RESUMEN
DNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype.
Asunto(s)
Metilación de ADN , Proteína 2 de Unión a Metil-CpG/metabolismo , Neuronas/metabolismo , Animales , Islas de CpG , Técnicas de Sustitución del Gen , Células HeLa , Humanos , Masculino , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Ratones Transgénicos , Mutación , Células 3T3 NIH , Neuronas/patología , Dominios Proteicos , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patologíaRESUMEN
Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.
Asunto(s)
Composición de Base , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Ratones , Ratones Mutantes , Células Madre Embrionarias de Ratones/citología , Mutación , Neuronas/citología , Factores de Transcripción/genética , Regulación hacia Arriba , Dedos de ZincRESUMEN
MeCP2 is an abundant protein in mature nerve cells, where it binds to DNA sequences containing methylated cytosine. Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome (RTT), provoking intensive study of the underlying molecular mechanisms. Multiple functions have been proposed, one of which involves a regulatory role in splicing. Here we leverage the recent availability of high-quality transcriptomic data sets to probe quantitatively the potential influence of MeCP2 on alternative splicing. Using a variety of machine learning approaches that can capture both linear and non-linear associations, we show that widely different levels of MeCP2 have a minimal effect on alternative splicing in three different systems. Alternative splicing was also apparently indifferent to developmental changes in DNA methylation levels. Our results suggest that regulation of splicing is not a major function of MeCP2. They also highlight the importance of multi-variate quantitative analyses in the formulation of biological hypotheses.
Asunto(s)
Empalme Alternativo/genética , Proteína 2 de Unión a Metil-CpG/genética , Síndrome de Rett/genética , Transcriptoma/genética , Animales , Encéfalo/metabolismo , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , ADN Metiltransferasa 3A , Modelos Animales de Enfermedad , Humanos , Metilación , Ratones , Ratones Noqueados , Mutación/genética , Neuronas/metabolismo , Neuronas/patología , Unión Proteica/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patología , ADN Metiltransferasa 3BRESUMEN
Patterns of gene expression are primarily determined by proteins that locally enhance or repress transcription. While many transcription factors target a restricted number of genes, others appear to modulate transcription levels globally. An example is MeCP2, an abundant methylated-DNA binding protein that is mutated in the neurological disorder Rett syndrome. Despite much research, the molecular mechanism by which MeCP2 regulates gene expression is not fully resolved. Here, we integrate quantitative, multidimensional experimental analysis and mathematical modeling to indicate that MeCP2 is a global transcriptional regulator whose binding to DNA creates "slow sites" in gene bodies. We hypothesize that waves of slowed-down RNA polymerase II formed behind these sites travel backward and indirectly affect initiation, reminiscent of defect-induced shockwaves in nonequilibrium physics transport models. This mechanism differs from conventional gene-regulation mechanisms, which often involve direct modulation of transcription initiation. Our findings point to a genome-wide function of DNA methylation that may account for the reversibility of Rett syndrome in mice. Moreover, our combined theoretical and experimental approach provides a general method for understanding how global gene-expression patterns are choreographed.
Asunto(s)
Metilación de ADN , Modelos Teóricos , ARN Polimerasa II/metabolismo , Animales , Línea Celular , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Unión Proteica , Elongación de la Transcripción Genética , Iniciación de la Transcripción Genética , Activación TranscripcionalRESUMEN
Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome, and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.
Asunto(s)
Síndrome de Retracción de Duane , Factores de Transcripción , Humanos , Síndrome de Retracción de Duane/genética , Síndrome de Retracción de Duane/metabolismo , Síndrome de Retracción de Duane/patología , Mutación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
The DNA-binding protein MeCP2 is reported to bind methylated cytosine in CG and CA motifs in genomic DNA, but it was recently proposed that arrays of tandemly repeated CA containing either methylated or hydroxymethylated cytosine are the primary targets for MeCP2 binding and function. Here we investigated the predictions of this hypothesis using a range of published datasets. We failed to detect enrichment of cytosine modification at genomic CA repeat arrays in mouse brain regions and found no evidence for preferential MeCP2 binding at CA repeats. Moreover, we did not observe a correlation between the CA repeat density near genes and their degree of transcriptional deregulation when MeCP2 was absent. Our results do not provide support for the hypothesis that CA repeats are key mediators of MeCP2 function. Instead, we found that CA repeats are subject to CAC methylation to a degree that is typical of the surrounding genome and contribute modestly to MeCP2-mediated modulation of gene expression in accordance with their content of this canonical target motif.
Asunto(s)
Proteína 2 de Unión a Metil-CpG , Animales , Citosina/metabolismo , ADN/metabolismo , Metilación de ADN , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Neuronas/metabolismoRESUMEN
High-throughput sequencing SELEX (HT-SELEX) is a powerful technique for unbiased determination of preferred target motifs of DNA-binding proteins in vitro. The procedure depends upon selection of DNA binding sites from a random library of oligonucleotides by purifying protein-DNA complexes and amplifying bound DNA using the polymerase chain reaction. Here, we describe an optimized step-by-step protocol for HT-SELEX compatible with Illumina sequencing. We also introduce a bioinformatic pipeline (eme_selex) facilitating the detection of promiscuous DNA binding by analyzing the enrichment of all possible k-mers. For complete details on the use and execution of this protocol, please refer to Pantier et al. (2021).
Asunto(s)
Proteínas de Unión al ADN , Técnica SELEX de Producción de Aptámeros , ADN/genética , Proteínas de Unión al ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Oligonucleótidos , Técnica SELEX de Producción de Aptámeros/métodosRESUMEN
Most human genes are associated with promoters embedded in non-methylated, G + C-rich CpG islands (CGIs). Not all CGIs are found at annotated promoters, however, raising the possibility that many serve as promoters for transcripts that do not code for proteins. To test this hypothesis, we searched for novel transcripts in embryonic stem cells (ESCs) that originate within orphan CGIs. Among several candidates, we detected a transcript that included three members of the let-7 micro-RNA family: Let-7a-1, let-7f-1, and let-7d. Deletion of the CGI prevented expression of the precursor RNA and depleted the included miRNAs. Mice homozygous for this mutation were sub-viable and showed growth and other defects. The results suggest that despite the identity of their seed sequences, members of the let-7 miRNA family exert distinct functions that cannot be complemented by other members.