Early-Life Gene Expression in Neurons Modulates Lasting Epigenetic States.

In mammals, the environment plays a critical role in promoting the final steps in neuronal development during the early postnatal period. While epigenetic factors are thought to contribute to this process, the underlying molecular mechanisms remain poorly understood. Here, we show that in the brain during early life, the DNA methyltransferase DNMT3A transiently binds across transcribed regions of lowly expressed genes, and its binding specifies the pattern of DNA methylation at CA sequences (mCA) within these genes. We find that DNMT3A occupancy and mCA deposition within the transcribed regions of genes is negatively regulated by gene transcription and may be modified by early-life experience. Once deposited, mCA is bound by the methyl-DNA-binding protein MECP2 and functions in a rheostat-like manner to fine-tune the cell-type-specific transcription of genes that are critical for brain function.

NSD1 deposits histone H3 lysine 36 dimethylation to pattern non-CG DNA methylation in neurons.

During postnatal development, the DNA methyltransferase DNMT3A deposits high levels of non-CG cytosine methylation in neurons. This methylation is critical for transcriptional regulation, and loss of this mark is implicated in DNMT3A-associated neurodevelopmental disorders (NDDs). Here, we show in mice that genome topology and gene expression converge to shape histone H3 lysine 36 dimethylation (H3K36me2) profiles, which in turn recruit DNMT3A and pattern neuronal non-CG methylation. We show that NSD1, an H3K36 methyltransferase mutated in NDD, is required for the patterning of megabase-scale H3K36me2 and non-CG methylation in neurons. We find that brain-specific deletion of NSD1 causes altered DNA methylation that overlaps with DNMT3A disorder models to drive convergent dysregulation of key neuronal genes that may underlie shared phenotypes in NSD1- and DNMT3A-associated NDDs. Our findings indicate that H3K36me2 deposited by NSD1 is important for neuronal non-CG DNA methylation and suggest that the H3K36me2-DNMT3A-non-CG-methylation pathway is likely disrupted in NSD1-associated NDDs.

A Shortcut to Activity-Dependent Transcription.

Neuronal activity results in the rapid induction of gene transcription through a series of defined molecular events. Madabhushi et al. describe an unexpected role for the cutting of promoter DNA by topoisomerase IIB to facilitate transcription of activity-induced genes.

APC7 mediates ubiquitin signaling in constitutive heterochromatin in the developing mammalian brain.

Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons in vivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.

Npas4 regulates excitatory-inhibitory balance within neural circuits through cell-type-specific gene programs.

The nervous system adapts to experience by inducing a transcriptional program that controls important aspects of synaptic plasticity. Although the molecular mechanisms of experience-dependent plasticity are well characterized in excitatory neurons, the mechanisms that regulate this process in inhibitory neurons are only poorly understood. Here, we describe a transcriptional program that is induced by neuronal activity in inhibitory neurons. We find that, while neuronal activity induces expression of early-response transcription factors such as Npas4 in both excitatory and inhibitory neurons, Npas4 activates distinct programs of late-response genes in inhibitory and excitatory neurons. These late-response genes differentially regulate synaptic input to these two types of neurons, promoting inhibition onto excitatory neurons while inducing excitation onto inhibitory neurons. These findings suggest that the functional outcomes of activity-induced transcriptional responses are adapted in a cell-type-specific manner to achieve a circuit-wide homeostatic response.

MeCP2 Represses Enhancers through Chromosome Topology-Associated DNA Methylation.

The genomes of mammalian neurons contain uniquely high levels of non-CG DNA methylation that can be bound by the Rett syndrome protein, MeCP2, to regulate gene expression. How patterns of non-CG methylation are established in neurons and the mechanism by which this methylation works with MeCP2 to control gene expression is unclear. Here, we find that genes repressed by MeCP2 are often located within megabase-scale regions of high non-CG methylation that correspond with topologically associating domains of chromatin folding. MeCP2 represses enhancers found in these domains that are enriched for non-CG and CG methylation, with the strongest repression occurring for enhancers located within MeCP2-repressed genes. These alterations in enhancer activity provide a mechanism for how MeCP2 disruption in disease can lead to widespread changes in gene expression. Hence, we find that DNA topology can shape non-CG DNA methylation across the genome to dictate MeCP2-mediated enhancer regulation in the brain.

Emerging Insights into the Distinctive Neuronal Methylome.

The genomes of mammalian neurons are enriched for unique forms of DNA methylation, including exceptionally high levels of non-CG methylation. Here, we review recent studies defining how non-CG methylation accumulates in neurons and is read out by the critical regulator of neuronal transcription, MeCP2. We discuss the role of gene expression and genome architecture in establishing non-CG methylation and highlight emerging mechanistic insights into how non-CG methylation and MeCP2 control transcription. Further, we describe the cell type-specific functions of this methylation and explore growing evidence that disruption of this regulatory pathway contributes to neurodevelopmental disorders. These findings uncover how the distinctive epigenome in neurons facilitates the development and function of the complex mammalian brain.

Functions of Gtf2i and Gtf2ird1 in the developing brain: transcription, DNA binding and long-term behavioral consequences.

Gtf2ird1 and Gtf2i are two transcription factors (TFs) among the 28 genes deleted in Williams syndrome, and prior mouse models of each TF show behavioral phenotypes. Here we identify their genomic binding sites in the developing brain and test for additive effects of their mutation on transcription and behavior. GTF2IRD1 binding targets were enriched for transcriptional and chromatin regulators and mediators of ubiquitination. GTF2I targets were enriched for signal transduction proteins, including regulators of phosphorylation and WNT. Both TFs are highly enriched at promoters, strongly overlap CTCF binding and topological associating domain boundaries and moderately overlap each other, suggesting epistatic effects. Shared TF targets are enriched for reactive oxygen species-responsive genes, synaptic proteins and transcription regulators such as chromatin modifiers, including a significant number of highly constrained genes and known ASD genes. We next used single and double mutants to test whether mutating both TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 mutants, though with the caveat that our Gtf2ird1 mutants, like others previously reported, do produce low levels of a truncated protein product. Despite little difference in DNA binding and transcriptome-wide expression, homozygous Gtf2ird1 mutation caused balance, marble burying and conditioned fear phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone was sufficient for the observed phenotypes.

Disruption of DNA-methylation-dependent long gene repression in Rett syndrome.

Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism. MECP2 encodes a methyl-DNA-binding protein that has been proposed to function as a transcriptional repressor, but despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 protein regulates transcription. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain.

ME-Class2 reveals context dependent regulatory roles for 5-hydroxymethylcytosine.

Since the discovery of 5-hydroxymethylcytosine (5hmC) as a prominent DNA modification found in mammalian genomes, an emergent question has been what role this mark plays in gene regulation. 5hmC is hypothesized to function as an intermediate in the demethylation of 5-methylcytosine (5mC) and in the reactivation of silenced promoters and enhancers. Further, weak positive correlations are observed between gene body 5hmC and gene expression. We previously demonstrated that ME-Class is an effective tool to understand relationships between whole-genome bisulfite sequencing data and expression. In this work, we present ME-Class2, a machine-learning based tool to perform integrative 5mCG, 5hmCG and expression analysis. Using ME-Class2 we analyze whole-genome single-base resolution 5mCG and 5hmCG datasets from 20 primary tissue and cell samples to reveal relationships between 5hmCG and expression. Our analysis indicates that conversion of 5mCG to 5hmCG within 2 kb of the transcription start site associates with distinct functions depending on the summed level of 5mCG + 5hmCG. Unchanged levels of 5mCG + 5hmCG (conversion from 5mCG to stable 5hmCG) associate with repression. Meanwhile, decreases in 5mCG + 5hmCG (5hmCG-mediated demethylation) associate with gene activation. Our results demonstrate that ME-Class2 will prove invaluable to interpret genome-wide 5mC and 5hmC datasets and guide mechanistic studies into the function of 5hmCG.

The Transcriptional Regulator SnoN Promotes the Proliferation of Cerebellar Granule Neuron Precursors in the Postnatal Mouse Brain.

Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional KO of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal male and female mouse brain. In laser capture microdissection followed by RNA-Seq, designed to profile gene expression specifically in the external granule layer of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursors in vivo Remarkably, bioinformatics analyses reveal that SnoN-regulated genes contain binding sites for the transcription factors N-myc and Pax6, which promote the proliferation and differentiation of granule neuron precursors, respectively. Accordingly, we uncover novel physical interactions of SnoN with N-myc and Pax6 in cells. In behavior analyses, conditional KO of SnoN impairs cerebellar-dependent learning in a delayed eye-blink conditioning paradigm, suggesting that SnoN-regulation of granule neuron precursor proliferation bears functional consequences at the organismal level. Our findings define a novel function and mechanism for the major transcriptional regulator SnoN in the control of granule neuron precursor proliferation in the mammalian brain.SIGNIFICANCE STATEMENT This study reports the discovery that the transcriptional regulator SnoN plays a crucial role in the proliferation of cerebellar granule neuron precursors in the postnatal mouse brain. Conditional KO of SnoN in granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cycle exit specifically at later stages of cerebellar development, with biological consequences of impaired cerebellar-dependent learning. Genomics and bioinformatics analyses reveal that SnoN promotes the expression of cell proliferation genes and concomitantly represses cell differentiation genes in vivo Although SnoN has been implicated in distinct aspects of the development of postmitotic neurons, this study identifies a novel function for SnoN in neuronal precursors in the mammalian brain.

LONGO: an R package for interactive gene length dependent analysis for neuronal identity.

Motivation: Reprogramming somatic cells into neurons holds great promise to model neuronal development and disease. The efficiency and success rate of neuronal reprogramming, however, may vary between different conversion platforms and cell types, thereby necessitating an unbiased, systematic approach to estimate neuronal identity of converted cells. Recent studies have demonstrated that long genes (>100 kb from transcription start to end) are highly enriched in neurons, which provides an opportunity to identify neurons based on the expression of these long genes. Results: We have developed a versatile R package, LONGO, to analyze gene expression based on gene length. We propose a systematic analysis of long gene expression (LGE) with a metric termed the long gene quotient (LQ) that quantifies LGE in RNA-seq or microarray data to validate neuronal identity at the single-cell and population levels. This unique feature of neurons provides an opportunity to utilize measurements of LGE in transcriptome data to quickly and easily distinguish neurons from non-neuronal cells. By combining this conceptual advancement and statistical tool in a user-friendly and interactive software package, we intend to encourage and simplify further investigation into LGE, particularly as it applies to validating and improving neuronal differentiation and reprogramming methodologies. Availability and implementation: LONGO is freely available for download at https://github.com/biohpc/longo. Supplementary information: Supplementary data are available at Bioinformatics online.

Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR.

Rett syndrome (RTT) is an X-linked human neurodevelopmental disorder with features of autism and severe neurological dysfunction in females. RTT is caused by mutations in methyl-CpG-binding protein 2 (MeCP2), a nuclear protein that, in neurons, regulates transcription, is expressed at high levels similar to that of histones, and binds to methylated cytosines broadly across the genome. By phosphotryptic mapping, we identify three sites (S86, S274 and T308) of activity-dependent MeCP2 phosphorylation. Phosphorylation of these sites is differentially induced by neuronal activity, brain-derived neurotrophic factor, or agents that elevate the intracellular level of 3',5'-cyclic AMP (cAMP), indicating that MeCP2 may function as an epigenetic regulator of gene expression that integrates diverse signals from the environment. Here we show that the phosphorylation of T308 blocks the interaction of the repressor domain of MeCP2 with the nuclear receptor co-repressor (NCoR) complex and suppresses the ability of MeCP2 to repress transcription. In knock-in mice bearing the common human RTT missense mutation R306C, neuronal activity fails to induce MeCP2 T308 phosphorylation, suggesting that the loss of T308 phosphorylation might contribute to RTT. Consistent with this possibility, the mutation of MeCP2 T308A in mice leads to a decrease in the induction of a subset of activity-regulated genes and to RTT-like symptoms. These findings indicate that the activity-dependent phosphorylation of MeCP2 at T308 regulates the interaction of MeCP2 with the NCoR complex, and that RTT in humans may be due, in part, to the loss of activity-dependent MeCP2 T308 phosphorylation and a disruption of the phosphorylation-regulated interaction of MeCP2 with the NCoR complex.

DNA methylation in the gene body influences MeCP2-mediated gene repression.

Rett syndrome is a severe neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein gene (MECP2). MeCP2 is a methyl-cytosine binding protein that is proposed to function as a transcriptional repressor. However, multiple gene expression studies comparing wild-type and MeCP2-deficient neurons have failed to identify gene expression changes consistent with loss of a classical transcriptional repressor. Recent work suggests that one function of MeCP2 in neurons is to temper the expression of the longest genes in the genome by binding to methylated CA dinucleotides (mCA) within transcribed regions of these genes. Here we explore the mechanism of mCA and MeCP2 in fine tuning the expression of long genes. We find that mCA is not only highly enriched within the body of genes normally repressed by MeCP2, but also enriched within extended megabase-scale regions surrounding MeCP2-repressed genes. Whereas enrichment of mCA exists in a broad region around these genes, mCA together with mCG within gene bodies appears to be the primary driver of gene repression by MeCP2. Disruption of methylation at CA sites within the brain results in depletion of MeCP2 across genes that normally contain a high density of gene-body mCA. We further find that the degree of gene repression by MeCP2 is proportional to the total number of methylated cytosine MeCP2 binding sites across the body of a gene. These findings suggest a model in which MeCP2 tunes gene expression in neurons by binding within the transcribed regions of genes to impede the elongation of RNA polymerase.

Reading the unique DNA methylation landscape of the brain: Non-CpG methylation, hydroxymethylation, and MeCP2.

DNA methylation at CpG dinucleotides is an important epigenetic regulator common to virtually all mammalian cell types, but recent evidence indicates that during early postnatal development neuronal genomes also accumulate uniquely high levels of two alternative forms of methylation, non-CpG methylation and hydroxymethylation. Here we discuss the distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers of DNA methylation. We review studies of one critical reader of DNA methylation in the brain, the Rett syndrome protein methyl CpG-binding protein 2 (MeCP2), and discuss how differential binding affinity of MeCP2 for non-CpG and hydroxymethylation may affect the function of this methyl-binding protein in the nervous system.

mut-16 and other mutator class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans.

Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In Caenorhabditis elegans, mutator (mut) class genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well-understood. To characterize the endogenous small RNAs dependent on mut class genes, small RNA populations from a null allele of mut-16 as well as a regulatory mut-16(mg461) allele that disables only somatic RNAi were subjected to deep sequencing. Additionally, each of the mut class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. The results also reveal essential roles for mut-2 and mut-7 in the ERGO-1 class 26G siRNA pathway and less critical roles for mut-8, mut-14, and mut-15. We show that transposons are hypersusceptible to mut-16-dependent silencing and identify a requirement for the siRNA machinery in piRNA biogenesis from Tc1 transposons. We also show that the soma-specific mut-16(mg461) mutant allele is present in multiple C. elegans laboratory strains.

Non-CG DNA methylation and MeCP2 stabilize repeated tuning of long genes that distinguish closely related neuron types.

The extraordinary diversity of neuron types in the mammalian brain is delineated at the highest resolution by subtle gene expression differences that may require specialized molecular mechanisms to be maintained. Neurons uniquely express the longest genes in the genome and utilize neuron-enriched non-CG DNA methylation (mCA) together with the Rett syndrome protein, MeCP2, to control gene expression, but the function of these unique gene structures and machinery in regulating finely resolved neuron type-specific gene programs has not been explored. Here, we employ epigenomic and spatial transcriptomic analyses to discover a major role for mCA and MeCP2 in maintaining neuron type-specific gene programs at the finest scale of cellular resolution. We uncover differential susceptibility to MeCP2 loss in neuronal populations depending on global mCA levels and dissect methylation patterns and intragenic enhancer repression that drive overlapping and distinct gene regulation between neuron types. Strikingly, we show that mCA and MeCP2 regulate genes that are repeatedly tuned to differentiate neuron types at the highest cellular resolution, including spatially resolved, vision-dependent gene programs in the visual cortex. These repeatedly tuned genes display genomic characteristics, including long length, numerous intragenic enhancers, and enrichment for mCA, that predispose them to regulation by MeCP2. Thus, long gene regulation by the MeCP2 pathway maintains differential gene expression between closely-related neurons to facilitate the exceptional cellular diversity in the complex mammalian brain.

NSD1 deposits histone H3 lysine 36 dimethylation to pattern non-CG DNA methylation in neurons.

During postnatal development the DNA methyltransferase DNMT3A deposits high levels of non-CG cytosine methylation in neurons. This unique methylation is critical for transcriptional regulation in the mature mammalian brain, and loss of this mark is implicated in DNMT3A-associated neurodevelopmental disorders (NDDs). The mechanisms determining genomic non-CG methylation profiles are not well defined however, and it is unknown if this pathway is disrupted in additional NDDs. Here we show that genome topology and gene expression converge to shape histone H3 lysine 36 dimethylation (H3K36me2) profiles, which in turn recruit DNMT3A and pattern neuronal non-CG methylation. We show that NSD1, the H3K36 methyltransferase mutated in the NDD, Sotos syndrome, is required for megabase-scale patterning of H3K36me2 and non-CG methylation in neurons. We find that brain-specific deletion of NSD1 causes alterations in DNA methylation that overlap with models of DNMT3A disorders and define convergent disruption in the expression of key neuronal genes in these models that may contribute to shared phenotypes in NSD1- and DNMT3A-associated NDD. Our findings indicate that H3K36me2 deposited by NSD1 is an important determinant of neuronal non-CG DNA methylation and implicates disruption of this methylation in Sotos syndrome. Highlights: Topology-associated DNA methylation and gene expression independently contribute to neuronal gene body and enhancer non-CG DNA methylation patterns.Topology-associated H3K36me2 patterns and local enrichment of H3K4 methylation impact deposition of non-CG methylation by DNMT3A. Disruption of NSD1 in vivo leads to alterations in H3K36me2, DNA methylation, and gene expression that overlap with models of DNMT3A disorders.

MeCP2 represses the activity of topoisomerase IIß in long neuronal genes.

A unique signature of neurons is the high expression of the longest genes in the genome. These genes have essential neuronal functions, and disruption of their expression has been implicated in neurological disorders. DNA topoisomerases resolve DNA topological constraints and facilitate neuronal long gene expression. Conversely, the Rett syndrome protein, methyl-CpG-binding protein 2 (MeCP2), can transcriptionally repress long genes. How these factors regulate long genes is not well understood, and whether they interact is not known. Here, we identify and map a functional interaction between MeCP2 and topoisomerase IIß (TOP2ß) in mouse neurons. We profile neuronal TOP2ß activity genome wide, detecting enrichment at regulatory regions and gene bodies of long genes, including MeCP2-regulated genes. We show that loss and overexpression of MeCP2 alter TOP2ß activity at MeCP2-regulated genes. These findings uncover a mechanism of TOP2ß inhibition by MeCP2 in neurons and implicate TOP2ß dysregulation in disorders caused by MeCP2 disruption.

Towards a comprehensive regulatory map of Mammalian Genomes.

Genome mapping studies have generated a nearly complete collection of genes for the human genome, but we still lack an equivalently vetted inventory of human regulatory sequences. Cis-regulatory modules (CRMs) play important roles in controlling when, where, and how much a gene is expressed. We developed a training data-free CRM-prediction algorithm, the Mammalian Regulatory MOdule Detector (MrMOD) for accurate CRM prediction in mammalian genomes. MrMOD provides genome position-fixed CRM models similar to the fixed gene models for the mouse and human genomes using only genomic sequences as the inputs with one adjustable parameter - the significance p-value. Importantly, MrMOD predicts a comprehensive set of high-resolution CRMs in the mouse and human genomes including all types of regulatory modules not limited to any tissue, cell type, developmental stage, or condition. We computationally validated MrMOD predictions used a compendium of 21 orthogonal experimental data sets including thousands of experimentally defined CRMs and millions of putative regulatory elements derived from hundreds of different tissues, cell types, and stimulus conditions obtained from multiple databases. In ovo transgenic reporter assay demonstrates the power of our prediction in guiding experimental design. We analyzed CRMs located in the chromosome 17 using unsupervised machine learning and identified groups of CRMs with multiple lines of evidence supporting their functionality, linking CRMs with upstream binding transcription factors and downstream target genes. Our work provides a comprehensive base pair resolution annotation of the functional regulatory elements and non-functional regions in the mammalian genomes.