This investigation was approved by the Medical Ethics Committee of the Capital Institute of Pediatrics (No. SHERLLM2014002). Written informed consent was obtained from all participants in this study. The 100 NTDs and 100 normal control sample brain tissues were obtained from patients in the Lüliang area of Shanxi Province in northern China [19, 20]. Medically aborted fetuses which had been diagnosed with encephalocele by B-mode ultrasound in the early stages of pregnancy were subjected; Any fetuses displaying pathological malformations or intrauterine growth retardation were excluded from the control group; the summary of samples information were recorded in Table S1. In this study, 3 samples with the highest plasma glucose levels (the fasting blood glucose value＞7 mmol/L) from 100 NTDs were selected, and 3 samples with normal glucose levels (＜7 mmol/L) were selected from 100 controls. The summary of these six samples information were shown in Table S2.
As described in our previous paper , diabetes female mice were induced in 7–9 week-old virgin FVB mice by twice intraperitoneal injections of 100 mg/kg body weight streptozotocin (STZ; S0130, Sigma), which were dissolved in 100 mM sodium citrate buffer (C1013, Solarbio) at pH 4.5 and performed at an interval of 1 week. The fasting blood glucose value＞14 mmol/L after STZ treatment were confirmed as having diabetes. Female mice were treated with the same volume of vehicle (100mM sodium citrate buffer) and the blood glucose value ＜14 mmol/L, were included in the control group. The dams were then mated with 8–11-week-old male FVB mice. In addition, we established a rescue model, in 7-9 week-old, hydroxycitric acid (HCA) (90045-23-1, Sanherb, China), which can competitively inhibits ACL [21-23], was fed to STZ induced female mice 2g/kg/d for 4 weeks. The day that a vaginal plug was detected was considered day 0.5 of gestation, the dams were euthanized by CO2 for subsequent experiments. The study was approved by the Committee of Medical Ethics of the Capital Institute of Pediatrics (Beijing, China) (SYXK 2008–0011).
Grouping: On gestational day 8.5, embryos from maternal diabetic mice were grouped as DM; maternal mice fed with HCA and injected with STZ simultaneously, their embryos grouped as DM+HCA; embryos from control female mice were grouped in control group; Gestational day 14.5, at this stage, embryos with NTDs malformation can be clearly identified. Hence, we classified the NTDs fetus bred from diabetic mice to DM-NTDs group; the fetus bred from diabetic mice but without NTDs phenotype as DM group; embryos from maternal normal mice as control group. Details of the dissection information were shown in Table S3.
Glucose level detection
The human maternal plasma glucose content were detected by the laboratory department of our hospital. For mice, we collected the tail blood to measure by the blood glucose test strip, all tests were measured twice and the mean value was calculated.
Cell culture and glucose treatment
As described in our previous paper , NE4C cells (ATCC number: SCRC-CRL-2925™)  purchased from the Chinese Academy of Science, were cultured on plates pre-coated with 15 mg/ml poly L-lysine 2 h before passage in Eagle's MEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (10099, Gibco), 1% GlutaMAX and 1% nonessential amino acids at 37 °C in 5% CO2. To stimulate high glucose exposure, cells were pre-incubated in serum-free medium for 24 h, and then 5mM (normal control) or 25mM (high) glucose (G7020, Sigma) were added in each group and both with FBS reintroduced, cells were further cultured 24 h for following experiments.
Acl overexpression and knockdown in NE4C cells
Acl wildtype plasmid obtained from Origene (LC420122) for Acl overexpression. Acl knockdown in NE4C cells were achieved by shRNA virus infection. Interfering sequence were designed by Sangon Biotech with pSGU6/GFP/Neo plasmid vector. A negative shRNA was used as a control. Cells were transfected with overexpression/shRNAs plasmids using the Lipo3000 reagent (Thermofisher) according to the manufacturer's instructions. In addition, we used an inhibitor of Acl: SB-204990 (154566-12-8, Sigma) at 0, 1, 5, 10 μmol/L for 24h. The overexpression and knockdown effects were verified by either western blotting or by immunofluorescence staining.
Core histone proteins were extracted from NE4C cells/mice brain tissue using an acid extraction protocol . The samples were first homogenized in lysis buffer (10 ml solution containing 10 mM Tris-Cl (pH 8.0), 1 mM KCl, 1.5 mM MgCl2 and 1 mM dithiothreitol (DTT)) and chilled on ice. Protease and phosphatase inhibitors were added immediately before cell lysis, and nuclei were isolated by centrifugation (1,500 g for 10 min). For the preparation of histones, nuclei were incubated with four volumes of 0.2 mol/L sulfuric acid overnight at 4 °C. The supernatant was precipitated with 33% trichloroacetic acid followed by centrifugation (12,000 g for 20 min). The obtained pellet was washed with cold acetone and subsequently dissolved in distilled water. The samples were stored at −80 °C before analysis.
Brain tissues or cells were deproteinized by adding 200 μL of ice-cold 1 M perchloric acid (PCA) and homogenized thoroughly on ice using a Tissue-Tearor (Model 985370-395, Biospec Products Inc.). Then, the tissue was centrifuged at 10,000 g for 10 min to remove insoluble material. Acetyl-CoA level was measured with a kit (MAK039, Sigma). A 3M potassium bicarbonate solution was used to neutralize the supernatant until pH reached 6–8. Then, 10 μL sample and 40 μL buffer were added in the well, followed by a quenching and quench removal step. The fluorescence intensity was measured at λex = 535/λem = 587 nm by a machine (Thermoskan Flash, Thermo Scientific, USA).
In-solution tryptic digestion
As described in our previous paper , 50μg of total protein mixture was extracted from mice brain tissue and digested as follows. Disulfide bonds were reduced with 10mM (final concentration) DTT for 60 min at 37 °C. Then, alkylation was carried out by adding 40mM (final concentration) iodoacetamide for 60 min at room temperature in the dark. The alkylation reaction was quenched by treatment with 40mM DTT for 15 min. After diluting urea to less than 1 M with 25mM NH4HCO3, sequence-grade trypsin was added at a ratio of 1:50 (enzyme: total protein), and proteins were then digested at 37 °C for 24 h. The tryptic digestion was quenched by adding 1.0% trifluoroacetic acid, and the solution was then centrifuged at 13,000 g for 10 min to remove insoluble material. The supernatant was collected for subsequent experiments.
Identification and quantification of protein by Nano‐HPLC/MS/MS
Nano‐HPLC/MS/MS analyses were performed on a QE-HF mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with an UltiMate 3000 RSLCnano System (Dionex, Germering, Germany). Full-scan MS spectra in the m/z range of 350–2000 were acquired using an Orbitrap. Twenty of the most intense ions were isolated for MS/MS analysis. The raw data were processed using Proteome Discoverer (version 220.127.116.11, Thermo Scientific) by searching a database of human histones (www.uniprot.org, accessed October 2015) . Peptides were generated from a semi-tryptic digestion with up to four missed cleavages, using carbamidomethylation of cysteines as a fixed modification and oxidation of methionines as a variable modification. Target histone lysine acetylation was searched at 42.0 Da . The precursor mass tolerance was 10 ppm, and the product ions were searched at a tolerance of 0.025 Da. Peptide spectral matches were validated using a percolator based on q-values at a 1% false discovery rate (FDR). Modified peptides that passed the FDR were exported to a text file and processed by PRM. The area of the peaks was used to represent the number of modifications (as shown in our previous paper) .
Parallel reaction monitoring (PRM)
Raw data were searched against the corresponding histone database. The mass inclusion list included the mass, charge, polarity and time from the start and end. The full scan method was used as described above. The PRM method employed an Orbitrap resolution of 30,000 (at m/z 350) and a target automatic gain control value of 2×105. The precursor ions of each peptide were duplexed using ±0.8 m/z unit windows. Each sample was analyzed in triplicate (as shown in our previous paper) .
PRM data analysis
Protein quantification was manually processed within the Xcalibur Qual Browser (version 18.104.22.168; Thermo Fisher Scientific) using Skyline (version 22.214.171.12419; AB Sciex). In the Xcalibur Qual Browser, determination of the area under the curve of selected fragment ions was based on the presence of product ion signals within ±2.5 min of the expected retention time, with a mass error within ±5 ppm. In Skyline, .raw files were used as input to generate and extract the modified peptide normalized area at a 0.05 m/z ion match tolerance for each PRM spectrum (as shown in our previous paper) .
Western blotting (WB)
Total lysate total proteins or histone samples were resolved by 4-12% SDS-PAGE and subjected to western blot assays with the primary antibody: ACL (4332, Cell Signaling Technology (CST), 1:1000); Gadd45g(5174, CST, 1:1000); Sirt2 (12672, CST, 1:1000); Ddit3 (2895, CST, 1:1000); Gapdh (4970, CST, 1:1000) ; H3K4ac (ab176799, Abcam, 1:1000); H3K9ac (ab32129, Abcam, 1:1000); H3K14ac (ab52946, Abcam, 1:1000); H3K27ac (ab4729, Abcam, 1:1000); H3 (ab1791, Abcam, 1:1000). Followed by incubation with an anti-rabbit horseradish peroxidase-conjugated antibody (SC-2048, Zhongshan Jinqiao, 1:5000) and detection with a West Pico ECL kit (Thermo Scientific). The band intensities were determined using Image Lab software and expressed relative to Gapdh (for total proteins) / H3 (for histones).
A SimpleChIP® Enzymatic Chromatin IP Kit (9003, CST) was used for the ChIP assays, in accordance with the manufacturer’s protocol. 4% Formaldehyde cross-linked chromatin was obtained from about 8×107 NE4C cells. Cross-linked chromatin was immunoprecipitated with antibodies to H3K27ac overnight at 4°C. Normal rabbit IgG were used as negative control. Immunoprecipitated DNA was analyzed by sequencing. Indepth whole-genome DNA sequencing was performed by BGI (www.genomics.org.cn, BGI, Shenzhen, China). The raw sequencing image data were examined by the Illumina analysis pipeline, aligned to the Mus musculus reference genome (UCSC, mm9) using Bowtie 2, and further analyzed by MACS (Model-based Analysis for ChIP-Seq; https://github.com/taoliu/MACS). Enriched binding peaks were generated after filtering through control input.
ChIP‑qPCR analysis was performed using an ABI 7500 system (Applied Biosystems). The primers (Thermo Fisher Scientific, Inc.) used for ChIP‑qPCRs are listed in Table S4. The relative enrichment of the histone modification was determined using the 2(input‑Ct ) NTDs /2(input‑Ct)Control method.
RNA extraction and RNA-seq analysis
Total RNA was extracted from cultured cells or frozen tissue samples at −80°C using TRIzol® (15596‑026, Invitrogen). Library construction and sequencing were performed on a BGISEQ-500 by CapitalBio Technology (Beijing, China). Clean-tags were mapped to the reference genome and genes available at the Mice Genome. For gene expression analysis, the matched reads were calculated and then normalized to fragments per kilobase million (FPKM). The significance of the differential expression of genes was defined by the bioinformatics service of BGI according to the combination of the absolute value of log2 Ratio ≥ 1 and FDR ≤ 0.001.
RNA was reverse transcribed using a First Strand cDNA Synthesis kit (K1612; Beijing TransGen Biotech). The cDNA samples were analyzed using an Applied Biosystems 7500 Real‑Time PCR system (Thermo Fisher Scientific, Inc.) and a 2X PCR UltraSYBR Mixture kit (CW0956; CWBIO) according to the manufacturer's protocol. The expression levels of the target genes were normalized to glyceraldehyde 3‑phosphate dehydrogenase (Gapdh). The fold change in expression was determined using the 2‑ΔΔCt method. The primer sequences designed using Primer 5 and synthetic by Sangon Biotech, Inc. are listed in Table S5. The PCR thermocycling steps were as follows: 95˚C for 10 min; then 40 cycles of 95˚C for 15 sec, 60˚C for 1 min; and 72˚C for 5 min.
Immunofluorescence staining (IF)
Cells were fixed in 4% paraformaldehyde for 15 min, and permeabilized in 0.1% Triton/PBS for 10 min in a 4 ℃ icebox. After incubation with 5% BSA for 1 hr at room temperature, primary antibody Acl (4332, CST, 1:150) was added. Secondary antibody goat anti-rabbit IgG H&L (Alexa Fluor 594) (ab150080, Abcam, 1:500) was used. Nuclei were labeled with DAPI (P36935, Life technologies). Leica TCS SP8 confocal microscope was used to image the cells, and Image J software was used for analysis (Version 1.44)
IHC studies were performed on 3μm sections obtained from brain tissues pre-fixation with 4% paraformaldehyde. Bioscience™ (00-4956-58, Invitrogen) were used for antigen retrieval. Then sections were blocked with 5% bovine serum albumin in PBS and incubated with the primary antibodies anti-Acl, anti-Gadd45g, anti-Ddit3 overnight at 4 ◦C. After washing with 0.1 M PBST, pH 7.4, 5 min, 3 times, antibodies were detected using the Polink-2 Plus polymer horseradish peroxidase detection system for goat primary antibodies (ZSGB-BIO) according to the manufacturer's instructions. Quality assessment was performed on each batch of slides by including a negative control in which the primary antibody was replaced by 10% normal goat serum to preclude nonspecific signals. The levels of Acl, Gadd45g and Ddit3 were analyzed by gray-scale analysis (ZEN 2012 ZEISS COMPANY).
All experiments were repeated at least three times. GraphPad Prism 8.0.1 (GraphPad Software, USA) was mainly used in the data analysis. Student's t-tests and one-way ANOVA analyses were used to determine the statistical differences among the groups. Data are presented as the mean ± SD. A p-value <0.05 was regarded as statistically significant.