Prenatal ethanol exposure alters adult hippocampal VGLUT2 expression with concomitant changes in promoter DNA methylation, H3K4 trimethylation and miR-467b-5p levels

Animals and prenatal ethanol exposure

Animal work was conducted in accordance with the Australian code for the care and
use of animals for scientific purposes, and was approved by an Animal Ethics Committee
at The University of Queensland (MMRI/120/12/NHMRC). Prenatal ethanol exposure in
inbred C57BL/6J mice was performed as described previously 26]. The model is based on voluntary maternal consumption of 10 % (v/v) ethanol from
fertilisation to 8.5 dpc. Briefly, adult (8-week-old) C57BL/6J males and C57BL/6J
females (6–7 weeks) were purchased from the Animal Resources Centre (Perth, Australia)
and habituated to a 12-h light/12-h dark cycle for a week before mating. Males were
paired with a single, nulliparous female overnight and females were checked each morning
for the presence of a vaginal plug (defined as 0.5 dpc). At 0.5 dpc, the male and
female were separated and females were randomly assigned to either the ethanol-exposed
group [10 % (v/v) ethanol] or control group (water). Pregnant females were allowed
free access to both food and liquid, and the volume of liquid consumed was measured
to the nearest 0.2 ml every 24 h. Following 8 days of exposure, ethanol-exposed females
were given water for the remainder of the experiment. Female body weight was measured
at 0.5 and 8.5 dpc.

Microarray-based analysis of gene expression

Hippocampi were dissected from male ethanol-exposed and control offspring (n = 6 per group) at P87, snap-frozen in liquid nitrogen and stored at ?80 °C. Total
RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Netherlands). For each sample,
500 ng of total RNA was used for complementary RNA (cRNA) synthesis using the TotalPrep
RNA Amplification kit (Illumina, USA) and 1.5 µg of cRNA was then hybridised to MouseWG-6
v2.0 Expression BeadChips (Illumina, USA) at 58 °C for 16 h according to the manufacturer’s
instructions. Data were quantile normalised using Genome Studio v1.9.0 (Illumina,
USA) and differential expression was determined using GeneSpring GX11 (Agilent, USA)
on default settings. Genes with uncorrected P values less than 0.05 and expression fold changes greater than 1.5 were selected
for validation experiments.

Availability of supporting data

The microarray data sets supporting the results of this article are available in the
Gene Expression Omnibus (GEO) repository under accession number GSE60000 (http://www.ncbi.nlm.nih.gov/geo/).

qPCR validation of differential gene expression

RNA (250 ng) was reverse transcribed in a 20 µl reaction using 100 ng of random hexamer
primers (Life Technologies, USA), 500 µM dNTP mix, 1× first strand buffer, 5 mM DTT,
40 units of RNaseOUT (Life Technologies, USA) and 200 units of SuperScript III Reverse
Transcriptase (Life Technologies, USA). The cDNA was then diluted 1 in 10 and 4 µl
was used in the qPCR with 1× SYBR green MasterMix (Life Technologies, USA), 0.3 µM
of forward/reverse primers (Additional file 1: Table S3) and 0.2 µl of ROX dye (Life Technologies, USA). Cycling parameters were
50 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing
and extension at 60 °C for 1 min. Each reaction was performed in triplicate. Gapdh and Hprt1 were used as housekeepers, and similar results were obtained with both genes. Analysis
was performed using the 2
^???CT
method.

Clonal bisulphite sequencing

A total of 1 µg of genomic DNA was converted with sodium bisulphite using the EpiTect
Bisulfite kit (Qiagen, Netherlands) according to the manufacturer’s instructions.
A region (BS1) extending from ?144 to ?20 bp relative to the transcriptional start
site identified by Li and colleagues in the mouse brain 31] was then amplified using a semi-nested PCR strategy. The first round PCR was conducted
in a 50 µl reaction that contained 3 µl of converted genomic DNA, 1× BIOTAQ NH
₄
buffer, 1.5 mM MgCl
₂
, 200 µM dNTPs, 0.3 µM of each primer (BS_F and BS_R1, Additional file 1: Table S3) and 1 U BIOTAQ DNA Polymerase (Bioline, UK). Cycling parameters involved
denaturation at 94 °C for 5 min followed by 29 cycles of denaturation at 94 °C for
1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min. A final extension
step was carried out at 72 °C for 3 min. First round PCR product (1 µl) was used as
template for a second round PCR using primers BS_F and BS_R2 (Additional file 1: Table S3) and the same reaction and cycling conditions as the first round. Two independent
semi-nested PCRs were performed per sample to reduce the impact of PCR bias on the
results 46]. PCR products were gel-purified, subcloned into the pGEM-T vector (Promega, USA)
and transformed into E. coli DH5? competent cells according to standard protocols. Two to four clones were sequenced
per PCR and were analysed using BiQ Analyzer with default settings 47].

In vitro reporter assays of promoter function

The Slc17a6 promoter (?144 to +68 bp) was amplified from mouse hippocampal genomic DNA using
the primers PM_F and PM_R (Additional file 1: Table S3) and ligated to the pGL4.14 [luc2/Hygro] vector (Promega, USA) using SfiI restriction enzyme sites. A CpG methyltransferase (M.SssI), HpaII methyltransferase or HhaI methyltransferase (New England Biolabs, USA) were used either alone or in combination
to methylate the promoter-containing construct and vector only according to the manufacturer’s
instructions. pGL4.14-based plasmids (45 ng) and pGL4.74[hRluc/TK] (5 ng) were transiently transfected into CAD cells using Lipofectamine 2000 (Life
Technologies, USA) according to the manufacturer’s protocol. CAD cells were grown
in DMEM:HAMS F12 (1:1) with 8 % foetal bovine serum and 2 mM glutamine. pGL4.74[hRluc/TK] contains Renilla luciferase and was used to control for transfection efficiency. Transfections were
performed in quadruplicate. Luciferase assays were carried out 36 h after transfection
using the Dual-Glo luciferase assay system (Promega, USA) according to the manufacturer’s
instructions.

ChIP-qPCR

ChIP-qPCR was performed using the ChIP-IT High-Sensitivity Kit and ChIP-IT qPCR Analysis
Kit (Active Motif, USA) according to the manufacturer’s instructions. Chromatin was
prepared from the pooled hippocampi of twelve males per group. Slc17a6 ChIP-qPCR primers, ChIP_F and ChIP_R, are listed in Table S3 (Additional file 1). The H3K4me3 and H3K27me3 antibodies, Mouse Negative Control Primer Set 1, Positive
Control Primer Set Gapdh–2 and Positive Control Primer Set Pax-2 were all purchased from Active Motif. Reactions were performed in triplicate. Data
were analysed using an Excel-based ChIP-IT qPCR Analysis Spreadsheet according to
the manufacturer’s instructions 48].

Western blotting

Individual hippocampi were homogenised in a urea protein lysis buffer, centrifuged
at 13,000 rpm for 5 min at 4 °C and the supernatants were and stored at ?80 °C. Total
protein concentration was determined using the BCA Protein Assay (Thermo Fisher Scientific,
USA) following the manufacturer’s instructions. 10 µg of total protein was electrophoresed
on a NuPage 4–12 % Bis–Tris gel (Life Technologies, USA) and transferred onto PDVF
membrane using the Hoefer miniVE (Hoefer, USA). The membrane was incubated in Odyssey
blocking buffer (LI-COR Biosciences, USA) at room temperature for 90 min, followed
by incubation with a primary guinea pig polyclonal anti-VGLUT2 antibody (135404; Synaptic
Systems, Germany) diluted with 5 ml Odyssey blocking buffer at a ratio of 1:1000 and
0.1 % Tween 20 (Sigma-Aldrich, USA) at 4 °C overnight with gentle rolling. The membrane
was then washed four times with PBS containing 0.1 % Tween 20 (5 min each at room
temperature). A fluorescent-labelled secondary antibody, IRDye 680RD Donkey anti-guinea
pig (LI-COR Biosciences, USA) diluted with Odyssey blocking buffer at a ratio of 1:10,000,
supplemented with 0.01 % SDS, was then incubated with the membrane in the dark at
room temperature for 45 min. The membrane was washed again and scanned using the Odyssey
system (LI-COR Biosciences, USA). After scanning, the membrane was rinsed with blocking
buffer and incubated with a primary mouse monoclonal anti-GAPDH antibody (MAB374;
Millipore, USA) at room temperature for an hour. A different fluorescent-labelled
secondary antibody, IRDye 800RD Donkey anti-mouse (LI-COR Biosciences, USA), was incubated
with the membrane at room temperature for 45 min followed by four washes as mentioned
above. The membrane was re-scanned and VGLUT2 levels were calculated relative to GAPDH
levels.

MicroRNA profiling

Small RNA was isolated from the hippocampus using the miRNeasy mini kit (Qiagen, Netherlands).
RNA (200 ng) was reverse transcribed using the miScript II RT kit (Qiagen, Netherlands)
and genome-wide miRNA profiling was conducted using the miScript miRNA PCR Array (MIMM-3216ZE-12;
Qiagen, Netherlands) according to the manufacturer’s instructions. A total of 944
different mature miRNAs as well as six housekeeping genes were assayed. Data were
normalised to the average Ct of the two most stably expressed housekeepers: RNU6–2 and SNORD95, and analysed by the 2
^???CT
method using an Excel-based miScript PCR Array Data Analysis Template 49].

TaqMan qPCR validation of differential miRNA expression

cDNA was prepared using reverse transcription primers provided with TaqMan MicroRNA
Assays (Life Technologies, USA) and the TaqMan MicroRNA Reverse Transcription kit
(Life Technologies, USA) according to the manufacturer’s instructions. The cDNA was
then diluted 10 times with RNase-free water and 4.5 ?l was mixed with 5 ?l TaqMan
Universal Master Mix II, no UNG (Life Technologies, USA) and 1× TaqMan MicroRNA Assay
to obtain a 10 µl reaction. PCR was performed on a ViiA 7 (Applied Biosystems, USA)
using 10 min denaturation at 95 °C, followed by 40 repeats of denaturation at 95 °C
for 15 s and annealing and extension at 60 °C for 60 s. Reactions were performed in
triplicate. Data were normalised to RNU6–2 and relative expression was calculated by the 2
^???CT
method.

Luciferase reporter assay of miRNA–target mRNA interaction

Plasmid constructs containing the putative miR-467b-5p target site (position 374–380
of Slc17a6 3?UTR, NCBI:NM_080853) (Target) or a mutated target site (Scramble), containing seven
mismatches in the seed region, were engineered using the pmirGLO vector system (Promega, USA) according to the manufacturer’s instructions. pmirGLO contains both a firefly luciferase reporter gene (luc2) and a Renilla luciferase reporter gene (hRluc–neo fusion). Inserts with PmeI (5?) and XbaI (3?) sticky ends were generated by annealing 2 ?g of sense oligonucleotides and
2 ?g of antisense oligonucleotides (Additional file 1: Table S3) in 46 ?l of annealing buffer at 37 °C for 15 min after denaturation at
90 °C for 3 min. Annealed products, Target or Scramble, were ligated to linearized
pmirGLO that had been digested with PmeI and XbaI. The constructs or vector only (50 ng) were transfected into 2 × 10
⁴
CAD cells using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer’s
protocol. Co-transfection with a miR-467b-5p mimic or an inhibitor (3.3 pmol) was
performed using the same conditions. Transfected cells were cultured for 24 h and
then subjected to a luciferase assay using the Dual-Glo luciferase assay system (Promega,
USA) according to the manufacturer’s instructions. Assays were done in quadruplicate.
Firefly luciferase activity was normalised to Renilla luciferase activity.

Analysis of alcohol-sensitive miRNAs in serum

Serum was isolated from 500 ?l of whole blood by centrifugation at 8000 rcf for 15 min.
Serum samples (100 ?l) were mixed with 3.5 µl of a synthetic spike-in control, Caenorhabditis elegans microRNA 39 (Syn-cel-miR-39) (1.6 × 10
⁸
copies/µl), before isolation of total RNA using miRNeasy mini kit according to the
serum RNA extraction protocol (Qiagen, Netherlands). Total RNA (170 ng) was reverse
transcribed into cDNA using the TaqMan microRNA reverse transcription kit (Life Technologies,
USA) following manufacturer’s instructions. Pre-amplification was performed using
1× TaqMan PreAmp Master Mix (Life Technologies, USA), 0.0075× pre-amplification primer
pool and 2.5 ?l of RT products. Cycling parameters were 95 °C for 10 min, 55 °C for
2 min and 72 °C for 2 min, followed by 12 cycles of denaturation at 95 °C for 15 s
and annealing and extension at 60 °C for 4 min with a final deactivation at 99.9 °C
for 10 min. To amplify the miRNAs of interest, 2.5 µl of pre-amplification products
(diluted 40 times) was amplified using 1× TaqMan microRNA assay in the presence of
1× TaqMan Universal Master mix II (No AmpErase UNG) (Life Technologies, USA) following
manufacturer’s instructions. Reactions were performed in triplicate. Data were normalised
using Syn-cel-miR-39 and analysed using the 2
^???CT
method.

Statistics

All statistical analyses were conducted using R 50].