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Abnormal epigenetic regulation of the gene expression levels of Wnt2b and Wnt7b: Implications for neural tube defects

The association between Wnt genes and neural tube defects (NTDs) is recognized, however, it remains to be fully elucidated. Our previous study demonstrated that epigenetic mechanisms are affected in human NTDs. Therefore, the present study aimed to evaluate whether Wnt2b and Wnt7b are susceptible to abnormal epigenetic modification in NTDs, using chromatin immunoprecipitation assays to evaluate histone enrichments and the MassARRAY platform to detect the methylation levels of target regions within Wnt genes. The results demonstrated that the transcriptional activities of Wnt2b and Wnt7b were abnormally upregulated in mouse fetuses with NTDs and, in the GC-rich promoters of these genes, histone 3 lysine 4 (H3K4) acetylation was enriched, whereas H3K27 trimethylation was reduced. Furthermore, several CpG sites in the altered histone modification of target regions were significantly hypomethylated. The present study also detected abnormal epigenetic modifications of these Wnt genes in human NTDs. In conclusion, the present study detected abnormal upregulation in the levels of Wnt2b and Wnt7b, and hypothesized that the alterations may be due to the ectopic opening of chromatin structure. These results improve understanding of the dysregulation of epigenetic modification of Wnt genes in NTDs.


Introduction
Abnormal alterations in Wnt genes are associated with neural tube defects (NTDs) (1,2). NTDs occur when the neural tube fails to close at the early stage of neural development, which can result in spina bifida, myelomeningocele (also known as spina bifida aperta), exencephaly or craniorachischisis (3). A previous study demonstrated that a null mutation of Wnt3a, a canonical Wnt, led to spina bifida aperta in mice (4). Wnt5a -/-, a non-canonical Wnt, mice did not suffer from NTDs; however, mice with double deletions of the Wnt5a -/and Ltap/Vangl2 genes exhibited craniorachischisis (5). Furthermore, knockout mutations of the canonical Wnt3 or non-canonical Wnt genes, Wnt5a and Wnt7a, induce deformities of the anteroposterior (A/P) axis of the neural tube (6). These findings suggest that, in addition to roles of several known Wnt genes in the etiology of NTDs, other Wnt members may be involved in the pathogenesis of NTDs.
Wnt2b is a canonical Wnt, which is expressed in the primitive streak at embryonic day (E) 7-7.75, followed by the dorsal midline of the telencephalon and mesencephalon at E8. 5-9.5, and in the cortical hem at E11. 5-17.5 in murine embryos. This specific spatio-temporal expression of Wnt2b suggests that it functions in gastrulation, neurulation and cerebral cortex patterning (7,8). In addition, Wnt7b, which is associated with canonical and non-canonical Wnt signaling, is expressed at E5.5, and is expressed in the forebrain, and the ventral and intermediate spinal cord at the developmental stages during which patterning and neural specification occur (9). Notably, Wnt7b can assist in ensuring correct A/P guidance, and is involved in dorsoventral patterning of the amphibian embryonic neural tube (10). Although Wnt2b and Wnt7b mutations have not induced NTDs in mice (6), they are essential during neural tube patterning; therefore, the present study hypothesized that the expression levels of these two genes are likely to be disrupted in NTDs.
Epigenetic modifications typically regulate gene expression; histone 3 lysine 4 acetylation (H3K4ac) and trimethylation of H3 lysine 4 (H3K4me3), are commonly located in active promoters and are associated with gene activation, whereas H3K27me3 suppresses transcription (11). DNA methylation is predominantly catalyzed by DNA methyltransferase through the addition of a methyl group to cytosine residues on dinucleotide CpG islands, and is frequently distributed in the core promoter close to the transcription start site (TSS); once the promoter CpG islands are methylated, these genes are often transcriptionally inactivated (12). Our previous studies demonstrated that epigenetic modifications were significantly altered in patients with NTDs (13,14); however, the detailed mechanism and target genes remain to be elucidated.
The present study aimed to investigate dysregulation of epigenetic modification of Wnt genes in NTDs, using a mouse model of spina bifida. It was hypothesized that in NTDs, dysregulation of epigenetic modification is involved in the regulation of Wnt2b and Wnt7b gene expression.

Materials and methods
Animals. All animals experiments were performed in accordance with the standards of the Capital Institute of Pediatrics Ethics Committee (Beijing, China; permit no. SYXK 2008-0011). A total of 40 adult C57BL/6J mice (20 male and 20 female) were housed in light-and temperature-controlled rooms (12/12 h 1ight/dark cycle; 23±1˚C) and maintained on pure mouse feed and tap water provided ad libitum. Adult virgin females (above 9 weeks of age and 21 g in weight) were mated for 2 h with males (1 male per 2 females, 8:00-10:00 am) of the same stock. A vaginal plug observed at 10:00 AM was considered as embryonic day 0 (E0). The mice were administered with a 20 mg/kg body weight dose of retinoic acid (RA; suspended in olive oil; Sigma-Aldrich, St. Louis, MO, USA) on E8.0-0 h, E8.0-6 h and E8.0-12 h. Mice treated with isovolumetric pure olive oil (Agric, Crete, Greece) at these same time points served as control groups, as described in our previous study (15). E18.0 fetuses were delivered by cesarean section, and 21 cases of spina bifida were selected ( Fig. 1A and B). As the spinal cord of individual mice provides insufficient material for RNA extraction, the mice were randomly divided into three groups and the samples in each group were pooled. A total of 10 normal spinal cords were randomly pooled to create three control groups, two of which contained 3 mice and one contained 4 mice.
Human subjects. Aborted NTD case subjects were obtained from Fenyang Hospital and Linxian County Hospital (Shanxi, China). The surgical details were, as previously described (14). The spinal cord tissues were used in the following experiments. A total of four NTD-affected fetuses and four age-matched controls were used in the present study. The clinical phenotypes of the four cases of NTD were as follows: One case of thoracic meningomyelocele (24 weeks gestation; female), one case of anencephalic and occipital encephalomeningocele (17 weeks gestation; female), and two cases of thoracic and lumbar spina bifida (20 and 26 weeks gestation, respectively; female). The present study was approved by the Committee of Medical Ethics at the Capital Institute of Pediatrics (SHERLLM2014002). Written informed consent was obtained from the parents on behalf of the fetuses.
RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated from the samples using TRIzol ® (cat. no. 15596-026; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and was reverse transcribed using a First Strand cDNA Synthesis kit (cat. no. K1612; Beijing TransGen Biotech Co., Ltd., Beijing, China). 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 (cat. no. CW0956; CWBIO, Beijing, China) containing carboxy-X-rhodamine, 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 -ΔΔCq method (16). The primer sequences designed using Primer 5 and provided by Thermo Fisher Scientific, Inc. are listed in Table I. 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.
DNA methylation analyses. Following ultrasonic sonication (P-141008; Diagenode SA, Seraing, Belgium) of mouse and human spinal cord tissues, genomic DNA was extracted from the homogenates using a Genomic DNA Miniprep kit (Axygen Scientific, Inc., Union City, CA, USA). A total of 500 ng genomic DNA from each sample was bisulfite-treated using an EZ DNA Methylation-Gold™ kit (cat. no. D5005; Zymo Research, Irvine, CA, USA). The Sequenom MassARRAY platform (CapitalBio Corporation, Beijing, China) was used to perform quantitative methylation analysis of Wnt2b and Wnt7b (13). The primers used to detect DNA methylation levels of the target regions are presented in Table III.
Statistical analysis. Statistical analyses of the transcripts, histone modification enrichment and DNA methylation levels of Wnt genes were performed at least three times. Data were

Altered transcription levels of Wnt2b and Wnt7b in a mouse model of RA-induced spina bifida.
To determine into the possible changes occurring in the expression levels of Wnt2b and Wnt7b during the development of RA-induced NTDs, the mRNA expression levels were determined. The results indicated that, in the mice with spina bifida, the mRNA expression levels of Wnt2b and Wnt7b were 3.1-and 2.8-fold higher, respectively, compared with the control mice (Fig. 1C). These results were consistent with those of a previous study, which detected the transcriptional upregulation of WNT2 (including WNT2B) and WNT7B in NT2/NTera2 cells following RA treatment (17). These results indicated that the transcription of Wnt2b and Wnt7b were abnormally activated in RA-induced spina bifida.
Abnormal histone modifications are present in the promoter regions of Wnt2b and Wnt7b. Due to the ectopic alterations in mRNA expression levels, epigenetic factors in the GC-rich promoter regions of Wnt2b and Wnt7b were examined, according to the current UCSC database (July 2007 NCBI37/mm9; http:// genome.ucsc.edu/). As shown in Fig. 2A and B, H3K4me3 and H3K27me3 modifications target Wnt2b and Wnt7b in embryonic stem cells and the brain in the central nervous system, whereas H3K9me3 modifications are weak. Furthermore, in the cerebellum and mouse embryonic fibroblasts, these histone modification enrichments are markedly lower, indicating that the histone modifications, which occur in Wnt2b and Wnt7b are stable and spatio-temporal specific. Therefore, the present study aimed to determine whether H3K27me3 or H3K4me bind  to the target regions of samples by performing ChIP assays. As H3K4ac is also a transcriptional activation marker (18), the enrichment of this modification was also assessed. In the target regions of Wnt2b and Wnt7b in the RA-treated NTD mice, enrichments of H3K4ac were upregulated, whereas H3K27me3 enrichments were significantly downregulated in the chr3: 104765115-104765239 segment of Wnt2b (Fig. 3A). The enrichment of H3K4me was undetectable in the target regions of the two genes (data not shown). The abnormally attenuated H3K27me3 and enhanced H3K4ac enrichment of Wnt2b and Wnt7b indicated their roles in the dysregulated increased transcription observed in the mice with NTDs.  (Fig. 3B and C). For Wnt7b, the chr15: 85411571-72 CpG site (407 bp upstream of the TSS) was also hypomethylated (Fig. 3D). These results suggested that, in NTDs, the increased mRNA expression levels of Wnt2b

DNA methylation levels of Wnt2b and
and Wnt7b may also be partially due to hypomethylation of specific CpG-units in the promoter regions. The combination of the ChIP assay and DNA methylation assay data suggested that, in the mouse model of RA-induced spina bifida, increased H3K4ac and decreased H3K27me3 enrichment, and hypomethylation of CpG islands in the promoter regions potentially led to enhanced mRNA expression (Fig. 4).
Aberrant epigenetic modification of WNT2B and WNT7B in human NTDs. As abnormal epigenetic modifications of Wnt2b and Wnt7b were detected in the mice with NTDs, the present study subsequently aimed to validate whether similar changes were present in the human NTD samples. In H1 embryonic stem cells, at the early stage prior to neural development, the GC-rich promoters of the two WNT genes were enriched for H3K27me3 ( Fig. 5A and B), and were bound to a series of enzymes for its catalysis, including enhancer of zeste homolog 2 (EZH2) and suppressor of zeste homolog 12 (SUZ12). However, low levels of HDAC2-6 were detected, which is known to regulate the deacetylation of H3. Furthermore, astrocytes (NH-A), which are a type of mature neuronal cell associated with the later stage of neural development, exhibited reduced H3K27me3 and EZH2 enrichment (19), indicating that H3K27me3 and H3K4ac may preferentially function in the early stage of neural development.
In the human spinal cord samples, the GC-rich promoter regions of WNT2B and WNT7B were enriched for H3K4ac and H3K27me3. However, in the NTD samples, H3K4ac enrichment was downregulated, compared with the controls,

A B
and there were no differences in H3K27me3 enrichment between the groups (Fig. 6A). Furthermore, several CpG-units of WNT2B and WNT7B were significantly hypomethylated, compared with the control group, particularly those close to the TSS (Figs. 6B-D and 7). These results indicated that aberrant epigenetic modification of WNT2B and WNT7B occurred in the human NTDs.

Discussion
Previous studies have demonstrated that H3K27me3 binding assists in maintaining gene silencing in multi-cellular organisms, and is associated with the timed suppression of developmental regulatory genes (20,21). By contrast, H3K4ac is considered to be involved in the activation of gene transcription (18), whereas methylated DNA makes it difficult for histones to form nucleosomes, resulting in chromosomal instability and modified accessibility of regulatory proteins, which alter the pattern of gene expression (12,22). The present study hypothesized that the aberrant DNA methylation and histone modification profiles observed in NTDs may affect the binding ability of RNA polymerase and key transcription factors to promoter regions on genes, including EZH2, SUZ12, HDAC2, HDAC6, which in turn regulate histone modification and affect transcription initiation (19). In addition, H3K4ac and H3K27me3 modifications of the target genes in the present study were observed to function preferentially at the early stage of neural development, however not in the later stages. Furthermore, taking the data from our previous study into account, which reported DNA hypomethylation of long interspersed nuclear element-1 and low expression levels of H3K79me2 in human NTDs (13,14), the present study reiterates the hypothesis that abnormal epigenetic modifications have a destructive role in the etiology of NTDs. It remains uncertain how alterations in Wnt2b and Wnt7b affect normal neural development. As ligands, Wnt2b and Wnt7b can function through the canonical β-catenin pathway, which maintains the proliferative state of neural progenitor cells (23,24). In addition, Wnt7b can signal through the non-canonical pathway (planar cell polarity), affect convergent extension movements prior to the closure of the neural tube, and control A/P axis formation and neural patterning (25). Therefore, the upregulated expression levels of Wnt2b and Wnt7b may lead to excessive signaling and result in disordered neural development, suggesting that aberrant Wnt gene expression may be involved in the etiology of NTDs. However, the Wnt5a genetic knockout mice demonstrated that individual deficiencies in Wnt genes do not induce an NTD phenotype (5). This suggests that, C when multiple Wnt ligands are altered at one time, they may potentially contribute to the etiology of disease. However, further studies are required to confirm this hypothesis.
In conclusion, the present study provided novel insight into the synergistic function of transcriptional dysregulation and epigenetic modifications. Specifically, abnormal epigenetic A B D C Figure 6. Alterations of epigenetic modification in human NTDs. (A) In WNT2B and WNT7B, H3K4ac enrichment was reduced in NTDs, whereas no differences in H3K27me3 modification were observed between the NTD and control groups. DNA methylation levels of the specific CpG sites within (B and C) WNT2B and (D) WNT7B target regions in human patients with NTDs, compared with in the controls. * P<0.05; data are presented as the mean ± standard error. NTD, neural tube defect; H3K, histone 3 lysine; me3, trimethylation; ac, acetylation; TSS, transcription start site.

A B
modifications in the Wnt genes were detected following exposure to RA and may significantly contribute to the development and etiology of NTDs.