CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests a role in male anxiety and ethanol intake

McEwan, Andrew R.; Davidson, Courtney; Hay, Elizabeth; Turnbull, Yvonne; Erickson, Johanna Celene; Marini, Pietro; Wilson, David; McIntosh, Andrew M.; Adams, Mark; Murgatroyd, Chris; Barrett, Perry; Delibegović, Mirela; Clarke, Toni‐Kim; MacKenzie, Alasdair

doi:10.1038/s41380-020-0707-7

Cited by 11 publications

(31 citation statements)

References 48 publications

(68 reference statements)

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“…We have previously shown that the GAL5.1 enhancer controls anxiety and alcohol intake [ 24 ]. This provided us with a unique opportunity to determine the effects of environmental factors such as dietary changes and early life stress on 5mC/5hmC levels within an enhancer region shown to control behaviours with a direct effect on human health.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to examining the effects of environmental factors on levels of 5mC/5hmC within GAL5.1 we also explored the effects of DNA methylation on the activity of GAL5.1 as well as its response to known stimuli such as PKC stimulation and expression of EGR1 [ 23 , 24 ]. To address this question, we first cloned the human GAL5.1 (GG) enhancer into the pCpGfree-promoter-Lucia luciferase vector that lacks CpG dinucleotides and contains the EF-1α promoter.…”

Section: Resultsmentioning

confidence: 99%

“…Mice containing a disrupted GAL5.1 enhancer [ 24 ] were maintained as a colony on a heterozygous C57BL/6 background which was mated to produce homozygous wildtype and homozygous GAL5.1 KO age matched and sex matched individuals. These were identified using PCR of earclip DNA [ 24 ]. Once identified, animals were assigned random numbers to hide their genotype from the operators of subsequent tests.…”

Section: Methodsmentioning

confidence: 99%

“…In order to address this knowledge gap we previously used comparative genomics to identify a highly conserved enhancer sequence (GAL5.1) that lay 42 kilobases (kb) 5′ of the GAL gene transcriptional start site in humans. We then demonstrated its activity in galanin expressing cells of the hypothalamus including the periventricular nucleus (PVN), dorsomedial hypothalamus (DMH) and arcuate nucleus (ARC) [ 23 , 24 ]. The analysis of two polymorphisms in the human GAL5.1 enhancer using the UK Biobank cohort suggested a mechanistic link between allelic variants of these polymorphisms and alcohol abuse when stratified against sex (male) and anxiety [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

“…We then demonstrated its activity in galanin expressing cells of the hypothalamus including the periventricular nucleus (PVN), dorsomedial hypothalamus (DMH) and arcuate nucleus (ARC) [ 23 , 24 ]. The analysis of two polymorphisms in the human GAL5.1 enhancer using the UK Biobank cohort suggested a mechanistic link between allelic variants of these polymorphisms and alcohol abuse when stratified against sex (male) and anxiety [ 24 ]. Subsequent use of CRISPR genome editing, to disrupt the mouse GAL5.1 enhancer (mGAL5.1KO), demonstrated a role for mGAL5.1 in the tissue specific expression of the Gal gene in hypothalamus and amygdala as well as the modulation of alcohol intake and male anxiety-like behaviour [ 24 ].…”

Section: Introductionmentioning

confidence: 99%

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The anxiety and ethanol intake controlling GAL5.1 enhancer is epigenetically modulated by, and controls preference for, high-fat diet

McEwan

Erickson

Davidson

et al. 2020

Cell. Mol. Life Sci.

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Excess maternal fat intake and obesity increase offspring susceptibility to conditions such as chronic anxiety and substance abuse. We hypothesised that environmentally modulated DNA methylation changes (5mC/5hmC) in regulatory regions of the genome that modulate mood and consumptive behaviours could contribute to susceptibility to these conditions. We explored the effects of environmental factors on 5mC/5hmC levels within the GAL5.1 enhancer that controls anxiety-related behaviours and alcohol intake. We first observed that 5mC/5hmC levels within the GAL5.1 enhancer differed significantly in different parts of the brain. Moreover, we noted that early life stress had no significant effect of 5mC/5hmC levels within GAL5.1. In contrast, we identified that allowing access of pregnant mothers to high-fat diet (> 60% calories from fat) had a significant effect on 5mC/5hmC levels within GAL5.1 in hypothalamus and amygdala of resulting male offspring. Cell transfection-based studies using GAL5.1 reporter plasmids showed that 5mC has a significant repressive effect on GAL5.1 activity and its response to known stimuli, such as EGR1 transcription factor expression and PKC agonism. Intriguingly, CRISPR-driven disruption of GAL5.1 from the mouse genome, although having negligible effects on metabolism or general appetite, significantly decreased intake of high-fat diet suggesting that GAL5.1, in addition to being epigenetically modulated by high-fat diet, also actively contributes to the consumption of high-fat diet suggesting its involvement in an environmentally influenced regulatory loop. Furthermore, considering that GAL5.1 also controls alcohol preference and anxiety these studies may provide a first glimpse into an epigenetically controlled mechanism that links maternal high-fat diet with transgenerational susceptibility to alcohol abuse and anxiety.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%