Prader–Willi syndrome (PWS), a genetic disorder of obesity, intellectual disability and sleep abnormalities, is caused by loss of non-coding RNAs on paternal chromosome 15q11-q13. The imprinted minimal PWS locus encompasses a long non-coding RNA (lncRNA) transcript processed into multiple SNORD116 small nucleolar RNAs and the spliced exons of the host gene, 116HG. However, both the molecular function and the disease relevance of the spliced lncRNA 116HG are unknown. Here, we show that 116HG forms a subnuclear RNA cloud that co-purifies with the transcriptional activator RBBP5 and active metabolic genes, remains tethered to the site of its transcription and increases in size in post-natal neurons and during sleep. Snord116del mice lacking 116HG exhibited increased energy expenditure corresponding to the dysregulation of diurnally expressed Mtor and circadian genes Clock, Cry1 and Per2. These combined genomic and metabolic analyses demonstrate that 116HG regulates the diurnal energy expenditure of the brain. These novel molecular insights into the energy imbalance in PWS should lead to improved therapies and understanding of lncRNA roles in complex neurodevelopmental and metabolic disorders.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AScausing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuronspecific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.rader-Willi syndrome (PWS) and Angelman syndrome (AS) are imprinted neurodevelopmental disorders caused by oppositely inherited deficiencies of chromosome 15q11-q13. AS and PWS are both characterized by hypotonia at birth, disordered sleep, autistic features, and intellectual disabilities, but the diseases differentiate into phenotypically distinct syndromes in early childhood (1, 2). Seizures, ataxia, and inappropriate laughter characterize AS, whereas hyperphagia leading to obesity and obsessive-compulsive behaviors characterize PWS. Maternal mutations in UBE3A/Ube3a in humans and mice have identified the loss of function of this ubiquitin E3 ligase encoding gene as the cause of AS (3, 4). For PWS, small deletions of the HBII-85/ SNORD116 locus (5-7) and two mouse models of Snord116 deletions (8, 9) have identified the minimal causative deficiency to be the paternally expressed, highly repetitive, long noncoding RNA (lncRNA) that is processed into multiple small nucleolar RNAs (snoRNAs) and spliced nuclear retained host genes (116HG and 115HG) (10,11).A recent drug screen discovered that topoisomerase inhibitors, including topotecan, reduce Ube3a-ATS by an unknown mechanism to reverse the silencing of paternal Ube3a in mouse neurons and brain (12). Topotec...

Summary Rare variants enriched for functions in chromatin regulation and neuronal synapses have been linked to autism. How chromatin and DNA methylation interact with environmental exposures at synaptic genes in autism etiologies is currently unclear. Using whole genome bisulfite sequencing in brain tissue and a neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, we find significant global DNA hypomethylation that is enriched over autism candidate genes and impacts gene expression. The cumulative effect of multiple chromosomal duplications and exposure to the pervasive persistent organic pollutant PCB 95 altered methylation of >1,000 genes. Hypomethylated genes were enriched for H2A.Z, increased maternal UBE3A in Dup15q corresponded to reduced levels of RING1B, and bivalently modified H2A.Z was altered by PCB 95 and duplication. These results demonstrate the compounding effects of genetic and environmental insults on the neuronal methylome that converge upon dysregulation of chromatin and synaptic genes.

Prader-Willi syndrome (PWS) is an imprinted neurodevelopmental disease caused by a loss of paternal genes on chromosome 15q11-q13. It is characterized by cognitive impairments, developmental delay, sleep abnormalities, and hyperphagia often leading to obesity. Clinical research has shown that a lack of expression of SNORD116, a paternally expressed imprinted gene cluster that encodes multiple copies of a small nucleolar RNA (snoRNA) in both humans and mice, is most likely responsible for many PWS symptoms seen in humans. The majority of previous research using PWS preclinical models focused on characterization of the hyperphagic and metabolic phenotypes. However, a crucial understudied clinical phenotype is cognitive impairments and thus we investigated the learning and memory abilities using a model of PWS, with a heterozygous deletion in Snord116. We utilized the novel object recognition task, which doesn't require external motivation, or exhaustive swim training. Automated findings were further confirmed with manual scoring by a highly trained blinded investigator. We discovered deficits in Snord116+/- mutant mice in the novel object recognition, location memory and tone cue fear conditioning assays when compared to age-, sex- matched, littermate control Snord116+/+ mice. Further, we confirmed that despite physical neo-natal developmental delays, Snord116+/- mice had normal exploratory and motor abilities. These results show that the Snord116+/- deletion murine model is a valuable preclinical model for investigating learning and memory impairments in individuals with PWS without common confounding phenotypes.

Supplementary Figure 2: ASD DMRs heatmap by child outcome continuous measurements of cognition and autism severity versus potential cofounding variables.The plot shows a heatmap of ASD DMRs (y-axis) and the association of % methylation at each DMR with other measured variables. The first 5 child outcome variables on the x-axis includes 4 sub-categories of Mullen scores as well as composite score and autism severity score from the ADOS. Significant associations are red (p < 0.05). While ASD DMRs were highly associated with autism severity and to a lesser degree with early learning Mullen's scores, other potential confounding variables from MARBLES exhibited only rare associations with individual ASD DMRs.