RESUMEN
Long interspersed elements-1 (LINE-1, L1) are retrotransposons that hold the capacity of self-propagation in the genome with potential mutagenic outcomes. How somatic cells restrict L1 activity and how this process becomes dysfunctional during aging and in cancer cells is poorly understood. L1s are enriched at lamin-associated domains, heterochromatic regions of the nuclear periphery. Whether this association is necessary for their repression has been elusive. Here we show that the sirtuin family member SIRT7 participates in the epigenetic transcriptional repression of L1 genome-wide in both mouse and human cells. SIRT7 depletion leads to increased L1 expression and retrotransposition. Mechanistically, we identify a novel interplay between SIRT7 and Lamin A/C in L1 repression. Our results demonstrate that SIRT7-mediated H3K18 deacetylation regulates L1 expression and promotes L1 association with elements of the nuclear lamina. The failure of such activity might contribute to the observed genome instability and compromised viability in SIRT7 knockout mice. Overall, our results reveal a novel function of SIRT7 on chromatin organization by mediating the anchoring of L1 to the nuclear envelope, and a new functional link of the nuclear lamina with transcriptional repression.
Asunto(s)
Genoma , Lamina Tipo A/genética , Elementos de Nucleótido Esparcido Largo , Sirtuinas/genética , Transcripción Genética , Animales , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Epigénesis Genética , Fibroblastos/citología , Fibroblastos/metabolismo , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Lamina Tipo A/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Miocardio/citología , Miocardio/metabolismo , Lámina Nuclear/metabolismo , Lámina Nuclear/ultraestructura , Sirtuinas/deficiencia , Sirtuinas/metabolismo , Testículo/citología , Testículo/metabolismoRESUMEN
Long interspersed element type 1 (LINE-1; L1) mobilizes during early embryogenesis, neurogenesis, and germ cell development, accounting for 25% of disease-causing heritable insertions and 98% of somatic insertions in cancer. To better understand the regulation and impact of L1 mobilization in the genome, reliable methods for measuring L1 copy number variation (CNV) are needed. Here we present a comprehensive analysis of a droplet digital PCR (ddPCR) based method for quantifying endogenous mouse L1. We provide experimental evidence that ddPCR assays can be designed to target specific L1 subfamilies using diagnostic single nucleotide polymorphisms (SNPs). The target and off-target L1 subfamilies form distinct droplet clusters, which were experimentally verified using both synthetic gene fragments and endogenous L1 derived plasmid clones. We further provide a roadmap for in silico assay design and evaluation of target specificity, ddPCR testing, and optimization for L1 CNV quantification. The assay can achieve a sensitivity of 5% CNV with 8 technical replicates. With 24 technical replicates, it can detect 2% CNV because of the increased precision. The same approach will serve as a guide for the development of ddPCR based assays for quantifying human L1 copy number and any other high copy genomic target sequences.
Asunto(s)
Elementos de Nucleótido Esparcido Largo/genética , Reacción en Cadena de la Polimerasa , Animales , RatonesRESUMEN
The PIWI-interacting RNA (piRNA) pathway is essential for retrotransposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter. Importantly, DNA methylation dynamics of a single-copy transgene were indistinguishable from those of endogenous L1s. Analysis of Mov10l1-/- testes established that de novo methylation of the L1 transgene required the intact piRNA pathway. Consistent with loss of DNA methylation and programmed reduction of H3K9me2 at meiotic onset, the transgene showed 1,400-fold increase in RNA expression and consequently 70-fold increase in retrotransposition in postnatal day 14 Mov10l1-/- germ cells compared with the wild-type. Analysis of adult Mov10l1-/- germ-cell fractions indicated a stage-specific increase of retrotransposition in the early meiotic prophase. However, extrapolation of the transgene data to endogenous L1s suggests that it is unlikely insertional mutagenesis alone accounts for the Mov10l1-/- phenotype. Indeed, pharmacological inhibition of reverse transcription did not rescue the meiotic defect. Cumulatively, these results establish the occurrence of productive L1 mobilization in the absence of an intact piRNA pathway but leave open the possibility of processes preceding L1 integration in triggering meiotic checkpoints and germ-cell death. Additionally, our data suggest that many heritable L1 insertions originate from individuals with partially compromised piRNA defense.
Asunto(s)
Meiosis , ARN Interferente Pequeño/metabolismo , Retroelementos , Transgenes , Regiones no Traducidas 5' , Animales , Codón , Metilación de ADN , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Masculino , Metilación , Ratones , Ratones Transgénicos , Sistemas de Lectura Abierta , Fenotipo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Espermatocitos/metabolismo , Espermatogénesis , Testículo/metabolismoRESUMEN
Influenza B virus (FLUBV) is an important pathogen that infects humans and causes seasonal influenza epidemics. To date, little is known about defective genomes of FLUBV and their roles in viral replication. In this study, by using a next-generation sequencing approach, we analyzed total mRNAs extracted from A549 cells infected with B/Brisbane/60/2008 virus (Victoria lineage), and identified four defective FLUBV genomes with two (PB1∆A and PB1∆B) from the polymerase basic subunit 1 (PB1) segment and the other two (M∆A and M∆B) from the matrix (M) protein-encoding segment. These defective genomes contained significant deletions in the central regions with each having the potential for encoding a novel polypeptide. Significantly, each of the discovered defective RNAs can potently inhibit the replication of B/Yamanashi/166/98 (Yamagata lineage). Furthermore, PB1∆A was able to interfere modestly with influenza A virus (FLUAV) replication. In summary, our study provides important initial insights into FLUBV defective-interfering genomes, which can be further explored to achieve better understanding of the replication, pathogenesis and evolution of FLUBV.
Asunto(s)
Virus Defectuosos/genética , Virus de la Influenza B/genética , Gripe Humana/virología , ARN Viral/genética , Células A549 , Virus Defectuosos/fisiología , Genoma Viral , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Virus de la Influenza B/fisiología , Proteínas Virales/genética , Replicación ViralRESUMEN
Long interspersed elements (LINEs), through both self-mobilization and trans-mobilization of short interspersed elements and processed pseudogenes, have made an indelible impact on the structure and function of the human genome. One consequence is the creation of new CpG islands (CGIs). In fact, more than half of all CGIs in the genome are associated with repetitive DNA, three-quarters of which are derived from retrotransposons. However, little is known about the epigenetic impact of newly inserted CGIs. We utilized a transgenic LINE-1 mouse model and tracked DNA methylation dynamics of individual germline insertions during mouse development. The retrotransposed GFP marker sequence, a strong CGI, is hypomethylated in male germ cells but hypermethylated in somatic tissues, regardless of genomic location. The GFP marker is similarly methylated when delivered into the genome via the Sleeping Beauty DNA transposon, suggesting that the observed methylation pattern may be independent of the mode of insertion. Comparative analyses between insertion- and non-insertion-containing alleles further reveal a graded influence of the retrotransposed CGI on flanking CpG sites, a phenomenon that we described as "sloping shores." Computational analyses of human and mouse methylomic data at single-base resolution confirm that sloping shores are universal for hypomethylated CGIs in sperm and somatic tissues. Additionally, the slope of a hypomethylated CGI can be affected by closely positioned CGI neighbors. Finally, by tracing sloping shore dynamics through embryonic and germ cell reprogramming, we found evidence of bookmarking, a mechanism that likely determines which CGIs will be eventually hyper- or hypomethylated.
Asunto(s)
Islas de CpG , Elementos de Nucleótido Esparcido Largo , Ratones Transgénicos/crecimiento & desarrollo , Ratones Transgénicos/genética , Animales , Biología Computacional/métodos , Metilación de ADN , Elementos Transponibles de ADN , Epigénesis Genética , Genoma , Humanos , Masculino , Ratones , Espermatozoides/crecimiento & desarrolloAsunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Epigénesis Genética , Regulación de la Expresión Génica/fisiología , Mamíferos/metabolismo , Espermatogénesis/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Metilación de ADN , Humanos , Masculino , Mamíferos/genética , ARN/genética , ARN/metabolismo , Espermatogénesis/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: The internal promoter in L1 5'UTR is critical for autonomous L1 transcription and initiating retrotransposition. Unlike the human genome, which features one contemporarily active subfamily, four subfamilies (A_I, Gf_I and Tf_I/II) have been amplifying in the mouse genome in the last one million years. Moreover, mouse L1 5'UTRs are organized into tandem repeats called monomers, which are separated from ORF1 by a tether domain. In this study, we aim to compare promoter activities across young mouse L1 subfamilies and investigate the contribution of individual monomers and the tether sequence. RESULTS: We observed an inverse relationship between subfamily age and the average number of monomers among evolutionarily young mouse L1 subfamilies. The youngest subgroup (A_I and Tf_I/II) on average carry 3-4 monomers in the 5'UTR. Using a single-vector dual-luciferase reporter assay, we compared promoter activities across six L1 subfamilies (A_I/II, Gf_I and Tf_I/II/III) and established their antisense promoter activities in a mouse embryonic fibroblast cell line and a mouse embryonal carcinoma cell line. Using consensus promoter sequences for three subfamilies (A_I, Gf_I and Tf_I), we dissected the differential roles of individual monomers and the tether domain in L1 promoter activity. We validated that, across multiple subfamilies, the second monomer consistently enhances the overall promoter activity. For individual promoter components, monomer 2 is consistently more active than the corresponding monomer 1 and/or the tether for each subfamily. Importantly, we revealed intricate interactions between monomer 2, monomer 1 and tether domains in a subfamily-specific manner. Furthermore, using three-monomer 5'UTRs, we established a complex nonlinear relationship between the length of the outmost monomer and the overall promoter activity. CONCLUSIONS: The laboratory mouse is an important mammalian model system for human diseases as well as L1 biology. Our study extends previous findings and represents an important step toward a better understanding of the molecular mechanism controlling mouse L1 transcription as well as L1's impact on development and disease.
RESUMEN
Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.
Asunto(s)
Silenciador del Gen , Leucemia Mieloide/genética , Elementos de Nucleótido Esparcido Largo/genética , Animales , Sistemas CRISPR-Cas/genética , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Genoma Humano , Inestabilidad Genómica , Hematopoyesis/genética , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/genéticaRESUMEN
Influenza D virus (IDV) utilizes bovines as a primary reservoir with periodical spillover to other mammalian hosts. By using traditional hemagglutination assay coupled with sialoglycan microarray (SGM) platform and functional assays, we demonstrated that IDV is more efficient in recognizing both 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac2) and 9-O-acetylated N-glycolylneuraminic acid (Neu5Gc9Ac) than influenza C virus (ICV), a ubiquitous human pathogen. ICV seems to strongly prefer Neu5,9Ac2 over Neu5Gc9Ac. Since Neu5Gc9Ac is different from Neu5,9Ac2 only by an additional oxygen in the group at the C5 position, our results reveal that the hydroxyl group in Neu5Gc9Ac plays a critical role in determining receptor binding specificity, which as a result may discriminate IDV from ICV in communicating with 9-O-acetylated SAs. These findings shall provide a framework for further investigation towards better understanding of how newly discovered multiple-species-infecting IDV exploits natural 9-O-acetylated SA variations to expand its host range.
Asunto(s)
Gammainfluenzavirus/metabolismo , Gripe Humana/metabolismo , Polisacáridos/metabolismo , Receptores Virales/metabolismo , Thogotovirus/metabolismo , Humanos , Gripe Humana/virología , Gammainfluenzavirus/genética , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/química , Receptores Virales/química , Ácidos Siálicos/metabolismo , Thogotovirus/clasificación , Thogotovirus/genética , Thogotovirus/aislamiento & purificaciónRESUMEN
BACKGROUND: Transposable elements (TEs) make up > 50% of the human genome, and the majority of retrotransposon insertions are truncated and many are located in introns. However, the effects of retrotransposition on the host genes remain incompletely known. RESULTS: We report here that insertion of a chimeric L1 (cL1), but not IAP solo LTR, into intron 6 of Axin1 using CRIPSR/Cas9 induced the kinky tail phenotype with ~ 80% penetrance in heterozygous Axin cL1 mice. Both penetrant (with kinky tails) and silent (without kinky tails) Axin cL1 mice, regardless of sex, could transmit the phenotype to subsequent generations with similar penetrance (~ 80%). Further analyses revealed that a longer Axin1 transcript isoform containing partial cL1-targeted intron was present in penetrant, but absent in silent and wild type mice, and the production of this unique Axin1 transcript appeared to correlate with altered levels of an activating histone modification, H3K9ac. CONCLUSIONS: The mechanism for Axin cL1 mice is different from those previously identified in mice with spontaneous retrotransposition of IAP, e.g., Axin Fu and A vy , both of which have been associated with DNA methylation changes. Our data suggest that Axin1 locus is sensitive to genetic and epigenetic alteration by retrotransposons and thus, ideally suited for studying the effects of new retrotransposition events on target gene function in mice.