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DNA Methylation in the Gene Body Influences Mecp2-Mediated Gene Repression

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DNA Methylation in the Gene Body Influences Mecp2-Mediated Gene Repression DNA methylation in the gene body influences MeCP2-mediated gene repression Benyam Kindea, Dennis Y. Wub, Michael E. Greenberga,1, and Harrison W. Gabelb,1 aDepartment of Neurobiology, Harvard Medical School, Boston, MA 02115; and bDepartment of Neuroscience, Washington University School of Medicine, St. Louis, MO 63110 Contributed by Michael E. Greenberg, November 21, 2016 (sent for review June 22, 2016; reviewed by Anne Brunet and Li-Huei Tsai) Rett syndrome is a severe neurodevelopmental disorder caused by MBD of MeCP2 as essential for the high-affinity interaction be- mutations in the methyl-CpG binding protein gene (MECP2). MeCP2 tween MeCP2 and methylcytosine (6, 7). For many years methyl- is a methyl-cytosine binding protein that is proposed to function as ation of cytosines in the CpG dinucleotide context (mCG) has been a transcriptional repressor. However, multiple gene expression stud- thought to represent the majority of DNA methylation in mam- ies comparing wild-type and MeCP2-deficient neurons have failed to malian cells and to be the major site of MeCP2 binding in neurons. identify gene expression changes consistent with loss of a classical It has recently been shown that in the brain, high levels of non- transcriptional repressor. Recent work suggests that one function of CG methylation (predominantly mCA) also contribute to the “ ” MeCP2 in neurons is to temper the expression of the longest genes neuronal methylome, with the number of mCA sites at late stages of neuronal maturation approaching the number of mCG in the genome by binding to methylated CA dinucleotides (mCA) – within transcribed regions of these genes. Here we explore the sites (8 10). We, and others, have recently investigated whether mechanism of mCA and MeCP2 in fine tuning the expression of long MeCP2 binds mCA sites and demonstrated that MeCP2 binds to genes. We find that mCA is not only highly enriched within mCA and symmetrically methylated CG with similarly high affinity the body of genes normally repressed by MeCP2, but also enriched (9, 11, 12). Thus, the number of possible sites of MeCP2 binding in neurons increases significantly as mCA is laid down in the post- within extended megabase-scale regions surrounding MeCP2-repressed natal period. Given that the mCA mark is deposited at the time genes. Whereas enrichment of mCA exists in a broad region around that MeCP2 levels increase postnatally, and when the phenotype of these genes, mCA together with mCG within gene bodies appears to RTT syndrome is first observed in MeCP2 mutant mice, it has NEUROSCIENCE be the primary driver of gene repression by MeCP2. Disruption of been suggested that the disruption of MeCP2 binding to mCA in methylation at CA sites within the brain results in depletion of neurons may be a key event in the etiology of RTT. Consistent with MeCP2 across genes that normally contain a high density of gene- this possibility, mutations that disrupt the function of Dnmt3a, the body mCA. We further find that the degree of gene repression by de novo methyltransferase enzyme responsible for depositing mCA MeCP2 is proportional to the total number of methylated cytosine in the brain, results in severe neurological deficits in mice that are MeCP2 binding sites across the body of a gene. These findings sug- reminiscent of phenotypes observed in MeCP2 KO mice (13). gest a model in which MeCP2 tunes gene expression in neurons by Furthermore, mutations in DNMT3A have been linked to in- binding within the transcribed regions of genes to impede the elon- tellectual disability and autism spectrum disorder in humans (14). gation of RNA polymerase. Considerable evidence supports the conclusion that when bound to mC sequences, MeCP2 functions as a repressor of transcription. DNA methylation | Rett syndrome | MeCP2 | transcription Biochemical studies have demonstrated that the TRD of MeCP2 interacts with NCoR/SMRT and Sin3a corepressor complexes (5, ett syndrome (RTT) is a severe neurodevelopmental disorder 15). Notably, one of the most common non-MBD MeCP2 missense Rcharacterized by developmental stagnation and regression, stereotyped hand movements, seizures, and autism spectrum-like Significance behavior (1). RTT is caused by mutations in the gene encoding the methyl-CpG binding protein 2 (MECP2) (1), and the monogenic Mutations in the methyl-CpG binding protein 2 (MECP2) lead to nature of RTT provides the unique opportunity to investigate the the severe neurological disorder Rett syndrome, but our un- molecular basis of a complex human neurodevelopmental disor- derstanding of how MeCP2 regulates gene expression in the brain der. One particularly useful approach for studying RTT has been has been limited. Recently we uncovered evidence that MeCP2 to generate mouse models that harbor RTT-causing mutations in controls transcription of very long genes with critical neuronal MeCP2. These RTT-like mice recapitulate many features of RTT functions by binding a unique form of DNA methylation, enriched seen in humans, displaying defects in neural circuit excitatory– in neurons. Here, we provide evidence that MeCP2 represses inhibitory balance, increased incidence of seizures, motor dis- transcription by binding within transcribed regions of genes. We coordination, and breathing abnormalities (2, 3). show that this repressive effect is proportional to the total num- The onset of symptoms in girls with RTT and in mouse models of ber of methylated DNA binding sites for MeCP2 within each gene. the disorder occurs during a period of postnatal brain development Our findings suggest a model in which MeCP2 represses tran- in which MeCP2 accumulates to exceedingly high levels in neurons scription of long neuronal genes that contain many methylated of the brain, such that the number of MeCP2 molecules in neurons binding sites by impeding transcriptional elongation. approaches the number of nucleosomes in adult neuronal nuclei (4). Whereas MeCP2 is expressed to some extent in most cells of the Author contributions: B.K., M.E.G., and H.W.G. designed research; B.K., D.Y.W., and body, MeCP2 protein levels are approximately sevenfold higher in H.W.G. performed research; B.K., D.Y.W., and H.W.G. analyzed data; and B.K., M.E.G., and neurons (4). Brain-specific disruption of MeCP2 is sufficient to cause H.W.G. wrote the paper. the vast majority of RTT-like phenotypes in mice, providing evidence Reviewers: A.B., Stanford University; and L.-H.T., Massachusetts Institute of Technology. that RTT is predominantly a disorder of neuronal dysfunction (2, 3). The authors declare no conflict of interest. Key molecular functions of MeCP2 have been highlighted by the Data deposition: The data reported in this paper have been deposited in Gene Expression observation that RTT-causing mutations largely cluster into two Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE90704). functional domains: the methyl-DNA binding domain (MBD) and 1To whom correspondence may be addressed. Email: [email protected] or michael_ the transcriptional repressor domain (TRD) (5). Bird and col- [email protected]. leagues first identified MeCP2 on the basis of its high-affinity This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. binding to DNA containing mCG sequences and identified the 1073/pnas.1618737114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1618737114 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 mutations that leads to RTT, MeCP2 R306C, disrupts the in- broadly across the genome. In the latter case, mCA would be teraction between MeCP2 and NCoR, suggesting that a key predicted to recruit MeCP2 throughout the broad domain of function of MeCP2 is to mediate transcriptional repression (5, 16). mCA and could potentially repress the transcription of genes that Despite evidence that MeCP2 functions as a silencer of tran- happen to reside within the mCA domain. At these sites, MeCP2 scription, identifying the specific targets of MeCP2 has proven to might function as a classical repressor that inhibits transcription by be difficult both because MeCP2 binds broadly across the entire binding to specific noncoding regulatory sequences or by com- neuronal genome (4, 12, 17, 18), and because the changes in pacting the DNA throughout broad genomic domains. Alterna- gene expression that occur in the absence of MeCP2 are small tively, binding of MeCP2 within the gene body might function (11, 12, 17, 19–23). These unique challenges have made it dif- to retard the movement of the RNA polymerase II complex. ficult to identify which changes in gene expression in the absence To begin to explore these possibilities, we examined the DNA of MeCP2 are direct consequences of MeCP2 loss and which are methylation (mCG and mCA) and MeCP2 binding profiles in and secondary effects of overall cellular dysfunction. around genes that have been consistently implicated as repressed As a strategy for identifying the direct targets of MeCP2 ac- or activated by the presence of MeCP2 across multiple studies tion, we recently sought to identify common features of genes (12), comparing these profiles to the average profiles for all other that might distinguish whether or not a gene will be misregulated genes in the genome. For this analysis, we calculated mCA or as a direct consequence of the absence of MeCP2. These anal- mCG levels as the number of unconverted cytosines sequenced yses revealed that at a genome-wide level, MeCP2 functions to during whole genome bisulfite sequencing analysis (8) within a – temper the expression of genes in a gene-length associated 1-kb window of the genome divided by the total number of cyto- manner, possibly by binding to mCA sequences within the sine positions sequenced within that window; we then plotted the transcribed region of these genes (12). Consistent with this idea, average values for windows across gene loci (SI Experimental the disruption of MeCP2 or Dnmt3a leads to up-regulation of Procedures).
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