how intrinsically disordered region biophysical characteristics are affected by evolution.

ZNF91 is an endogenous repressor of the molecular phenotype associated with X-linked dystonia-parkinsonism (XDP).

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder resulting from an inherited intronic SINE-Alu-VNTR (SVA) retrotransposon in the TAF1 gene that causes dysregulation of TAF1 transcription. The specific mechanism underlying this dysregulation remains unclear, but it is hypothesized to involve the formation of G-quadruplexes (G4) structures within the XDP-SVA that impede transcription. In this study, we show that ZNF91, a critical repressor of SVA retrotransposons, specifically binds to G4-forming DNA sequences. Further, we found that genetic deletion of ZNF91 exacerbates the molecular phenotype associated with the XDP-SVA insertion in patient cells, while no difference was observed when ZNF91 was deleted from isogenic control cells. Additionally, we observed a significant age-related reduction in ZNF91 expression in whole blood and brain, indicating a progressive loss of repression of the XDP-SVA in XDP. These findings indicate that ZNF91 plays a crucial role in controlling the molecular phenotype associated with XDP. Since ZNF91 binds to G4-forming DNA sequences in SVAs, this suggests that interactions between ZNF91 and G4-forming sequences in the XDP-SVA minimize the severity of the molecular phenotype. Our results showing that ZNF91 expression levels significantly decrease with age provide a potential explanation for the age-related progressive neurodegenerative character of XDP. Collectively, our study provides important insights into the protective role of ZNF91 in XDP pathogenesis and suggests that restoring ZNF91 expression, destabilization of G4s, or targeted repression of the XDP-SVA could be future therapeutic strategies to prevent or treat XDP.

Evolution of retrocopies in the context of HUSH silencing.

Retrotransposition is one of the main factors responsible for gene duplication and thus genome evolution. However, the sequences that undergo this process are not only an excellent source of biological diversity, but in certain cases also pose a threat to the integrity of the DNA. One of the mechanisms that protects against the incorporation of mobile elements is the HUSH complex, which is responsible for silencing long, intronless, transcriptionally active transposed sequences that are rich in adenine on the sense strand. In this study, broad sets of human and porcine retrocopies were analysed with respect to the above factors, taking into account evolution of these molecules. Analysis of expression pattern, genomic structure, transcript length, and nucleotide substitution frequency showed the strong relationship between the expression level and exon length as well as the protective nature of introns. The results of the studies also showed that there is no direct correlation between the expression level and adenine content. However, protein-coding retrocopies, which have a lower adenine content, have a significantly higher expression level than the adenine-rich non-coding but expressed retrocopies. Therefore, although the mechanism of HUSH silencing may be an important part of the regulation of retrocopy expression, it is one component of a more complex molecular network that remains to be elucidated.

Duplications and Retrogenes Are Numerous and Widespread in Modern Canine Genomic Assemblies.

Recent years have seen a dramatic increase in the number of canine genome assemblies available. Duplications are an important source of evolutionary novelty and are also prone to misassembly. We explored the duplication content of nine canine genome assemblies using both genome self-alignment and read-depth approaches. We find that 8.58% of the genome is duplicated in the canFam4 assembly, derived from the German Shepherd Dog Mischka, including 90.15% of unplaced contigs. Highlighting the continued difficulty in properly assembling duplications, less than half of read-depth and assembly alignment duplications overlap, but the mCanLor1.2 Greenland wolf assembly shows greater concordance. Further study shows the presence of multiple segments that have alignments to four or more duplicate copies. These high-recurrence duplications correspond to gene retrocopies. We identified 3,892 candidate retrocopies from 1,316 parental genes in the canFam4 assembly and find that ∼8.82% of duplicated base pairs involve a retrocopy, confirming this mechanism as a major driver of gene duplication in canines. Similar patterns are found across eight other recent canine genome assemblies, with metrics supporting a greater quality of the PacBio HiFi mCanLor1.2 assembly. Comparison between the wolf and other canine assemblies found that 92% of retrocopy insertions are shared between assemblies. By calculating the number of generations since genome divergence, we estimate that new retrocopy insertions appear, on average, in 1 out of 3,514 births. Our analyses illustrate the impact of retrogene formation on canine genomes and highlight the variable representation of duplicated sequences among recently completed canine assemblies.

Competition between two HUSH complexes orchestrates the immune response to retroelement invasion.

The human silencing hub (HUSH) preserves genome integrity through the epigenetic repression of invasive genetic elements. However, despite our understanding of HUSH as an obligate complex of three subunits, only loss of MPP8 or Periphilin, but not TASOR, triggers interferon signaling following derepression of endogenous retroelements. Here, we resolve this paradox by characterizing a second HUSH complex that shares MPP8 and Periphilin but assembles around TASOR2, an uncharacterized paralog of TASOR. Whereas HUSH represses LINE-1 retroelements marked by the repressive histone modification H3K9me3, HUSH2 is recruited by the transcription factor IRF2 to repress interferon-stimulated genes. Mechanistically, HUSH-mediated retroelement silencing sequesters the limited pool of the shared subunits MPP8 and Periphilin, preventing TASOR2 from forming HUSH2 complexes and hence relieving the HUSH2-mediated repression of interferon-stimulated genes. Thus, competition between two HUSH complexes intertwines retroelement silencing with the induction of an immune response, coupling epigenetic and immune aspects of genome defense.

ChAHP2 and ChAHP control diverse retrotransposons by complementary activities.

Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against short interspersed element (SINE) retrotransposons that is composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, in which ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to endogenous retroviruses (ERVs) and long interspersed elements (LINEs) via HP1ß-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a manner mechanistically equivalent to that of ChAHP2 and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both nonautonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.

Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, precise RNA-mediated insertion of transgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

Perturbation of periodic spot-generation balance leads to diversified pigmentation patterning of harlequin Phalaenopsis orchids: in silico prediction.

BACKGROUND: A retrotransposon HORT1 in the promoter of the anthocyanin activator gene PeMYB11, microRNA858 (miR858) that targets PeMYB11, and a repressor PeMYBx have been implicated in pigmentation patterning diversity of harlequin Phalaenopsis orchids. However, the interrelationship among them remains to be elucidated. RESULTS: To understand how these factors interact to generate anthocyanin spots in Phalaenopsis, we successfully developed a mathematical model based on the known reaction-diffusion system to simulate their interplay and refined the conceptual biological model. Intriguingly, the expression of both PeMYBx and PeMYB11 were in phase for purple spot formation, even though they showed adverse effects on anthocyanin accumulations. An increase in the self-activation rate of PeMYB11 resulted in the increased size of purple spots, but no effects on spot fusion. Decreased degradation rate of miR858 in the purple regions, led to disruption of the formation of spotted pigmentation patterning and a full-red pigmentation pattern. Significantly, the reduced miR858 level promotes the fusion of large dark purple dots induced by the solo-LTR of HORT1, eventually generating the purple patches. In addition, the spatially heterogeneous insertion of HORT1 caused by the remnant solo-LTR of HORT1 derived from random homologous unequal recombination of HORT1 in individual cells of floral organs could explain the diverse pigmentation patterning of harlequin Phalaenopsis. CONCLUSIONS: This devised model explains how HORT1 and miR858 regulate the formation of the pigmentation patterning and holds great promise for developing efficient and innovative approaches to breeding harlequin Phalaenopsis orchids.

Evolutional heterochromatin condensation delineates chromocenter formation and retrotransposon silencing in plants.

Heterochromatic condensates (chromocenters) are critical for maintaining the silencing of heterochromatin. It is therefore puzzling that the presence of chromocenters is variable across plant species. Here we reveal that variations in the plant heterochromatin protein ADCP1 confer a diversity in chromocenter formation via phase separation. ADCP1 physically interacts with the high mobility group protein HMGA to form a complex and mediates heterochromatin condensation by multivalent interactions. The loss of intrinsically disordered regions (IDRs) in ADCP1 homologues during evolution has led to the absence of prominent chromocenter formation in various plant species, and introduction of IDR-containing ADCP1 with HMGA promotes heterochromatin condensation and retrotransposon silencing. Moreover, plants in the Cucurbitaceae group have evolved an IDR-containing chimaera of ADCP1 and HMGA, which remarkably enables formation of chromocenters. Together, our work uncovers a coevolved mechanism of phase separation in packing heterochromatin and silencing retrotransposons.

Differential Conservation and Loss of Chicken Repeat 1 (CR1) Retrotransposons in Squamates Reveal Lineage-Specific Genome Dynamics Across Reptiles.

Transposable elements (TEs) are repetitive DNA sequences which create mutations and generate genetic diversity across the tree of life. In amniote vertebrates, TEs have been mainly studied in mammals and birds, whose genomes generally display low TE diversity. Squamates (Order Squamata; including ∼11,000 extant species of lizards and snakes) show as much variation in TE abundance and activity as they do in species and phenotypes. Despite this high TE activity, squamate genomes are remarkably uniform in size. We hypothesize that novel, lineage-specific genome dynamics have evolved over the course of squamate evolution. To understand the interplay between TEs and host genomes, we analyzed the evolutionary history of the chicken repeat 1 (CR1) retrotransposon, a TE family found in most tetrapod genomes which is the dominant TE in most reptiles. We compared 113 squamate genomes to the genomes of turtles, crocodilians, and birds and used ancestral state reconstruction to identify shifts in the rate of CR1 copy number evolution across reptiles. We analyzed the repeat landscapes of CR1 in squamate genomes and determined that shifts in the rate of CR1 copy number evolution are associated with lineage-specific variation in CR1 activity. We then used phylogenetic reconstruction of CR1 subfamilies across amniotes to reveal both recent and ancient CR1 subclades across the squamate tree of life. The patterns of CR1 evolution in squamates contrast other amniotes, suggesting key differences in how TEs interact with different host genomes and at different points across evolutionary history.

All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Retrotransposon-mediated disruption of a chitin synthase gene confers insect resistance to Bacillus thuringiensis Vip3Aa toxin.

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.

The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator.

In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription.

A sequence of SVA retrotransposon insertions in ASIP shaped human pigmentation.

Retrotransposons comprise about 45% of the human genome1, but their contributions to human trait variation and evolution are only beginning to be explored2,3. Here, we find that a sequence of SVA retrotransposon insertions in an early intron of the ASIP (agouti signaling protein) gene has probably shaped human pigmentation several times. In the UK Biobank (n = 169,641), a recent 3.3-kb SVA insertion polymorphism associated strongly with lighter skin pigmentation (0.22 [0.21-0.23] s.d.; P = 2.8 × 10-351) and increased skin cancer risk (odds ratio = 1.23 [1.18-1.27]; P = 1.3 × 10-28), appearing to underlie one of the strongest common genetic influences on these phenotypes within European populations4-6. ASIP expression in skin displayed the same association pattern, with the SVA insertion allele exhibiting 2.2-fold (1.9-2.6) increased expression. This effect had an unusual apparent mechanism: an earlier, nonpolymorphic, human-specific SVA retrotransposon 3.9 kb upstream appeared to have caused ASIP hypofunction by nonproductive splicing, which the new (polymorphic) SVA insertion largely eliminated. Extended haplotype homozygosity indicated that the insertion allele has risen to allele frequencies up to 11% in European populations over the past several thousand years. These results indicate that a sequence of retrotransposon insertions contributed to a species-wide increase, then a local decrease, of human pigmentation.

Comprehensive analysis of the Xya riparia genome uncovers the dominance of DNA transposons, LTR/Gypsy elements, and their evolutionary dynamics.

Transposable elements (TEs) are DNA sequences that can move or replicate within a genome, and their study has become increasingly important in understanding genome evolution and function. The Tridactylidae family, including Xya riparia (pygmy mole cricket), harbors a variety of transposable elements (TEs) that have been insufficiently investigated. Further research is required to fully understand their diversity and evolutionary characteristics. Hence, we conducted a comprehensive repeatome analysis of X. riparia species using the chromosome-level assembled genome. The study aimed to comprehensively analyze the abundance, distribution, and age of transposable elements (TEs) in the genome. The results indicated that the genome was 1.67 Gb, with 731.63 Mb of repetitive sequences, comprising 27% of Class II (443.25 Mb), 16% of Class I (268.45 Mb), and 1% of unknown TEs (19.92 Mb). The study found that DNA transposons dominate the genome, accounting for approximately 60% of the total repeat size, with retrotransposons and unknown elements accounting for 37% and 3% of the genome, respectively. The members of the Gypsy superfamily were the most abundant amongst retrotransposons, accounting for 63% of them. The transposable superfamilies (LTR/Gypsy, DNA/nMITE, DNA/hAT, and DNA/Helitron) collectively constituted almost 70% of the total repeat size of all six chromosomes. The study further unveiled a significant linear correlation (Pearson correlation: r = 0.99, p-value = 0.00003) between the size of the chromosomes and the repetitive sequences. The average age of DNA transposon and retrotransposon insertions ranges from 25 My (million years) to 5 My. The satellitome analysis discovered 13 satellite DNA families that comprise about 0.15% of the entire genome. In addition, the transcriptional analysis of TEs found that DNA transposons were more transcriptionally active than retrotransposons. Overall, the study suggests that the genome of X. riparia is complex, characterized by a substantial portion of repetitive elements. These findings not only enhance our understanding of TE evolution within the Tridactylidae family but also provide a foundation for future investigations into the genomic intricacies of related species.

A catalogue of recombination coldspots in interspecific tomato hybrids.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

LTR retrotransposon-derived LncRNA LINC01446 promotes hepatocellular carcinoma progression and angiogenesis by regulating the SRPK2/SRSF1/VEGF axis.

The causal link between long terminal repeat (LTR) retrotransposon-derived lncRNAs and hepatocellular carcinoma (HCC) remains elusive and whether these cancer-exclusive lncRNAs contribute to the effectiveness of current HCC therapies is yet to explore. Here, we investigated the activation of LTR retrotransposon-derived lncRNAs in a broad range of liver diseases. We found that LTR retrotransposon-derived lncRNAs are mainly activated in HCC and is correlated with the proliferation status of HCC. Furthermore, we discovered that an LTR retrotransposon-derived lncRNA, LINC01446, exhibits specific expression in HCC. HCC patients with higher LINC01446 expression had shorter overall survival times. In vitro and in vivo assays showed that LINC01446 promoted HCC growth and angiogenesis. Mechanistically, LINC01446 bound to serine/arginine protein kinase 2 (SRPK2) and activated its downstream target, serine/arginine splicing factor 1 (SRSF1). Furthermore, activation of the SRPK2-SRSF1 axis increased the splicing and expression of VEGF isoform A165 (VEGFA165). Notably, inhibiting LINC01446 expression dramatically impaired tumor growth in vivo and resulted in better therapeutic outcomes when combined with antiangiogenic agents. In addition, we found that the transcription factor MESI2 bound to the cryptic MLT2B3 LTR promoter and drove LINC01446 transcription in HCC cells. Taken together, our findings demonstrate that LTR retrotransposon-derived LINC01446 promotes the progression of HCC by activating the SRPK2/SRSF1/VEGFA165 axis and highlight targeting LINC01446 as a potential therapeutic strategy for HCC patients.

The transcription factor PAX5 activates human LINE1 retrotransposons to induce cellular senescence.

As a hallmark of senescent cells, the derepression of Long Interspersed Elements 1 (LINE1) transcription results in accumulated LINE1 cDNA, which triggers the secretion of the senescence-associated secretory phenotype (SASP) and paracrine senescence in a cGAS-STING pathway-dependent manner. However, transcription factors that govern senescence-associated LINE1 reactivation remain ill-defined. Here, we predict several transcription factors that bind to human LINE1 elements to regulate their transcription by analyzing the conserved binding motifs in the 5'-untranslated regions (UTR) of the commonly upregulated LINE1 elements in different types of senescent cells. Further analysis reveals that PAX5 directly binds to LINE1 5'-UTR and the binding is enhanced in senescent cells. The enrichment of PAX5 at the 5'-UTR promotes cellular senescence and SASP by activating LINE1. We also demonstrate that the longevity gene SIRT6 suppresses PAX5 transcription by directly binding to the PAX5 promoter, and overexpressing PAX5 abrogates the suppressive effect of SIRT6 on stress-dependent cellular senescence. Our work suggests that PAX5 could serve as a potential target for drug development aiming to suppress LINE1 activation and treat senescence-associated diseases.

Pioneer Transcription Factors: The First Domino in Zygotic Genome Activation.

Zygotic genome activation (ZGA) is a pivotal event in mammalian embryogenesis, marking the transition from maternal to zygotic control of development. During the ZGA process that is characterized by the intricate cascade of gene expression, who tipped the first domino in a meticulously arranged sequence is a subject of paramount interest. Recently, Dux, Obox and Nr5a2 were identified as pioneer transcription factors that reside at the top of transcriptional hierarchy. Through co-option of retrotransposon elements as hubs for transcriptional activation, these pioneer transcription factors rewire the gene regulatory network, thus initiating ZGA. In this review, we provide a snapshot of the mechanisms underlying the functions of these pioneer transcription factors. We propose that ZGA is the starting point where the embryo's own genome begins to influence development trajectory, therefore in-depth dissecting the functions of pioneer transcription factors during ZGA will form a cornerstone of our understanding for early embryonic development, which will pave the way for advancing our grasp of mammalian developmental biology and optimizing in vitro production (IVP) techniques.

Comprehensive Identification and Characterization of HML-9 Group in Chimpanzee Genome.

Endogenous retroviruses (ERVs) are related to long terminal repeat (LTR) retrotransposons, comprising gene sequences of exogenous retroviruses integrated into the host genome and inherited according to Mendelian law. They are considered to have contributed greatly to the evolution of host genome structure and function. We previously characterized HERV-K HML-9 in the human genome. However, the biological function of this type of element in the genome of the chimpanzee, which is the closest living relative of humans, largely remains elusive. Therefore, the current study aims to characterize HML-9 in the chimpanzee genome and to compare the results with those in the human genome. Firstly, we report the distribution and genetic structural characterization of the 26 proviral elements and 38 solo LTR elements of HML-9 in the chimpanzee genome. The results showed that the distribution of these elements displayed a non-random integration pattern, and only six elements maintained a relatively complete structure. Then, we analyze their phylogeny and reveal that the identified elements all cluster together with HML-9 references and with those identified in the human genome. The HML-9 integration time was estimated based on the 2-LTR approach, and the results showed that HML-9 elements were integrated into the chimpanzee genome between 14 and 36 million years ago and into the human genome between 18 and 49 mya. In addition, conserved motifs, cis-regulatory regions, and enriched PBS sequence features in the chimpanzee genome were predicted based on bioinformatics. The results show that pathways significantly enriched for ERV LTR-regulated genes found in the chimpanzee genome are closely associated with disease development, including neurological and neurodevelopmental psychiatric disorders. In summary, the identification, characterization, and genomics of HML-9 presented here not only contribute to our understanding of the role of ERVs in primate evolution but also to our understanding of their biofunctional significance.