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1.
Methods Mol Biol ; 2577: 147-159, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36173571

RESUMO

Long Interspersed Element-1 (LINE-1, L1) is a retrotransposon that has the ability to amplify its copy in the genome autonomously. L1Hs is a human-specific active subtype of L1 reported to amplify its copy in neural progenitor cells causing genomic mosaicism. This chapter describes a new method named NECO-seq (Novel Elements Concentrated-sequence) to identify the genomic locus of L1Hs insertions at the single-cell level. This protocol contains the steps of (1) preparation of neuronal cell nuclei from a postmortem human brain, (2) whole genome amplification from single neural nuclei (snWGA), (3) single nucleotide polymorphisms (SNPs) genotyping for quality control of snWGA products, (4) library preparation for next-generation sequencing to enrich the genomic locus of L1Hs insertions, and (5) bioinformatic analysis to detect novel somatic L1Hs insertions. This method can detect approximately 97% of L1Hs originally existing in reference human genome and approximately 10-20 newly inserted L1Hs copies in a neuronal cell of a postmortem human brain.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Retroelementos , Encéfalo , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Retroelementos/genética
2.
Gene ; 850: 146943, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36198378

RESUMO

Long interspersed element-1 (LINE-1, L1) transposable element (TE) composes about 17% of the human genome. However, genetic and biochemical interactions between L1 and hepatitis B virus (HBV) remain poorly understood. In this study, I found that HBV restricts L1 retrotransposition in a reverse transcriptase (RT)-independent manner. Notably, HBV polymerase (Pol) strongly inhibited L1 retrotransposition. Indeed, the ribonuclease H (RNase H) domain was essential for inhibition of L1 retrotransposition. The L1 ORF1p RNA-binding protein predominantly localized into cytoplasmic RNA granule termed P-body. However, HBV Pol hijacked L1 ORF1p from P-body through an interaction with L1 ORF1p, when both proteins were co-expressed. Furthermore, HBV Pol repressed the L1 5' untranslated region (UTR). Altogether, HBV seems to restrict L1 mobility at multiple steps. Thus, these results suggest a novel function or activity of HBV Pol in regulation of L1 retrotransposition.


Assuntos
Elementos de DNA Transponíveis , Vírus da Hepatite B , Elementos Nucleotídeos Longos e Dispersos , DNA Polimerase Dirigida por RNA , Humanos , Regiões 5' não Traduzidas , Elementos de DNA Transponíveis/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Ribonuclease H/genética , Ribonuclease H/metabolismo , Proteínas de Ligação a RNA/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo
3.
Sci Adv ; 8(47): eabq3806, 2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36417507

RESUMO

Transposable elements (TEs) are genomic parasites that propagate within the host genome and introduce mutations. Long interspersed nuclear element-1 (LINE-1 or L1) is the major TE class, which occupies nearly 20% of the mouse genome. L1 is highly active in mammalian preimplantation embryos, posing a major threat to genome integrity, but the mechanism of stage-specific protection against L1 retrotransposition is unknown. Here, we show that TAR DNA-binding protein 43 (TDP-43), mutations in which constitute a major risk factor for amyotrophic lateral sclerosis, inhibits L1 retrotransposition in mouse embryonic stem cells (mESCs) and preimplantation embryos. Knockdown of TDP-43 resulted in massive genomic L1 expansion and impaired cell growth in preimplantation embryos and ESCs. Functional analysis demonstrated that TDP-43 interacts with L1 open reading frame 1 protein (L1 ORF1p) to mediate genomic protection, and loss of this interaction led to derepression of L1 retrotransposition. Our results identify TDP-43 as a guardian of the embryonic genome.


Assuntos
Proteínas de Ligação a DNA , Elementos Nucleotídeos Longos e Dispersos , Animais , Camundongos , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Células-Tronco Embrionárias Murinas , Fases de Leitura Aberta , Mamíferos/genética
4.
Genes (Basel) ; 13(11)2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36360249

RESUMO

BACKGROUND: Methotrexate (MTX) is considered the first choice among disease-modifying anti-rheumatic drugs (DMARDs) for rheumatoid arthritis (RA) treatment. However, response to it varies as approximately 40% of the patients do not respond and would lose the most effective period of treatment time. Therefore, having a predictive biomarker before starting MTX treatment is of utmost importance. Methylation of long interspersed nucleotide element-1 (LINE-1) is generally considered a surrogate marker for global genomic methylation, which has been reported to associate with disease activity after MTX therapy. METHODS: We performed a prospective study on 273 naïve early RA (ERA) patients who were treated with MTX, followed up to 12 months, and classified according to their therapy response. The baseline LINE-1 methylation levels in peripheral blood mononuclear cells (PBMC) of cases were assessed by bisulfite pyrosequencing. RESULTS: Baseline LINE-1 methylation level per se turned out not to predict the response to the therapy, nor did age, sex, body mass index, or smoking status. However, if cases were stratified according to positivity to rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) or seronegativity, we observed an opposite association between baseline LINE-1 methylation levels and optimal response to MTX therapy among responders. The best response to MTX therapy was associated with hypermethylated LINE-1 among double-positive ERA cases (p-value: 0.002) and with hypomethylated LINE-1 in seronegative ERA patients (p-value: 0.01). CONCLUSION: The LINE-1 methylation level in PBMCs of naïve ERA cases associates with the degree of response to MTX therapy in an opposite way depending on the presence of RF and ACPA antibodies. Our results suggest LINE-1 methylation level as a new epigenetic biomarker for predicting the degree of response to MTX in both double-positive and seronegative ERA patients.


Assuntos
Antirreumáticos , Artrite Reumatoide , Humanos , Metotrexato/uso terapêutico , Leucócitos Mononucleares , Estudos Prospectivos , Metilação , Elementos Nucleotídeos Longos e Dispersos/genética , Resultado do Tratamento , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Antirreumáticos/uso terapêutico , Biomarcadores , Anticorpos/uso terapêutico
5.
Int J Mol Sci ; 23(19)2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36232908

RESUMO

Long interspersed nuclear element 1 (LINE-1) bisulfite pyrosequencing is a widely used technique for genome-wide methylation analyses. We aimed to investigate the effects of experimental and biological factors on its results to improve the comparability. LINE-1 bisulfite pyrosequencing was performed on colorectal tissue (n = 222), buffy coat (n = 39), and plasma samples (n = 9) of healthy individuals and patients with colorectal tumors. Significantly altered methylation was observed between investigated LINE-1 CpG positions of non-tumorous tissues (p ≤ 0.01). Formalin-fixed, paraffin-embedded biopsies (73.0 ± 5.3%) resulted in lower methylation than fresh frozen samples (76.1 ± 2.8%) (p ≤ 0.01). DNA specimens after long-term storage showed higher methylation levels (+3.2%, p ≤ 0.01). In blood collection tubes with preservatives, cfDNA and buffy coat methylation significantly changed compared to K3EDTA tubes (p ≤ 0.05). Lower methylation was detected in older (>40 years, 76.8 ± 1.7%) vs. younger (78.1 ± 1.0%) female patients (p ≤ 0.05), and also in adenomatous tissues with MTHFR 677CT, or 1298AC mutations vs. wild-type (p ≤ 0.05) comparisons. Based on our findings, it is highly recommended to consider the application of standard DNA samples in the case of a possible clinical screening approach, as well as in experimental research studies.


Assuntos
Ácidos Nucleicos Livres , Neoplasias Colorretais , Idoso , Fatores Biológicos , Biópsia , Ácidos Nucleicos Livres/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA/genética , Metilação de DNA , Feminino , Formaldeído , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Sulfitos
6.
Genes (Basel) ; 13(10)2022 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-36292627

RESUMO

This work focuses on the distribution of LINE-1 (a Long Interspersed Nuclear Element) in primates and its role during evolution and as a constituent of the architecture of primate genomes. To pinpoint the LINE-1 repeat distribution and its role among primates, LINE-1 probes were mapped onto chromosomes of Homo sapiens (Hominidae, Catarrhini), Sapajus apella, and Cebus capucinus (Cebidae, Platyrrhini) using fluorescence in situ hybridisation (FISH). The choice of platyrrhine species are due to the fact they are taxa characterised by a high level of rearrangements; for this reason, they could be a useful model for the study of LINE-1 and chromosome evolution. LINE-1 accumulation was found in the two Cebidae at the centromere of almost all acrocentric chromosomes 16-22 and on some bi-armed chromosomes. LINE-1 pattern was similar in the two species but only for chromosomes 6, 8, 10, and 18, due to intrachromosomal rearrangements in agreement with what was previously hypothesised as through g banding. LINE-1 interstitial accumulation was found in humans on the 1, 8, 9, 13-15, and X chromosomes; on chromosomes 8, 9, and 13-15, the signal was also at the centromeric position. This is in agreement with recent and complete molecular sequence analysis of human chromosomes 8 and some acrocentric ones. Thus, the hypothesis regarding a link between LINE-1 and centromeres as well as a link with rearrangements are discussed. Indeed, data analysis leads us to support a link between LINE-1 and inter- and intrachromosomal rearrangements, as well as a link between LINE-1 and structural functions at centromeres in primates.


Assuntos
Cebidae , Animais , Humanos , Cebidae/genética , Retroelementos/genética , Cariotipagem , Cebus/genética , Cromossomo X , Elementos Nucleotídeos Longos e Dispersos/genética
7.
Am J Hum Genet ; 109(10): 1850-1866, 2022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36150389

RESUMO

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval.


Assuntos
Azoospermia , Infertilidade Masculina , Animais , Azoospermia/genética , Humanos , Infertilidade Masculina/genética , Elementos Nucleotídeos Longos e Dispersos , Masculino , Camundongos , RNA Interferente Pequeno/metabolismo , Sêmen , Espermatogênese/genética , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Testículo/patologia
8.
BMC Bioinformatics ; 23(1): 375, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36100885

RESUMO

BACKGROUND: Long interspersed element 1 (LINE-1 or L1) retrotransposons are mobile elements that constitute 17-20% of the human genome. Strong correlations between abnormal L1 expression and several human diseases have been reported. This has motivated increasing interest in accurate quantification of the number of L1 copies present in any given biologic specimen. A main obstacle toward this aim is that L1s are relatively long DNA segments with regions of high variability, or largely present in the human genome as truncated fragments. These particularities render traditional alignment strategies, such as seed-and-extend inefficient, as the number of segments that are similar to L1s explodes exponentially. This study uses the pattern matching methodology for more accurate identification of L1s. We validate experimentally the superiority of pattern matching for L1 detection over alternative methods and discuss some of its potential applications. RESULTS: Pattern matching detected full-length L1 copies with high precision, reasonable computational time, and no prior input information. It also detected truncated and significantly altered copies of L1 with relatively high precision. The method was effectively used to annotate L1s in a target genome and to calculate copy number variation with respect to a reference genome. Crucial to the success of implementation was the selection of a small set of k-mer probes from a set of sequences presenting a stable pattern of distribution in the genome. As in seed-and-extend methods, the pattern matching algorithm sowed these k-mer probes, but instead of using heuristic extensions around the seeds, the analysis was based on distribution patterns within the genome. The desired level of precision could be adjusted, with some loss of recall. CONCLUSION: Pattern matching is more efficient than seed-and-extend methods for the detection of L1 segments whose characterization depends on a finite set of sequences with common areas of low variability. We propose that pattern matching may help establish correlations between L1 copy number and disease states associated with L1 mobilization and evolution.


Assuntos
Variações do Número de Cópias de DNA , Genoma Humano , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos
9.
Nucleic Acids Res ; 50(18): 10680-10694, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36169232

RESUMO

Condensin I and condensin II are multi-subunit complexes that are known for their individual roles in genome organization and preventing genomic instability. However, interactions between condensin I and condensin II subunits and cooperative roles for condensin I and condensin II, outside of their genome organizing functions, have not been reported. We previously discovered that condensin II cooperates with Gamma Interferon Activated Inhibitor of Translation (GAIT) proteins to associate with Long INterspersed Element-1 (LINE-1 or L1) RNA and repress L1 protein expression and the retrotransposition of engineered L1 retrotransposition in cultured human cells. Here, we report that the L1 3'UTR is required for condensin II and GAIT association with L1 RNA, and deletion of the L1 RNA 3'UTR results in increased L1 protein expression and retrotransposition. Interestingly, like condensin II, we report that condensin I also binds GAIT proteins, associates with the L1 RNA 3'UTR, and represses L1 retrotransposition. We provide evidence that the condensin I protein, NCAPD2, is required for condensin II and GAIT protein association with L1 RNA. Furthermore, condensin I and condensin II subunits interact to form a L1-dependent super condensin complex (SCC) which is located primarily within the cytoplasm of both transformed and primary epithelial cells. These data suggest that increases in L1 expression in epithelial cells promote cytoplasmic condensin protein associations that facilitate a feedback loop in which condensins may cooperate to mediate L1 repression.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Complexos Multiproteicos/metabolismo , Regiões 3' não Traduzidas , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA , Humanos , Interferon gama/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética
10.
Neuron ; 110(20): 3278-3287.e8, 2022 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-36070749

RESUMO

Dysregulation of long interspersed nuclear element 1 (LINE-1, L1), a dominant class of transposable elements in the human genome, has been linked to neurodegenerative diseases, but whether elevated L1 expression is sufficient to cause neurodegeneration has not been directly tested. Here, we show that the cerebellar expression of L1 is significantly elevated in ataxia telangiectasia patients and strongly anti-correlated with the expression of epigenetic silencers. To examine the role of L1 in the disease etiology, we developed an approach for direct targeting of the L1 promoter for overexpression in mice. We demonstrated that L1 activation in the cerebellum led to Purkinje cell dysfunctions and degeneration and was sufficient to cause ataxia. Treatment with a nucleoside reverse transcriptase inhibitor blunted ataxia progression by reducing DNA damage, attenuating gliosis, and reversing deficits of molecular regulators for calcium homeostasis in Purkinje cells. Our study provides the first direct evidence that L1 activation can drive neurodegeneration.


Assuntos
Elementos de DNA Transponíveis , Inibidores da Transcriptase Reversa , Animais , Humanos , Camundongos , Ataxia/metabolismo , Cálcio/metabolismo , Cerebelo/metabolismo , Nucleosídeos/metabolismo , Células de Purkinje/fisiologia , Inibidores da Transcriptase Reversa/metabolismo , Elementos Nucleotídeos Longos e Dispersos
11.
Gene ; 843: 146799, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-35963498

RESUMO

The genetics of an individual is a crucial factor in understanding the risk of developing the neurodegenerative disease amyotrophic lateral sclerosis (ALS). There is still a large proportion of the heritability of ALS, particularly in sporadic cases, to be understood. Among others, active transposable elements drive inter-individual variability, and in humans long interspersed element 1 (LINE1, L1), Alu and SINE-VNTR-Alu (SVA) retrotransposons are a source of polymorphic insertions in the population. We undertook a pilot study to characterise the landscape of non-reference retrotransposon insertion polymorphisms (non-ref RIPs) in 15 control and 15 ALS individuals' whole genomes from Project MinE, an international project to identify potential genetic causes of ALS. The combination of two bioinformatics tools (mobile element locator tool (MELT) and TEBreak) identified on average 1250 Alu, 232 L1 and 77 SVA non-ref RIPs per genome across the 30 analysed. Further PCR validation of individual polymorphic retrotransposon insertions showed a similar level of accuracy for MELT and TEBreak. Our preliminary study did not identify a specific RIP or a significant difference in the total number of non-ref RIPs in ALS compared to control genomes. The use of multiple bioinformatic tools improved the accuracy of non-ref RIP detection and our study highlights the potential importance of studying these elements further in ALS.


Assuntos
Esclerose Amiotrófica Lateral , Doenças Neurodegenerativas , Esclerose Amiotrófica Lateral/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Doenças Neurodegenerativas/genética , Projetos Piloto , Retroelementos/genética , Sequenciamento Completo do Genoma
12.
Sci Transl Med ; 14(657): eabl6057, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35947677

RESUMO

Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 (Long Interspersed Nuclear Element-1; L1) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N-methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16, p21, activating transcription factor 3 (ATF3), matrix metallopeptidase 13 (MMP13), interleukin 1a (IL1a), BTG anti-proliferation factor 2 (BTG2), and growth arrest and DNA damage inducible beta (GADD45b). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.


Assuntos
Senilidade Prematura , Síndrome de Cockayne , Proteínas Imediatamente Precoces , Progéria , Senilidade Prematura/genética , Animais , Antígenos de Diferenciação , Heterocromatina , Histonas/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Lisina/metabolismo , Camundongos , Fenótipo , Progéria/genética , RNA , Telômero/genética , Proteínas Supressoras de Tumor/genética
13.
Sci Rep ; 12(1): 13970, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35978033

RESUMO

Long interspersed nucleotide element-1 (LINE-1) and Alu elements are retrotransposons whose abilities cause abnormal gene expression and genomic instability. Several studies have focused on DNA methylation profiling of gene regions, but the locus-specific methylation of LINE-1 and Alu elements has not been identified in autism spectrum disorder (ASD). Here we interrogated locus- and family-specific methylation profiles of LINE-1 and Alu elements in ASD whole blood using publicly-available Illumina Infinium 450 K methylation datasets from heterogeneous ASD and ASD variants (Chromodomain Helicase DNA-binding 8 (CHD8) and 16p11.2del). Total DNA methylation of repetitive elements were notably hypomethylated exclusively in ASD with CHD8 variants. Methylation alteration in a family-specific manner including L1P, L1H, HAL, AluJ, and AluS families were observed in the heterogeneous ASD and ASD with CHD8 variants. Moreover, LINE-1 and Alu methylation within target genes is inversely related to the expression level in each ASD variant. The DNA methylation signatures of the LINE-1 and Alu elements in ASD whole blood, as well as their associations with the expression of ASD-related genes, have been identified. If confirmed in future larger studies, these findings may contribute to the identification of epigenomic biomarkers of ASD.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Elementos Alu/genética , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Metilação de DNA , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética
14.
Cell ; 185(16): 3025-3040.e6, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35882231

RESUMO

Non-allelic recombination between homologous repetitive elements contributes to evolution and human genetic disorders. Here, we combine short- and long-DNA read sequencing of repeat elements with a new bioinformatics pipeline to show that somatic recombination of Alu and L1 elements is widespread in the human genome. Our analysis uncovers tissue-specific non-allelic homologous recombination hallmarks; moreover, we find that centromeres and cancer-associated genes are enriched for retroelements that may act as recombination hotspots. We compare recombination profiles in human-induced pluripotent stem cells and differentiated neurons and find that the neuron-specific recombination of repeat elements accompanies chromatin changes during cell-fate determination. Finally, we report that somatic recombination profiles are altered in Parkinson's and Alzheimer's disease, suggesting a link between retroelement recombination and genomic instability in neurodegeneration. This work highlights a significant contribution of the somatic recombination of repeat elements to genomic diversity in health and disease.


Assuntos
Genoma Humano , Retroelementos , Elementos Alu/genética , Recombinação Homóloga , Humanos , Elementos Nucleotídeos Longos e Dispersos , Sequências Repetitivas de Ácido Nucleico
15.
J Exp Med ; 219(8)2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35802137

RESUMO

Ionizing radiations (IR) alter hematopoietic stem cell (HSC) function on the long term, but the mechanisms underlying these effects are still poorly understood. We recently showed that IR induces the derepression of L1Md, the mouse young subfamilies of LINE-1/L1 retroelements. L1 contributes to gene regulatory networks. However, how L1Md are derepressed and impact HSC gene expression are not known. Here, we show that IR triggers genome-wide H3K9me3 decrease that occurs mainly at L1Md. Loss of H3K9me3 at intronic L1Md harboring NF-κB binding sites motifs but not at promoters is associated with the repression of HSC-specific genes. This is correlated with reduced NFKB1 repressor expression. TNF-α treatment rescued all these effects and prevented IR-induced HSC loss of function in vivo. This TNF-α/NF-κB/H3K9me3/L1Md axis might be important to maintain HSCs while allowing expression of immune genes during myeloid regeneration or damage-induced bone marrow ablation.


Assuntos
Células-Tronco Hematopoéticas , Histonas , Elementos Nucleotídeos Longos e Dispersos , NF-kappa B , Fator de Necrose Tumoral alfa , Animais , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Camundongos , NF-kappa B/metabolismo , Radiação Ionizante , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
16.
BMC Res Notes ; 15(1): 245, 2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-35799274

RESUMO

OBJECTIVE: Long INterspersed Element-1 (L1) is an autonomous transposable element in the genome. L1 transcripts that are not reverse transcribed back into the genome can accumulate in the cytoplasm and activate an inflammatory response via the cyclic GMP-AMP (cGAS)-STING pathway. We examined skeletal muscle L1 markers as well as STING protein levels in 10 older individuals (63 ± 11 y, BMI = 30.2 ± 6.8 kg/m2) with end-stage osteoarthritis (OA) undergoing total hip (THA, n = 4) or knee (TKA, n = 6) arthroplasty versus 10 young, healthy comparators (Y, 22 ± 2 y, BMI = 23.2 ± 2.5 kg/m2). For OA, muscle was collected from surgical (SX) and contralateral (CTL) sides whereas single vastus lateralis samples were collected from Y. RESULTS: L1 mRNA was higher in CTL and SX compared to Y (p < 0.001 and p = 0.001, respectively). Protein expression was higher in SX versus Y for ORF1p (p = 0.002) and STING (p = 0.022). While these data are preliminary due to limited n-sizes and the lack of a BMI-matched younger control group, higher L1 mRNA expression, ORF1p and STING protein are evident in older versus younger adults. More research is needed to determine whether cGAS-STING signaling contributes to heightened muscle inflammation during aging and/or OA.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Músculo Esquelético , Osteoartrite , Idoso , Biomarcadores/metabolismo , Humanos , Articulação do Joelho/metabolismo , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Nucleotidiltransferases/metabolismo , Osteoartrite/genética , RNA Mensageiro/genética , Adulto Jovem
18.
Nucleic Acids Res ; 50(15): 8690-8699, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-35871298

RESUMO

Long interspersed nuclear element 1 (L1) parasitized most vertebrates and constitutes ∼20% of the human genome. It encodes ORF1p and ORF2p which form an L1-ribonucleoprotein (RNP) with their encoding transcript that is copied into genomic DNA (retrotransposition). ORF1p binds single-stranded nucleic acid (ssNA) and exhibits NA chaperone activity. All vertebrate ORF1ps contain a coiled coil (CC) domain and we previously showed that a CC-retrotransposition null mutant prevented formation of stably bound ORF1p complexes on ssNA. Here, we compared CC variants using our recently improved method that measures ORF1p binding to ssDNA at different forces. Bound proteins decrease ssDNA contour length and at low force, retrotransposition-competent ORF1ps (111p and m14p) exhibit two shortening phases: the first is rapid, coincident with ORF1p binding; the second is slower, consistent with formation of tightly compacted complexes by NA-bound ORF1p. In contrast, two retrotransposition-null CC variants (151p and m15p) did not attain the second tightly compacted state. The C-terminal half of the ORF1p trimer (not the CC) contains the residues that mediate NA-binding. Our demonstrating that the CC governs the ability of NA-bound retrotransposition-competent trimers to form tightly compacted complexes reveals the biochemical phenotype of these coiled coil mutants.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Animais , DNA/química , DNA de Cadeia Simples/genética , Humanos , Ácidos Nucleicos , Fases de Leitura Aberta , Ribonucleoproteínas/metabolismo
19.
EMBO Rep ; 23(9): e54458, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35856394

RESUMO

LINE-1 (L1) retroelements have retained their ability to mobilize. Mechanisms regulating L1 mobility include DNA methylation in somatic cells and the piRNA pathway in the germline. During preimplantation stages of mouse embryonic development, however, both pathways are inactivated leading to a window necessitating alternate means of L1 regulation. We previously reported an increase in L1 levels in Dicer_KO mouse embryonic stem cells (mESCs), which was accompanied by only a marginal increase in retrotransposition, suggesting additional mechanisms suppressing L1 mobility. Here, we demonstrate that L1 ribonucleoprotein complexes (L1 RNP) accumulate as aggregates in the cytoplasm of Dicer_KO mESCs along with the RNA helicase MOV10. The combined overexpression of L1 ORF1p and MOV10 is sufficient to create L1 RNP aggregates. In Dicer_KO mESCs, MOV10 is upregulated due to the loss of its direct regulation by miRNAs. The newly discovered posttranscriptional regulation of Mov10, and its role in preventing L1 retrotransposition by driving cytosolic aggregation, provides routes to explore for therapy in disease conditions where L1s are upregulated.


Assuntos
Desenvolvimento Embrionário , MicroRNAs , Animais , Elementos Nucleotídeos Longos e Dispersos , Camundongos , MicroRNAs/metabolismo , RNA Interferente Pequeno/metabolismo , Retroelementos/genética
20.
Genome Res ; 32(7): 1298-1314, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35728967

RESUMO

The retrotransposon LINE-1 (L1) is central to the recent evolutionary history of the human genome and continues to drive genetic diversity and germline pathogenesis. However, the spatiotemporal extent and biological significance of somatic L1 activity are poorly defined and are virtually unexplored in other primates. From a single L1 lineage active at the divergence of apes and Old World monkeys, successive L1 subfamilies have emerged in each descendant primate germline. As revealed by case studies, the presently active human L1 subfamily can also mobilize during embryonic and brain development in vivo. It is unknown whether nonhuman primate L1s can similarly generate somatic insertions in the brain. Here we applied approximately 40× single-cell whole-genome sequencing (scWGS), as well as retrotransposon capture sequencing (RC-seq), to 20 hippocampal neurons from two rhesus macaques (Macaca mulatta). In one animal, we detected and PCR-validated a somatic L1 insertion that generated target site duplications, carried a short 5' transduction, and was present in ∼7% of hippocampal neurons but absent from cerebellum and nonbrain tissues. The corresponding donor L1 allele was exceptionally mobile in vitro and was embedded in PRDM4, a gene expressed throughout development and in neural stem cells. Nanopore long-read methylome and RNA-seq transcriptome analyses indicated young retrotransposon subfamily activation in the early embryo, followed by repression in adult tissues. These data highlight endogenous macaque L1 retrotransposition potential, provide prototypical evidence of L1-mediated somatic mosaicism in a nonhuman primate, and allude to L1 mobility in the brain over the past 30 million years of human evolution.


Assuntos
Encéfalo , Elementos Nucleotídeos Longos e Dispersos , Retroelementos , Animais , Proteínas de Ligação a DNA/genética , Macaca mulatta/genética , Neurônios , Retroelementos/genética , Fatores de Transcrição/genética
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