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1.
Proc Natl Acad Sci U S A ; 114(40): 10654-10659, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28923964

RESUMO

X chromosome inactivation is an epigenetic dosage compensation mechanism in female mammals driven by the long noncoding RNA, Xist. Although recent genomic and proteomic approaches have provided a more global view of Xist's function, how Xist RNA localizes to the inactive X chromosome (Xi) and spreads in cis remains unclear. Here, we report that the CDKN1-interacting zinc finger protein CIZ1 is critical for localization of Xist RNA to the Xi chromosome territory. Stochastic optical reconstruction microscopy (STORM) shows a tight association of CIZ1 with Xist RNA at the single-molecule level. CIZ1 interacts with a specific region within Xist exon 7-namely, the highly repetitive Repeat E motif. Using genetic analysis, we show that loss of CIZ1 or deletion of Repeat E in female cells phenocopies one another in causing Xist RNA to delocalize from the Xi and disperse into the nucleoplasm. Interestingly, this interaction is exquisitely sensitive to CIZ1 levels, as overexpression of CIZ1 likewise results in Xist delocalization. As a consequence, this delocalization is accompanied by a decrease in H3K27me3 on the Xi. Our data reveal that CIZ1 plays a major role in ensuring stable association of Xist RNA within the Xi territory.


Assuntos
Cromossomos de Mamíferos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares , RNA Longo não Codificante , Sequências Repetitivas de Ácido Nucleico , Cromossomo X , Animais , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Motivos de Nucleotídeos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromossomo X/genética , Cromossomo X/metabolismo
2.
PLoS One ; 12(8): e0182568, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28796844

RESUMO

In mammals, monoallelic gene expression can result from X-chromosome inactivation, genomic imprinting, and random monoallelic expression (RMAE). Epigenetic regulation of RMAE is not fully understood. Here we analyze allelic imbalance in chromatin state of autosomal genes using ChIP-seq in a clonal cell line. We identify approximately 3.7% of autosomal genes that show significant differences between chromatin states of two alleles. Allelic regulation is represented among several functional gene categories including histones, chromatin modifiers, and multiple early developmental regulators. Most cases of allelic skew are produced by quantitative differences between two allelic chromatic states that belong to the same gross type (active, silent, or bivalent). Combinations of allelic states of different types are possible but less frequent. When different chromatin marks are skewed on the same gene, their skew is coordinated as a result of quantitative relationships between these marks on each individual allele. Finally, combination of allele-specific densities of chromatin marks is a quantitative predictor of allelic skew in gene expression.


Assuntos
Desequilíbrio Alélico , Cromatina/genética , Alelos , Animais , Linhagem Celular , Epigênese Genética , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Genoma , Impressão Genômica , Masculino , Camundongos , Camundongos da Linhagem 129
3.
Science ; 356(6343)2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28619887

RESUMO

Chen et al (Reports, 28 October 2016, p. 468) proposed that an interaction between Xist RNA and Lamin B receptor (LBR) is necessary and sufficient for Xist spreading during X-chromosome inactivation. We reanalyzed their data and found that reported genotypes of mutants are not supported by the sequencing data. These inconsistencies preclude assessment of the role of LBR in Xist spreading.


Assuntos
Inativação Gênica , Lâmina Nuclear , RNA Longo não Codificante/genética , RNA não Traduzido/genética , Cromossomo X , Inativação do Cromossomo X
4.
Mol Cell ; 57(2): 361-75, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25578877

RESUMO

CTCF is a master regulator that plays important roles in genome architecture and gene expression. How CTCF is recruited in a locus-specific manner is not fully understood. Evidence from epigenetic processes, such as X chromosome inactivation (XCI), indicates that CTCF associates functionally with RNA. Using genome-wide approaches to investigate the relationship between its RNA interactome and epigenomic landscape, here we report that CTCF binds thousands of transcripts in mouse embryonic stem cells, many in close proximity to CTCF's genomic binding sites. CTCF is a specific and high-affinity RNA-binding protein (Kd < 1 nM). During XCI, CTCF differentially binds the active and inactive X chromosomes and interacts directly with Tsix, Xite, and Xist RNAs. Tsix and Xite RNAs target CTCF to the X inactivation center, thereby inducing homologous X chromosome pairing. Our work elucidates one mechanism by which CTCF is recruited in a locus-specific manner and implicates CTCF-RNA interactions in long-range chromosomal interactions.


Assuntos
RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Cromossomo X/genética , Animais , Fator de Ligação a CCCTC , Células Cultivadas , Pareamento Cromossômico , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Loci Gênicos , Camundongos , Ligação Proteica
5.
Cell ; 159(4): 869-83, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25417162

RESUMO

X chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). PRC2 is also targeted to other sites throughout the genome to effect transcriptional repression. Using XCI as a model, we apply an unbiased proteomics approach to isolate Xist and PRC2 regulators and identified ATRX. ATRX unexpectedly functions as a high-affinity RNA-binding protein that directly interacts with RepA/Xist RNA to promote loading of PRC2 in vivo. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X chromosome. Moreover, epigenomic profiling reveals that genome-wide targeting of PRC2 depends on ATRX, as loss of ATRX leads to spatial redistribution of PRC2 and derepression of Polycomb responsive genes. Thus, ATRX is a required specificity determinant for PRC2 targeting and function.


Assuntos
DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/metabolismo , Inativação do Cromossomo X , Animais , DNA Helicases/isolamento & purificação , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Proteínas Nucleares/isolamento & purificação , Proteína Nuclear Ligada ao X
7.
Cell ; 153(7): 1537-51, 2013 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-23791181

RESUMO

In mammals, dosage compensation between XX and XY individuals occurs through X chromosome inactivation (XCI). The noncoding Xist RNA is expressed and initiates XCI only when more than one X chromosome is present. Current models invoke a dependency on the X-to-autosome ratio (X:A), but molecular factors remain poorly defined. Here, we demonstrate that molecular titration between an X-encoded RNA and an autosomally encoded protein dictates Xist induction. In pre-XCI cells, CTCF protein represses Xist transcription. At the onset of XCI, Jpx RNA is upregulated, binds CTCF, and extricates CTCF from one Xist allele. We demonstrate that CTCF is an RNA-binding protein and is titrated away from the Xist promoter by Jpx RNA. Thus, Jpx activates Xist by evicting CTCF. The functional antagonism via molecular titration reveals a role for long noncoding RNA in epigenetic regulation.


Assuntos
RNA Longo não Codificante/metabolismo , Proteínas Repressoras/metabolismo , Regulação para Cima , Inativação do Cromossomo X , Animais , Fator de Ligação a CCCTC , Cromossomos de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Cromossomo X/metabolismo
8.
Genome Res ; 22(10): 1864-76, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22948768

RESUMO

X chromosome inactivation (XCI) achieves dosage balance in mammals by repressing one of two X chromosomes in females. During XCI, the long noncoding Xist RNA and Polycomb proteins spread along the inactive X (Xi) to initiate chromosome-wide silencing. Although inactivation is known to commence at the X-inactivation center (Xic), how it propagates remains unknown. Here, we examine allele-specific binding of Polycomb repressive complex 2 (PRC2) and chromatin composition during XCI and generate a chromosome-wide profile of Xi and Xa (active X) at nucleosome-resolution. Initially, Polycomb proteins are localized to ∼150 strong sites along the X and concentrated predominantly within bivalent domains coinciding with CpG islands ("canonical sites"). As XCI proceeds, ∼4000 noncanonical sites are recruited, most of which are intergenic, nonbivalent, and lack CpG islands. Polycomb sites are depleted of LINE repeats but enriched for SINEs and simple repeats. Noncanonical sites cluster around the ∼150 strong sites, and their H3K27me3 levels reflect a graded concentration originating from strong sites. This suggests that PRC2 and H3K27 methylation spread along a gradient unique to XCI. We propose that XCI is governed by a hierarchy of defined Polycomb stations that spread H3K27 methylation in cis.


Assuntos
Proteínas do Grupo Polycomb/metabolismo , Inativação do Cromossomo X , Alelos , Animais , Sítios de Ligação , Linhagem Celular , Imunoprecipitação da Cromatina , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/química , Domínios e Motivos de Interação entre Proteínas , Sequências Repetitivas de Ácido Nucleico , Cromossomo X
9.
Curr Opin Genet Dev ; 22(2): 62-71, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22424802

RESUMO

Equalization of X linked gene expression is necessary in mammalian cells due to the presence of two X chromosomes in females and one in males. To achieve this, all female cells inactivate one of the two X chromosomes during development. This process, termed X chromosome inactivation (XCI), is a quintessential epigenetic phenomenon and involves a complex interplay between noncoding RNAs and protein factors. Progress in this area of study has consequently resulted in new approaches to study epigenetics and regulatory RNA function. Here we will discuss recent developments in the field that have advanced our understanding of XCI and its regulatory mechanisms.


Assuntos
Inativação do Cromossomo X , Animais , Dano ao DNA , Dosagem de Genes , Regulação da Expressão Gênica , Inativação Gênica , Humanos , RNA não Traduzido/genética
10.
Cell ; 146(1): 119-33, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21729784

RESUMO

The long noncoding Xist RNA inactivates one X chromosome in the female mammal. Current models posit that Xist induces silencing as it spreads along X and recruits Polycomb complexes. However, the mechanisms for Xist loading and spreading are currently unknown. Here, we define the nucleation center for Xist RNA and show that YY1 docks Xist particles onto the X chromosome. YY1 is a "bivalent" protein, capable of binding both RNA and DNA through different sequence motifs. Xist's exclusive attachment to the inactive X is determined by an epigenetically regulated trio of YY1 sites as well as allelic origin. Specific YY1-to-RNA and YY1-to-DNA contacts are required to load Xist particles onto X. YY1 interacts with Xist RNA through Repeat C. We propose that YY1 acts as adaptor between regulatory RNA and chromatin targets.


Assuntos
RNA não Traduzido/metabolismo , Inativação do Cromossomo X , Cromossomo X/genética , Fator de Transcrição YY1/metabolismo , Animais , Feminino , Camundongos , Proteínas do Grupo Polycomb , RNA Longo não Codificante , RNA não Traduzido/química , Proteínas Repressoras/metabolismo , Transgenes
11.
Semin Cell Dev Biol ; 22(4): 336-42, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21376830

RESUMO

Acquisition of the pluripotent state coincides with epigenetic reprogramming of the X-chromosome. Female embryonic stem cells are characterized by the presence of two active X-chromosomes, cell differentiation by inactivation of one of the two Xs, and induced pluripotent stem cells by reactivation of the inactivated X-chromosome in the originating somatic cell. The tight linkage between X- and stem cell reprogramming occurs through pluripotency factors acting on noncoding genes of the X-inactivation center. This review article will discuss the latest advances in our understanding at the molecular level. Mouse embryonic stem cells provide a standard for defining the pluripotent ground state, which is characterized by low levels of the noncoding Xist RNA and the absence of heterochromatin marks on the X-chromosome. Human pluripotent stem cells, however, exhibit X-chromosome epigenetic instability that may have implications for their use in regenerative medicine. XIST RNA and heterochromatin marks on the X-chromosome indicate whether human pluripotent stem cells are developmentally 'naïve', with characteristics of the pluripotent ground state. X-chromosome status and determination thereof via noncoding RNA expression thus provide valuable benchmarks of the epigenetic quality of pluripotent stem cells, an important consideration given their enormous potential for stem cell therapy.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA não Traduzido/metabolismo , Cromossomo X/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Camundongos , RNA não Traduzido/genética , Inativação do Cromossomo X
12.
Biochem Biophys Res Commun ; 365(3): 575-82, 2008 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-17997977

RESUMO

In model organisms, MCM10 is required for forming the pre-initiation complex for initiation of chromosome replication and is involved in the elongation step. To investigate the role of MCM10 in human chromosome replication, we used small interfering RNA (siRNA) in MCM10-knockdown experiments and found that knockdown accumulated S and G2 phase cells. The chromosome replication of MCM10-knockdown cells was slowed during early and mid S phases, although Cdc45, Polalpha, and PCNA proteins were loaded onto the chromatin, and was aberrant during late S phase. Our results indicate that MCM10 is essential for the efficient elongation step of chromosome replication.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Cromossomos Humanos/genética , Replicação do DNA/genética , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , DNA Polimerase I/análise , DNA Polimerase I/metabolismo , Replicação do DNA/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Fase G2/genética , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo , Antígeno Nuclear de Célula em Proliferação/análise , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno/farmacologia , Fase S/efeitos dos fármacos , Fase S/genética
13.
J Biol Chem ; 282(20): 14882-90, 2007 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-17293600

RESUMO

Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G(1) to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G(1)/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G(1)/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.


Assuntos
Proteínas de Transporte/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Fase G1/fisiologia , Proteínas Nucleares/metabolismo , Fase S/fisiologia , Linhagem Celular , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Dano ao DNA , Humanos , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Ativadas por p21
14.
Proc Natl Acad Sci U S A ; 102(18): 6419-24, 2005 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-15845769

RESUMO

Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that fire reproducibly. Early replication was correlated with high gene density. An acute transition of gene density from early to late replicating areas suggests that discrete chromatin states dictate early versus late replication. Surprisingly, at least 60% of the interrogated chromosomal segments replicate equally in all quarters of S phase, suggesting that large stretches of chromosomes are replicated by inefficient, variably located and asynchronous origins and forks, producing a pan-S phase pattern of replication. Thus, at least for aneuploid cancer cells, a typical discrete time of replication in S phase is not seen for large segments of the chromosomes.


Assuntos
Cromossomos Humanos/genética , Replicação do DNA/fisiologia , Origem de Replicação/genética , Fase S/genética , Análise por Conglomerados , Replicação do DNA/genética , DNA Complementar/genética , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Fatores de Tempo
15.
Mol Cell ; 11(4): 997-1008, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12718885

RESUMO

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Transformação Celular Neoplásica/genética , Replicação do DNA/genética , Células Eucarióticas/metabolismo , Genes cdc/fisiologia , Proteína Supressora de Tumor p53/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Quinase do Ponto de Checagem 2 , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Ciclina A/genética , Ciclina A/metabolismo , Ciclina A/farmacologia , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo
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