Allelic contribution of Nrxn1α to autism-relevant behavioral phenotypes in mice.

Copy number variations (CNVs) in the Neurexin 1 (NRXN1) gene, which encodes a presynaptic protein involved in neurotransmitter release, are some of the most frequently observed single-gene variants associated with autism spectrum disorder (ASD). To address the functional contribution of NRXN1 CNVs to behavioral phenotypes relevant to ASD, we carried out systematic behavioral phenotyping of an allelic series of Nrxn1 mouse models: one carrying promoter and exon 1 deletion abolishing Nrxn1α transcription, one carrying exon 9 deletion disrupting Nrxn1α protein translation, and one carrying an intronic deletion with no observable effect on Nrxn1α expression. We found that homozygous loss of Nrxn1α resulted in enhanced aggression in males, reduced affiliative social behaviors in females, and significantly altered circadian activities in both sexes. Heterozygous or homozygous loss of Nrxn1α affected the preference for social novelty in male mice, and notably, enhanced repetitive motor skills and motor coordination in both sexes. In contrast, mice bearing an intronic deletion of Nrxn1 did not display alterations in any of the behaviors assessed. These findings demonstrate the importance of Nrxn1α gene dosage in regulating social, circadian, and motor functions, and the variables of sex and genomic positioning of CNVs in the expression of autism-related phenotypes. Importantly, mice with heterozygous loss of Nrxn1, as found in numerous autistic individuals, show an elevated propensity to manifest autism-related phenotypes, supporting the use of models with this genomic architecture to study ASD etiology and assess additional genetic variants associated with autism.

Long genes linked to autism spectrum disorders harbor broad enhancer-like chromatin domains.

Genetic variants associated with autism spectrum disorders (ASDs) are enriched in genes encoding synaptic proteins and chromatin regulators. Although the role of synaptic proteins in ASDs is widely studied, the mechanism by which chromatin regulators contribute to ASD risk remains poorly understood. Upon profiling and analyzing the transcriptional and epigenomic features of genes expressed in the cortex, we uncovered a unique set of long genes that contain broad enhancer-like chromatin domains (BELDs) spanning across their entire gene bodies. Analyses of these BELD genes show that they are highly transcribed with frequent RNA polymerase II (Pol II) initiation and low Pol II pausing, and they exhibit frequent chromatin-chromatin interactions within their gene bodies. These BELD features are conserved from rodents to humans, are enriched in genes involved in synaptic function, and appear post-natally concomitant with synapse development. Importantly, we find that BELD genes are highly implicated in neurodevelopmental disorders, particularly ASDs, and that their expression is preferentially down-regulated in individuals with idiopathic autism. Finally, we find that the transcription of BELD genes is particularly sensitive to alternations in ASD-associated chromatin regulators. These findings suggest that the epigenomic regulation of BELD genes is important for post-natal cortical development and lend support to a model by which mutations in chromatin regulators causally contribute to ASDs by preferentially impairing BELD gene transcription.

Bromodomain protein Brd3 associates with acetylated GATA1 to promote its chromatin occupancy at erythroid target genes.

Acetylation of histones triggers association with bromodomain-containing proteins that regulate diverse chromatin-related processes. Although acetylation of transcription factors has been appreciated for some time, the mechanistic consequences are less well understood. The hematopoietic transcription factor GATA1 is acetylated at conserved lysines that are required for its stable association with chromatin. We show that the BET family protein Brd3 binds via its first bromodomain (BD1) to GATA1 in an acetylation-dependent manner in vitro and in vivo. Mutation of a single residue in BD1 that is involved in acetyl-lysine binding abrogated recruitment of Brd3 by GATA1, demonstrating that acetylation of GATA1 is essential for Brd3 association with chromatin. Notably, Brd3 is recruited by GATA1 to both active and repressed target genes in a fashion seemingly independent of histone acetylation. Anti-Brd3 ChIP followed by massively parallel sequencing in GATA1-deficient erythroid precursor cells and those that are GATA1 replete revealed that GATA1 is a major determinant of Brd3 recruitment to genomic targets within chromatin. A pharmacologic compound that occupies the acetyl-lysine binding pockets of Brd3 bromodomains disrupts the Brd3-GATA1 interaction, diminishes the chromatin occupancy of both proteins, and inhibits erythroid maturation. Together these findings provide a mechanism for GATA1 acetylation and suggest that Brd3 "reads" acetyl marks on nuclear factors to promote their stable association with chromatin.

Disentangling chromatin architecture to gain insights into the etiology of brain disorders.

Chromatin organization, together with DNA and histone modifications, is directly linked to the spatiotemporal control of gene expression that specifies and maintains cell type-specific functions. This is particularly important in the brain where hundreds of cell types with distinct functions reside. Recent advances in molecular and computational technologies have enabled the query of chromatin architecture at unprecedented resolution and detail. Here, we review recent studies on the emerging importance of chromatin architecture in the pathogenesis of brain disorders, with emphasis on schizophrenia, autism spectrum disorders (ASD), and unstable repeat expansion disorders. These studies provide molecular insights into how these brain disorders arise at the level of chromatin architecture and implicate new therapeutic directions.

Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC.

Efforts to manipulate locus-specific histone acetylation to assess their causal role in gene expression and cellular and behavioural phenotypes have been impeded by a lack of experimental tools. The Cas9 nuclease has been adapted to target epigenomic modifications, but a detailed description of the parameters of such synthetic epigenome remodellers is still lacking. Here we describe a Cas9-based histone deacetylase (HDAC) and the design principles required to achieve locus-specific histone deacetylation. We assess its range of activity and specificity, and analyse target gene expression in two different cell types to investigate cellular context-dependent effects. Our findings demonstrate that the chromatin environment is an important element to consider when utilizing this synthetic HDAC.

Elevating expression of MeCP2 T158M rescues DNA binding and Rett syndrome-like phenotypes.

Mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome (RTT), a neurological disorder affecting cognitive development, respiration, and motor function. Genetic restoration of MeCP2 expression reverses RTT-like phenotypes in mice, highlighting the need to search for therapeutic approaches. Here, we have developed knockin mice recapitulating the most common RTT-associated missense mutation, MeCP2 T158M. We found that the T158M mutation impaired MECP2 binding to methylated DNA and destabilized MeCP2 protein in an age-dependent manner, leading to the development of RTT-like phenotypes in these mice. Genetic elevation of MeCP2 T158M expression ameliorated multiple RTT-like features, including motor dysfunction and breathing irregularities, in both male and female mice. These improvements were accompanied by increased binding of MeCP2 T158M to DNA. Further, we found that the ubiquitin/proteasome pathway was responsible for MeCP2 T158M degradation and that proteasome inhibition increased MeCP2 T158M levels. Together, these findings demonstrate that increasing MeCP2 T158M protein expression is sufficient to mitigate RTT-like phenotypes and support the targeting of MeCP2 T158M expression or stability as an alternative therapeutic approach.

Biotin tagging of MeCP2 in mice reveals contextual insights into the Rett syndrome transcriptome.

Mutations in MECP2 cause Rett syndrome (RTT), an X-linked neurological disorder characterized by regressive loss of neurodevelopmental milestones and acquired psychomotor deficits. However, the cellular heterogeneity of the brain impedes an understanding of how MECP2 mutations contribute to RTT. Here we developed a Cre-inducible method for cell-type-specific biotin tagging of MeCP2 in mice. Combining this approach with an allelic series of knock-in mice carrying frequent RTT-associated mutations (encoding T158M and R106W) enabled the selective profiling of RTT-associated nuclear transcriptomes in excitatory and inhibitory cortical neurons. We found that most gene-expression changes were largely specific to each RTT-associated mutation and cell type. Lowly expressed cell-type-enriched genes were preferentially disrupted by MeCP2 mutations, with upregulated and downregulated genes reflecting distinct functional categories. Subcellular RNA analysis in MeCP2-mutant neurons further revealed reductions in the nascent transcription of long genes and uncovered widespread post-transcriptional compensation at the cellular level. Finally, we overcame X-linked cellular mosaicism in female RTT models and identified distinct gene-expression changes between neighboring wild-type and mutant neurons, providing contextual insights into RTT etiology that support personalized therapeutic interventions.

Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization.

BACKGROUND: Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are closely related members of the B. cereus-group of bacilli. Suppressive subtractive hybridization (SSH) was used to identify specific chromosomal sequences unique to B. anthracis. RESULTS: Two SSH libraries were generated. Genomic DNA from plasmid-cured B. anthracis was used as the tester DNA in both libraries, while genomic DNA from either B. cereus or B. thuringiensis served as the driver DNA. Progressive screening of the libraries by colony filter and Southern blot analyses identified 29 different clones that were specific for the B. anthracis chromosome relative not only to the respective driver DNAs, but also to seven other different strains of B. cereus and B. thuringiensis included in the process. The nucleotide sequences of the clones were compared with those found in genomic databases, revealing that over half of the clones were located into 2 regions on the B. anthracis chromosome. CONCLUSIONS: Genes encoding potential cell wall synthesis proteins dominated one region, while bacteriophage-related sequences dominated the other region. The latter supports the hypothesis that acquisition of these bacteriophage sequences occurred during or after speciation of B. anthracis relative to B. cereus and B. thuringiensis. This study provides insight into the chromosomal differences between B. anthracis and its closest phylogenetic relatives.

Structural basis and specificity of acetylated transcription factor GATA1 recognition by BET family bromodomain protein Brd3.

Recent data demonstrate that small synthetic compounds specifically targeting bromodomain proteins can modulate the expression of cancer-related or inflammatory genes. Although these studies have focused on the ability of bromodomains to recognize acetylated histones, it is increasingly becoming clear that histone-like modifications exist on other important proteins, such as transcription factors. However, our understanding of the molecular mechanisms through which these modifications modulate protein function is far from complete. The transcription factor GATA1 can be acetylated at lysine residues adjacent to the zinc finger domains, and this acetylation is essential for the normal chromatin occupancy of GATA1. We have recently identified the bromodomain-containing protein Brd3 as a cofactor that interacts with acetylated GATA1 and shown that this interaction is essential for the targeting of GATA1 to chromatin. Here we describe the structural basis for this interaction. Our data reveal for the first time the molecular details of an interaction between a transcription factor bearing multiple acetylation modifications and its cognate recognition module. We also show that this interaction can be inhibited by an acetyllysine mimic, highlighting the importance of further increasing the specificity of compounds that target bromodomain and extraterminal (BET) bromodomains in order to fully realize their therapeutic potential.

Acetylation of GATA-1 is required for chromatin occupancy.

All 3 hematopoietic GATA transcription factors, GATA-1, GATA-2, and GATA-3, are acetylated, although the in vivo role of this modification remains unclear. We examined the functions of an acetylation-defective mutant of GATA-1 in maturing erythroid cells. We found that removal of the acetylation sites in GATA-1 does not impair its nuclear localization, steady-state protein levels, or its ability to bind naked GATA elements in vitro. However, chromatin immunoprecipitation (ChIP) experiments revealed that mutant GATA-1 was dramatically impaired in binding to all examined cellular target sites in vivo, including genes that are normally activated and repressed by GATA-1. Together, these results suggest that acetylation regulates chromatin occupancy of GATA-1. These findings point to a novel function for transcription factor acetylation, perhaps by facilitating protein interactions required for stable association with chromatin templates in vivo.

Comparative secretome analyses of three Bacillus anthracis strains with variant plasmid contents.

Bacillus anthracis, the causative agent of anthrax, secretes numerous proteins into the extracellular environment during infection. A comparative proteomic approach was employed to elucidate the differences among the extracellular proteomes (secretomes) of three isogenic strains of B. anthracis that differed solely in their plasmid contents. The strains utilized were the wild-type virulent B. anthracis RA3 (pXO1(+) pXO2(+)) and its two nonpathogenic derivative strains: the toxigenic, nonencapsulated RA3R (pXO1(+) pXO2(-)) and the totally cured, nontoxigenic, nonencapsulated RA3:00 (pXO1(-) pXO2(-)). Comparative proteomics using two-dimensional gel electrophoresis followed by computer-assisted gel image analysis was performed to reveal unique, up-regulated, or down-regulated secretome proteins among the strains. In total, 57 protein spots, representing 26 different proteins encoded on the chromosome or pXO1, were identified by peptide mass fingerprinting. S-layer-derived proteins, such as Sap and EA1, were most frequently observed. Many sporulation-associated enzymes were found to be overexpressed in strains containing pXO1(+). This study also provides evidence that pXO2 is necessary for the maximal expression of the pXO1-encoded toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). Several newly identified putative virulence factors were observed; these include enolase, a high-affinity zinc uptake transporter, the peroxide stress-related alkyl hydroperoxide reductase, isocitrate lyase, and the cell surface protein A.