RESUMO
Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.
Assuntos
Fatores de Transcrição Maf/genética , RNA Longo não Codificante/genética , Linfócitos T/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Humanos , Fatores de Transcrição Maf/imunologia , RNA Longo não Codificante/imunologia , Transcrição Gênica/genética , Transcrição Gênica/imunologia , Transcriptoma/genética , Transcriptoma/imunologiaRESUMO
Repetitive sequences account for more than 50% of the human genome. Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease associated with reduction in the copy number of the D4Z4 repeat mapping to 4q35. By an unknown mechanism, D4Z4 deletion causes an epigenetic switch leading to de-repression of 4q35 genes. Here we show that the Polycomb group of epigenetic repressors targets D4Z4 in healthy subjects and that D4Z4 deletion is associated with reduced Polycomb silencing in FSHD patients. We identify DBE-T, a chromatin-associated noncoding RNA produced selectively in FSHD patients that coordinates de-repression of 4q35 genes. DBE-T recruits the Trithorax group protein Ash1L to the FSHD locus, driving histone H3 lysine 36 dimethylation, chromatin remodeling, and 4q35 gene transcription. This study provides insights into the biological function of repetitive sequences in regulating gene expression and shows how mutations of such elements can influence the progression of a human genetic disease.
Assuntos
Epigênese Genética , Distrofia Muscular Facioescapuloumeral/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , RNA não Traduzido/metabolismo , Proteínas Repressoras/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetinae , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/fisiopatologia , Proteínas do Grupo Polycomb , Elementos de Resposta , Fatores de Transcrição/metabolismoRESUMO
NF1 microdeletion syndrome, accounting for 5-11% of NF1 patients, is caused by a deletion in the NF1 region and it is generally characterized by a severe phenotype. Although 70% of NF1 microdeletion patients presents the same 1.4 Mb type-I deletion, some patients may show additional clinical features. Therefore, the contribution of several pathogenic mechanisms, besides haploinsufficiency of some genes within the deletion interval, is expected and needs to be defined. We investigated an altered expression of deletion flanking genes by qPCR in patients with type-1 NF1 deletion, compared to healthy donors, possibly contributing to the clinical traits of NF1 microdeletion syndrome. In addition, the 1.4-Mb deletion leads to changes in the 3D chromatin structure in the 17q11.2 region. Specifically, this deletion alters DNA-DNA interactions in the regions flanking the breakpoints, as demonstrated by our 4C-seq analysis. This alteration likely causes position effect on the expression of deletion flanking genes.Interestingly, 4C-seq analysis revealed that in microdeletion patients, an interaction was established between the RHOT1 promoter and the SLC6A4 gene, which showed increased expression. We performed NGS on putative modifier genes, and identified two "likely pathogenic" rare variants in RAS pathway, possibly contributing to incidental phenotypic features.This study provides new insights into understanding the pathogenesis of NF1 microdeletion syndrome and suggests a novel pathomechanism that contributes to the expression phenotype in addition to haploinsufficiency of genes located within the deletion.This is a pivotal approach that can be applied to unravel microdeletion syndromes, improving precision medicine, prognosis and patients' follow-up.
Assuntos
Deleção Cromossômica , Epigênese Genética , Haploinsuficiência , Neurofibromatose 1 , Humanos , Neurofibromatose 1/genética , Feminino , Masculino , Neurofibromina 1/genética , Cromossomos Humanos Par 17/genética , Fenótipo , Criança , Regiões Promotoras GenéticasRESUMO
Recent branching (100 MYA) of the mammalian evolutionary tree has enhanced brain complexity and functions at the putative cost of increased emotional circuitry vulnerability. Thus, to better understand psychopathology, a burden for the modern society, novel approaches should exploit evolutionary aspects of psychiatric-relevant molecular pathways. A handful of genes is nowadays tightly associated to psychiatric disorders. Among them, neuronal-enriched RbFOX1 modifies the activity of synaptic regulators in response to neuronal activity, keeping excitability within healthy domains. We here dissect a higher primates-restricted interaction between RbFOX1 and the transcriptional corepressor Lysine Specific Demethylase 1 (LSD1/KDM1A). A single nucleotide variation (AA to AG) in LSD1 gene appeared in higher primates and humans, endowing RbFOX1 with the ability to promote the alternative usage of a novel 3' AG splice site, which extends LSD1 exon E9 in the upstream intron (E9-long). Exon E9-long regulates LSD1 levels by Nonsense-Mediated mRNA Decay. As reintroduction of the archaic LSD1 variant (AA) abolishes E9-long splicing, the novel 3' AG splice site is necessary for RbFOX1 to control LSD1 levels. LSD1 is a homeostatic immediate early genes (IEGs) regulator playing a relevant part in environmental stress-response. In primates and humans, inclusion of LSD1 as RbFOX1 target provides RbFOX1 with the additional ability to regulate the IEGs. These data, together with extensive RbFOX1 involvement in psychiatric disorders and its stress-dependent regulation in male mice, suggest the RbFOX1-LSD1-IEGs axis as an evolutionary recent psychiatric-relevant pathway. Notably, outside the nervous system, RbFOX2-dependent LSD1 modulation could be a candidate deregulated mechanism in cancer.SIGNIFICANCE STATEMENT To be better understood, anxiety and depression need large human genetics studies aimed at further resolving the often ambiguous, aberrant neuronal pathomechanisms that impact corticolimbic circuitry physiology. Several genetic associations of the alternative splicing regulator RbFOX1 with psychiatric conditions suggest homeostatic unbalance as a neuronal signature of psychopathology. Here we move a step forward, characterizing a disease-relevant higher primates-specific pathway by which RbFOX1 acquires the ability to regulate neuronal levels of Lysine Specific Demethylase 1, an epigenetic modulator of environmental stress response. Thus, two brain-enriched enzymes, independently shown to homeostatically protect neurons with a clear readout in terms of emotional behavior in lower mammals, establish in higher primates and humans a new functional cooperation enhancing the complexity of environmental adaptation and stress vulnerability.
Assuntos
Processamento Alternativo , Lisina , Processamento Alternativo/genética , Animais , Encéfalo/metabolismo , Histona Desmetilases/genética , Humanos , Lisina/metabolismo , Masculino , Mamíferos , Camundongos , Primatas , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/genéticaRESUMO
The genome is no longer deemed as a fixed and inert item but rather as a moldable matter that is continuously evolving and adapting. Within this frame, Transposable Elements (TEs), ubiquitous, mobile, repetitive elements, are considered an alive portion of the genomes to date, whose functions, although long considered "dark", are now coming to light. Here we will review that, besides the detrimental effects that TE mobilization can induce, TEs have shaped genomes in their current form, promoting genome sizing, genomic rearrangements and shuffling of DNA sequences. Although TEs are mostly represented in the genomes by evolutionarily old, short, degenerated, and sedentary fossils, they have been thoroughly co-opted by the hosts as a prolific and original source of regulatory instruments for the control of gene transcription and genome organization in the nuclear space. For these reasons, the deregulation of TE expression and/or activity is implicated in the onset and progression of several diseases. It is likely that we have just revealed the outermost layers of TE functions. Further studies on this portion of the genome are required to unlock novel regulatory functions that could also be exploited for diagnostic and therapeutic approaches.
Assuntos
Elementos de DNA Transponíveis , Regulação da Expressão Gênica , Elementos de DNA Transponíveis/genética , Tamanho do Genoma , Fases de Leitura , Evolução MolecularRESUMO
Despite increasing insights in genome structure organization, the role of DNA repetitive elements, accounting for more than two thirds of the human genome, remains elusive. Facioscapulohumeral muscular dystrophy (FSHD) is associated with deletion of D4Z4 repeat array below 11 units at 4q35.2. It is known that the deletion alters chromatin structure in cis, leading to gene up-regulation. Here we show a genome-wide role of 4q-D4Z4 array in modulating gene expression via 3D nuclear contacts. We have developed an integrated strategy of 4q-D4Z4-specific 4C-seq and chromatin segmentation analyses, showing that 4q-D4Z4 3D interactome and chromatin states of interacting genes are impaired in FSHD1 condition; in particular, genes that have lost the 4q-D4Z4 interaction and with a more active chromatin state are enriched for muscle atrophy transcriptional signature. Expression level of these genes is restored by the interaction with an ectopic 4q-D4Z4 array, suggesting that the repeat directly modulates the transcription of contacted targets. Of note, the up-regulation of atrophic genes is a common feature of several FSHD1 and FSHD2 patients, indicating that we have identified a core set of deregulated genes involved in FSHD pathophysiology.
Assuntos
Cromatina/genética , Cromossomos Humanos Par 4 , Distrofia Muscular Facioescapuloumeral/genética , Sequências de Repetição em Tandem , Transcrição Gênica , Biomarcadores , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Expressão Ectópica do Gene , Epistasia Genética , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Proteínas Musculares/genética , Distrofia Muscular Facioescapuloumeral/diagnóstico , Proteínas Ligases SKP Culina F-Box/genéticaRESUMO
BACKGROUND: Preterm birth affects almost 9-11% of newborns and is one of the leading causes of childhood neurodevelopmental disabilities; the underlying molecular networks are poorly defined. In neurons, retrotransposons LINE-1 (L1) are an active source of genomic mosaicism that is deregulated in several neurological disorders; early life experience has been shown to regulate L1 activity in mice. METHODS: Very preterm infants were randomized to receive standard care or early intervention. L1 methylation was measured at birth and at hospital discharge. At 12 and 36 months, infants' neurodevelopment was evaluated with the Griffiths Scales. L1 methylation and CNVs were measured in mouse brain areas at embryonic and postnatal stages. RESULTS: Here we report that L1 promoter is hypomethylated in preterm infants at birth and that an early intervention program, based on enhanced maternal care and positive multisensory stimulation, restores L1 methylation levels comparable to healthy newborns and ameliorates neurodevelopment in childhood. We further show that L1 activity is fine-tuned in the perinatal mouse brain, suggesting a sensitive and vulnerable window for the L1 epigenetic setting. CONCLUSIONS: Our results open the field on the inspection of L1 activity as a novel molecular and predictive approach to infants' prematurity-related neurodevelopmental outcomes. TRIAL REGISTRATION: ClinicalTrial.gov ( NCT02983513 ). Registered on 6 December 2016, retrospectively registered.
Assuntos
Desenvolvimento Infantil/fisiologia , Metilação de DNA/fisiologia , Cuidado do Lactente/métodos , Recém-Nascido Prematuro/fisiologia , Transtornos do Neurodesenvolvimento/prevenção & controle , Feminino , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro/crescimento & desenvolvimento , Metilação , Alta do Paciente/estatística & dados numéricos , Gravidez , Nascimento PrematuroRESUMO
Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle degeneration and currently there are few therapeutic options. The identification of new drug targets and their validation in model systems of DMD could be a promising approach to make progress in finding new treatments for this lethal disease. Histone deacetylases (HDACs) play key roles in myogenesis and the therapeutic approach targeting HDACs in DMD is in an advanced phase of clinical trial. Here, we show that the expression of HDAC8, one of the members of the HDAC family, is increased in DMD patients and dystrophic zebrafish. The selective inhibition of HDAC8 with the PCI-34051 inhibitor rescues skeletal muscle defects, similarly to the treatment with the pan-HDAC inhibitor Givinostat. Through acetylation profile of zebrafish with HDAC8 dysregulation, we identified new HDAC8 targets involved in cytoskeleton organization such as tubulin that, when acetylated, is a marker of stable microtubules. Our work provides evidence of HDAC8 overexpression in DMD patients and zebrafish and supports its specific inhibition as a new valuable therapeutic approach in the treatment of this pathology.
Assuntos
Diferenciação Celular , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos , Indóis , Desenvolvimento Muscular , Músculo Esquelético , Distrofia Muscular de Duchenne , Proteínas Repressoras , Proteínas de Peixe-Zebra , Animais , Humanos , Acetilação , Animais Geneticamente Modificados , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/enzimologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
Assuntos
Atlas como Assunto , Anotação de Sequência Molecular , Regiões Promotoras Genéticas/genética , Transcriptoma/genética , Animais , Linhagem Celular , Células Cultivadas , Análise por Conglomerados , Sequência Conservada/genética , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Genes Essenciais/genética , Genoma/genética , Humanos , Camundongos , Fases de Leitura Aberta/genética , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica/genéticaRESUMO
Cells and tissues are continuously exposed to a changing microenvironment, hence the necessity of a flexible modulation of gene expression that in complex organism have been achieved through specialized chromatin mechanisms. Chromatin-based cell memory enables cells to maintain their identity by fixing lineage specific transcriptional programs, ensuring their faithful transmission through cell division; in particular PcG-based memory system evolved to maintain the silenced state of developmental and cell cycle genes. In evolution the complexity of this system have increased, particularly in vertebrates, indicating combinatorial and dynamic properties of Polycomb proteins, in some cases even overflowing outside the cell nucleus. Therefore, their function may not be limited to the imposition of rigid states of genetic programs, but on the ability to recognize signals and allow plastic transcriptional changes in response to different stimuli. Here, we discuss the most novel PcG mediated memory functions in facing and responding to the challenges posed by a fluctuating environment.
Assuntos
Epigênese Genética/genética , Proteínas do Grupo Polycomb/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Cromatina/genética , Cromatina/metabolismo , Proteínas do Grupo Polycomb/genéticaRESUMO
: Transposable elements (TEs), which cover ~45% of the human genome, although firstly considered as "selfish" DNA, are nowadays recognized as driving forces in eukaryotic genome evolution. This capability resides in generating a plethora of sophisticated RNA regulatory networks that influence the cell type specific transcriptome in health and disease. Indeed, TEs are transcribed and their RNAs mediate multi-layered transcriptional regulatory functions in cellular identity establishment, but also in the regulation of cellular plasticity and adaptability to environmental cues, as occurs in the immune response. Moreover, TEs transcriptional deregulation also evolved to promote pathogenesis, as in autoimmune and inflammatory diseases and cancers. Importantly, many of these findings have been achieved through the employment of Next Generation Sequencing (NGS) technologies and bioinformatic tools that are in continuous improvement to overcome the limitations of analyzing TEs sequences. However, they are highly homologous, and their annotation is still ambiguous. Here, we will review some of the most recent findings, questions and improvements to study at high resolution this intriguing portion of the human genome in health and diseases, opening the scenario to novel therapeutic opportunities.
Assuntos
Elementos de DNA Transponíveis/genética , Genoma Humano/genética , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
The nucleophosmin 1 gene (NPM1) is the most frequently mutated gene in acute myeloid leukemia. Notably, NPM1 mutations are always accompanied by additional mutations such as those in cohesin genes RAD21, SMC1A, SMC3, and STAG2 but not in the cohesin regulator, nipped B-like (NIPBL). In this work, we analyzed a cohort of adult patients with acute myeloid leukemia and NPM1 mutation and observed a specific reduction in the expression of NIPBL but not in other cohesin genes. In our zebrafish model, overexpression of the mutated form of NPM1 also induced downregulation of nipblb, the zebrafish ortholog of human NIPBL To investigate the hematopoietic phenotype and the interaction between mutated NPM1 and nipblb, we generated a zebrafish model with nipblb downregulation which showed an increased number of myeloid progenitors. This phenotype was due to hyper-activation of the canonical Wnt pathway: myeloid cells blocked in an undifferentiated state could be rescued when the Wnt pathway was inhibited by dkk1b mRNA injection or indomethacin administration. Our results reveal, for the first time, a role for NIPBL during zebrafish hematopoiesis and suggest that an interplay between NIPBL/NPM1 may regulate myeloid differentiation in zebrafish and humans through the canonical Wnt pathway and that dysregulation of these interactions may drive leukemic transformation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/patologia , Mutação , Proteínas Nucleares/genética , Adulto , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Hematopoese , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Nucleofosmina , Fenótipo , Via de Sinalização Wnt , Peixe-Zebra , CoesinasRESUMO
Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience. In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution. Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different contexts.
Assuntos
Imunofluorescência , Humanos , Imunofluorescência/métodos , Núcleo Celular/química , Núcleo Celular/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Linfócitos T/química , Processamento de Imagem Assistida por Computador/métodosRESUMO
We performed a detailed genomic investigation of the chimpanzee locus syntenic to human chromosome 4q35.2, associated to the facioscapulohumeral dystrophy. Two contigs of approximately 150 kb and 200 kb were derived from PTR chromosomes 4q35 and 3p12, respectively: both regions showed a very similar sequence organization, including D4Z4 and Beta satellite linked clusters. Starting from these findings, we derived a hypothetical evolutionary history of human 4q35, 10q26 and 3p12 chromosome regions focusing on the D4Z4-Beta satellite linked organization. The D4Z4 unit showed an open reading frame (DUX4) at both PTR 4q35 and 3p12 regions; furthermore some subregions of the Beta satellite unit showed a high degree of conservation between chimpanzee and humans. In conclusion, this paper provides evidence that at the 4q subtelomere the linkage between D4Z4 and Beta satellite arrays is a feature that appeared late during evolution and is conserved between chimpanzee and humans.
Assuntos
Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 4/genética , Evolução Molecular , Distrofia Muscular Facioescapuloumeral/genética , Pan troglodytes/genética , Animais , Sequência de Bases , Southern Blotting , Mapeamento de Sequências Contíguas , Ligação Genética , Biblioteca Genômica , Proteínas de Homeodomínio/genética , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Família Multigênica/genética , Análise de Sequência de DNA , Especificidade da Espécie , Sintenia/genéticaRESUMO
How gene expression is controlled to preserve human T cell quiescence is poorly understood. Here we show that non-canonical splicing variants containing long interspersed nuclear element 1 (LINE1) enforce naive CD4+ T cell quiescence. LINE1-containing transcripts are derived from CD4+ T cell-specific genes upregulated during T cell activation. In naive CD4+ T cells, LINE1-containing transcripts are regulated by the transcription factor IRF4 and kept at chromatin by nucleolin; these transcripts act in cis, hampering levels of histone 3 (H3) lysine 36 trimethyl (H3K36me3) and stalling gene expression. T cell activation induces LINE1-containing transcript downregulation by the splicing suppressor PTBP1 and promotes expression of the corresponding protein-coding genes by the elongating factor GTF2F1 through mTORC1. Dysfunctional T cells, exhausted in vitro or tumor-infiltrating lymphocytes (TILs), accumulate LINE1-containing transcripts at chromatin. Remarkably, depletion of LINE1-containing transcripts restores TIL effector function. Our study identifies a role for LINE1 elements in maintaining T cell quiescence and suggests that an abundance of LINE1-containing transcripts is critical for T cell effector function and exhaustion.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Cromatina/metabolismo , Regulação da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Splicing de RNA , Linfócitos T CD4-Positivos/imunologia , Cromatina/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Histonas/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfoproteínas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica , NucleolinaRESUMO
Epigenetic mechanisms govern the quality, the stability, and the responsiveness of transcriptional programs to the environment. This regulation is ensured via the concerted action of different players (transcription factors, "reader" and "writer" enzymes, histone marks, structural proteins, noncoding regulatory RNAs) that flow in the 3D organization of the genome. Indeed, nuclear architecture participates in the punctual and cell-type-specific regulation of transcription. Hence, the fine dissection of these mechanisms will allow a deeper understanding of the gene expression machinery. In this chapter, we propose a challenging imaging-based method to study the reciprocal interactions between chromatin-associated RNAs, genomic loci, and chromatin compartment with a procedure of 3D COMBO chrRNA-DNA-ImmunoFISH, specifically developed to preserve the nuclear integrity and topology of human primary T cells. We believe that our protocol will contribute to the improvement of epigenetic studies on the 3D nuclear structure of T cell subsets, possibly shedding light on the still hidden epigenetic players responsible for the great plasticity and functional diversification exerted by T cells.
Assuntos
Núcleo Celular/metabolismo , Epigênese Genética/genética , Células Cultivadas , Elementos de DNA Transponíveis/genética , Humanos , Linfócitos T/metabolismoRESUMO
We present an algorithm, and its MATLAB implementation, based on mathematical methods to detect and localize 3D multicolor DNA FISH spots in fluorescence cell image z-stacks. This algorithm provides a method to measure the relative positioning of spots in the nucleus and inter-spot distances with the aim to enrich our understanding of the 3D spatial organization of the genome within the cell nucleus.
Assuntos
Algoritmos , Núcleo Celular/metabolismo , Genoma/genética , Animais , Núcleo Celular/genética , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects. RESULTS: In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation. CONCLUSION: We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Desenvolvimento Muscular/genética , Distrofia Muscular Facioescapuloumeral/genética , Mioblastos Esqueléticos/metabolismo , Proteínas Nucleares/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Cromossomos Humanos Par 4/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji , Masculino , Proteínas dos Microfilamentos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Facioescapuloumeral/metabolismo , Mioblastos Esqueléticos/ultraestrutura , Proteínas Nucleares/genética , Oxirredutases N-Desmetilantes/metabolismo , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA , Proteínas Repressoras/metabolismo , Sequências de Repetição em Tandem/fisiologiaRESUMO
A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.
Assuntos
Núcleo Celular/metabolismo , DNA/metabolismo , Imageamento Tridimensional/métodos , Hibridização in Situ Fluorescente/métodos , HumanosRESUMO
Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are imprinting-related disorders associated with genetic/epigenetic alterations of the 11p15.5 region, which harbours two clusters of imprinted genes (IGs). 11p15.5 IGs are regulated by the methylation status of imprinting control regions ICR1 and ICR2. 3D chromatin structure is thought to play a pivotal role in gene expression control; however, chromatin architecture models are still poorly defined in most cases, particularly for IGs. Our study aimed at elucidating 11p15.5 3D structure, via 3C and 3D FISH analyses of cell lines derived from healthy, BWS or SRS children. We found that, in healthy cells, IGF2/H19 and CDKN1C/KCNQ1OT1 domains fold in complex chromatin conformations, that facilitate the control of IGs mediated by distant enhancers. In patient-derived cell lines, we observed a profound impairment of such a chromatin architecture. Specifically, we identified a cross-talk between IGF2/H19 and CDKN1C/KCNQ1OT1 domains, consisting in in cis, monoallelic interactions, that are present in healthy cells but lost in patient cell lines: an inter-domain association that sees ICR2 move close to IGF2 on one allele, and to H19 on the other. Moreover, an intra-domain association within the CDKN1C/KCNQ1OT1 locus seems to be crucial for maintaining the 3D organization of the region.