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
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
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 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
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
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
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
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.