Domains of methylated CAC and CG target MeCP2 to tune transcription in the brain
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE84671 | GEO | 2017/05/04
SECONDARY ACCESSION(S): PRJNA330796
REPOSITORIES: GEO
Similar Datasets
Project description:Domains of methylated CAC and CG target MeCP2 to tune transcription in the brain
| PRJNA330796 | ENA
Project description:Mutations in the gene encoding the methyl-CG binding protein MeCP2 cause several neurological disorders including Rett syndrome. The di-nucleotide methyl-CG (mCG) is the classical MeCP2 DNA recognition sequence, but additional methylated sequence targets have been reported. Here we show by in vitro and in vivo analyses that MeCP2 binding to non-CG methylated sites in brain is largely confined to the tri-nucleotide sequence mCAC. MeCP2 binding to chromosomal DNA in mouse brain is proportional to mCAC + mCG density and unexpectedly defines large genomic domains within which transcription is sensitive to MeCP2 occupancy. Our results suggest that MeCP2 integrates patterns of mCAC and mCG in the brain to restrain transcription of genes critical for neuronal function.
| S-EPMC5446194 | biostudies-literature
Project description:Epigenetic mechanisms, such as DNA methylation, regulate transcriptional programs to afford the genome flexibility in responding to developmental and environmental cues in health and disease. A prime example involving epigenetic dysfunction is the postnatal neurodevelopmental disorder Rett syndrome (RTT), which is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2). Despite decades of research, it remains unclear how MeCP2 regulates transcription or why RTT features appear 6-18 months after birth. Here we report integrated analyses of genomic binding of MeCP2, gene-expression data, and patterns of DNA methylation. In addition to the expected high-affinity binding to methylated cytosine in the CG context (mCG), we find a distinct epigenetic pattern of substantial MeCP2 binding to methylated cytosine in the non-CG context (mCH, where H = A, C, or T) in the adult brain. Unexpectedly, we discovered that genes that acquire elevated mCH after birth become preferentially misregulated in mouse models of MeCP2 disorders, suggesting that MeCP2 binding at mCH loci is key for regulating neuronal gene expression in vivo. This pattern is unique to the maturing and adult nervous system, as it requires the increase in mCH after birth to guide differential MeCP2 binding among mCG, mCH, and nonmethylated DNA elements. Notably, MeCP2 binds mCH with higher affinity than nonmethylated identical DNA sequences to influence the level of Bdnf, a gene implicated in the pathophysiology of RTT. This study thus provides insight into the molecular mechanism governing MeCP2 targeting and sheds light on the delayed onset of RTT symptoms.
| S-EPMC4418849 | biostudies-literature
Project description: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.
| S-EPMC7980222 | biostudies-literature
Project description:CG-NAP, also known as AKAP450, is an anchoring/adaptor protein that streamlines signal transduction in various cell types by localizing signaling proteins and enzymes with their substrates. Great efforts are being devoted to elucidating functional roles of this protein and associated macromolecular signaling complex. Increasing understanding of pathways involved in regulating T lymphocytes suggests that CG-NAP can facilitate dynamic interactions between kinases and their substrates and thus fine-tune T cell motility and effector functions. As a result, new binding partners of CG-NAP are continually being uncovered. Here, we review recent advances in CG-NAP research, focusing on its interactions with kinases in T cells with an emphasis on the possible role of this anchoring protein as a target for therapeutic intervention in immune-mediated diseases.
| S-EPMC6861388 | biostudies-literature
Project description:We have investigated the association of DNA methylation and proteins interpreting methylation state with the distinctive closed and open chromatin structural domains that are directly observable in the lampbrush chromosomes (LBCs) of amphibian oocytes. To establish the distribution in LBCs of MeCP2, one of the key proteins binding 5-methylcytosine-modified DNA (5mC), we expressed HA-tagged MeCP2 constructs in Xenopus laevis oocytes. Full-length MeCP2 was predominantly targeted to the closed, transcriptionally inactive chromomere domains in a pattern proportional to chromomeric DNA density and consistent with a global role in determining chromatin state. A minor fraction of HA-MeCP2 was also found to associate with a distinctive structural domain, namely a short region at the bases of some of the extended lateral loops. Expression in oocytes of deleted constructs and of point mutants derived from Rett syndrome patients demonstrated that the association of MeCP2 with LBCs was determined by its 5mC-binding domain. We also examined more directly the distribution of 5mC by immunostaining Xenopus and axolotl LBCs and confirmed the pattern suggested by MeCP2 targeting of intense staining of the chromomeres and of some loop bases. In addition, we found in the longer loops of axolotl LBCs that short interstitial regions could also be clearly stained for 5mC. These 5mC regions corresponded precisely to unusual segments of active transcription units from which RNA polymerase II (pol II) and nascent transcripts were simultaneously absent. We also examined by immunostaining the distribution in lampbrush chromatin of the oxidized 5mC derivative, 5-hydroxymethylcytosine (5hmC). Although in general, the pattern resembled that obtained for 5mC, one antibody against 5hmC produced intense staining of restricted chromosomal foci. These foci corresponded to a third type of lampbrush chromatin domain, the transcriptionally active but less extended structures formed by clusters of genes transcribed by pol III. This raises the possibility that 5hmC may play a role in establishing the distinctive patterns of gene repression and activation that characterize specific pol III-transcribed gene families in amphibian genomes.
| S-EPMC3565088 | biostudies-literature
Project description:DNA methylation at gene promoters in a CG context is associated with transcriptional repression, including at genes silenced on the inactive X chromosome in females. Non-CG methylation (mCH) is a distinct feature of the neuronal epigenome that is differentially distributed between males and females on the X chromosome. However, little is known about differences in mCH on the active (Xa) and inactive (Xi) X chromosomes because stochastic X-chromosome inactivation (XCI) confounds allele-specific epigenomic profiling. We used whole-genome bisulfite sequencing in a mouse model with nonrandom XCI to examine allele-specific DNA methylation in frontal cortex. Xi was largely devoid of mCH, whereas Xa contained abundant mCH similar to the male X chromosome and the autosomes. In contrast to the repressive association of DNA methylation at CG dinucleotides (mCG), mCH accumulates on Xi in domains with transcriptional activity, including the bodies of most genes that escape XCI and at the X-inactivation center, validating this epigenetic mark as a signature of transcriptional activity. Escape genes showing CH hypermethylation were the only genes with CG-hypomethylated promoters on Xi, a well-known mark of active transcription. Finally, we found extensive allele-specific mCH and mCG at autosomal imprinted regions, some with a negative correlation between methylation in the two contexts, further supporting their distinct functions. Our findings show that neuronal mCH functions independently of mCG and is a highly dynamic epigenomic correlate of allele-specific gene regulation.
| S-EPMC5389278 | biostudies-literature
Project description:Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2'-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me(2) and H3K27me(3) in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.
| S-EPMC3326294 | biostudies-literature
Project description:Human leukosialin (CD43) is expressed in a cell lineage-specific as well as a differentiation stage-specific fashion. The leukosialin promoter, made up of an Sp1 binding site and a sequence similar to that of an initiator, possesses high transcriptional potential. Previous data have demonstrated that the leukosialin gene is down-regulated in nonproducing cells by DNA methylation. In this paper the repressive mechanism of DNA methylation in expression systems is reported. In vitro DNA methylation with SssI (CpG) methylase of leukosialin-chloramphenicol acetyltransferase (CAT) constructs drastically reduced transcriptional activities in stable transfection systems with the human HeLa and Jurkat cell lines. On the other hand, the transcriptional repression by in vitro methylation was less pronounced in Drosophila melanogaster cells, which lack genomic methylation. In these cells, Sp1 could transactivate equally well both the unmethylated and methylated leukosialin promoter. In order to test whether one of the methyl-CpG-binding proteins, MeCP2, is responsible for transcriptional repression of the leukosialin gene, I isolated the human MeCP2 cDNA (encoding 486 amino acid residues) and expressed it in Drosophila cells. I found that MeCP2 substantially inhibited Sp1-activated transcription when the leukosialin promoter was methylated. The level of repression was directly proportional to the amount of MeCP2 expression vector transfected. Analysis of C-terminal deletion mutants of MeCP2 showed that repressive activity of Sp1 transactivation is localized to the N-terminal region consisting of amino acid residues 1 to 193, which encompass the methyl-binding domain. These results suggest that interference with Sp1 transactivation by MeCP2 is an important factor in the down-regulation of leukosialin gene expression by DNA methylation.
| S-EPMC109134 | biostudies-literature
Project description:To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer occurring three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD, and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. The electrophoretic mobility shift assay (EMSA) confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50× methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TFs, which may be functional in vivo.
| S-EPMC3668366 | biostudies-literature