Annie Vogel Ciernia

Snord116-dependent diurnal rhythm of DNA methylation in mouse cortex

Rhythmic oscillations of physiological processes depend on integrating the circadian clock and diurnal environment. DNA methylation is epigenetically responsive to daily rhythms, as a subset of CpG dinucleotides in brain exhibit diurnal rhythmic methylation. Here, we show a major genetic effect on rhythmic methylation in a mouse Snord116 deletion model of the imprinted disorder Prader–Willi syndrome (PWS). More than 23,000 diurnally rhythmic CpGs are identified in wild-type cortex, with nearly all lost or phase-shifted in PWS. Circadian dysregulation of a second imprinted Snord cluster at the Temple/Kagami-Ogata syndrome locus is observed at the level of methylation, transcription, and chromatin, providing mechanistic evidence of cross-talk. Genes identified by diurnal epigenetic changes in PWS mice overlapped rhythmic and PWS-specific genes in human brain and are enriched for PWS-relevant phenotypes and pathways. These results support the proposed evolutionary relationship between imprinting and sleep, and suggest possible chronotherapy in the treatment of PWS and related disorders.Many genes have oscillating gene expression pattern in circadian centers of the brain. This study shows cortical diurnal DNA methylation oscillation in a mouse model of Prader-Willi syndrome, and describes corresponding changes in gene expression and chromatin compaction.

Early motor phenotype detection in a female mouse model of Rett syndrome is improved by cross-fostering

&NA; Rett syndrome (RTT) is an X‐linked neurodevelopmental disorder caused by mutations in the gene encoding methyl CpG binding protein 2 (MeCP2) that occur sporadically in 1:10,000 female births. RTT is characterized by a period of largely normal development followed by regression in language and motor skills at 6‐18 months of age. Mecp2 mutant mice recapitulate many of the clinical features of RTT, but the majority of behavioral assessments have been conducted in male Mecp2 hemizygous null mice as offspring of heterozygous dams. Given that RTT patients are predominantly female, we conducted a systematic analysis of developmental milestones, sensory abilities, and motor deficits, following the longitudinal decline of function from early postnatal to adult ages in female Mecp2 heterozygotes of the conventional Bird line (Mecp2tm1.1bird‐/+), as compared to their female wildtype littermate controls. Further, we assessed the impact of postnatal maternal environment on developmental milestones and behavioral phenotypes. Cross‐fostering to CD1 dams accelerated several developmental milestones independent of genotype, and induced earlier onset of weight gain in adult female Mecp2tm1.1bird‐/+ mice. Cross‐fostering improved the sensitivity of a number of motor behaviors that resulted in observable deficits in Mecp2tm1.1bird‐/+ mice at much earlier (6‐7 weeks) ages than were previously reported (6‐9 months). Our findings indicate that female Mecp2tm1.1bird‐/+ mice recapitulate many of the motor aspects of RTT syndrome earlier than previously appreciated. In addition, rearing conditions may impact the phenotypic severity and improve the ability to detect genotype differences in female Mecp2 mutant mice.

Microglia from offspring of dams with allergic asthma exhibit epigenomic alterations in genes dysregulated in autism

Dysregulation in immune responses during pregnancy increases the risk of a having a child with an autism spectrum disorder (ASD). Asthma is one of the most common chronic diseases among pregnant women, and symptoms often worsen during pregnancy. We recently developed a mouse model of maternal allergic asthma (MAA) that induces changes in sociability, repetitive, and perseverative behaviors in the offspring. Since epigenetic changes help a static genome adapt to the maternal environment, activation of the immune system may epigenetically alter fetal microglia, the brains resident immune cells. We therefore tested the hypothesis that epigenomic alterations to microglia may be involved in behavioral abnormalities observed in MAA offspring. We used the genome‐wide approaches of whole genome bisulfite sequencing to examine DNA methylation and RNA sequencing to examine gene expression in microglia from juvenile MAA offspring. Differentially methylated regions were enriched for immune signaling pathways and important microglial developmental transcription factor binding motifs. Differential expression analysis identified genes involved in controlling microglial sensitivity to the environment and shaping neuronal connections in the developing brain. Differentially expressed genes significantly overlapped genes with altered expression in human ASD cortex, supporting a role for microglia in the pathogenesis of ASD.

Mutation of neuron-specific chromatin remodeling subunit BAF53b: rescue of plasticity and memory by manipulating actin remodeling

Recent human exome-sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several intellectual disabilities and cognitive disorders, including autism. However, it remains unclear how mutations in BAF complexes result in impaired cognitive function. Post-mitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Subdomain 2 of BAF53b is essential for the differentiation of neuronal precursor cells into neurons. We generated transgenic mice lacking subdomain 2 of Baf53b (BAF53bΔSB2). Long-term synaptic potentiation (LTP) and long-term memory, both of which are associated with phosphorylation of the actin severing protein cofilin, were assessed in these animals. A phosphorylation mimic of cofilin was stereotaxically delivered into the hippocampus of BAF53bΔSB2 mice in an effort to rescue LTP and memory. BAF53bΔSB2 mutant mice show impairments in phosphorylation of synaptic cofilin, LTP, and memory. Both the synaptic plasticity and memory deficits are rescued by overexpression of a phosphorylation mimetic of cofilin. Baseline physiology and behavior were not affected by the mutation or the experimental treatment. This study suggests a potential link between nBAF function, actin cytoskeletal remodeling at the dendritic spine, and memory formation. This work shows that a targeted manipulation of synaptic function can rescue adult plasticity and memory deficits caused by manipulations of nBAF, and thereby provides potential novel avenues for therapeutic development for multiple intellectual disability disorders.

UBE3A-mediated regulation of imprinted genes and epigenome-wide marks in human neurons

ABSTRACT The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including UBE3A, which is maternally expressed exclusively in neurons. UBE3A encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of UBE3A in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition.

Whole genome bisulfite sequencing of Down syndrome brain reveals regional DNA hypermethylation and novel disease insights

Down Syndrome (DS) is the most common genetic cause of intellectual disability, where an extra copy of human chromosome 21 (HSA21) produces differential genome-wide DNA methylation profiles. Although DNA methylation has been examined across the genome at select regulatory regions in a variety of DS tissues and cells, the differentially methylated regions (DMRs) have yet to be examined in an unbiased sequencing-based approach. Here, we present the first analysis of DMRs from whole-genome bisulfite sequencing (WGBS) data of human DS and matched control brain, specifically frontal cortex. While we did not observe global differences in DNA methylation, we identified 3,152 DMRs across the entire genome, which were primarily hypermethylated in DS. DS-DMRs were significantly enriched at CpG islands and de-enriched at specific gene body and regulatory regions. Functionally, the hypermethylated DS-DMRs were enriched for one-carbon metabolism, membrane transport, and glutamatergic synaptic signaling, while the hypomethylated DMRs were enriched for proline isomerization, glial immune response, and apoptosis. Furthermore, in a cross-tissue comparison to previous studies of DNA methylation from diverse DS tissues and reference epigenomes, hypermethylated DS DMRs showed a strong cross-tissue concordance, while a more tissue-specific pattern was observed for the hypomethylated DS DMRs. Overall, this approach highlights the utility of applying low-coverage WGBS to clinical samples and demonstrates novel gene pathways potentially relevant to DS treatments. These results also provide new insights into the genome-wide effects of genetic alterations on DNA methylation profiles indicative of altered neurodevelopment.

Epigenomic convergence of genetic and immune risk factors in autism brain

Autism spectrum disorders (ASD) are characterized by impairments in social communication and increased repetitive behaviors. ASD etiology is complex, involving multiple genetic and environmental risks. Epigenetic modifications are poised at the interface between genes and environment and are predicted to reveal insight into the gene networks, cell types, and developmental timing of ASD etiology. Here, whole-genome bisulfite sequencing (WGBS) was used to examine DNA methylation in ASD and control frontal cortex samples. Systems biology approaches were leveraged to integrate methylation differences with relevant genomic datasets, revealing ASD-specific differentially methylated regions (DMRs) are significantly enriched for known neuronal and microglial regulatory elements, including cell-type-specific enhancers and transcription factor binding sites. ASD DMRs were also significantly enriched for known ASD genetic risk factors, including both common inherited and rare de novo variants. Weighted gene co-expression network analysis (WGCNA) revealed enrichment of ASD DMRs within developmental expression modules of brain and isolated microglia. Microglial modules identified dysregulated genes in maternal immune activation models of ASD. Weighted gene body co-methylation network analysis revealed a module characterized by hypomethylation of clustered protocadherin genes. Together, these results demonstrate an epigenomic signature of ASD in frontal cortex shared with known genetic and immune etiological risk. Epigenomic insights into cell types and gene pathways will aid in defining therapeutic targets and early biomarkers at the interface of genetic and environmental ASD risk factors.Neurodevelopmental disorders (NDDs) impact 7% to 14% of all children in developed countries and are one of the leading causes of lifelong disability. Epigenetic modifications are poised at the interface between genes and environment and are predicted to reveal insight into the gene networks, cell types, and developmental timing of NDD etiology. Whole-genome bisulfite sequencing was used to examine DNA methylation in 49 human cortex samples from three different NDDs (autism spectrum disorder, Rett syndrome, and Dup15q syndrome) and matched controls. Integration of methylation differences across NDDs with relevant genomic and genetic datasets revealed differentially methylated regions (DMRs) unique to each type of NDD but with shared regulatory functions in neurons and microglia. DMRs were significantly enriched for known NDD genetic risk factors, including both common inherited and rare de novo variants. Weighted region co-methylation network analysis revealed a module related to NDD diagnosis and enriched for microglial regulatory regions. Together, these results demonstrate an epigenomic signature of NDDs in human cortex shared with known genetic and immune etiological risk. Epigenomic insights into cell types and gene regulatory regions will aid in defining therapeutic targets and early biomarkers at the interface of genetic and environmental NDD risk factors.

Epigenetic regulation of the circadian gene Per1 in the hippocampus mediates age-related changes in memory and synaptic plasticity

Aging is accompanied by impairments in both circadian rhythmicity and long-term memory. Although it is clear that memory performance is affected by circadian cycling, it is unknown whether age-related disruption of the circadian clock causes impaired hippocampal memory. Here, we show that the repressive histone deacetylase HDAC3 restricts long-term memory, synaptic plasticity, and learning-induced expression of the circadian gene Per1 in the aging hippocampus without affecting rhythmic circadian activity patterns. We also demonstrate that hippocampal Per1 is critical for long-term memory formation. Together, our data challenge the traditional idea that alterations in the core circadian clock drive circadian-related changes in memory formation and instead argue for a more autonomous role for circadian clock gene function in hippocampal cells to gate the likelihood of long-term memory formation.

Epigenetic regulation of the circadian gene Per1 contributes to age-related changes in hippocampal memory

Aging is accompanied by impairments in both circadian rhythmicity and long-term memory. Although it is clear that memory performance is affected by circadian cycling, it is unknown whether age-related disruption of the circadian clock causes impaired hippocampal memory. Here, we show that the repressive histone deacetylase HDAC3 restricts long-term memory, synaptic plasticity, and experience-induced expression of the circadian gene Per1 in the aging hippocampus without affecting rhythmic circadian activity patterns. We also demonstrate that hippocampal Per1 is critical for long-term memory formation. Together, our data challenge the traditional idea that alterations in the core circadian clock drive circadian-related changes in memory formation and instead argue for a more autonomous role for circadian clock gene function in hippocampal cells to gate the likelihood of long-term memory formation.Circadian rhythms are known to modulate memory, but it’s not known whether clock genes in the hippocampus are required for memory consolidation. Here, the authors show that epigenetic regulation of clock gene Period1 in the hippocampus regulates memory and contributes to age-related memory decline, independent of circadian rhythms.

MeCP2 isoform e1 mutant mice recapitulate motor and metabolic phenotypes of Rett syndrome

&NA; Mutations in the X‐linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2‐e1 and MeCP2‐e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform‐specific MeCP2‐e1‐deficient male mouse model of a human RTT mutation that lacks MeCP2‐e1 while preserving expression of MeCP2‐e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2‐e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2‐e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age‐dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2‐e1 mutants exhibited significantly increased body fat compared to sex‐matched wild‐type littermates prior to weight differences. Mecp2e1‐/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild‐type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2‐e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2‐e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.