Hippocampal epigenetic changes in a mouse model of Fetal Alcohol Spectrum Disorders

Fetal alcohol spectrum disorders (FASD) refers to the neurological, developmental, and behavioural abnormalities arising from in utero ethanol exposure. These abnormities included attention deficit, anxiety, and learning and memory impairment persisting into adulthood. The molecular mechanisms of such persistent behavioural changes remain unknown and are an area of intense research. In this thesis, mice were exposed to ethanol during the third trimester equivalent, the peak of synaptic development. Following this exposure, genome-wide epigenetic and gene expression and changes in the hippocampus were assessed in adult (70 day old) mice. In the first experiment, genome-wide trimethylation of histone H3 at histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) were assessed using chromatin immunoprecipitation (ChIP) microarray (ChIP-chip). Cell-cell signalling genes were enriched for changes in both methylations. It included the protocadherin (Pcdh) genes, which confer neuronal identity and may be important for synaptic development. Changes in methylation also occurred at imprinted genes and lipid-metabolism genes The second experiment assessed DNA methylation using methylated DNA immunoprecipitation (MeDIP) microarray (MeDIP-chip). The screen identified genes involved in peroxisome biogenesis, which metabolize lipids and generate free-radicals. This was also true when the histone and DNA methylation changes were considered together. Combined analysis of affected genes from each experiment implicated free-radical scavenging genes. Identification of this novel interplay between epigenetic and oxidative stress genes may provide insight into diagnostic or therapeutic interventions. In general, the results support a role of epigenetic mechanisms in long-term FASD phenotypes. Finally, the third experiment examined gene expression and miRNA microarrays identified 59 and 60 differentially expressed genes and miRNAs between ethanol-exposed and control mice. These genes primarily affect free radical scavenging genes. Differential expression of five genes in this pathway was confirmed with droplet digital PCR (ddPCR), including the transcription factor Tcf7l2 and the apoptosis regulator Casp3. The affected genes also included other oxidative stress proteins, olfactory receptors, and biosynthetic enzymes that may contribute to FASD-related abnormalities

X-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by HDAC inhibition

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD.'' All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations

Coordinated Tcf7l2 regulation in a mouse model implicates Wnt signaling in Fetal Alcohol Spectrum Disorders

Mouse models of fetal alcohol spectrum disorders (FASD) have repeatedly identified genes with long-term changes in expression, DNA methylation, noncoding RNA and histone modifications in response to neurodevelopmental alcohol exposure. Articulation of FASD is achieved via alcoholâ s effect on gene expression, likely involving epigenetic regulation. The list of genes affected is large and heterogeneous, depending on experimental protocol. We present reanalysis and synthesis of results highlighting Wnt transcription factor 7 like 2 (Tcf7l2) gene as uniquely compatible with hippocampal DNA methylation, histone modifications, and gene expression changes in a coordinated response to neurodevelopmental alcohol exposure. We data-mined literature for Tcf7l2 alterations in response to prenatal alcohol exposure. Four studies identified changes in brain Tcf7l2 expression in different FASD models. Further, we performed an in silico TCF7L2 binding site analysis for FASD mouse model datasets. Seven of these published gene lists were significantly enriched for TCF7L2 binding, indicating potential functional relationships. Finally, TCF7L2 is involved in regulation of hundreds of genes, with a role in brain development, myelination, and neuronal function. Tcf7l2 may be involved in neurological defects associated with alcohol exposure via dysregulation of many genes through Wnt signaling. Further functional work is warranted to validate this model for FASD.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Alteration of gene expression, DNA methylation, and histone methylation in free radical scavenging networks in adult mouse hippocampus following fetal alcohol exposure

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse\u27s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as Free radical scavenging . We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylationaffected genes was Peroxisome biogenesis ; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD

Potential origins of observed epigenetic and gene expression hippocampal profile in response to neonatal ethanol exposure.

<p>It is well established that in the brain ethanol leads to increased ROS, leading to oxidative damage, which contributes to altered behaviour. The epigenetic and gene expression changes identified here (represented by the Circos plot from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154836#pone.0154836.g001" target="_blank">Fig 1</a>) may have arisen from: a) the direct action of ethanol during the exposure period, which may then act to perpetuate ethanol-induced oxidative damage; b) an early response to ethanol-induced oxidative cellular damage, acting to ameliorate or compensate for this damage; c) an later response to accumulating oxidative damage over the early life of the mouse, prior to 70 days of age.</p

Pathways significantly enriched with DMR- or RDHM-proximal genes.

<p>Pathways significantly enriched with DMR- or RDHM-proximal genes.</p

Schematic of peroxisome Biogenesis pathway from Partek pathway.

<p>Proteins are arranged into functional groups. Proteins whose genes bear DMRs or RDHMs in their promoter are colored: genes with a change predicted to increase gene expression are shown in red, those predicted to decrease are shown in green, conflicting marks are shown in yellow. Score determined in Partek was 5.4 (right-tailed Fisher’s exact test).</p

Droplet digital PCR (ddPCR) confirmation of differential gene expression.

<p>Data are normalized to a gene of interest relative quantity of 1.00 for the control group. n = 14, 7 ethanol-exposed and 7 control mice. Data are mean ± standard error. *<i>p</i><0.05 (Student’s t-test).</p

Top IPA network for gene expression changes “Free Radical Scavenging, Gene expression, Dermatological Diseases and Conditions”.

<p>Red nodes represent proteins whose transcripts were increased in ethanol-exposed mice vs. controls, green nodes represent those that were decreased in ethanol exposed mice. Score determined in IPA was 31 (right-tailed Fisher’s Exact Test).</p