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1.
Cell ; 185(16): 3025-3040.e6, 2022 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-35882231

RESUMEN

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.


Asunto(s)
Genoma Humano , Retroelementos , Elementos Alu/genética , Recombinación Homóloga , Humanos , Elementos de Nucleótido Esparcido Largo , Secuencias Repetitivas de Ácidos Nucleicos
2.
Cell ; 174(2): 448-464.e24, 2018 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-30007417

RESUMEN

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.


Asunto(s)
Chara/genética , Genoma de Planta , Evolución Biológica , Pared Celular/metabolismo , Chara/crecimiento & desarrollo , Embryophyta/genética , Redes Reguladoras de Genes , Pentosiltransferasa/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma
3.
Cell ; 171(6): 1316-1325.e12, 2017 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-29129375

RESUMEN

Alternative promoter usage is a proteome-expanding mechanism that allows multiple pre-mRNAs to be transcribed from a single gene. The impact of this mechanism on the proteome and whether it is positively exploited in normal organismal responses remain unclear. We found that the plant photoreceptor phytochrome induces genome-wide changes in alternative promoter selection in Arabidopsis thaliana. Through this mechanism, protein isoforms with different N termini are produced that display light-dependent differences in localization. For instance, shade-grown plants accumulate a cytoplasmic isoform of glycerate kinase (GLYK), an essential photorespiration enzyme that was previously thought to localize exclusively to the chloroplast. Cytoplasmic GLYK constitutes a photorespiratory bypass that alleviates fluctuating light-induced photoinhibition. Therefore, phytochrome controls alternative promoter selection to modulate protein localization in response to changing light conditions. This study suggests that alternative promoter usage represents another ubiquitous layer of gene expression regulation in eukaryotes that contributes to diversification of the proteome.


Asunto(s)
Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Fitocromo/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Luz , Regiones Promotoras Genéticas
4.
Cell ; 161(5): 1058-1073, 2015 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-26000482

RESUMEN

Regnase-1 and Roquin are RNA binding proteins essential for degradation of inflammation-related mRNAs and maintenance of immune homeostasis. However, their mechanistic relationship has yet to be clarified. Here, we show that, although Regnase-1 and Roquin regulate an overlapping set of mRNAs via a common stem-loop structure, they function in distinct subcellular locations: ribosome/endoplasmic reticulum and processing-body/stress granules, respectively. Moreover, Regnase-1 specifically cleaves and degrades translationally active mRNAs and requires the helicase activity of UPF1, similar to the decay mechanisms of nonsense mRNAs. In contrast, Roquin controls translationally inactive mRNAs, independent of UPF1. Defects in both Regnase-1 and Roquin lead to large increases in their target mRNAs, although Regnase-1 tends to control the early phase of inflammation when mRNAs are more actively translated. Our findings reveal that differential regulation of mRNAs by Regnase-1 and Roquin depends on their translation status and enables elaborate control of inflammation.


Asunto(s)
Inflamación/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Secuencia de Bases , Codón de Terminación , Células HeLa , Humanos , Inflamación/genética , Inflamación/inmunología , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Conformación de Ácido Nucleico , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/química , Proteínas Ribosómicas/metabolismo , Transactivadores/metabolismo
5.
Nature ; 627(8002): 221-228, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38383791

RESUMEN

Epigenomes enable the rectification of disordered cancer gene expression, thereby providing new targets for pharmacological interventions. The clinical utility of targeting histone H3 lysine trimethylation (H3K27me3) as an epigenetic hallmark has been demonstrated1-7. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumour cells remain unclear. Here we show the potency and mechanisms of action and resistance of the EZH1-EZH2 dual inhibitor valemetostat in clinical trials of patients with adult T cell leukaemia/lymphoma. Administration of valemetostat reduced tumour size and demonstrated durable clinical response in aggressive lymphomas with multiple genetic mutations. Integrative single-cell analyses showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, including tumour suppressor genes. Nevertheless, subsequent long-term treatment encounters the emergence of resistant clones with reconstructed aggregate chromatin that closely resemble the pre-dose state. Acquired mutations at the PRC2-compound interface result in the propagation of clones with increased H3K27me3 expression. In patients free of PRC2 mutations, TET2 mutation or elevated DNMT3A expression causes similar chromatin recondensation through de novo DNA methylation in the H3K27me3-associated regions. We identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Targeting epigenetic drivers and chromatin homeostasis may provide opportunities for further sustained epigenetic cancer therapies.


Asunto(s)
Histonas , Linfoma , Adulto , Humanos , Histonas/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Metilación , Cromatina/genética
6.
Cell ; 153(5): 1036-49, 2013 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-23706741

RESUMEN

Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase that destabilizes a set of mRNAs, including Il6 and Il12b, through cleavage of their 3' UTRs. Although Regnase-1 inactivation leads to development of an autoimmune disease characterized by T cell activation and hyperimmunoglobulinemia in mice, the mechanism of Regnase-1-mediated immune regulation has remained unclear. We show that Regnase-1 is essential for preventing aberrant effector CD4(+) T cell generation cell autonomously. Moreover, in T cells, Regnase-1 regulates the mRNAs of a set of genes, including c-Rel, Ox40, and Il2, through cleavage of their 3' UTRs. Interestingly, T cell receptor (TCR) stimulation leads to cleavage of Regnase-1 at R111 by Malt1/paracaspase, freeing T cells from Regnase-1-mediated suppression. Furthermore, Malt1 protease activity is critical for controlling the mRNA stability of T cell effector genes. Collectively, these results indicate that dynamic control of Regnase-1 expression in T cells is critical for controlling T cell activation.


Asunto(s)
Caspasas/metabolismo , Activación de Linfocitos , Proteínas de Neoplasias/metabolismo , Ribonucleasas/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Humanos , Interleucina-2/genética , Células Jurkat , Glicoproteínas de Membrana/genética , Ratones , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Ligando OX40 , Proteínas Proto-Oncogénicas c-rel/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Factores de Necrosis Tumoral/genética
7.
Nature ; 578(7794): 311-316, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31996847

RESUMEN

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.


Asunto(s)
Secuencias de Aminoácidos , Secuencia de Consenso , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Fosfolipasa D/química , Fosfolipasa D/metabolismo , ARN Interferente Pequeño/biosíntesis , Adenosina Trifosfato/metabolismo , Animales , Secuencia de Bases , Bombyx , Línea Celular , Sistema Libre de Células , Técnicas de Inactivación de Genes , Proteínas de Insectos/genética , Metilación , Ratones , ARN Helicasas/metabolismo
8.
Nucleic Acids Res ; 52(11): 6158-6170, 2024 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-38567720

RESUMEN

In mice, transcription from the zygotic genome is initiated at the mid-one-cell stage, and occurs promiscuously in many areas of the genome, including intergenic regions. Regulated transcription from selected genes is established during the two-cell stage. This dramatic change in the gene expression pattern marks the initiation of the gene expression program and is essential for early development. We investigated the involvement of the histone variants H3.1/3.2 in the regulation of changes in gene expression pattern during the two-cell stage. Immunocytochemistry analysis showed low nuclear deposition of H3.1/3.2 in the one-cell stage, followed by a rapid increase in the late two-cell stage. Where chromatin structure is normally closed between the one- and two-cell stages, it remained open until the late two-cell stage when H3.1/3.2 were knocked down by small interfering RNA. Hi-C analysis showed that the formation of the topologically associating domain was disrupted in H3.1/3.2 knockdown (KD) embryos. Promiscuous transcription was also maintained in the late two-cell stage in H3.1/3.2 KD embryos. These results demonstrate that H3.1/3.2 are involved in the initial process of the gene expression program after fertilization, through the formation of a closed chromatin structure to execute regulated gene expression during the two-cell stage.


Asunto(s)
Cromatina , Regulación del Desarrollo de la Expresión Génica , Histonas , Animales , Ratones , Histonas/metabolismo , Cromatina/metabolismo , Transcripción Genética , Cigoto/metabolismo , Técnicas de Silenciamiento del Gen , Femenino
9.
Nucleic Acids Res ; 52(2): e7, 2024 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-37994784

RESUMEN

Precise detection of the transcriptional start site (TSS) is a key for characterizing transcriptional regulation of genes and for annotation of newly sequenced genomes. Here, we describe the development of an improved method, designated 'TSS-seq2.' This method is an iterative improvement of TSS-seq, a previously published enzymatic cap-structure conversion method to detect TSSs in base sequences. By modifying the original procedure, including by introducing split ligation at the key cap-selection step, the yield and the accuracy of the reaction has been substantially improved. For example, TSS-seq2 can be conducted using as little as 5 ng of total RNA with an overall accuracy of 96%; this yield a less-biased and more precise detection of TSS. We then applied TSS-seq2 for TSS analysis of four plant species that had not yet been analyzed by any previous TSS method.


Asunto(s)
Análisis de Secuencia de ARN , Sitio de Iniciación de la Transcripción , Secuencia de Bases , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Análisis de Secuencia de ARN/métodos
10.
Genes Dev ; 32(2): 112-126, 2018 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-29440259

RESUMEN

Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that ∼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.


Asunto(s)
Linfocitos B/metabolismo , Células Madre Multipotentes/metabolismo , Transcripción Genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Linaje de la Célula/genética , Células Cultivadas , Epigénesis Genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Código de Histonas , Factor 4 Similar a Kruppel , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción PAX5/fisiología , Análisis de la Célula Individual , Transactivadores/fisiología , Transcriptoma
11.
Plant J ; 118(6): 2037-2054, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38577750

RESUMEN

Knotted1-like homeodomain (KNOX) proteins are essential in regulating plant organ differentiation. Land plants, including tomato (Solanum lycopersicum), have two classes of the KNOX protein family, namely, class I (KNOX I) and class II KNOX (KNOX II). While tomato KNOX I proteins are known to stimulate chloroplast development in fruit, affecting fruit coloration, the role of KNOX II proteins in this context remains unclear. In this study, we employ CRISPR/Cas9 to generate knockout mutants of the KNOX II member, SlKN5. These mutants display increased leaf complexity, a phenotype commonly associated with reduced KNOX II activity, as well as enhanced accumulation of chloroplasts and chlorophylls in smaller cells within young, unripe fruit. RNA-seq data analyses indicate that SlKN5 suppresses the transcriptions of genes involved in chloroplast biogenesis, chlorophyll biosynthesis, and gibberellin catabolism. Furthermore, protein-protein interaction assays reveal that SlKN5 physically interacts with three transcriptional repressors from the BLH1-clade of BEL1-like homeodomain (BLH) protein family, SlBLH4, SlBLH5, and SlBLH7, with SlBLH7 showing the strongest interaction. CRISPR/Cas9-mediated knockout of these SlBLH genes confirmed their overlapping roles in suppressing chloroplast biogenesis, chlorophyll biosynthesis, and lycopene cyclization. Transient assays further demonstrate that the SlKN5-SlBLH7 interaction enhances binding capacity to regulatory regions of key chloroplast- and chlorophyll-related genes, including SlAPRR2-like1, SlCAB-1C, and SlGUN4. Collectively, our findings elucidate that the KNOX II SlKN5-SlBLH regulatory modules serve to inhibit fruit greening and subsequently promote lycopene accumulation, thereby fine-tuning the color transition from immature green fruit to mature red fruit.


Asunto(s)
Frutas , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio , Proteínas de Plantas , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/crecimiento & desarrollo , Frutas/genética , Frutas/metabolismo , Frutas/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Cloroplastos/metabolismo , Sistemas CRISPR-Cas , Clorofila/metabolismo , Plantas Modificadas Genéticamente
12.
EMBO J ; 40(14): e106434, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34152017

RESUMEN

Alternative splicing of pre-mRNAs can regulate gene expression levels by coupling with nonsense-mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS-NMD) in an organism, we performed long-read RNA sequencing of poly(A)+ RNAs from an NMD-deficient mutant strain of Caenorhabditis elegans, and obtained full-length sequences for mRNA isoforms from 259 high-confidence AS-NMD genes. Among them are the S-adenosyl-L-methionine (SAM) synthetase (sams) genes sams-3 and sams-4. SAM synthetase activity autoregulates sams gene expression through AS-NMD in a negative feedback loop. We furthermore find that METT-10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3' splice site (3'SS) in␣vitro. Direct RNA sequencing coupled with machine learning confirms m6 A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m6 A modification at the 3'SS of the sams genes.


Asunto(s)
Empalme Alternativo/genética , Homeostasis/genética , Ligasas/genética , Metionina Adenosiltransferasa/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Mensajero/genética , S-Adenosilmetionina/metabolismo , Animales , Caenorhabditis elegans/genética , Metiltransferasas/genética , Precursores del ARN/genética
13.
Proc Natl Acad Sci U S A ; 119(33): e2204338119, 2022 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-35939709

RESUMEN

Despite the recent discovery of tissue regeneration enhancers in highly regenerative animals, upstream and downstream genetic programs connected by these enhancers still remain unclear. Here, we performed a genome-wide analysis of enhancers and associated genes in regenerating nephric tubules of Xenopus laevis. Putative enhancers were identified using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) analyses. Their target genes were predicted based on their proximity to enhancers on genomic DNA and consistency of their transcriptome profiles to ATAC-seq/ChIP-seq profiles of the enhancers. Motif enrichment analysis identified the central role of Krüppel-like factors (Klf) in the enhancer. Klf15, a member of the Klf family, directly binds enhancers and stimulates expression of regenerative genes, including adrenoreceptor alpha 1A (adra1a), whereas inhibition of Klf15 activity results in failure of nephric tubule regeneration. Moreover, pharmacological inhibition of Adra1a-signaling suppresses nephric tubule regeneration, while its activation promotes nephric tubule regeneration and restores organ size. These results indicate that Klf15-dependent adrenergic receptor signaling through regeneration enhancers plays a central role in the genetic network for kidney regeneration.


Asunto(s)
Elementos de Facilitación Genéticos , Túbulos Renales , Factores de Transcripción de Tipo Kruppel , Receptores Adrenérgicos , Regeneración , Animales , Cromatina/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Túbulos Renales/fisiología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Receptores Adrenérgicos/metabolismo , Regeneración/genética , Xenopus laevis
14.
Circulation ; 147(3): 223-238, 2023 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-36503256

RESUMEN

BACKGROUND: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. METHODS: We generated a novel Tcf21iCre/reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21iCre/Tomato/MGTH2A and Tcf21iCre/mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. RESULTS: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. CONCLUSIONS: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure.


Asunto(s)
Insuficiencia Cardíaca , Infarto del Miocardio , Ratones , Animales , Miocitos Cardíacos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Fibrosis , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Fibroblastos/metabolismo , Reprogramación Celular
15.
J Neurochem ; 168(4): 342-354, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37994470

RESUMEN

Skeletal muscle fiber is a large syncytium with multiple and evenly distributed nuclei. Adult subsynaptic myonuclei beneath the neuromuscular junction (NMJ) express specific genes, the products of which coordinately function in the maintenance of the pre- and post-synaptic regions. However, the gene expression profiles that promote the NMJ formation during embryogenesis remain largely unexplored. We performed single-nucleus RNA sequencing (snRNA-seq) analysis of embryonic and neonatal mouse diaphragms, and found that each myonucleus had a distinct transcriptome pattern during the NMJ formation. Among the previously reported NMJ-constituting genes, Dok7, Chrna1, and Chrnd are specifically expressed in subsynaptic myonuclei at E18.5. In the E18.5 diaphragm, ca. 10.7% of the myonuclei express genes for the NMJ formation (Dok7, Chrna1, and Chrnd) together with four representative ß-catenin regulators (Amotl2, Ptprk, Fam53b, and Tcf7l2). Additionally, the temporal gene expression patterns of these seven genes are synchronized in differentiating C2C12 myoblasts. Amotl2 and Ptprk are expressed in the sarcoplasm, where ß-catenin serves as a structural protein to organize the membrane-anchored NMJ structure. In contrast, Fam53b and Tcf7l2 are expressed in the myonucleus, where ß-catenin serves as a transcriptional coactivator in Wnt/ß-catenin signaling at the NMJ. In C2C12 myotubes, knockdown of Amotl2 or Ptprk markedly, and that of Fam53b and Tcf7l2 less efficiently, impair the clustering of acetylcholine receptors. In contrast, knockdown of Fam53b and Tcf7l2, but not of Amotl2 or Ptprk, impairs the gene expression of Slit2 encoding an axonal attractant for motor neurons, which is required for the maturation of motor nerve terminal. Thus, Amotl2 and Ptprk exert different roles at the NM compared to Fam53b and Tcf7l2. Additionally, Wnt ligands originating from the spinal motor neurons and the perichondrium/chondrocyte are likely to work remotely on the subsynaptic nuclei and the myotendinous junctional nuclei, respectively. We conclude that snRNA-seq analysis of embryonic/neonatal diaphragms reveal a novel coordinated expression profile especially in the Wnt/ß-catenin signaling that regulate the formation of the embryonic NMJ.


Asunto(s)
Transcriptoma , beta Catenina , Ratones , Animales , beta Catenina/metabolismo , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Vía de Señalización Wnt/genética , ARN Nuclear Pequeño/metabolismo , Desarrollo Embrionario , Músculo Esquelético/metabolismo , Receptores Colinérgicos/metabolismo
16.
Cancer Sci ; 115(2): 529-539, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38083992

RESUMEN

Biomarkers that could detect the postoperative recurrence of upper tract urothelial carcinoma (UTUC) have not been established. In this prospective study, we aim to evaluate the utility of individualized circulating tumor DNA (ctDNA) monitoring using digital PCR (dPCR) as a tumor recurrence biomarker for UTUC in the perioperative period. Twenty-three patients who underwent radical nephroureterectomy (RNU) were included. In each patient, whole exome sequencing by next-generation sequencing and TERT promoter sequencing of tumor DNA were carried out. Case-specific gene mutations were selected from sequencing analysis to examine ctDNA by dPCR analysis. We also prospectively collected plasma and urine ctDNA from each patient. The longitudinal variant allele frequencies of ctDNA during the perioperative period were plotted. Case-specific gene mutations were detected in 22 cases (96%) from ctDNA in the preoperative samples. Frequently detected genes were TERT (39%), FGFR3 (26%), TP53 (22%), and HRAS (13%). In all cases, we obtained plasma and urine samples for 241 time points and undertook individualized ctDNA monitoring for 2 years after RNU. Ten patients with intravesical recurrence had case-specific ctDNA detected in urine at the time of recurrence. The mean lead time of urinary ctDNA in intravesical recurrence was 60 days (range, 0-202 days). Two patients with distal metastasis had case-specific ctDNA in plasma at the time of metastasis. In UTUC, tumor-specific gene mutations can be monitored postoperatively as ctDNA in plasma and urine. Individualized ctDNA might be a minimally invasive biomarker for the early detection of postoperative recurrence.


Asunto(s)
Carcinoma de Células Transicionales , ADN Tumoral Circulante , Neoplasias de la Vejiga Urinaria , Humanos , Carcinoma de Células Transicionales/diagnóstico , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/cirugía , ADN Tumoral Circulante/genética , Estudios Prospectivos , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Biomarcadores , Biomarcadores de Tumor/genética
17.
Cancer Sci ; 115(7): 2360-2370, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38659235

RESUMEN

N6-methyladenosine (m6A) is an RNA modification involved in RNA processing and widely found in transcripts. In cancer cells, m6A is upregulated, contributing to their malignant transformation. In this study, we analyzed gene expression and m6A modification in cancer tissues, ducts, and acinar cells derived from pancreatic cancer patients using MeRIP-seq. We found that dozens of RNAs highly modified by m6A were detected in cancer tissues compared with ducts and acinar cells. Among them, the m6A-activated mRNA TCEAL8 was observed, for the first time, as a potential marker gene in pancreatic cancer. Spatially resolved transcriptomic analysis showed that TCEAL8 was highly expressed in specific cells, and activation of cancer-related signaling pathways was observed relative to TCEAL8-negative cells. Furthermore, among TCEAL8-positive cells, the cells expressing the m6A-modifying enzyme gene METTL3 showed co-activation of Notch and mTOR signaling, also known to be involved in cancer metastasis. Overall, these results suggest that m6A-activated TCEAL8 is a novel marker gene involved in the malignant transformation of pancreatic cancer.


Asunto(s)
Adenosina , Biomarcadores de Tumor , Regulación Neoplásica de la Expresión Génica , Metiltransferasas , Neoplasias Pancreáticas , ARN Mensajero , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Línea Celular Tumoral , Receptores Notch/genética , Receptores Notch/metabolismo , Perfilación de la Expresión Génica/métodos
18.
Genome Res ; 31(6): 1060-1068, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-34006571

RESUMEN

DNA methylation is an important factor regulating gene expression in organisms. However, whether DNA methylation plays a key role in adaptive evolution is unknown. Here, we show evidence of naturally selected DNA methylation in Arabidopsis thaliana In comparison with single nucleotide polymorphisms, three types of methylation-methylated CGs (mCGs), mCHGs, and mCHHs-contributed highly to variable gene expression levels among an A thaliana population. Such variably expressed genes largely affect a large variation of specialized metabolic quantities. Among the three types of methylations, only mCGs located in promoter regions of genes associated with specialized metabolites show a selective sweep signature in the A thaliana population. Thus, naturally selected mCGs appear to be key mutations that cause the expressional diversity associated with specialized metabolites during plant evolution.


Asunto(s)
Arabidopsis , Epigenómica , Genoma de Planta , Arabidopsis/genética , Arabidopsis/metabolismo , Metilación de ADN , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Mutación
19.
Development ; 148(8)2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33766931

RESUMEN

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We have previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X-chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility.


Asunto(s)
Fertilidad , Nucleoproteínas/metabolismo , Espermátides/metabolismo , Espermatogénesis , Células Madre/metabolismo , Animales , Femenino , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Nucleoproteínas/genética , Espermatogonias/metabolismo
20.
Genes Cells ; 28(7): 482-495, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37073980

RESUMEN

Histone methylation plays a vital role in retinal development. However, the role of histone H3K36 methylation in retinal development is not clear. We examined the role of H3K36 methylation by loss-of-function analysis of H3K36me1/2 demethylases, Fbxl10, and Fbxl11. We analyzed the effect of knockout of these genes in the developing and mature retina on retinal development. Knockout of Fbxl10 specifically in the developing retina did not result in gross developmental abnormalities. Although adult rod photoreceptor-specific knockout of Fbxl11 in mature retinas did not result in morphological abnormalities, Fbxl11 knockout in developing retinas increased apoptosis, suppressed the proliferation of retinal progenitor cells, and resulted in microphthalmia. Morphological analysis revealed perturbed differentiation of rod photoreceptor and bipolar cells. RNA-seq of retinas at P7 showed markedly decreased expression of genes characterizing rod photoreceptor and bipolar cells in Fbxl11-knockout retinas. In addition, perturbation of alternative splicing increased intron retention in Fbxl11-knockout retinas. Genome-wide evaluation of the H3K36 methylation status revealed that Fbxl11 knockout altered the distribution of H3K36me2/3 in genes important for rod photoreceptor development. Taken together, we show that Fbxl11 plays pivotal roles in the development of retinal late-born cell types and may contribute to tight control of H3K36 methylation during retinal development.


Asunto(s)
Histona Demetilasas , Histonas , Diferenciación Celular/genética , Histona Demetilasas/genética , Histonas/genética , Histonas/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones , Animales , Ratones
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