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Ube3a CRISPR-deletion rat model study design. Reciprocal crosses with Ube3a-deletion carriers (gray) produced the maternal-derived (red) and paternalderived (blue) deletion. Hypothalamus and cortex were harvested from female rat pups at P2 and P9 in triplicate with litter-matched controls and utilized for RNA-seq and WGBS.
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UBE3A encodes a E3 ubiquitin ligase whose loss from the maternal allele causes the neurodevelopmental disorder Angelman syndrome. Previous studies of UBE3A function have not examined full Ube3a deletion in mouse, the complexity of imprinted gene networks in brain, nor the molecular basis of systems-level cognitive dysfunctions in Angelman syndrome....
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... Ube3a mutant mice have been extensively used as models of AS (30)(31)(32)(33)(34), none of these are complete knockouts of the gene. To produce a more robust model of AS and to better understand the functional consequences of maternal UBE3A loss, an AS rat model was designed to completely delete Ube3a by targeting CRISPR/Cas9 nucleases to sites flanking Ube3a ( Supplementary Material Fig. S1). ...
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... devised a breeding strategy to produce both maternally and paternally derived Ube3a deletions and harvested hypothalamus and cortex from deletion animals and litter-matched controls at postnatal day 2 (P2) and postnatal day 9 (P9) (Fig. 1). These two timepoints were chosen because Ube3a was expected to become imprinted within the first week of life, based on prior studies in mouse (35,36), and this was also the time window when deficits in pup ultrasonic vocalization and developmental delays were observed in maternal Ube3a deletion rat pups when compared to wild-type ...
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... gene modules were detected and tested for correlation with either time point or genotype in separate analyses of hypothalamus (Fig. 3) and cortex (Supplementary Material Fig. S10). Positively correlated modules in red represent increased expression or methylation in Ube3a deletion samples compared to wild-type or P9 samples compared to P2. Correlations colored in blue were negative, indicating decreased expression or methylation in Ube3a deletion samples or at P9 relative to P2. ...
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... expected, significant genotype modules were only detected in maternal samples from WGCNA and were enriched in Wnt signaling, neuron differentiation and maintenance, epigenetic regulation, synaptic activity and circadian entrainment for negatively correlated modules, and calcium signaling, chromatin organization and cAMP signaling for positively correlated modules (Fig. 3A). GO enrichment for negatively correlated modules detected in cortex samples were similar to those observed in hypothalamus modules for time, but the maternal genotype module was distinct in highlighting ubiquitin functions, dendrite and spliceosome in cortex ( Supplementary Material Fig. S11). A control data set comparing wild-type samples from maternal and paternal litters produced three significant modules for parent-of-origin in hypothalamus and one in cortex with genes enriched in ubiquitin activity ( Supplementary Material Fig. S12). ...
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... enrichment for negatively correlated modules detected in cortex samples were similar to those observed in hypothalamus modules for time, but the maternal genotype module was distinct in highlighting ubiquitin functions, dendrite and spliceosome in cortex ( Supplementary Material Fig. S11). A control data set comparing wild-type samples from maternal and paternal litters produced three significant modules for parent-of-origin in hypothalamus and one in cortex with genes enriched in ubiquitin activity ( Supplementary Material Fig. S12). GO enrichment terms identified by WGCNA were highly similar to those observed by the simpler pairwise analysis (Supplementary Material Fig. S13). ...
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... control data set comparing wild-type samples from maternal and paternal litters produced three significant modules for parent-of-origin in hypothalamus and one in cortex with genes enriched in ubiquitin activity ( Supplementary Material Fig. S12). GO enrichment terms identified by WGCNA were highly similar to those observed by the simpler pairwise analysis (Supplementary Material Fig. S13). ...
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... contrast, the control co-methylation WGCNA from wild-type maternal versus paternal samples not only produced no significant modules but also no clustered modules were detected. Furthermore, GO enrichment terms identified by WGCNA were highly similar to those observed by the pairwise analysis of differentially methylated genes ( Supplementary Material Fig. S13). ...
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... directly compare the WGCNA results for the co-expression modules and co-methylation modules as well as across parent-of-origin of Ube3a deletion, we performed cluster profiling of the GO results for each analysis. For individual GO terms, four distinct clusters were observed for downregulated (negatively correlated modules) specific GO terms ( Supplementary Material Fig. S14), while the upregulated (positively correlated modules) GO profile clustered into two groups ( Supplementary Material Fig. S15). Cluster profiling by KEGG pathways revealed five distinct clusters for downregulated pathways (Fig. 5A): Group I clusters by downregulated pathway terms that were specific to maternal, but not paternal, genotype effects, some of which also overlapped with maternal genotype and time-associated co-methylation and paternal timeassociated co-methylation terms. ...
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... directly compare the WGCNA results for the co-expression modules and co-methylation modules as well as across parent-of-origin of Ube3a deletion, we performed cluster profiling of the GO results for each analysis. For individual GO terms, four distinct clusters were observed for downregulated (negatively correlated modules) specific GO terms ( Supplementary Material Fig. S14), while the upregulated (positively correlated modules) GO profile clustered into two groups ( Supplementary Material Fig. S15). Cluster profiling by KEGG pathways revealed five distinct clusters for downregulated pathways (Fig. 5A): Group I clusters by downregulated pathway terms that were specific to maternal, but not paternal, genotype effects, some of which also overlapped with maternal genotype and time-associated co-methylation and paternal timeassociated co-methylation terms. ...
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Background:
Angelman syndrome (AS) is a neurodevelopmental disorder that is caused by maternal genetic deficiency of a gene that encodes E6-AP ubiquitin-protein ligase (gene symbol UBE3A) mapping to chromosome 15q11-q13. AS leads to stiff and jerky gait, excess laughter, seizures, and severe intellectual disability. In some parts of the brain, the...
Citations
... Other putative UBE3A substrates have been reviewed elsewhere [3,39,40]. Of these, signi cant interest was at some point directed towards ARC and Ephexin-5, two proteins claimed to share a highly variable "UBE3A-target sequence" with Sacsin, a protein whose ubiquitination fraction was claimed to be increased by UBE3A on a proteomic dataset referred to by Greenberg and colleagues [41,42]. ...
Angelman Syndrome (AS) is a neurodevelopmental disorder with complex symptomatology caused by the loss of maternal allele expression of one single gene in the brain, the ubiquitin E3 ligase UBE3A . The underlying genetic basis of AS, and the phenotypes observed in both humans and in animal models of AS, have previously been extensively described. However, the molecular mechanisms regulated by UBE3A ubiquitination in the brain remain highly elusive. Previous studies have reported a number of proteins whose abundance or activity are altered in AS models, implicating various signalling pathways in the physiopathology of AS. However, the identified pathways could well be altered further downstream of UBE3A ubiquitination events. We provide the first proteomic report of UBE3A-mediated ubiquitination events in a mammalian brain. For this we have combined the bioUb mouse model with a new mouse strain moderately increasing UBE3A levels. Several proteins known to be involved in the trafficking and maintenance of neurotransmitter receptors as well as proteins relaying the signals of these synaptic receptors are shown here to be ubiquitinated by UBE3A. The identified proteins have roles in higher mental function, long term potentiation, seizures and neurodevelopmental disorders, being involved in the BDNF, RAS/ERK and TSC/mTOR signalling pathways. A reduced ubiquitination of these proteins is expected when UBE3A levels are lower, so their identification could be key to opening novel therapeutic strategies for treating Angelman Syndrome. Further work will be required to characterize how UBE3A timely orchestrates each of these multiple regulatory events in different neuronal subtypes within the human brain.
... The underlying etiology of AS is the lack of expression of the maternally inherited UBE3A gene in the 15q11-13 imprinted region chromosome 15q11-13 [7,8]. UBE3A encodes a HECT domain E3 ubiquitin ligase that targets substrate proteins, transferring the ubiquitin Genes 2022, 13, 1447 2 of 9 to the proteins targeted for degradation by the ubiquitin-proteasome system [9,10]. The most common subtype that occurs in approximately 65-75% of affected individuals is the "deletion subtype", followed by UBE3A mutations (8-11%), paternal uniparental disomy for chromosome15 (UPD 15pat, 3-7%), and imprinting defects (3%) [11]. ...
Angelman syndrome (AS) is a neurodevelopmental genetic disorder, but there has been limited analysis of a large cohort of Chinese children with Angelman syndrome. This study aims to assess the phenotype and genotype of Chinese children with Angelman syndrome. We retrospectively analyzed data through a detailed online survey combined with an on-site study. Furthermore, phenotype analysis stratified by deletion and non-deletion groups was carried out. The responses of family members of 695 individuals with AS revealed that 577 patients (83.02%) had maternal deletions, 65 patients (9.35%) carried UBE3A mutations, 31 (4.46%) patients had UPD15pat (one patient with UPD15pat constituted by a mosaic), 10 patients (1.44%) had imprinting defects and 12 (1.58%) patients only showed abnormal methylation without further detection. We identified 50 different pathogenic variants in this cohort, although 18 of these variants were unreported. Recurrent variant c.2507_2510del (p.K836Rfs*4) was found in 7 patients. In the deletion group, patients were diagnosed at an earlier age, had a more severe clinical phenotype, a higher rate of epilepsy with more multiple seizure types, and more frequently combined medication. Strabismus and sleep disturbances were both common in deletion and non-deletion groups. The top three resources invested in caring for AS children are: daily involvement in patient care, rehabilitation cost, and anti-epileptic treatment. Our study showed the genetic composition of Chinese children with 83.02% of maternal deletions, and the mutation spectrum for UBE3A variants was expanded. Developmental outcomes are associated with genotype, and this was confirmed by deletion patients having a worse clinical phenotype and complex epilepsy.
... UBE3A interacts with most of the components of the proteasome [29] regulating the activity of signal transduction pathways such as Wnt signaling that regulates central nervous system development [30][31][32] and synaptic plasticity in both excitatory and inhibitory GABAergic axon terminals [33][34][35][36]. At the nucleus, UBE3A has been shown to regulate chromatin structure, DNA methylation and transcriptional regulation [37][38][39][40]. Interestingly, 8 out of the 10 genes found mutated in this study are mainly involved in synapsis (VAMP2, SYNGAP1, SLC6A1 and KCNQ3) [41][42][43][44] and chromatin remodeling or transcription regulation (TBL1XR1, SATB2, SMARCE1 and ASXL3) [45][46][47][48]. ...
... UBE3A has been shown to be present in euchromatin-rich nuclear domains indicating that it may influence neuronal physiology by regulating chromatin and gene transcription [59]. RNA-seq studies of UBE3A loss in rat cortex, mice hippocampus and SH-SY5Y cells have shown differential gene expression of KCNQ3 [60], SMARCE1, HSF2 [38], SPTAN1 and SATB2 [39] suggesting that these genes may be transcriptionally regulated by UBE3A. Moreover, UBE3A gain and loss in human SH-SY5Y cells has been shown to have significant effects on DNA methylation and chromatin modification in genes involved in transcriptional regulation and brain development including SATB2, ASXL3, SMARCE1 and TBL1XR1 [38]. ...
Angelman syndrome (AS) is a neurogenetic disorder characterized by severe developmental delay with absence of speech, happy disposition, frequent laughter, hyperactivity, stereotypies, ataxia and seizures with specific EEG abnormalities. There is a 10–15% of patients with an AS phenotype whose genetic cause remains unknown (Angelman-like syndrome, AS-like). Whole-exome sequencing (WES) was performed on a cohort of 14 patients with clinical features of AS and no molecular diagnosis. As a result, we identified 10 de novo and 1 X-linked pathogenic/likely pathogenic variants in 10 neurodevelopmental genes ( SYNGAP1 , VAMP2 , TBL1XR1 , ASXL3 , SATB2 , SMARCE1 , SPTAN1 , KCNQ3 , SLC6A1 and LAS1L ) and one deleterious de novo variant in a candidate gene ( HSF2 ). Our results highlight the wide genetic heterogeneity in AS-like patients and expands the differential diagnosis.
... Chromatin folding plays also a major role in the regulation of imprinted gene expression as exemplified at the Igf2/H19 locus where chromatin conformation is intimately linked to access to shared enhancers [16,[22][23][24][25]. ncRNAs are also central in the control of imprinted gene expression in time and space [13,26]. For example, Ube3A, which encodes an ubiquitin ligase involved in synaptic physiology [27][28][29] and whose function is lost in Angelman syndrome [30], is maternally expressed in the hippocampus while it is biallelic in the liver [29,31]. This is due to the presence in the hippocampus while absence in the liver of an antisense RNA transcript, Ube3a-ATS that cis-represses Ube3A paternal allele [32,33]. ...
Imprinted genes are a group of~150 genes that are preferentially expressed from one parental allele owing to epigenetic marks asymmetrically distributed on inherited maternal and paternal chromosomes. Altered imprinted gene expression causes human brain disorders such as Prader-Willi and Angelman syndromes and additional rare brain diseases. Research data principally obtained from the mouse model revealed how imprinted genes act in the normal and pathological brain. However, a better understanding of imprinted gene functions calls for building detailed maps of their parent-of-origin-dependent expression and of associated epigenetic signatures. Here we review current methods for quantifying genomic imprinting at tissue and cell resolutions, with a special emphasis on methods to detect parent-of-origin dependent expression and their applications to the brain. We first focus on bulk RNA-sequencing, the main method to detect parent-of-origin-dependent expression transcriptome-wide. We discuss the benefits and caveats of bulk RNA-sequencing and provide a guideline to use it on F1 hybrid mice. We then review methods for detecting parent-of-origin-dependent expression at cell resolution, including single-cell RNA-seq, genetic reporters, and molecular probes. Finally, we provide an overview of single-cell epigenomics technologies that profile additional features of genomic imprinting, including DNA methylation, histone modifications and chromatin conformation and their combination into sc-multimodal omics approaches, which are expected to yield important insights into genomic imprinting in individual brain cells.
... familial ASD (Autism Spectrum Disorders) worldwide [31]. UBE3A/E6AP was involved in many biological processes, such as Wnt signaling, circadian rhythms, imprinted gene networks, immunity responses, synapse plasticity and early brain development [32,33]. As UBE3A related neurodevelopmental diseases with incompletely understood mechanisms, suggesting yet undiscovered roles of UBE3A-mediated ubiquitination signal in diverse pathophysiological contexts [34,35]. ...
Global identification of substrates for PTMs (post-translational modifications) represents a critical but yet dauntingly challenging task in understanding biology and disease pathology. Here we presented a synthetic biology approach, namely ‘YESS’, which coupled Y2H (yeast two hybrid) interactome screening with PTMs reactions reconstituted in bacteria for substrates identification and validation, followed by the functional validation in mammalian cells. Specifically, the sequence-independent Gateway® cloning technique was adopted to afford simultaneous transfer of multiple hit ORFs (open reading frames) between the YESS sub-systems. In proof-of-evidence applications of YESS, novel substrates were identified for UBE3A and UFL1, the E3 ligases for ubiquitination and ufmylation, respectively. Therefore, the YESS approach could serve as a potentially powerful tool to study cellular signaling mediated by different PTMs.
... Besides these mouse models with constitutional deletions reproducing human defects, more sophisticated models based on conditional KOs, including those in specific neurons or models of UBE3A reinstatement, have been advanced to study UBE3A function at particular cellular/developmental stages [41][42][43][44] that will be detailed in the next sections. Apart from mouse models, Lopez et al. [45] have recently generated the first CRISPR/Cas9-engineered rat AS model bearing a complete Ube3a deletion, expanding the current AS animal models to other rodent species. Fig. 1. ...
Angelman syndrome (AS) is an incurable neurodevelopmental disease caused by loss of function of the maternally inherited UBE3A gene. AS is characterized by a defined set of symptoms, namely severe developmental delay, speech impairment, uncontrolled laughter, and ataxia. Current understanding of the pathophysiology of AS relies mostly on studies using the murine model of the disease, although alternative models based on patient‐derived stem cells are now emerging. Here, we summarize the literature of the last decade concerning the three major brain areas that have been the subject of study in the context of AS: hippocampus, cortex, and the cerebellum. Our comprehensive analysis highlights the major phenotypes ascribed to the different brain areas. Moreover, we also discuss the major drawbacks of current models and point out future directions for research in the context of AS, which will hopefully lead us to an effective treatment of this condition in humans.
Autism spectrum disorders (ASD) is a heterogeneous group of neurodevelopmental disorders characterized by synaptic dysfunction and defects in dendritic spine morphology. In the past decade, an extensive list of genes associated with ASD has been identified by genome-wide sequencing initiatives. Several of these genes functionally converge in the regulation of the Wnt/β-catenin signaling pathway, a conserved cascade essential for stem cell pluripotency and cell fate decisions during development. Here, we review current information regarding the transcriptional program of Wnt/β-catenin signaling in ASD. First, we discuss that Wnt/β-catenin gain and loss of function studies recapitulate brain developmental abnormalities associated with ASD. Second, transcriptomic approaches using patient-derived induced pluripotent stem cells (iPSC) cells, featuring mutations in high confidence ASD genes, reveal a significant dysregulation in the expression of Wnt signaling components. Finally, we focus on the activity of chromatin-remodeling proteins and transcription factors considered high confidence ASD genes, including CHD8, ARID1B, ADNP, and TBR1, that regulate Wnt/β-catenin-dependent transcriptional activity in multiple cell types, including pyramidal neurons, interneurons and oligodendrocytes, cells which are becoming increasingly relevant in the study of ASD. We conclude that the level of Wnt/β-catenin signaling activation could explain the high phenotypical heterogeneity of ASD and be instrumental in the development of new diagnostics tools and therapies.
Angelman syndrome (AS) is an incurable neurodevelopmental disease characterized by serious developmental delay, impaired speech, motor incoordination, and frequent atypical episodes of smiling and laughter. This disease is caused by loss of function of the maternally inherited UBE3A allele. This gene is submitted to imprinting regulation, being exclusively expressed from the maternal allele in mature neurons. Paternal UBE3A silencing involves the SNHG14 antisense noncoding RNA that is expressed exclusively from the paternal allele in neurons. UBE3A encodes for an E3 ubiquitin ligase involved in targeting specific proteins to proteasomal degradation being also potentially implicated in other molecular pathways. Most of our current understanding of the pathophysiology of AS has been gathered from seminal studies using maternal UBE3A null mouse models. However, several hurdles subsist to the direct translation of mouse studies to human patients, urging for the need of humanized models for this disease. The advent of induced pluripotent stem cells (iPSCs) and the continuing improvement of protocols for neuronal differentiation, including three-dimensional brain-in-a-dish models, created an alternative system to study molecular and cellular mechanisms underlying the pathology of neurodevelopmental diseases such as AS. Here, we document the available iPSC lines derived from AS patients that cover all the (epi) genetic causes of the disease and sum up the new knowledge brought by their study so far. Although still in its infancy, we believe iPSC technology will prove to be an important model system not only to understand the pathophysiological mechanisms underlying this disease but also as a valuable platform for drug screening/development in the context of AS therapy.
