To initiate studies on how protein-protein interaction (or “interactome”) networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico , the current version of the Worm Interactome (WI5) map contains ∼5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.

Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism1. MECP2 encodes a methyl-DNA-binding protein2 that has been proposed to function as a transcriptional repressor, but despite numerous studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 regulates transcription3–9. 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.

Summary 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.

Caenorhabditis elegans homologues of the retinoblastoma (Rb) tumour suppressor complex specify cell lineage during development. Here we show that mutations in Rb pathway components enhance RNA interference (RNAi) and cause somatic cells to express genes and elaborate perinuclear structures normally limited to germline-specific P granules. Furthermore, particular gene inactivations that disrupt RNAi reverse the cell lineage transformations of Rb pathway mutants. These findings suggest that mutations in Rb pathway components cause cells to revert to patterns of gene expression normally restricted to germ cells. Rb may act by a similar mechanism to transform mammalian cells.

SUMMARY Autism spectrum disorders such as Rett syndrome (RTT) have been hypothesized to arise from defects in experience-dependent synapse maturation. RTT is caused by mutations in MECP2, a nuclear protein that becomes phosphorylated at S421 in response to neuronal activation. We show here that disruption of MeCP2 S421 phosphorylation in vivo results in defects in synapse development and behavior, implicating activity-dependent regulation of MeCP2 in brain development and RTT. We investigated the mechanism by which S421 phosphorylation regulates MeCP2 function and show by chromatin immunoprecipitation-sequencing that this modification occurs on MeCP2 bound across the genome. The phosphorylation of MeCP2 S421 appears not to regulate the expression of specific genes; rather, MeCP2 functions as a histone-like factor whose phosphorylation may facilitate a genome-wide response of chromatin to neuronal activity during nervous system development. We propose that RTT results in part from a loss of this experience-dependent chromatin remodeling.

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.

SUMMARY In mammals during the early postnatal period the environment plays a critical role in promoting the final steps in the neuronal development. 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.

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.M ethylation of cytosines at the carbon 5 position (5-methylcytosine, mC) constitutes the most common covalent modification of vertebrate genomic DNA. Traditionally, cytosine methylation in vertebrate genomes has been viewed as largely restricted to CpG dinucleotide (CG) sequences, providing a stable epigenetic mark that mediates long-term transcriptional silencing. Indeed, 60-90% of all CGs are methylated in mammalian genomes, and CG methylation (mCG) has been shown to play critical roles in genomic imprinting, X-chromosome inactivation, cellular differentiation, and development (1). In addition, the disruption of cellular DNA methylation patterns has been linked to human disease, including multiple cancers (2, 3).Evidence that DNA methylation has a uniquely important role in the brain emerged almost two decades ago with the discovery of the prominent methyl-DNA-binding protein, methyl-CpG-binding protein 2 (MeCP2), and the later identification that mutations in MeCP2 give rise to the X-linked neurological disorder Rett syndrome (RTT) (4-6). Subsequent studies also have identified neurodevelopmental disorders associated with mutations in DNA methyltransferases (7), suggesting that both the enzymatic "writers" of DNA methylation patterns and the "readers" of these marks have important roles in the brain. In this context, new studies from several laboratories have uncovered extensive cytosine modification in the brain beyond mCG. Non-CG methylation (CH methylation or mCH, in which H = A, C, or T) is now appreciated to accumulate in the human and mouse brain postnatally, reaching levels similar to that of mCG in the neuronal genome (8, 9). Moreover, oxidation of mC by the ten-eleven translocation (Tet) family of dioxygenases leads to the selective accumulation of 5-hydroxymethylcytosine (hmC) in the adult brain, together with its more highly oxidized derivatives 5-formylcytosine and 5-carboxylcytosine (10, 11). This finding suggests that hmC may act as an intermediate in an active DNA demethylation pathway, though growing evidence also suggests that hmC may serve as a stable neuronal epigenetic mark in its own right (12).The discovery of these previously unidentified brain-enriched forms of DNA methylation provides...