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. 2023 Mar 3:16:1082104.
doi: 10.3389/fnmol.2023.1082104. eCollection 2023.

Adolescent binge ethanol impacts H3K36me3 regulation of synaptic genes

Emily R Brocato  1 , Jennifer T Wolstenholme  1   2
Affiliations

Adolescent binge ethanol impacts H3K36me3 regulation of synaptic genes

Emily R Brocato et al. Front Mol Neurosci. .

Abstract

Adolescence is marked in part by the ongoing development of the prefrontal cortex (PFC). Binge ethanol use during this critical stage in neurodevelopment induces significant structural changes to the PFC, as well as cognitive and behavioral deficits that can last into adulthood. Previous studies showed that adolescent binge ethanol causes lasting deficits in working memory, decreases in the expression of chromatin remodeling genes responsible for the methylation of histone 3 lysine 36 (H3K36), and global decreases in H3K36 in the PFC. H3K36me3 is present within the coding region of actively-transcribed genes, and safeguards against aberrant, cryptic transcription by RNA Polymerase II. We hypothesize that altered methylation of H3K36 could play a role in adolescent binge ethanol-induced memory deficits. To investigate this at the molecular level, ethanol (4 g/kg, i.g.) or water was administered intermittently to adolescent mice. RNA-and ChIP-sequencing were then performed within the same tissue to determine gene expression changes and identify genes and loci where H3K36me3 was disrupted by ethanol. We further assessed ethanol-induced changes at the transcription level with differential exon-use and cryptic transcription analysis - a hallmark of decreased H3K36me3. Here, we found ethanol-induced changes to the gene expression and H3K36me3-regulation of synaptic-related genes in all our analyses. Notably, H3K36me3 was differentially trimethylated between ethanol and control conditions at synaptic-related genes, and Snap25 and Cplx1 showed evidence of cryptic transcription in males and females treated with ethanol during adolescence. Our results provide preliminary evidence that ethanol-induced changes to H3K36me3 during adolescent neurodevelopment may be linked to synaptic dysregulation at the transcriptional level, which may explain the reported ethanol-induced changes to PFC synaptic function.

Keywords: ChIP-seq; H3K36me3; PFC; RNA-seq; adolescent ethanol; alcohol; cryptic transcription; epigenetics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Methods. Adolescent DBA/2J mice were dosed with water or ethanol (4 g/kg) from PND 29–42. PFC was collected 24 h after the last dose of ethanol. Three PFCs from each treatment group were homogenized together, divided, and subjected to ChIP-seq and RNA-seq analysis.
Figure 2
Figure 2
GO analysis of DEGs. (A) Number of genes differentially expressed due to adolescent binge ethanol in males and females at p < 0.01. Fisher’s exact test determined male and female gene overlap to be significant, p = 5.7e-34. (B) GO analysis of DEGs unique to males. (C) GO analysis of DEGs unique to females. (D) GO analysis of DEGs that are shared between males and females.
Figure 3
Figure 3
GO analysis of differential exon use (DEU) genes. (A) Number of genes showing differential exon use due to adolescent binge ethanol in males and females at p < 0.001. Fisher’s exact test determined male and female gene overlap to be significant, p = 1.7e-11. (B) GO analysis of genes with differentially used exons unique to males. (C) GO analysis of genes with differentially used exons unique to females. (D) GO analysis of genes with differentially used exons that are shared between males and females.
Figure 4
Figure 4
GO analysis of cryptically transcribed (CT) genes. (A) Number of genes cryptically transcribed due to adolescent binge ethanol in males and females at p < 0.05. Fisher’s exact test determined male and female gene overlap to be significant, p = 8.2e-09. (B) GO analysis of cryptically transcribed genes with unique to males. (C) GO analysis of cryptically transcribed genes unique to females. Snap25, Cplx1, Ccdc124, and Per1 were cryptically transcribed in both males and females and were excluded from the male and female GO analyses to identify categories unique to each sex.
Figure 5
Figure 5
GO analysis of genes showing differentially bound H3K36me3 regions (DBR). (A) Number of genes showing differential binding of H3K36me3 due to adolescent binge ethanol in males and females at p < 0.05. Fisher’s exact test determined male and female gene overlap to be significant, p = 2.9e-131. (B) GO analysis of genes showing differential binding of H3K36me3 unique to males. (C) GO analysis of genes showing differential binding of H3K36me3 unique to females. (D) GO analysis of genes showing differential binding of H3K36me3 that are shared between males and females.
Figure 6
Figure 6
Adolescent binge ethanol alters the regulation and expression of genes relating to synaptic function. Our differential gene expression, differential exon usage, cryptic transcription and differential H3K36me3 bound loci analyses identified a number of gene ontology categories related to synaptic structure and function. These changes are likely reflected in presynaptic and postsynaptic cells as well as within the extracellular matrix and may be why persistent cognitive deficits occur after adolescent binge ethanol exposure. Categories shown were significantly over-represented in at least one analysis in both males and females, p < 0.05. Extracellular matrix organization – GO:0030198, Synaptic vesicle cycle – GO:0099504, Synapse assembly – GO:0007416, Negative regulation of synaptic transmission – GO:0050805, Synaptic transmission, glutamatergic – GO:0035249, Postsynapse organization – GO:0099173, Calmodulin binding – GO:0005516, Action potential – GO:0001508.
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