RESUMO
Recent studies recognize a vast diversity of noncoding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using C0T-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (C0T-1 RNA), including LINE-1. C0T-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The C0T-1 RNA territory resists mechanical disruption and fractionates with the nonchromatin scaffold but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. C0T-1 RNA has several properties similar to XIST chromosomal RNA but is excluded from chromatin condensed by XIST. These findings impact two "black boxes" of genome science: the poorly understood diversity of noncoding RNA and the unexplained abundance of repetitive elements.
Assuntos
Cromossomos de Mamíferos/química , Eucromatina/química , Interfase , RNA não Traduzido/análise , Animais , Núcleo Celular/química , Humanos , Células Híbridas , Elementos Nucleotídeos Longos e Dispersos , Camundongos , RNA não Traduzido/genética , Sequências Repetitivas de Ácido Nucleico , Transcrição GênicaRESUMO
Here we provide a brief review of relevant background before presenting results of our investigation into the interplay between scaffold attachment factor A (SAF-A), chromatin-associated RNAs, and DNA condensation. SAF-A, also termed heterogenous nuclear protein U (hnRNP U), is a ubiquitous nuclear scaffold protein that was implicated in XIST RNA localization to the inactive X-chromosome (Xi) but also reported to maintain open DNA packaging in euchromatin. Here we use several means to perturb SAF-A and examine potential impacts on the broad association of RNAs on euchromatin, and on chromatin compaction. SAF-A has an N-terminal DNA binding domain and C-terminal RNA binding domain, and a prominent model has been that the protein provides a single-molecule bridge between XIST RNA and chromatin. Here analysis of the impact of SAF-A on broad RNA-chromatin interactions indicate greater biological complexity. We focus on SAF-A's role with repeat-rich C0T-1 hnRNA (repeat-rich heterogeneous nuclear RNA), shown recently to comprise mostly intronic sequences of pre-mRNAs and diverse long non-coding RNAs (lncRNAs). Our results show that SAF-A mutants cause dramatic changes to cytological chromatin condensation through dominant negative effects on C0T-1 RNA's association with euchromatin, and likely other nuclear scaffold factors. In contrast, depletion of SAF-A by RNA interference (RNAi) had no discernible impact on C0T-1 RNA, nor did it cause similarly marked chromatin changes as did three different SAF-A mutations. Overall results support the concept that repeat-rich, chromatin-associated RNAs interact with multiple RNA binding proteins (RBPs) in a complex dynamic meshwork that is integral to larger-scale chromatin architecture and collectively influences cytological-scale DNA condensation.
Assuntos
Cromatina , RNA Longo não Codificante , Cromatina/genética , Eucromatina , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Cromossomo XRESUMO
Down's syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down's syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a 'chromosome 21 Barr body'. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of 'chromosome therapy'.
Assuntos
Cromossomos Humanos Par 21/genética , Mecanismo Genético de Compensação de Dose , Síndrome de Down/genética , RNA Longo não Codificante/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Metilação de DNA , Síndrome de Down/terapia , Inativação Gênica , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Mutagênese Insercional , Neurogênese , RNA Longo não Codificante/genética , Cromatina Sexual/genética , Inativação do Cromossomo X/genéticaRESUMO
Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.
Assuntos
Cromossomos de Mamíferos/genética , Impressão Genômica , Mães , Proteínas Repressoras/metabolismo , Inativação do Cromossomo X/genética , Cromossomo X/genética , Animais , Animais Congênicos , Blastocisto/metabolismo , Linhagem Celular , Perda do Embrião/genética , Pai , Feminino , Inativação Gênica , Masculino , Camundongos , Camundongos Transgênicos , RNA Longo não Codificante , RNA não Traduzido/genética , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Ubiquitina-Proteína LigasesRESUMO
Interest has recently reawakened in whether loss of the heterochromatic X chromosome (Barr body) is prevalent in certain breast and ovarian cancers, and new insights into the mechanisms involved have emerged. Mitotic segregation errors commonly explain the loss of the inactive X chromosome (Xi), but compromise of Xi heterochromatin in some cancers may signal broader deficits of nuclear heterochromatin. The debated link between BRCA1 and Xi might reflect a general relationship between BRCA1 and heterochromatin, which could connect BRCA1 to both epigenetic and genetic instability. We suggest that heterochromatic instability is a common but largely unexplored mechanism, leading to widespread genomic misregulation and the evolution of some cancers.
Assuntos
Neoplasias da Mama/patologia , Neoplasias Ovarianas/patologia , Cromatina Sexual/patologia , Neoplasias da Mama/metabolismo , Cromossomos Humanos X , Feminino , Genes BRCA1 , Heterocromatina/metabolismo , Humanos , Neoplasias Ovarianas/metabolismo , Cromatina Sexual/metabolismoRESUMO
The linear DNA sequence of mammalian chromosomes is organized in large blocks of DNA with similar sequence properties, producing a pattern of dark and light staining bands on mitotic chromosomes. Cytogenetic banding is essentially invariant between people and cell-types and thus may be assumed unrelated to genome regulation. We investigate whether large blocks of Alu-rich R-bands and L1-rich G-bands provide a framework upon which functional genome architecture is built. We examine two models of large-scale chromatin condensation: X-chromosome inactivation and formation of senescence-associated heterochromatin foci (SAHFs). XIST RNA triggers gene silencing but also formation of the condensed Barr Body (BB), thought to reflect cumulative gene silencing. However, we find Alu-rich regions are depleted from the L1-rich BB, supporting it is a dense core but not the entire chromosome. Alu-rich bands are also gene-rich, affirming our earlier findings that genes localize at the outer periphery of the BB. SAHFs similarly form within each territory by coalescence of syntenic L1 regions depleted for highly Alu-rich DNA. Analysis of senescent cell Hi-C data also shows large contiguous blocks of G-band and R-band DNA remodel as a segmental unit. Entire dark-bands gain distal intrachromosomal interactions as L1-rich regions form the SAHF. Most striking is that sharp Alu peaks within R-bands resist these changes in condensation. We further show that Chr19, which is exceptionally Alu rich, fails to form a SAHF. Collective results show regulation of genome architecture corresponding to large blocks of DNA and demonstrate resistance of segments with high Alu to chromosome condensation.
RESUMO
XIST RNA triggers chromosome-wide gene silencing and condenses an active chromosome into a Barr body. Here, we use inducible human XIST to examine early steps in the process, showing that XIST modifies cytoarchitecture before widespread gene silencing. In just 2-4 h, barely visible transcripts populate the large "sparse zone" surrounding the smaller "dense zone"; importantly, density zones exhibit different chromatin impacts. Sparse transcripts immediately trigger immunofluorescence for H2AK119ub and CIZ1, a matrix protein. H3K27me3 appears hours later in the dense zone, which enlarges with chromosome condensation. Genes examined are silenced after compaction of the RNA/DNA territory. Insights into this come from the findings that the A-repeat alone can silence genes and rapidly, but only where dense RNA supports sustained histone deacetylation. We propose that sparse XIST RNA quickly impacts architectural elements to condense the largely non-coding chromosome, coalescing RNA density that facilitates an unstable, A-repeat-dependent step required for gene silencing.
Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromatina , Inativação Gênica , Cromossomo X/metabolismoRESUMO
RNAs play diverse roles in formation and function of subnuclear compartments, most of which are associated with active genes. NEAT1 and NEAT2/MALAT1 exemplify long non-coding RNAs (lncRNAs) known to function in nuclear bodies; however, we suggest that RNA biogenesis itself may underpin much nuclear compartmentalization. Recent studies show that active genes cluster with nuclear speckles on a genome-wide scale, significantly advancing earlier cytological evidence that speckles (aka SC-35 domains) are hubs of concentrated pre-mRNA metabolism. We propose the 'karyotype to hub' hypothesis to explain this organization: clustering of genes in the human karyotype may have evolved to facilitate the formation of efficient nuclear hubs, driven in part by the propensity of ribonucleoproteins (RNPs) to form large-scale condensates. The special capacity of highly repetitive RNAs to impact architecture is highlighted by recent findings that human satellite II RNA sequesters factors into abnormal nuclear bodies in disease, potentially co-opting a normal developmental mechanism.
Assuntos
Núcleo Celular/metabolismo , Genoma , RNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cariótipo , Proteínas de Ligação a RNA/metabolismoRESUMO
The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different "nuclear landscape" in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell-type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML-defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display approximately 1-3 large PML structures of two morphological types: long linear "rods" or elaborate "rosettes", which lack substantial SUMO-1, Daxx, and Sp100. These occur primarily between Day 0-2 of differentiation and become rare thereafter. PML rods may be "taut" between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a "gap" in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML-defined structures.
Assuntos
Núcleo Celular/ultraestrutura , Células-Tronco Embrionárias/citologia , Epigênese Genética , Humanos , Leucemia Promielocítica Aguda/etiologia , Leucemia Promielocítica Aguda/patologia , Células-Tronco Pluripotentes/citologiaRESUMO
The clinical and research value of human embryonic stem cells (hESC) depends upon maintaining their epigenetically naïve, fully undifferentiated state. Inactivation of one X chromosome in each cell of mammalian female embryos is a paradigm for one of the earliest steps in cell specialization through formation of facultative heterochromatin. Mouse ES cells are derived from the inner cell mass (ICM) of blastocyst stage embryos prior to X-inactivation, and cultured murine ES cells initiate this process only upon differentiation. Less is known about human X-inactivation during early development. To identify a human ES cell model for X-inactivation and study differences in the epigenetic state of hESC lines, we investigated X-inactivation in all growth competent, karyotypically normal, NIH approved, female hESC lines and several sublines. In the vast majority of undifferentiated cultures of nine lines examined, essentially all cells exhibit hallmarks of X-inactivation. However, subcultures of any hESC line can vary in X-inactivation status, comprising distinct sublines. Importantly, we identified rare sublines that have not yet inactivated Xi and retain competence to undergo X-inactivation upon differentiation. Other sublines exhibit defects in counting or maintenance of XIST expression on Xi. The few hESC sublines identified that have not yet inactivated Xi may reflect the earlier epigenetic state of the human ICM and represent the most promising source of NIH hESC for study of human X-inactivation. The many epigenetic anomalies seen indicate that maintenance of fully unspecialized cells, which have not formed Xi facultative heterochromatin, is a delicate epigenetic balance difficult to maintain in culture.
Assuntos
Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/fisiologia , Epigênese Genética , Inativação do Cromossomo X , Animais , Cromossomos Humanos X , Mecanismo Genético de Compensação de Dose , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Camundongos , RNA Longo não Codificante , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
This study reveals that high-copy satellite II (HSATII) sequences in the human genome can bind and impact distribution of chromatin regulatory proteins and that this goes awry in cancer. In many cancers, master regulatory proteins form two types of cancer-specific nuclear bodies, caused by locus-specific deregulation of HSATII. DNA demethylation at the 1q12 mega-satellite, common in cancer, causes PRC1 aggregation into prominent Cancer-Associated Polycomb (CAP) bodies. These loci remain silent, whereas HSATII loci with reduced PRC1 become derepressed, reflecting imbalanced distribution of UbH2A on these and other PcG-regulated loci. Large nuclear foci of HSATII RNA form and sequester copious MeCP2 into Cancer-Associated Satellite Transcript (CAST) bodies. Hence, HSATII DNA and RNA have an exceptional capacity to act as molecular sponges and sequester chromatin regulatory proteins into abnormal nuclear bodies in cancer. The compartmentalization of regulatory proteins within nuclear structure, triggered by demethylation of "junk" repeats, raises the possibility that this contributes to further compromise of the epigenome and neoplastic progression.
Assuntos
Desmetilação do DNA , DNA Satélite/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Neoplasias/genética , Proteínas do Grupo Polycomb/metabolismo , RNA/metabolismo , Proteína BRCA1/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Loci Gênicos , Humanos , Modelos Biológicos , Complexo Repressor Polycomb 1/metabolismo , Agregados Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Direct localization of specific genes, RNAs, and proteins has allowed the dissection of individual nuclear speckles in relation to the molecular biology of gene expression. Nuclear speckles (aka SC35 domains) are essentially ubiquitous structures enriched for most pre-mRNA metabolic factors, yet their relationship to gene expression has been poorly understood. Analyses of specific genes and their spliced or mature mRNA strongly support that SC35 domains are hubs of activity, not stores of inert factors detached from gene expression. We propose that SC35 domains are hubs that spatially link expression of specific pre-mRNAs to rapid recycling of copious RNA metabolic complexes, thereby facilitating expression of many highly active genes. In addition to increasing the efficiency of each step, sequential steps in gene expression are structurally integrated at each SC35 domain, consistent with other evidence that the biochemical machineries for transcription, splicing, and mRNA export are coupled. Transcription and splicing are subcompartmentalized at the periphery, with largely spliced mRNA entering the domain prior to export. In addition, new findings presented here begin to illuminate the structural underpinnings of a speckle by defining specific perturbations of phosphorylation that promote disassembly or assembly of an SC35 domain in relation to other components. Results thus far are consistent with the SC35 spliceosome assembly factor as an integral structural component. Conditions that disperse SC35 also disperse poly(A) RNA, whereas the splicing factor ASF/SF2 can be dispersed under conditions in which SC35 or SRm300 remain as intact components of a core domain.
Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Precursores de RNA/química , Precursores de RNA/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/fisiologia , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/genética , Estrutura Terciária de Proteína/genética , Precursores de RNA/biossíntese , Splicing de RNA/genética , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina , Spliceossomos/química , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Beginning with the precedent of XIST RNA as a 'chromosomal RNA' (cRNA), there is growing interest in the possibility that a diversity of non-coding RNAs may function in chromatin. We review findings which lead us to suggest that RNA is essentially a widespread component of interphase chromosomes. Further, RNA likely contributes to architecture and regulation, with repeat-rich 'junk' RNA in euchromatin (ecRNA) promoting a more open chromatin state. Thousands of low-abundance nuclear RNAs have been reported, however it remains a challenge to determine which of these may function in chromatin. Recent findings indicate that repetitive sequences are enriched in chromosome-associated non-coding RNAs, and repeat-rich RNA shows unusual properties, including localization and stability, with similarities to XIST RNA. We suggest two frontiers in genome biology are emerging and may intersect: the broad contribution of RNA to interphase chromosomes and the distinctive properties of repeat-rich intronic or intergenic junk sequences that may play a role in chromosome structure and regulation.
Assuntos
Cromatina/genética , Cromossomos/genética , Interfase/genética , RNA/genética , Sequência de Bases/genética , Eucromatina/genética , Genoma , RNA Longo não Codificante/genéticaRESUMO
Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results.
Assuntos
Núcleo Celular/genética , DNA/análise , Hibridização in Situ Fluorescente/métodos , RNA/análise , Animais , Sondas de DNA , Humanos , Indicadores e Reagentes , Sondas de OligonucleotídeosRESUMO
An inducible program of inflammatory gene expression is central to antimicrobial defenses. This response is controlled by a collaboration involving signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin-modifying factors. We have identified a long noncoding RNA (lncRNA) that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2, mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad-acting regulatory component of the circuit that controls the inflammatory response.
Assuntos
Regulação da Expressão Gênica , Imunidade Inata/genética , Inflamação/genética , Macrófagos/imunologia , Macrófagos/metabolismo , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Ciclo-Oxigenase 2/genética , Citocinas/genética , Citocinas/metabolismo , Citosol/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ativação de Macrófagos , Camundongos , Modelos Imunológicos , Interferência de RNA , RNA Longo não Codificante/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
How XIST RNA strictly localizes across the inactive X chromosome is unknown; however, prophase release of human XIST RNA provides a clue. Tests of inhibitors that mimic mitotic chromatin modifications implicated an indirect role of PP1 (protein phosphatase 1), potentially via its interphase repression of Aurora B kinase (AURKB), which phosphorylates H3 and chromosomal proteins at prophase. RNA interference to AURKB causes mitotic retention of XIST RNA, unlike other mitotic or broad kinase inhibitors. Thus, AURKB plays an unexpected role in regulating RNA binding to heterochromatin, independent of mechanics of mitosis. H3 phosphorylation (H3ph) was shown to precede XIST RNA release, whereas results exclude H1ph involvement. Of numerous Xi chromatin (chromosomal protein) hallmarks, ubiquitination closely follows XIST RNA retention or release. Surprisingly, H3S10ph staining (but not H3S28ph) is excluded from Xi and is potentially linked to ubiquitination. Results suggest a model of multiple distinct anchor points for XIST RNA. This study advances understanding of RNA chromosome binding and the roles of AURKB and demonstrates a novel approach to manipulate and study XIST RNA.
Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA não Traduzido/metabolismo , RNA/metabolismo , Animais , Aurora Quinase B , Aurora Quinases , Ciclo Celular/fisiologia , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/genética , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Indóis/metabolismo , Masculino , Complexo Mediador , Modelos Moleculares , Proteína Fosfatase 1/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA/genética , Interferência de RNA , RNA Longo não Codificante , RNA não Traduzido/genética , Sulfonamidas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransgenesRESUMO
Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.
Assuntos
Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Dano ao DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Fibroblastos/enzimologia , Deleção de Genes , Camundongos , Ribonuclease III/metabolismoRESUMO
The BRCA1 tumor suppressor involved in breast and ovarian cancer is linked to several fundamental cell regulatory processes. Recently, it was reported that BRCA1 supports localization of XIST RNA to the inactive X chromosome (Xi) in women. The apparent cytological overlap between BRCA1 and XIST RNA across the Xi raised the possibility a direct role of BRCA1 in localizing XIST. We report here that BRCA1 does not paint the Xi or XIST territory, as do markers of Xi facultative heterochromatin. A smaller BRCA1 accumulation abuts Xi, although this is not exclusive to Xi. In BRCA1 depleted normal and tumor cells, or BRCA1 reconstituted cells, BRCA1 status does not closely correlate with XIST localization, however in a BRCA1 inducible system over-expression correlated strongly with enhanced XIST expression. We confirm frequent loss of an Xi in tumor cells. In addition to mitotic loss of Xi, we find XIST RNA expression or localization frequently become compromised in cultured breast cancer cells, suggesting Xi heterochromatin may not be fully maintained. We demonstrate that complex epigenetic differences between tumor cell subpopulations can have striking effects on XIST transcription, accumulation, and localization, but this does not strictly correlate with BRCA1. Although BRCA1 can have indirect effects that impact XIST, our results do not indicate a direct and specific role in XIST RNA regulation. Rather, regulatory factors such as BRCA1 that have broad effects on chromatin or gene regulation can impact XIST RNA and the Xi. We provide preliminary evidence that this may occur as part of a wider failure of heterochromatin maintenance in some cancers.
Assuntos
Proteína BRCA1/genética , Cromossomos Humanos X/genética , Heterocromatina/genética , RNA não Traduzido/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Hibridização in Situ Fluorescente , Interferência de RNA , RNA Longo não Codificante , Inativação do Cromossomo XRESUMO
During embryogenesis, the XIST RNA is expressed from and localizes to one X chromosome in females and induces chromosome-wide silencing. Although many changes to inactive X heterochromatin are known, the functional relationships between different modifications are not well understood, and studies of the initiation of X-inactivation have been largely confined to mouse. We now present a model system for human XIST RNA function in which induction of an XIST cDNA in somatic cells results in localized XIST RNA and transcriptional silencing. Chromatin immunoprecipitation and immunohistochemistry shows that this silencing need only be accompanied by a subset of heterochromatic marks and that these can differ between integration sites. Surprisingly, silencing is XIST-dependent, remaining reversible over extended periods. Deletion analysis demonstrates that the first exon of human XIST is sufficient for both transcript localization and the induction of silencing and that, unlike the situation in mice, the conserved repeat region is essential for both functions. In addition to providing mechanistic insights into chromosome regulation and formation of facultative heterochromatin, this work provides a tractable model system for the study of chromosome silencing and suggests key differences from mouse embryonic X-inactivation.
Assuntos
Cromossomos Humanos X/genética , Mecanismo Genético de Compensação de Dose , RNA não Traduzido , Inativação do Cromossomo X , Imunoprecipitação da Cromatina , Cromossomos Humanos X/metabolismo , Metilação de DNA , DNA Complementar , Doxiciclina/farmacologia , Fibrossarcoma/patologia , Inativação Gênica , Heterocromatina , Histonas/química , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Modelos Genéticos , RNA Longo não Codificante , Análise de Sequência de DNARESUMO
Most of the human genome encodes neither protein nor known functional RNA, yet available approaches to seek meaningful information in the "noncoding" sequence are limited. The unique biology of the X chromosome, one of which is silenced in mammalian females, can yield clues into sequence motifs involved in chromosome packaging and function. Although autosomal chromatin has some capacity for inactivation, evidence indicates that sequences enriched on the X chromosome render it fully competent for silencing, except in specific regions that escape inactivation. Here we have used a linguistic approach by analyzing the frequency and distribution of nine base-pair genomic "words" throughout the human genome. Results identify previously unknown sequence differences on the human X chromosome. Notably, the dinucleotide repeats [AT]n, [AC]n, and [AG]n are significantly enriched across the X chromosome compared with autosomes. Moreover, a striking enrichment (>10-fold) of [GATA]n is revealed throughout the 10-Mb segment at Xp22 that escapes inactivation, and is confirmed by fluorescence in situ hybridization. A similar enrichment is found in other eutherian genomes. Our findings clearly demonstrate sequence differences relevant to the novel biology and evolution of the X chromosome. Furthermore, they implicate simple sequence repeats, linked to gene regulation and unusual DNA structures, in the regulation and formation of facultative heterochromatin. Results suggest a new paradigm whereby a regional escape from X inactivation is due to the presence of elements that prevent heterochromatinization, rather than the lack of other elements that promote it.