MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation.

Correlative evidence has suggested that the methyl-CpG-binding protein MeCP2 contributes to the formation of heterochromatin condensates via liquid-liquid phase separation. This interpretation has been reinforced by the observation that heterochromatin, DNA methylation and MeCP2 co-localise within prominent foci in mouse cells. The findings presented here revise this view. MeCP2 localisation is independent of heterochromatin as MeCP2 foci persist even when heterochromatin organisation is disrupted. Additionally, MeCP2 foci fail to show hallmarks of phase separation in live cells. Importantly, we find that mouse cellular models are highly atypical as MeCP2 distribution is diffuse in most mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which is a mouse subspecies lacking methylated satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form condensates and its localisation is independent of heterochromatin. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns.

Loss of CpG island immunity to DNA methylation induced by mutation.

The inheritance of acquired traits in mammals is a highly controversial topic in biology. Recently, Takahashi et al. (Cell 186:715-731, 2023) have reported that insertion of CpG-free DNA into a CpG island (CGI) can induce DNA methylation of the CGI and that this aberrant methylation pattern can be transmitted across generations, even after removal of the foreign DNA. These results were interpreted as evidence for transgenerational inheritance of acquired DNA methylation patterns. Here, we discuss several interpretational issues raised by this study and consider alternative explanations.

A screen for MeCP2-TBL1 interaction inhibitors using a luminescence-based assay.

Understanding the molecular pathology of neurodevelopmental disorders should aid the development of therapies for these conditions. In MeCP2 duplication syndrome (MDS)-a severe autism spectrum disorder-neuronal dysfunction is caused by increased levels of MeCP2. MeCP2 is a nuclear protein that binds to methylated DNA and recruits the nuclear co-repressor (NCoR) complex to chromatin via an interaction with the WD repeat-containing proteins TBL1 and TBLR1. The peptide motif in MeCP2 that binds to TBL1/TBLR1 is essential for the toxicity of excess MeCP2 in animal models of MDS, suggesting that small molecules capable of disrupting this interaction might be useful therapeutically. To facilitate the search for such compounds, we devised a simple and scalable NanoLuc luciferase complementation assay for measuring the interaction of MeCP2 with TBL1/TBLR1. The assay allowed excellent separation between positive and negative controls, and had low signal variance (Z-factor = 0.85). We interrogated compound libraries using this assay in combination with a counter-screen based on luciferase complementation by the two subunits of protein kinase A (PKA). Using this dual screening approach, we identified candidate inhibitors of the interaction between MeCP2 and TBL1/TBLR1. This work demonstrates the feasibility of future screens of large compound collections, which we anticipate will enable the development of small molecule therapeutics to ameliorate MDS.

Structure of SALL4 zinc finger domain reveals link between AT-rich DNA binding and Okihiro syndrome.

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.

A critique of the hypothesis that CA repeats are primary targets of neuronal MeCP2.

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.

Convergent cerebrospinal fluid proteomes and metabolic ontologies in humans and animal models of Rett syndrome.

MECP2 loss-of-function mutations cause Rett syndrome, a neurodevelopmental disorder resulting from a disrupted brain transcriptome. How these transcriptional defects are decoded into a disease proteome remains unknown. We studied the proteome of Rett cerebrospinal fluid (CSF) to identify consensus Rett proteome and ontologies shared across three species. Rett CSF proteomes enriched proteins annotated to HDL lipoproteins, complement, mitochondria, citrate/pyruvate metabolism, synapse compartments, and the neurosecretory protein VGF. We used shared Rett ontologies to select analytes for orthogonal quantification and functional validation. VGF and ontologically selected CSF proteins had genotypic discriminatory capacity as determined by receiver operating characteristic analysis in Mecp2 -/y and Mecp2 -/+ . Differentially expressed CSF proteins distinguished Rett from a related neurodevelopmental disorder, CDKL5 deficiency disorder. We propose that Mecp2 mutant CSF proteomes and ontologies inform putative mechanisms and biomarkers of disease. We suggest that Rett syndrome results from synapse and metabolism dysfunction.

Comparative analysis of potential broad-spectrum neuronal Cre drivers.

Cre/Lox technology is a powerful tool in the mouse genetics tool-box as it enables tissue-specific and inducible mutagenesis of specific gene loci. Correct interpretation of phenotypes depends upon knowledge of the Cre expression pattern in the chosen mouse driver line to ensure that appropriate cell types are targeted. For studies of the brain and neurological disease a pan-neuronal promoter that reliably drives efficient neuron-specific transgene expression would be valuable. Here we compare a widely used "pan-neuronal" mouse Cre driver line, Syn1-cre, with a little-known alternative, Snap25-IRES2-cre. Our results show that the Syn1-cre line broadly expresses in the brain but is indetectable in more than half of all neurons and weakly active in testes. In contrast the Snap25-IRES2-cre line expressed Cre in a high proportion of neurons (~85%) and was indetectable in all non-brain tissues that were analysed, including testes. Our findings suggest that for many purposes Snap25-IRES2-cre is superior to Syn1-cre as a potential pan-neuronal cre driver.

High-throughput sequencing SELEX for the determination of DNA-binding protein specificities in vitro.

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).

Genetic therapies for neurological disorders.

In recent years, it has become increasingly apparent that many neurological disorders are underpinned by a genetic aetiology. This has resulted in considerable efforts to develop therapeutic strategies which can treat the disease-causing mutation, either by supplying a functional copy of the mutated gene or editing the genomic sequence. In this review, we will discuss the main genetic strategies which are currently being explored for the treatment of monogenic neurological disorders, as well as some of the challenges they face. In addition, we will address some of the ethical difficulties which may arise.

CDKL5 deficiency disorder: a pathophysiology of neural maintenance.

Critical periods are developmental time windows in which functional properties of the brain are particularly susceptible to the organism's experience. It was thought that therapeutic strategies for neurodevelopmental disorders (NDDs) required early life intervention for successful treatment, but previous studies in a mouse model of Rett syndrome indicated that this may not be the case, as some genetic disorders result from disruptions of neuromaintenance. In this issue of the JCI, Terzic et al. provide evidence that defective neuromaintenance also underlies CDKL5 deficiency disorder (CDD). The authors used genetic mouse models to examine the role of CDKL5 protein. Notably, when CDKL5 protein was restored in late adolescent Cdkl5-deficient animals, CDD behavioral defects were reversed. These results suggest that genetically or pharmacologically restoring CDKL5 may treat CDD after symptom onset.

Neuronal non-CG methylation is an essential target for MeCP2 function.

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.

SALL4 controls cell fate in response to DNA base composition.

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.

Quantitative analysis questions the role of MeCP2 as a global regulator of alternative splicing.

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.

DNA Methylation: Mega-Year Inheritance with the Help of Darwin.

DNA methylation in a fungal pathogen has persisted for millions of years without the enzyme that can efficiently add methyl groups de novo. This spectacular example of 'epigenetic' inheritance is explained by a super-efficient maintenance enzyme plus natural selection.

Absence of MeCP2 binding to non-methylated GT-rich sequences in vivo.

MeCP2 is a nuclear protein that binds to sites of cytosine methylation in the genome. While most evidence confirms this epigenetic mark as the primary determinant of DNA binding, MeCP2 is also reported to have an affinity for non-methylated DNA sequences. Here we investigated the molecular basis and in vivo significance of its reported affinity for non-methylated GT-rich sequences. We confirmed this interaction with isolated domains of MeCP2 in vitro and defined a minimal target DNA sequence. Binding depends on pyrimidine 5' methyl groups provided by thymine and requires adjacent guanines and a correctly orientated A/T-rich flanking sequence. Unexpectedly, full-length MeCP2 protein failed to bind GT-rich sequences in vitro. To test for MeCP2 binding to these motifs in vivo, we analysed human neuronal cells using ChIP-seq and ATAC-seq technologies. While both methods robustly detected DNA methylation-dependent binding of MeCP2 to mCG and mCAC, neither showed evidence of MeCP2 binding to GT-rich motifs. The data suggest that GT binding is an in vitro phenomenon without in vivo relevance. Our findings argue that MeCP2 does not read unadorned DNA sequence and therefore support the notion that its primary role is to interpret epigenetic modifications of DNA.

The Selfishness of Law-Abiding Genes.

Selfish genes were once controversial, but it is now accepted that the genome contains parasitic elements in addition to a complement of conventional genes. This opinion article argues that 'law-abiding' genes also indulge in game playing to ensure their propagation, so that initially nonessential processes secure a genetic heritage. A gene-centered view of this kind can help to explain otherwise puzzling aspects of biology, including the complexity and stability of living systems.

The Molecular Basis of MeCP2 Function in the Brain.

MeCP2 is a reader of the DNA methylome that occupies a large proportion of the genome due to its high abundance and the frequency of its target sites. It has been the subject of extensive study because of its link with 'MECP2-related disorders', of which Rett syndrome is the most prevalent. This review integrates evidence from patient mutation data with results of experimental studies using mouse models, cell lines and in vitro systems to critically evaluate our understanding of MeCP2 protein function. Recent evidence challenges the idea that MeCP2 is a multifunctional hub that integrates diverse processes to underpin neuronal function, suggesting instead that its primary role is to recruit the NCoR1/2 co-repressor complex to methylated sites in the genome, leading to dampening of gene expression.

Quantitative modelling predicts the impact of DNA methylation on RNA polymerase II traffic.

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.

An Orphan CpG Island Drives Expression of a let-7 miRNA Precursor with an Important Role in Mouse Development.

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.

R-Loops Enhance Polycomb Repression at a Subset of Developmental Regulator Genes.

R-loops are three-stranded nucleic acid structures that form during transcription, especially over unmethylated CpG-rich promoters of active genes. In mouse embryonic stem cells (mESCs), CpG-rich developmental regulator genes are repressed by the Polycomb complexes PRC1 and PRC2. Here, we show that R-loops form at a subset of Polycomb target genes, and we investigate their contribution to Polycomb repression. At R-loop-positive genes, R-loop removal leads to decreased PRC1 and PRC2 recruitment and Pol II activation into a productive elongation state, accompanied by gene derepression at nascent and processed transcript levels. Stable removal of PRC2 derepresses R-loop-negative genes, as expected, but does not affect R-loops, PRC1 recruitment, or transcriptional repression of R-loop-positive genes. Our results highlight that Polycomb repression does not occur via one mechanism but consists of different layers of repression, some of which are gene specific. We uncover that one such mechanism is mediated by an interplay between R-loops and RING1B recruitment.