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1.
Mol Cell ; 84(15): 2801-2803, 2024 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-39121841

RESUMEN

In this issue of Molecular Cell, Danac et al.1 identify a second HUSH complex, HUSH2, that represses interferon-stimulated genes and, by competing for subunits with the canonical HUSH complex, couples LINE-1 (L1) retrotransposon transcription with immune activation.


Asunto(s)
Inmunidad Innata , Elementos de Nucleótido Esparcido Largo , Humanos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo
2.
Nat Genet ; 56(6): 1181-1192, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38769457

RESUMEN

Eukaryotic transcription factors (TFs) activate gene expression by recruiting cofactors to promoters. However, the relationships between TFs, promoters and their associated cofactors remain poorly understood. Here we combine GAL4-transactivation assays with comparative CRISPR-Cas9 screens to identify the cofactors used by nine different TFs and core promoters in human cells. Using this dataset, we associate TFs with cofactors, classify cofactors as ubiquitous or specific and discover transcriptional co-dependencies. Through a reductionistic, comparative approach, we demonstrate that TFs do not display discrete mechanisms of activation. Instead, each TF depends on a unique combination of cofactors, which influences distinct steps in transcription. By contrast, the influence of core promoters appears relatively discrete. Different promoter classes are constrained by either initiation or pause-release, which influences their dynamic range and compatibility with cofactors. Overall, our comparative cofactor screens characterize the interplay between TFs, cofactors and core promoters, identifying general principles by which they influence transcription.


Asunto(s)
Regiones Promotoras Genéticas , Factores de Transcripción , Activación Transcripcional , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Sistemas CRISPR-Cas , Transcripción Genética , Regulación de la Expresión Génica
3.
Nat Neurosci ; 27(7): 1274-1284, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38773348

RESUMEN

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.


Asunto(s)
Interneuronas , Elementos de Nucleótido Esparcido Largo , Parvalbúminas , Animales , Interneuronas/metabolismo , Interneuronas/fisiología , Ratones , Elementos de Nucleótido Esparcido Largo/genética , Parvalbúminas/metabolismo , Retroelementos/genética , Masculino , Neurogénesis/fisiología , Neurogénesis/genética , Ratones Endogámicos C57BL , Regulación del Desarrollo de la Expresión Génica/genética
4.
Biochem Soc Trans ; 52(3): 1431-1447, 2024 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-38747720

RESUMEN

Whole genome sequencing of viruses provides high-resolution molecular insights, enhancing our understanding of viral genome function and phylogeny. Beyond fundamental research, viral sequencing is increasingly vital for pathogen surveillance, epidemiology, and clinical applications. As sequencing methods rapidly evolve, the diversity of viral genomics applications and catalogued genomes continues to expand. Advances in long-read, single molecule, real-time sequencing methodologies present opportunities to sequence contiguous, haplotype resolved viral genomes in a range of research and applied settings. Here we present an overview of nucleic acid sequencing methods and their applications in studying viral genomes. We emphasise the advantages of different viral sequencing approaches, with a particular focus on the benefits of third-generation sequencing technologies in elucidating viral evolution, transmission networks, and pathogenesis.


Asunto(s)
Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Virus/genética , Genómica/métodos , Filogenia , Secuenciación Completa del Genoma/métodos , Análisis de Secuencia de ADN/métodos
5.
bioRxiv ; 2024 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-38352366

RESUMEN

The O- GlcNAc transferase OGT interacts robustly with all three mammalian TET methylcytosine dioxygenases. We show here that deletion of the Ogt gene in mouse embryonic stem cells (mESC) results in a widespread increase in the TET product 5-hydroxymethylcytosine (5hmC) in both euchromatic and heterochromatic compartments, with concomitant reduction of the TET substrate 5-methylcytosine (5mC) at the same genomic regions. mESC engineered to abolish the TET1-OGT interaction likewise displayed a genome-wide decrease of 5mC. DNA hypomethylation in OGT-deficient cells was accompanied by de-repression of transposable elements (TEs) predominantly located in heterochromatin, and this increase in TE expression was sometimes accompanied by increased cis -expression of genes and exons located 3' of the expressed TE. Thus, the TET-OGT interaction prevents DNA demethylation and TE expression in heterochromatin by restraining TET activity genome-wide. We suggest that OGT protects the genome against DNA hypomethylation and impaired heterochromatin integrity, preventing the aberrant increase in TE expression observed in cancer, autoimmune-inflammatory diseases, cellular senescence and ageing.

6.
Development ; 151(1)2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38063486

RESUMEN

Cholinergic signaling plays a crucial role in the regulation of adult hippocampal neurogenesis; however, the mechanisms by which acetylcholine mediates neurogenic effects are not completely understood. Here, we report the expression of muscarinic acetylcholine receptor subtype M4 (M4 mAChR) on a subpopulation of neural precursor cells (NPCs) in the adult mouse hippocampus, and demonstrate that its pharmacological stimulation promotes their proliferation, thereby enhancing the production of new neurons in vivo. Using a targeted ablation approach, we also show that medial septum (MS) and the diagonal band of Broca (DBB) cholinergic neurons support both the survival and morphological maturation of adult-born neurons in the mouse hippocampus. Although the systemic administration of an M4-selective allosteric potentiator fails to fully rescue the MS/DBB cholinergic lesion-induced decrease in hippocampal neurogenesis, it further exacerbates the impairment in the morphological maturation of adult-born neurons. Collectively, these findings reveal stage-specific roles of M4 mAChRs in regulating adult hippocampal neurogenesis, uncoupling their positive role in enhancing the production of new neurons from the M4-induced inhibition of their morphological maturation, at least in the context of cholinergic signaling dysfunction.


Asunto(s)
Células-Madre Neurales , Receptor Muscarínico M4 , Ratones , Animales , Receptor Muscarínico M4/metabolismo , Células-Madre Neurales/metabolismo , Hipocampo/metabolismo , Neurogénesis/genética , Colinérgicos/metabolismo , Colinérgicos/farmacología , Proliferación Celular
7.
Dev Cell ; 59(1): 91-107.e6, 2024 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-38091997

RESUMEN

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.


Asunto(s)
Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Animales , Humanos , Miocitos Cardíacos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Pez Cebra/metabolismo , Diferenciación Celular/genética , Proliferación Celular
8.
Genome Res ; 33(9): 1465-1481, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37798118

RESUMEN

Mice harbor ∼2800 intact copies of the retrotransposon Long Interspersed Element 1 (L1). The in vivo retrotransposition capacity of an L1 copy is defined by both its sequence integrity and epigenetic status, including DNA methylation of the monomeric units constituting young mouse L1 promoters. Locus-specific L1 methylation dynamics during development may therefore elucidate and explain spatiotemporal niches of endogenous retrotransposition but remain unresolved. Here, we interrogate the retrotransposition efficiency and epigenetic fate of source (donor) L1s, identified as mobile in vivo. We show that promoter monomer loss consistently attenuates the relative retrotransposition potential of their offspring (daughter) L1 insertions. We also observe that most donor/daughter L1 pairs are efficiently methylated upon differentiation in vivo and in vitro. We use Oxford Nanopore Technologies (ONT) long-read sequencing to resolve L1 methylation genome-wide and at individual L1 loci, revealing a distinctive "smile" pattern in methylation levels across the L1 promoter region. Using Pacific Biosciences (PacBio) SMRT sequencing of L1 5' RACE products, we then examine DNA methylation dynamics at the mouse L1 promoter in parallel with transcription start site (TSS) distribution at locus-specific resolution. Together, our results offer a novel perspective on the interplay between epigenetic repression, L1 evolution, and genome stability.


Asunto(s)
Desarrollo Embrionario , Elementos de Nucleótido Esparcido Largo , Ratones , Animales , Retroelementos/genética , Metilación de ADN , Regiones Promotoras Genéticas
9.
Nature ; 620(7975): 863-872, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-37587336

RESUMEN

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.


Asunto(s)
Reprogramación Celular , Epigénesis Genética , Células Madre Pluripotentes Inducidas , Humanos , Cromatina/genética , Cromatina/metabolismo , Desmetilación del ADN , Metilación de ADN , Elementos Transponibles de ADN , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Lamina Tipo B
10.
Trends Neurosci ; 46(6): 413-414, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37061454

RESUMEN

How does neurodegeneration spread in the brain? Leveraging TDP-43 fly models of amyotrophic lateral sclerosis (ALS), Chang and Dubnau recently reported that the endogenous retrovirus (ERV) mdg4 can trigger and transmit TDP-43 proteinopathy in vivo. Their results suggest that human ERVs could be targeted to develop future ALS therapies.


Asunto(s)
Esclerosis Amiotrófica Lateral , Retrovirus Endógenos , Proteinopatías TDP-43 , Humanos , Retrovirus Endógenos/genética , Esclerosis Amiotrófica Lateral/genética , Proteinopatías TDP-43/genética , Encéfalo
12.
Mol Cell ; 83(3): 320-323, 2023 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-36736305

RESUMEN

The Central Dogma has been a useful conceptualization of the transfer of genetic information, and our understanding of the detailed mechanisms involved in that transfer continues to evolve. Here, we speak to several scientists about their research, how it influences our understanding of information transfer, and questions for the future.

13.
Neurobiol Aging ; 123: 222-232, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36599749

RESUMEN

Accumulation of somatic mutations in human neurons is associated with aging and neurodegeneration. To shed light on the somatic mutational burden in Alzheimer's disease (AD) neurons and get more insight into the role of somatic mutations in AD pathogenesis, we performed single-neuron whole genome sequencing to detect genome-wide somatic mutations (single nucleotide variants (SNVs) and Indels) in 96 single prefrontal cortex neurons from 8 AD patients and 8 elderly controls. We found that the mutational burden is ∼3000 somatic mutations per neuron genome in elderly subjects. AD patients have increased somatic mutation burden in AD-related annotation categories, including AD risk genes and differentially expressed genes in AD neurons. Mutational signature analysis showed somatic SNVs (sSNVs) primarily caused by aging and oxidative DNA damage processes but no significant difference was detected between AD and controls. Additionally, functional somatic mutations identified in AD patients showed significant enrichment in several AD-related pathways, including AD pathway, Notch-signaling pathway and Calcium-signaling pathway. These findings provide genetic insights into how somatic mutations may alter the function of single neurons and exert their potential roles in the pathogenesis of AD.


Asunto(s)
Enfermedad de Alzheimer , Humanos , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secuenciación Completa del Genoma , Envejecimiento/genética , Neuronas/metabolismo , Mutación INDEL , Mutación/genética , Polimorfismo de Nucleótido Simple/genética
14.
Br J Cancer ; 128(7): 1236-1248, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36707636

RESUMEN

BACKGROUND: Molecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood. METHODS: We analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems. RESULTS: We observed positive associations between L1 and activated TGFß-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFß-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFß-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p. DISCUSSION: Our data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Retroelementos , Carcinoma Hepatocelular/genética , Regulación hacia Arriba , Neoplasias Hepáticas/genética , Elementos de Nucleótido Esparcido Largo/genética , Factor de Crecimiento Transformador beta/genética , Peptidilprolil Isomerasa de Interacción con NIMA/genética
15.
Methods Mol Biol ; 2607: 151-171, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36449163

RESUMEN

Over the past 20 years, high-throughput genomic assays have fundamentally changed how transposable elements (TEs) are studied. While short-read DNA sequencing has been at the heart of these efforts, novel technologies that generate longer reads are driving a shift in the field. Long-read sequencing now permits locus-specific approaches to locate individual TE insertions and understand their epigenetic and transcriptional regulation, while still profiling TE activity genome-wide. Here we provide detailed guidelines to implement Oxford Nanopore Technologies (ONT) sequencing to identify polymorphic TE insertions and profile TE epigenetic landscapes. Using human long interspersed element-1 (LINE-1, L1) as an example, we explain the procedures involved, including final visualization, and potential bottlenecks and pitfalls. ONT sequencing will be, in our view, a workhorse technology for the foreseeable future in the TE field.


Asunto(s)
Secuenciación de Nanoporos , Humanos , Elementos Transponibles de ADN/genética , Epigénesis Genética , Epigenómica , Elementos de Nucleótido Esparcido Largo/genética
16.
Nat Commun ; 13(1): 7470, 2022 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-36463236

RESUMEN

Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.


Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Ratones , Animales , Retroelementos/genética , Elementos Transponibles de ADN/genética , Mutación , Elementos de Nucleótido Esparcido Largo/genética
18.
Genome Res ; 32(7): 1298-1314, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35728967

RESUMEN

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.


Asunto(s)
Encéfalo , Elementos de Nucleótido Esparcido Largo , Retroelementos , Animales , Proteínas de Unión al ADN/genética , Macaca mulatta/genética , Neuronas , Retroelementos/genética , Factores de Transcripción/genética
19.
Mol Cell ; 82(9): 1610-1612, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35523126

RESUMEN

Garland et al. (2022) discover a physical and functional connection between the HUSH silencing complex and the NEXT nuclear RNA decay complex in mouse embryonic stem cells and conclude HUSH recruits NEXT to degrade prematurely terminated retrotransposon RNAs.


Asunto(s)
ARN , Retroelementos , Animales , Ratones , ARN/genética , Retroelementos/genética
20.
Nat Rev Genet ; 23(8): 458-459, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35361925
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