RESUMO
X chromosome inactivation (XCI) in mammals is mediated by Xist RNA which functions in cis to silence genes on a single X chromosome in XX female cells, thereby equalising levels of X-linked gene expression relative to XY males. XCI progresses over a period of several days, with some X-linked genes silencing faster than others. The chromosomal location of a gene is an important determinant of silencing rate, but uncharacterised gene-intrinsic features also mediate resistance or susceptibility to silencing. In this study, we examine mouse embryonic stem cell lines with an inducible Xist allele (iXist-ChrX mESCs) and integrate allele-specific data of gene silencing and decreasing inactive X (Xi) chromatin accessibility over time courses of Xist induction with cellular differentiation. Our analysis reveals that motifs bound by the transcription factor YY1 are associated with persistently accessible regulatory elements, including many promoters and enhancers of slow-silencing genes. We further show that YY1 is evicted relatively slowly from target sites on Xi, and that silencing of X-linked genes is increased upon YY1 degradation. Together our results suggest that YY1 acts as a barrier to Xist-mediated silencing until the late stages of the XCI process.
Assuntos
Inativação Gênica , RNA Longo não Codificante , Inativação do Cromossomo X , Fator de Transcrição YY1 , Animais , Feminino , Masculino , Camundongos , Alelos , Diferenciação Celular/genética , Linhagem Celular , Cromatina/metabolismo , Cromatina/genética , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromossomo X/genética , Cromossomo X/metabolismo , Inativação do Cromossomo X/genética , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/genéticaRESUMO
X chromosome inactivation (XCI) is mediated by the non-coding RNA Xist, which directs chromatin modification and gene silencing in cis. The RNA binding protein SPEN and associated corepressors have a central role in Xist-mediated gene silencing. Other silencing factors, notably the Polycomb system, have been reported to function downstream of SPEN. In recent work, we found that SPEN has an additional role in correct localization of Xist RNA in cis, indicating that its contribution to chromatin-mediated gene silencing needs to be reappraised. Making use of a SPEN separation-of-function mutation, we show that SPEN and Polycomb pathways, in fact, function in parallel to establish gene silencing. We also find that differentiation-dependent recruitment of the chromosomal protein SmcHD1 is required for silencing many X-linked genes. Our results provide important insights into the mechanism of X inactivation and the coordination of chromatin-based gene regulation with cellular differentiation and development.
Assuntos
Proteínas de Drosophila , RNA Longo não Codificante , Cromatina , Proteínas de Drosophila/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromossomo X , Inativação do Cromossomo X/genéticaRESUMO
CIZ1 forms large assemblies at the inactive X chromosome (Xi) in female fibroblasts in an Xist lncRNA-dependent manner and is required for accurate maintenance of polycomb targets genome-wide. Here we address requirements for assembly formation and show that CIZ1 undergoes two direct interactions with Xist, via independent N- and C-terminal domains. Interaction with Xist, assembly at Xi, and complexity of self-assemblies formed in vitro are modulated by two alternatively spliced glutamine-rich prion-like domains (PLD1 and 2). PLD2 is dispensable for accumulation at existing CIZ1-Xi assemblies in wild-type cells but is required in CIZ1-null cells where targeting, assembly, and enrichment for H3K27me3 and H2AK119ub occur de novo. In contrast, PLD1 is required for both de novo assembly and accumulation at preexisting assemblies and, in vitro, drives formation of a stable fibrillar network. Together they impart affinity for RNA and a complex relationship with repeat E of Xist. These data show that alternative splicing of two PLDs modulates CIZ1's ability to build large RNA-protein assemblies.
Assuntos
Proteínas Nucleares , Príons , RNA Longo não Codificante , Inativação do Cromossomo X , Processamento Alternativo , Animais , Feminino , Fibroblastos , Histonas , Camundongos , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
Transposable elements (TEs) regulate diverse biological processes, from early development to cancer. Expression of young TEs is difficult to measure with next-generation, single-cell sequencing technologies because their highly repetitive nature means that short complementary DNA reads cannot be unambiguously mapped to a specific locus. Single CELl LOng-read RNA-sequencing (CELLO-seq) combines long-read single cell RNA-sequencing with computational analyses to measure TE expression at unique loci. We used CELLO-seq to assess the widespread expression of TEs in two-cell mouse blastomeres as well as in human induced pluripotent stem cells. Across both species, old and young TEs showed evidence of locus-specific expression with simulations demonstrating that only a small number of very young elements in the mouse could not be mapped back to the reference with high confidence. Exploring the relationship between the expression of individual elements and putative regulators revealed large heterogeneity, with TEs within a class showing different patterns of correlation and suggesting distinct regulatory mechanisms.
Assuntos
Elementos de DNA Transponíveis , Células-Tronco Pluripotentes Induzidas , Animais , Elementos de DNA Transponíveis/genética , Humanos , Camundongos , RNARESUMO
RNA N 6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5' splice sites. m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream from a 5' splice site that is suppressed in the presence of m6A and upstream of a 5' splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m6A machinery and the regulation of RNA splicing.
Assuntos
Éxons , Íntrons , Splicing de RNA , Transcriptoma , Processamento Alternativo , Animais , Éxons/genética , Íntrons/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Sítios de Splice de RNARESUMO
X-inactive specific transcript (Xist) RNA directs the process of X chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual-color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, which enabled us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we find that the protein SPEN, a key factor for Xist-mediated gene silencing, has a distinct function in Xist RNA localization, stability, and coupling behaviors. Our results provide insights toward understanding the distinct dynamic properties of Xist RNA.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Inativação do Cromossomo X , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias , Inativação Gênica , Camundongos , Microscopia , Proteínas Nucleares/genética , Estabilidade de RNA , RNA Longo não Codificante/biossíntese , Proteínas de Ligação a RNA/genética , Análise Espacial , Transcrição Gênica , Cromossomo X/metabolismoRESUMO
Transcriptional fidelity depends on accurate promoter selection and initiation from the correct sites. In yeast, H3K36me3-mediated recruitment of the Rpd3S HDAC complex to gene bodies suppresses spurious transcription initiation. Here we describe an equivalent pathway in metazoans. PWWP2A/B is an H3K36me3 reader that forms a stable complex with HDAC1/2. We used CAGE-seq to profile all transcription initiation sites in wild-type mESCs and cells lacking PWWP2A/B. Loss of PWWP2A/B enhances spurious initiation from intragenic sites present in wild-type mESCs, and this effect is associated with increased levels of initiating Pol-II and histone acetylation. Spurious initiation events in Pwwp2a/b DKO mESCs do not overlap in genomic location or chromatin features with spurious sites that arise in Dnmt3b KO mESCs, previously reported to function in the suppression of intragenic transcriptional initiation, suggesting these pathways function cooperatively in maintaining the fidelity of transcription initiation in metazoans.
RESUMO
The X inactive-specific transcript (Xist) gene is the master regulator of X chromosome inactivation in mammals. Xist produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify underlying chromatin and silence X-linked genes in cis Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them.
Assuntos
Proteínas/metabolismo , RNA Longo não Codificante/metabolismo , Inativação do Cromossomo X/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica/fisiologia , Metiltransferases/metabolismo , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor de Lamina BRESUMO
Many intricate pathways contribute to the timely control of gene expression during development. Polycomb repressive complexes (PRC1 and PRC2) and long non-coding RNAs (lncRNAs) are players associated with gene repression in various developmental processes such as X chromosome inactivation (XCI) and genomic imprinting. Historically, lncRNAs were proposed to directly recruit PRC2. However, recent evidence suggests that promiscuous interactions between PRC2 and RNA fine-tune the function of the complex through a multiplicity of mechanisms. A PRC2-recruitment model was definitively overturned in the paradigm of XCI by Xist RNA, being replaced by a novel mechanism which puts PRC1 in the spotlight. This review focuses on these recent advances in understanding the interplay between RNA and Polycomb complexes for gene expression control.
Assuntos
Impressão Genômica/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , RNA Longo não Codificante/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Proteínas do Grupo Polycomb/genética , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
Background: X chromosome inactivation in mammals is regulated by the non-coding (nc) RNA, Xist, which represses the chromosome from which it is transcribed. High levels of the N6-methyladenosine (m6A) RNA modification occur within Xist exon I, close to the 5' end of the transcript, and also further 3', in Xist exon VII. The m6A modification is catalysed by the METTL3/14 complex that is directed to specific targets, including Xist, by the RNA binding protein RBM15/15B. m6A modification of Xist RNA has been reported to be important for Xist-mediated gene silencing. Methods: We use CRISPR/Cas9 mediated mutagenesis to delete sequences around the 5' m6A region in interspecific XX mouse embryonic stem cells (mESCs). Following induction of Xist RNA expression, we assay chromosome silencing using allelic RNA-seq and Xist m6A distribution using m6A-seq. Additionally, we use Xist RNA FISH to analyse the effect of deleting the 5' m6A region on the function of the endogenous Xist promoter. We purify epitope tagged RBM15 from mESCs, and then apply MS/MS analysis to define the RBM15 interactome. Results: We show that a deletion encompassing the entire Xist 5' m6A region results in a modest reduction in Xist-mediated silencing, and that the 5' m6A region overlaps essential DNA elements required for activation of the endogenous Xist promoter. Deletion of the Xist A-repeat, to which RBM15 binds, entirely abolishes deposition of m6A in the Xist 5' m6A region without affecting the modification in exon VII. We show that in mESCs, RBM15 interacts with the m6A complex, the SETD1B histone modifying complex, and several proteins linked to RNA metabolism. Conclusions: Our findings support that RBM15 binding to the Xist A-repeat recruits the m6A complex to the 5' Xist m6A region and that this region plays a role in Xist-mediated chromosome silencing.
RESUMO
The non-coding RNA Xist regulates the process of X chromosome inactivation, in which one of the two X chromosomes present in cells of early female mammalian embryos is selectively and coordinately shut down. Remarkably Xist RNA functions in cis, affecting only the chromosome from which it is transcribed. This feature is attributable to the unique propensity of Xist RNA to accumulate over the territory of the chromosome on which it is synthesized, contrasting with the majority of RNAs that are rapidly exported out of the cell nucleus. In this review I provide an overview of the progress that has been made towards understanding localized accumulation of Xist RNA, drawing attention to evidence that some other non-coding RNAs probably function in a highly analogous manner. I describe a simple model for localized accumulation of Xist RNA and discuss key unresolved questions that need to be addressed in future studies.
Assuntos
RNA Longo não Codificante/genética , Inativação do Cromossomo X , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Humanos , Modelos Biológicos , Matriz Nuclear , Proteínas Nucleares/metabolismo , Ligação ProteicaRESUMO
Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing.
Assuntos
RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Inativação do Cromossomo X , Cromossomo X/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA , Técnicas de Inativação de Genes , Inativação Gênica , Histonas/genética , Camundongos , Células-Tronco Embrionárias Murinas , Proteínas do Grupo Polycomb/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3' mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
Assuntos
RNA Mensageiro/biossíntese , Terminação da Transcrição Genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Humanos , Mutação , Poliadenilação , Ligação Proteica , Clivagem do RNA , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.
Assuntos
Proteínas Cromossômicas não Histona/genética , Metilação de DNA/genética , Ativação Transcricional/genética , Inativação do Cromossomo X , Alelos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Ilhas de CpG , Éxons/genética , Feminino , Fibroblastos , Técnicas de Inativação de Genes , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Mutação Puntual , Proteínas do Grupo Polycomb/metabolismoRESUMO
The biology of non-coding RNA (ncRNA) and the regulation of mammalian gene expression is a rapidly expanding field. In this review, we consider how recent advances in technology, enabling the precise mapping of modifications to RNA transcripts, has provided new opportunities to dissect post-transcriptional gene regulation. With this has come the realisation that in the absence of translation, the modification of ncRNAs may play a fundamental role in their regulation, protein interactome and subsequent downstream effector functions. We focus upon modification of RNA by N6-methyladenosine (m6A); its readers, writers and erasers, before considering the differing role of m6A modified lncRNAs MALAT1 and Xist. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.
Assuntos
Adenina/análogos & derivados , Processamento Pós-Transcricional do RNA , RNA não Traduzido/genética , Adenina/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , RNA não Traduzido/metabolismoRESUMO
We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Complexo Repressor Polycomb 1/metabolismo , RNA Longo não Codificante/metabolismo , Inativação do Cromossomo X/genética , Animais , Sequência de Bases , Diferenciação Celular , Feminino , Genoma , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oligonucleotídeos/metabolismo , Transporte ProteicoRESUMO
Xist, the master regulator of the X chromosome inactivation in mammals, is a 17 kb lncRNA that acts in cis to silence the majority of genes along the chromosome from which it is transcribed. The two key processes required for Xist RNA function, localisation in cis and recruitment of silencing factors, are genetically separable, at least in part. Recent studies have identified Xist RNA sequences and associated RNA-binding proteins (RBPs) that are important for these processes. Notably, several of the key Xist RNA elements correspond to local tandem repeats. In this review, I use examples to illustrate different modes whereby tandem repeat amplification has been exploited to allow orthodox RBPs to confer new functions for Xist-mediated chromosome inactivation. I further discuss the potential generality of tandem repeat expansion in the evolution of functional long non-coding RNAs (lncRNAs).
RESUMO
Transcriptional regulation by chromatin is a highly dynamic process directed through the recruitment and coordinated action of epigenetic modifiers and readers of these modifications. Using an unbiased proteomic approach to find interactors of H3K36me3, a modification enriched on active chromatin, here we identify PWWP2A and HDAC2 among the top interactors. PWWP2A and its paralog PWWP2B form a stable complex with NuRD subunits MTA1/2/3:HDAC1/2:RBBP4/7, but not with MBD2/3, p66α/ß, and CHD3/4. PWWP2A competes with MBD3 for binding to MTA1, thus defining a new variant NuRD complex that is mutually exclusive with the MBD2/3 containing NuRD. In mESCs, PWWP2A/B is most enriched at highly transcribed genes. Loss of PWWP2A/B leads to increases in histone acetylation predominantly at highly expressed genes, accompanied by decreases in Pol II elongation. Collectively, these findings suggest a role for PWWP2A/B in regulating transcription through the fine-tuning of histone acetylation dynamics at actively transcribed genes.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Acetilação , Animais , Proteínas Cromossômicas não Histona/genética , Metilação de DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Nucleossomos/metabolismo , ProteômicaRESUMO
In mammals, a dosage compensation mechanism exists to equalize gene expression levels between male and female. This process is initiated by Xist RNA, a long noncoding RNA that mediates the transcriptional silencing of a complete chromosome. The kinetics of events occurring on the future inactive X-chromosome has been described in detail over the last 20 years. More recently, parallel studies using advanced biochemical assays and genetic screens identified key factors critical for the silencing cascade. Here, we describe the procedure adopted in one of these studies, an shRNA-based loss-of-function screen in mouse embryonic stem cells (mESCs).The screen made use of a reporter cell line in which Xist-mediated silencing could be monitored by changes in GFP fluorescence. Loss of function was achieved using a custom made bar-coded pooled shRNA library. The screen aimed to identify shRNAs that lessen Xist mediated repression of the GFP reporter. The methods that were applied are of potential relevance for the development of related screens, for example to better understand how specific repressors silence one or several genes.