Abstract
Background
Neural tube defects (NTDs) are severe, common birth defects that result
from failure of normal neural tube closure during early embryogenesis.
Accumulating strong evidence indicates that genetic factors contribute
to NTDs etiology, among them, HOX genes play a key role in neural tube
closure. Although abnormal HOX gene expression can lead to NTDs, the
underlying pathological mechanisms have not fully been understood.
Method
We detected that H3K27me3 and expression of the Hox genes in a retinoic
acid (RA) induced mouse NTDs model on E8.5, E9.5 and E10.5 using
RNA-sequencing and chromatin immunoprecipitation sequencing assays.
Furthermore, we quantified 10 Hox genes using NanoString nCounter in
brain tissue of fetuses with 39 NTDs patients including anencephaly,
spina bifida, hydrocephaly and encephalocele.
Results
Here, our results showed differential expression in 26 genes with a
> 20-fold change in the level of expression, including 10 upregulated
Hox genes. RT-qPCR revealed that these 10 Hox genes were all
upregulated in RA-induced mouse NTDs as well as RA-treated embryonic
stem cells (ESCs). Using ChIP-seq assays, we demonstrate that a
decrease in H3K27me3 level upregulates the expression of Hox cluster
A–D in RA-induced mouse NTDs model on E10.5. Interestingly, RA
treatment led to attenuation of H3K27me3 due to cooperate between UTX
and Suz12, affecting Hox gene regulation. Further analysis, in human
anencephaly cases, upregulation of 10 HOX genes was observed, along
with aberrant levels of H3K27me3. Notably, HOXB4, HOXC4 and HOXD1
expression was negatively correlated with H3K27me3 levels.
Conclusion
Our results indicate that abnormal HOX gene expression induced by
aberrant H3K27me3 levels may be a risk factor for NTDs and highlight
the need for further analysis of genome-wide epigenetic modification in
NTDs.
Keywords: Neural tube defects, Retinoic acid, Hox genes, H3K27me3, UTX,
SUZ12
Background
The neural tube is considered as the precursor of the future central
nervous system (CNS) which comprises the brain and spinal cord [[45]1].
Neural tube formation requires cell migration especially in the neural
folds of the brain region, precise coordination between numerous
cellular and molecular processes, and extensive interactions between
the neural ectoderm, mesenchyme, and surface ectoderm across time and
space. Multiple genes that regulate multiple cellular and molecular
events need to work in concert, both temporally as well as spatially,
for proper NT closure [[46]2]. Failure to properly close the neural
tube can result in neural tube defects (NTDs), the severity of which
depends on the level of the body axis affected [[47]3]. The etiologies
of NTDs are complex and multifactorial; both genetic and environmental
factors appear to be involved [[48]4]. For example, imbalance of
nutrient intake (e.g., folate or vitamin A) is an important risk factor
during per-pregnancy. Vitamin A (all-trans retinol) and its active
metabolites, collectively called retinoids, exert potent effects on
stem cell differentiation and thus, the formation of the entire
organism, in part via the modulation of the epigenome. However, how
environmental factors affect the process of neural tube closure and
their interaction with genetic factors remain largely unknown.
Recently, hundreds of genes have been shown to be regulated by RA
during the processes of neuronal development. The Hox family includes
four clusters, Hoxa, b, c and d, which encode transcriptional
regulators that are highly conserved in vertebrates [[49]5]. During
embryogenesis, Hox genes exhibit temporal and spatial linearity of
expression [[50]6], and are essential for the anterior–posterior
(head–tail) axis and neural tube development [[51]7]. Although there is
no direct evidence supporting the link between variants in Hox genes
and NTDs in genetic studies [[52]8]; however, it has been shown that
aberrant expression of Hox genes can lead to NTDs [[53]9, [54]10].
Recently, deregulation of gene expression through epigenetic mechanisms
has been hypothesized to be a potential attribute for NTDs [[55]11,
[56]12] and results from recent studies indicate that HOX gene
hypomethylation is a risk factor for NTDs [[57]13].
Within chromatin, histone methylation regulates gene expression by
either recruiting chromatin modifiers or directly altering chromatin
structure, the biological consequence of which differs depending on the
specific site where modifications occur [[58]14]. Tri-methyl groups on
lysine 27 of histone H3 (H3K27me3) induces gene silencing in cells, and
such histone modification can pass through cell generations [[59]14].
Genome-wide mapping has revealed H3K27me3 occupancy in a large set of
genes related to cell fate and embryonic development, including
developmental transcription factors (such as Hox genes) and
cell-surface or extracellular proteins involved in cell fate regulation
and patterning (such as Wnt [[60]15]). These findings suggest that
H3K27me3-regulated Hox gene expression might be associated with NTDs.
H2K27me3 is critical mediator for transcription gene expression and
contributes to important biological processes including animal body
patterning. Our previous study indicated that folate deficiency
attenuated H3K79me2, affecting some NTDs-associated genes, and
interrupting early embryo development [[61]16]. Enrichment of H3K4me1
at the hotspots of DSB regions enhances the recruitment of upstream
binding factor to rRNA genes, resulting in the increase in
transcription of rRNA genes [[62]17]. It might provide the evidence
that the risk of NTDs may be mediated through effecting histone
methylation.
In this study, in an attempt to correlate the temporal and spatial
expression pattern of HOX genes to NTDs, we employed a retinoic acid
(RA)-induced mouse NTDs model. RNA-sequencing analysis produced
detailed information on RA-induced transcriptome changes in the mouse
embryo. Surprisingly, 26 genes were differentially expressed with
levels changing over 20-fold, including 10 upregulated Hox genes. Using
ChIP-seq assays, we demonstrate that a decrease in H3K27me3 level
upregulates the expression of Hox cluster A–D in RA-induced mouse NTDs
model on E10.5. Interestingly, RA treatment led to attenuation of
H3K27me3 due to cooperate between UTX and Suz12, affecting Hox gene
regulation. Further analysis, in human anencephaly cases, upregulation
of 10 HOX genes was observed, along with aberrant levels of H3K27me3.
Notably, HOXB4, HOXC4 and HOXD1 expression was negatively correlated
with H3K27me3 levels. Taken together, our results provide the evidence
that aberrant H3K27me3 levels is the link between abnormal HOX gene
expression and NTDs, which further our understanding of the aberrant
epigenetic modification of Hox genes in NTDs.
Results
A rapid RA-induced mouse NTDs model
RA, a derivative of vitamin A, is involved in neurulation and
subsequent neural tube patterning, and plays an important role in
mammalian development [[63]18]. The association between vitamin A and
birth defects comes from studies in which high doses were used. As a
well-known teratogen, administration of RA to embryos induces NTDs,
including spina bifida, exencephaly and anencephaly in several species.
Given that a dose of RA of 28 mg/kg of body weight has been previously
shown to cause significant anencephaly, we employed a modified rapid
RA-induced NTDs mouse model via gavage of excess RA at E7.5 [[64]19].
Treatment with RA was overwhelmingly teratogenic and led to 94% of WT
embryos showing defects with about two-thirds of the embryos showing
anencephaly. As shown in Fig. [65]1A, morphological changes to the
normal mouse embryo was prominent from E8.5 to E10.5 (Fig. [66]1A: a,
c, e). After RA treatment, the morphology of mouse embryos differed
significantly compared with that of control embryos. The defects were
typical of anencephaly previously described following treatment with RA
at this stage (Fig. [67]1A: b, d, f). On E8.5, RA-treated mouse embryos
were similar qualitatively and quantitatively in the control embryos.
On E9.5 and E10.5, RA-treated mouse embryos showed an anencephaly
phenotype, maybe accompanied by growth retardation, enlarged heart and
ventricular chambers, short tail, and unfinished turning of the neural
axis [[68]20] (Fig. [69]1A and Additional file [70]1: Figure S1A).
Fig. 1.
[71]Fig. 1
[72]Open in a new tab
RA-induced mouse NTDs accompanied by dynamic transcriptome changes. A
RA-induced anencephaly in C57BL/6 mouse embryos. a: Con-E8.5, b:
RA-E8.5, c: Con-E9.5, d: RA-E9.5, e: Con-E10.5, f: RA-E10.5. Arrow
indicated the abnormal section. B Differentially expressed genes (DEGs)
identified in Con-E8.5-vs-RA-E8.5, Con-E9.5-vs-RA-E9.5 and
Con-E10.5-vs-RA-E10.5 comparisons. C Intersection analysis of DEGs by
Venny analysis. The overlaps represent the genes co-expressed in
Con-E8.5-vs-RA-E8.5, Con-E9.5-vs-RA-E9.5 and Con-E10.5-vs-RA-E10.5
comparisons. D Hierarchical clustering plot showing representative
expression patterns of 196 DEGs during mouse neural tube development.
These genes were mainly classified into two categories. The top GO
terms and corresponding enrichment P values are shown on the right
side. E RT-qPCR validation of 26 DEGs identified by RNA-seq. Actb was
used as control. Data are shown as the mean (SD; n = 3). P < 0.05
indicates statistical significance. Twenty five genes have a
statistically significant change in expression but not the Ermn gene
In both cell lines and embryos, RA target genes (e.g., Hox genes) are
differentially induced by RA in a time- and RA concentration-dependent
manner, where the genes at the 3′ end of the complexes are activated
earlier and display the highest sensitivity to RA exposure [[73]21]. It
is hoped that understanding the molecular mechanisms of the RA response
in cell culture will provide important insights into spatial
colinearity in the embryo.
Dynamic transcriptome changes and increased Hox gene expression induced by RA
during neural tube development
To define alterations in gene expression profile accompanying
RA-induced morphology changes, RNA-Seq was performed on mouse cranial
tissue samples from E8.5, E9.5 and E10.5 embryos. Unsupervised
clustering analysis revealed significant changes in transcriptome
profile the time period between E8.5 and E10.5, during which
morphological characteristics of NTDs became evident and accompany many
genes changed (Additional file [74]1: Figure S1B). By comparing
libraries from control and RA-induced samples at each of the three
sample collecting time points, we identified a significant number of
DEGs including 296 upregulated and 291 down-regulated genes on E8.5;
1782 upregulated and 474 down-regulated genes on E9.5; and 1464
upregulated and 873 down-regulated genes on E10.5, respectively
(Fig. [75]1B). The majority of DEGs detected at each specific time
point (E8.5, E9.5, E10.5) presented a respective distinctive profiling,
however, certain degree of overlap of DEGs in samples from different
times was observed (Fig. [76]1C). Further analysis with Gene Ontology
(GO) indicated that these DEGs were enriched for GO terms of pattern
specification, regionalization, cell differentiation and nervous system
development and enriched in some pathways of
glycolysis/gluconeogenesis, pathways in cancer, Hedgehog signaling
pathway, etc. (Fig. [77]1D, Additional file [78]1: Figure S1C, D,
Additional file [79]2: Table S1, Additional file [80]3: Table S2).
Interestingly, variations in GO terms and DEG enrichment in a given
pathway were observed between samples from different time points,
suggesting the complex nature of NTDs at different development stage.
In addition, PPI network suggested the complex regulating mechanisms in
NTDs occurrence (Additional file [81]1: Figure S1E).
Overall, there were 196 DEGs with a greater than twofold change from
E8.5 to E10.5. Of the 196 genes, 26 showed a greater than 20-fold
change in expression which included 10 Hox genes that were strongly
upregulated on E9.5 and E10.5. Results from RT-qPCR analysis of these
26 DEGs in cranial neural tissue of E10.5 embryos were consistent with
the RNA-seq data (Fig. [82]1E). Taken together, our data suggest that
gene expression pattern changes in RA-induced mouse NTDs and Hox genes
may play key roles in this process.
Elevation of Hox gene expression caused by reduced levels of H3K27me3 in
RA-induced mouse NTDs
During vertebrate embryogenesis, Hox genes exhibit temporal and spatial
collinearity of expression, with the most centromeric Hox genes
activated first and in the more anterior body structures, and the more
telomeric Hox genes activated later and in the more posterior body
structures [[83]6]. To investigate the induction kinetics of Hox genes
within each cluster in RA-induced mouse NTDs embryos with time, the
expression of 37 Hox genes was compared between normal and RA-induced
mouse NTDs embryos from E8.5 to E10.5. RNA-seq profiling revealed that
the order of genes in the Hox clusters tended to temporally regulate,
with a downward trending in expression in normal embryos (Fig. [84]2a).
However, upon RA treatment, the Hox clusters displayed a significant
upregulation at each developmental stage in mouse NTDs embryos from
E8.5 to E10.5 (Fig. [85]2a). Since it was found that the expression of
10 Hox genes, including Hoxa4, Hoxa5, Hoxb4, Hoxb5, Hoxc4, Hoxc5,
Hoxd1, Hoxd3, Hoxd4, and Hoxd8, was increased more than 20-fold in the
RA-induced mouse NTDs embryos at E10.5 (Fig. [86]1E), we then evaluated
the change in level of expression of these 10 Hox genes on E8.5, E9.5,
and E10.5, respectively. In control embryos, the highest level of
expression was seen on E8.5 for all 10 Hoxs, with a downward trend
(Fig. [87]2b). Upon RA-induction, upregulation of expression was
evident in all 10 Hox genes, with the most prominent elevation in
expression appeared on E9.5 or E10.5 (Fig. [88]2b). RT-qPCR assays were
performed on cranial neural tissue of E10.5 mouse embryos, and the
results indicated that levels of 10 Hox mRNA increased significantly,
ranging between 29- and 347-fold, in NTDs embryos compared to that in
controls (Fig. [89]2c). This further confirms that RA treatment
upregulated expression of 10 selected Hox genes in mouse embryos,
suggesting that Hox genes might play key roles in mouse early
development.
Fig. 2.
[90]Fig. 2
[91]Open in a new tab
RA caused upregulation of Hox genes. a RNA-seq analysis of the dynamic
expression of the four Hox clusters in cranial neural tissue of
RA-induced mouse NTDs embryos from E8.5 to E10.5. b Dynamic expression
of the 10 Hox genes in cranial neural tissue of RA-induced mouse NTDs
embryos from E8.5 to E10.5. c Hox gene mRNA in cranial neural tissue of
RA-induced mouse NTDs embryos was measured by RT-qPCR. Actb was used as
control. Data are shown as the mean (SD; n = 3). *P < 0.05
Decrease of H3K27me3 accumulation at Hox loci in RA-induced mouse NTDs
Previously data from genomic analysis indicate that the presence of
H3K27me3 at transcriptional start sites is correlated with repression
of Hox gene expression [[92]7, [93]22]. We found that level of H3K27me3
and H3K27me2 was both decreased in RA-induced cranial neural tissue
from E10.5 embryos, whereas no reduction was observed for H3K27me1
(Fig. [94]3a and Additional file [95]4: Figure S2A). Next, we also
investigated whether the expression of H3K27me3 was abnormally altered
in cranial neural tissue by immunohistochemical (IHC) analysis. To this
end, we examined H3K27me3 expression levels in 3 pairs of RA-induced
cranial neural tissue samples and their matched normal tissues by
immunohistochemical analysis. The staining of total H3K27me3 decreased
in mouse NTDs compared with that in their normal tissues (Fig. [96]3b).
Fig. 3.
[97]Fig. 3
[98]Open in a new tab
RA caused reduced levels of H3K27me3 at Hox loci in mouse NTDs embryos.
a Cranial neural tissue of normal and RA-induced mouse NTDs was
harvested at E10.5, and analyzed by western blotting. Numbers at the
bottom were generated by quantification (ImageJ) of the H3K27me3/2/1
signal normalized to the H3 signal. b Immunohistochemistry staining was
performed on transverse sections, for detecting H3K27me3 in NC and NTDs
mouse embryos of E10.5. Scale bar: 50 μm. Areas of H3K27me3 positive
cells were quantified by using ImageJ. Data are shown as the mean (SD;
n = 5). *P < 0.05. c Comparison of average ChIP-Seq reads densities for
H3K27me3 between NC and NTDs mouse embryos. d ChIP-Seq enrichment
profiles for H3K27me3 levels in NC and NTDs mouse embryos. e ChIP-Seq
density profiles for H3K27me3 at the mouse Hoxa–d clusters in NC and
NTDs mouse embryos. f ChIP-Seq density profiles for H3K27me3 at the 10
Hox genes in NC and NTDs mouse embryos
To further explore the importance of histone H3K27me3 in neural tube
defects, ChIP-seq was carried out in RA-induced mouse NTD model on
E10.5. By analyzing equal numbers of reads from H3K27me3, a total of
52,759 and 13,059 peaks were detected in normal and NTDs mouse embryos,
respectively, using an anti-H3K27me3 antibody, with the genome rate of
0.34% and 0.07% by scanning through the entire mouse genome (Additional
file [99]4: Figure S2B). ChIP-seq of H3K27me3 target genes showed that
the enrichment level of H3K27me3 was significantly decreased in
RA-treated mouse on E10.5 compared with controls (Fig. [100]3c). By
analyzing equal numbers of reads from H3K27me3, it also identified a
remarkable reduction of H3K27me3 near transcription start sites in
RA-treated mouse on E10.5 (Fig. [101]3d). Many peaks of H3K27me3
binding Hoxa–d genes clusters are decreased in RA-treated mouse on
E10.5 (Fig. [102]3e). Consistent with this, we analyzed the specific
region and accumulation of H3K27me3 peaks in 10 Hox genes
(Fig. [103]3f). The result showed that accumulation of H3K27me3 in 10
Hox genes was reduced after RA treatment, which consistent with Hox
clusters activation events. ChIP GO analysis showed that among genes
targeted by H3K27me3 there was a bias toward genes related to the
developmental process, especially nervous system development
(Additional file [104]4: Figure S2C and Additional file [105]5: Table
S3).
Association of elevated Hox expression with depressed H3K27me3 in RA-induced
mouse ESC
Next, we detected that decrease in H3K27me3 and H3K27me2 level in
RA-treated mouse ESCs, but no decrease in H3K27me1 level (Fig. [106]4a
and Additional file [107]6: Figure S3A). Interestingly, it is likely
that RA efficiently reduced the levels of H3K27me3 > H3K27me2.
Immunofluorescence staining for H3K27me3 indicated that the loss of
H3K27me3 in RA-treated cells. H3K27me3-enriched foci appeared to be
localized to dense heterochromatic chromocenters in control cells,
while the H3K27me3 immunostaining distribution was largely diffuse or
speckled, with decreased signal intensity in RA-treated cells
(Fig. [108]4b). In addition, RA treatment also led to an increase in
the levels of all 10 selected Hox gene mRNAs in mouse ESCs (P < 0.05)
(Fig. [109]4c). Besides, chromatin immunoprecipitation (ChIP) assays
were performed on mouse ESCs to evaluate the enrichment of H3K27me3 to
the selected 10 Hox genes. As shown in Fig. [110]4d, enrichment of
H3K27me3 in these 7 Hox genes (Hoxa4, Hoxa5, Hoxb4, Hoxb5, Hoxc4, Hoxd1
and Hoxd8) sequences was significant attenuated after RA treatment. The
most significant attenuation was observed in enrichment of H3K27me3 in
Hoxb5 for which a 72% decrease in the union was observed
(Fig. [111]4d). By contrast, no change in enrichment of H3K27me3 in IgG
loci sequence was observed (Fig. [112]4d). To further verify the
effects of H3K27me2 enrichment on the Hox genes upon RA treatment, we
also performed quantitative ChIP assays. The results showed that RA has
no significant effects H3K27me2 enrichment on the sequences of most of
Hox genes (Additional file [113]6: Figure S3B) in mouse ESCs. It is
possible that H3K27me2 recruited to specific RA-inducible Hox genes.
Collectively, our data indicate that during RA treatment, the
enrichment of H3K27me3 on Hox genes (Hoxa4, Hoxa5, Hoxb4, Hoxb5, Hoxc4,
Hoxd1 and Hoxd8) were decreased, their expression level increased, and
the overall level of histone H3K27me3 was decreased.
Fig. 4.
[114]Fig. 4
[115]Open in a new tab
RA caused reduced levels of H3K27me3 at Hox genes in mouse ESCs. a
Mouse ESCs were harvested after RA (1 μM) treatment for 24 h, and
analyzed by western blotting. Numbers at the bottom were generated by
quantification (ImageJ) of the H3K27me3/2/1 signal normalized to the H3
signal. b Immunostaining for H3K27me3 in RA-treated Hep-G2 cell. Direct
immunofluorescence analysis was performed. Images were captured by
confocal microscope and the nuclei were stained with DAPI. Scale bar:
35 μm. Data are shown as the mean (SD; n = 5). *P < 0.05. c Hox gene
mRNA from mouse ESCs treated with RA was assessed by RT-qPCR. RA 1 μM,
24 h. Actb was used as control. Data are shown as mean (SD; n = 3).
*P < 0.05. d ChIP assays of H3K27me3 were performed using mouse ESCs
treated with 1 μM RA for 24 h. Mouse IgG was used as control.
Enrichment of Hox gene promoters was measured by qPCR
UTX activity is important for RA-induced Hox upregulation
Previously, it has been shown that UTX demethylates H3K27me3 at the Hox
loci, controls posterior development of zebrafish [[116]23]. We were
interested to explore the possibility that the decrease level of
H3K27me3 in RA-induced mouse NTDs embryos and RA-treated mouse ESCs was
due to the action of UTX, a key factor for embryonic development. No
significant difference in level of expression was observed between
samples from normal and NTDs embryos (Fig. [117]5a). Equivalent results
were obtained in RA-induced mouse ESCs (Fig. [118]5b). Interestingly,
the demethylase activity of UTX, which is capable of removing the
methyl groups from H3K27me3, increased in RA-induced NTDs mouse cranial
tissue and RA-treated ESCs (Fig. [119]5c, d).
Fig. 5.
[120]Fig. 5
[121]Open in a new tab
UTX activity plays the important role in RA-induced Hox upregulation. a
UTX mRNA in cranial neural tissue of RA-induced mouse NTDs was measured
by RT-qPCR. Actb was used as a loading control. Data are shown as the
mean (SD; n = 4). *P < 0.05. b UTX mRNA in mouse ESCs treated with RA
was measured by RT-qPCR. Actb was used as a loading control. Data are
shown as the mean (SD; n = 3). *P < 0.05. c UTX demethylase activity
was detected in cranial neural tissue of RA-induced NTD mouse embryos.
Data are shown as the mean (SD; n = 4). *P < 0.05. d UTX demethylase
activity was detected in mouse ESCs after RA treatment. Data are shown
as the mean (SD; n = 3). *P < 0.05. e ChIP assays of UTX were performed
using mouse ESCs treated with 1 μM RA for 24 h. Mouse IgG was used as
control. Enrichment of Hox gene promoters was measured by qPCR. f
GSK-J4 (UTX inhibitor) affected mRNA levels of Hox genes in RA-induced
mouse ESCs. Mouse ESCs were treated with GSK-J4 (30 nM) for 6 h. Then,
after 24 h of RA treatment, cells were collected and analyzed. Data are
shown as the mean (SD; n = 3)
Next, we examined the binding of UTX to the promoters of the selected
10 Hox genes using ChIP-qPCR assay. As shown in Fig. [122]5e, while no
changes were seen for the binding to UTX to promoter regions of IgG,
significantly increased UTX binding to the promoter regions of the
Hoxb5 and Hoxd4 genes were evident upon RA treatment in mouse ESCs.
Thus, RA treatment led to increased UTX activity, demethylation of
H3K27me3, and subsequently an attenuation of H3K27me3 enrichment to the
promoters of Hox genes.
To further confirm that an increase in UTX activity upon RA treatment
was responsible for the increase in the expression of the selected Hox
genes, we utilized a potent UTX inhibitor, GSK-J4, in our experiment.
GSK-J4 treatment resulted in Hoxa4, Hoxa5, Hoxb4, Hoxc4, Hoxc5, Hoxd4
genes were down-regulated significantly. However, Hoxb5, Hoxd1, Hoxd3,
Hoxd8 were slightly change but no significantly in UTX
inhibitor-treated mouse ESCs (line 1 vs line 3). We next investigated
whether UTX demethylase is involved in the RA-induced reduction of
H3K27me3 levels at Hox genes in mouse ESCs. The expression of the Hox
genes was increased in RA-treated mouse ESCs (line 1 vs line 2), but
was much less RA-induced in UTX inhibitor-treated mouse ESCs (line 2 vs
line 4) (Fig. [123]5f). Taken together, these data provide evidence
indicating that RA treatment of mouse embryos and ESCs causes a
decrease H3K27me3 methylation due to an increased demethylase activity
of UTX. Recruitment of UTX to Hox promoters coincides with
disappearance of H3K27me3, and Hox gene activation.
Suz12 is required for RA-induced Hox upregulation
Suz12 is physically associated with EZH2 and enhances H3K27me3 at its
target genes. It is essential for polycomb repressive complex 2 (PRC2)
activity and is required for embryonic stem cell differentiation, and
which inactivation results in early lethality and NTDs occurence of
mouse embryos [[124]24]. In our study, the level of Suz12 expression
was decreased significantly in NTDs embryos (Fig. [125]6a). RNA-seq
analysis showed that the Suz12 gene expression was lower in NTDs
embryos on E9.5 and E10.5 (Additional file [126]7: Figure S4A).
Equivalent results were obtained in RA-induced mouse ESCs
(Fig. [127]6b). In addition, Ezh2 expression was also decreased in
RA-induced mouse NTDs embryos and mouse ESCs (Additional file [128]7:
Figure S4B–D). We next investigated H3K27me3 by downregulating Suz12
under RA treatment. Level of H3K27me3 was decreased upon Suz12
depletion (line 1 vs line 3). Furthermore, depletion of Suz12
significantly decreased H3K27me3 after RA treatment (Fig. [129]6c).
And, overexpression of Suz12 rescued the RA-reduced in the expression
of the H3K27me3 (Additional file [130]7: Figure S4C). We next
investigated whether Suz12 is involved in the RA-induced Hox genes in
F9 cells. Hox genes were upregulated in Suz12-depleted F9 cells (line 1
vs line 3). We next investigated whether Suz12 is involved in the
reduction of H3K27me3 levels Hox genes in RA-treated F9 cells. The
inhibition of Suz12 expression by siRNA led to a strong increase in the
levels of mRNAs encoded by Hox genes both before and after RA treatment
(Fig. [131]6d). ChIP assay showed H3K27me3 enrichment in these 10 Hox
genes was significant decreased after knockdown of Suz12 and
responsible for the increase in the expression of the Hox genes. By
contrast, no enrichment of H3K27me3 in IgG loci was observed
(Fig. [132]6e). These data indicated that RA treatment of mouse embryos
and ESCs could also cause a decrease H3K27me3 methylation through
depressing Suz12, and led to Hox genes activation. Taken together,
these findings provide evidence indicating that Suz12 effect on level
of H3K27me3 at Hox genes.
Fig. 6.
[133]Fig. 6
[134]Open in a new tab
Suz12 is required for RA-induced Hox upregulation. a Suz12 mRNA in
cranial neural tissue of RA-induced mouse NTDs was measured by RT-qPCR.
Actb was used as a loading control. Data are shown as the mean (SD;
n = 4). *P < 0.05. b Suz12 mRNA in mouse ESCs treated with RA was
measured by RT-qPCR. Actb was used as a loading control. Data are shown
as the mean (SD; n = 3). *P < 0.05. c H3K27me3 level after upon RA
treatment was measured by Western blotting, respectively, by knockdown
of Suz12. F9 cells were knockdown of Suz12 for 24 h, and then treated
with RA. H3 was used as a loading control. d Knockdown of Suz12
affected mRNA level of Hox genes in RA-induced F9 cells. F9 cells were
knockdown of Suz12 for 24 h. Then, after 24 h of RA treatment, cells
were collected and analyzed. Data were shown as mean ± SD (n = 3).
*P < 0.05. e ChIP assays of H3K27me3 were performed using mouse F9
cells after siSuz12 transfection. Mouse IgG was used as control.
Enrichment of Hox gene promoters was measured by qPCR
Negative correlation of HOXB4, HOXC4 and HOXD1 expression with H3K27me3 in
human anencephaly
HOX genes encode highly conserved transcription factors expressed in
the brain and spinal cord that play a central role in establishing the
anterior–posterior body axis during embryogenesis. Their expression is
tightly regulated in a spatiotemporal and collinear manner, partly by
chromatin structure and epigenetic modifications. To investigate the
potential clinical relevance of expression levels of the selected HOX
genes in anencephaly, we first examined the mRNA level of HOX genes,
including HOXA4, HOXA5, HOXB4, HOXB5, HOXC4, HOXC5, HOXD1, HOXD3,
HOXD4, and HOXD8 in brain tissues from 39 NTD-affected and 39 normal
control human fetuses. The clinical phenotypes of the cases were 10
anencephaly, 10 spina bifida, 10 spina bifida combined with
hydrocephaly and 9 encephalocele. NanoString assays showed that
expression of the 10 HOX genes (HOXA4, HOXA5, HOXB4, HOXB5, HOXC4,
HOXC5, HOXD1, HOXD3, HOXD4, and HOXD8) was significantly upregulated in
anencephaly tissues compared with normal tissues (P < 0.05)
(Fig. [135]7a and Table [136]1). However, the results showed that in
spina bifida, hydrocephaly and encephalocele level of 10 HOX genes were
no significantly increased (Additional file [137]8: Figure S5A–C).
Western blot analysis of ten anencephaly subjects compared with age-
and gender-matched controls revealed that H3K27me3 levels decreased
obviously in six anencephaly samples (Fig. [138]7b, c). The average
level of H3K27me3 expression (normalized to H3) was found to be
significantly lower in NTDs samples (P = 0.052). Interestingly, genetic
studies could not show an association between variants in HOX genes and
NTDs. We sequenced in 163 stillborn or miscarried with NTDs. The number
of variants by minor allele frequency (MAF) and impact group in NTDs
sample (Additional file [139]8: Figure S5D–I). The relative expression
of H3K27me3 was decreased 30%. Importantly, significantly negative
correlations of HOXB4, HOXC4 and HOXD1 expression were observed with
H3K27me3 levels among the examined subjects (r[HOXB4]= − 0.468,
P = 0.038; r[HOXC4]= − 0.484, P = 0.031; r[HOXD1] = − 0.528, P = 0.017)
(Fig. [140]7d). In conclusion, these data identify the abnormal
upregulation of HOX genes, especially HOXB4, HOXC4 and HOXD1,
concomitant with decreased H3K27me3 levels in human anencephaly cases.
This work is the first to demonstrate that in NTDs, especially
anencephaly, increased HOX gene expression was accompanied by aberrant
H3K27me3 levels.
Fig. 7.
[141]Fig. 7
[142]Open in a new tab
HOX gene expression was upregulated and H3K27me3 levels were decreased
in human anencephaly. a HOX genes were detected with NanoString in the
brain tissues from human anencephaly and normal cases. Data are shown
as the mean (SD; n = 10). P < 0.05 indicates statistical significance.
b Detection of histone H3K27me3 modification in brain tissues from
human anencephaly and normal cases by western blotting. Total histone
H3 was used as a loading control. Data are shown as the mean (SD;
n = 10). c Quantification analysis of the H3K27me3 signal normalized to
the H3 signal between human anencephaly and normal cases. d Pearson’s
correlation analysis between HOXs expression and H3K27me3 level.
P < 0.05 indicates statistical significance
Table 1.
mRNA expression of 10 HOX genes in human anencephaly
Gene Con (
[MATH: X¯±S
:MATH]
) NTDs (
[MATH: X¯±S
:MATH]
) t P value
HOXA4 1.270 ± 0.867 2.835 ± 1.891 − 2.379 0.029
5HOXA5 1.357 ± 0.956 2.887 ± 1.368 − 2.899 0.010
HOXB4 1.234 ± 0.774 2.577 ± 1.463 − 2.567 0.019
HOXB5 1.705 ± 1.069 3.090 ± 1.334 − 2.562 0.020
HOXC4 0.963 ± 0.814 2.311 ± 0.831 − 3.664 0.002
HOXC5 1.174 ± 1.093 1.650 ± 1.459 − 2.561 0.020
HOXD1 2.085 ± 1.175 3.886 ± 1.800 − 2.650 0.016
HOXD3 1.599 ± 1.261 3.190 ± 1.638 − 2.496 0.023
HOXD4 1.759 ± 1.213 3.744 ± 1.781 − 2.913 0.009
HOXD8 1.822 ± 1.191 3.466 ± 1.249 − 3.012 0.007
[143]Open in a new tab
Data were shown as mean ± SD (n = 10). P < 0.05 means the difference
have statistical significance
Discussion
Neural tube defects (NTDs) are multifactorial disorders that arise from
a combination of genetic and environmental interactions. Many studies
have focused on screening for candidate NTD genes, leading to the
discovery of more than 200 genes known to cause NTDs in mouse [[144]25,
[145]26], although few of these have been validated in human NTDs.
Results from recent studies show that not only gene mutations but also
aberrant gene expression are important in NTDs [[146]27]. Previous work
from our laboratory has confirmed that abnormal expression of Wnt2b and
Wnt7b is involved in human NTDs [[147]28]. In this study, we attempted
to explore the role of abnormal expression of HOX genes in NTDs as well
as its underlining regulatory mechanism, based our data from
transcriptome profile analysis of RA-induced mouse NTDs embryos.
The clustered Hox genes are required for establishing the embryonic
anterior–posterior axis, and are important for embryogenesis. A unique
feature of the clustered Hox genes is the direct relationship between
their expression and function in time and space during development,
termed collinearity, disruption of which can result in abnormal neural
tube closure [[148]29, [149]30]. Each NTDs phenotype depends on a
different set of genes, such as Hox genes, which are activated in a
temporally collinear manner to drive the progressive specification of
different segments. Our RNA-seq data from this study showed the
expression of 10 Hox genes decreased during mouse neural tube
development, consistent with what has been reported from a previous
study [[150]31]. However, in RA-treated mouse embryo, all of these
genes were significantly upregulated. In our study, we selected a
window in time during development critical for normal neuronal tube
closure, between E8.5 and E10.5. In normal mouse embryo, we observed a
downward trending for the temporal expression of all Hox genes, from
E8.5 to E10.5. On E8.5, the difference in level expression of Hox genes
between control and RA-induced was insignificant, however, a drastic
increase in Hox gene expression was observed on E9.5 and 10.5.
Therefore, we reasoned that the accumulative disruption of the normal
Hox gene expression level on E8.5 through E10.5 may have a profound
effect of developmental processes leading to NTDs.
Components of histone modification have been shown recently to be
critical for normal brain function and development, and aberrant levels
of modification in these components contribute to nervous system
diseases [[151]32, [152]33]. Our previous data have also established
associations between histone modifications and DNA methylation in human
NTDs [[153]34, [154]35]. Among all NTD-related histone modification
components, a direct link between abnormal level of H3K27me3 and
upregulation of Wnt genes has been demonstrated in NTDs [[155]28]. In
the present study, decreased levels of H3K27me3 were found in both
RA-induced mouse anencephaly and sample from a number of human
anencephaly patients. In order to explore the possibility whether the
decreased level of H3K27me3 is associated with abnormal Hox gene
expression, we employed ChIP-qPCR on samples from RA-treated mouse ESCs
to evaluate the enrichment of H3K27me3 in Hox genes. Indeed, data from
ChIP-qPCR revealed that enrichment of H3K27me3 was decreased in all 10
Hox genes, suggesting that Hox upregulation was caused by decreased
H3K27me3 levels. In addition, mutation screens eliminated the
possibility that specific mutations on HOX genes may occur that causes
a diminished enrichment of H3K27me3 in the HOX genes in Chinese NTDs.
Lastly, we evaluated the expression and activity of H3K27 demethylases
in RA-treated samples since it direct affects H3K27me3 levels. The
expression level of UTX, the enzyme demethylates H3K27 at the Hox loci,
was unchanged during RA treatment. However, RA treatment led to an
increase in UTX enzymatic activities, enabling elevated demethylating
of H3K27me3. Previous results show that UTX binding correlates with
diminished levels of H3K27me3 at transcription start sites [[156]23].
Active regions of the Hox cluster are marked by long, continuous
H3K4me2/3 stretches devoid of H3K27me3. UTX is not the only H3K27
demethylase throughout this long stretch. For example, another H3K27
demethylase, JMJD3, is associated with the Hoxa7 and Hoxa11 loci during
bone marrow cell differentiation [[157]32]. This suggests multiple
H3K27 demethylases control expression of Hox gene clusters and it is
tempting to speculate that additional H3K27 demethylases involved in
transcriptional repression could more effectively regulate
transcription [[158]36].
One of the most intriguing questions remaining is that in our study,
HOX gene expression was only increased in human anencephaly cases, but
was not increased in encephalocele or spina bifida cases. Encephalocele
is a phenotype of NTD that is different from anencephaly, although the
lesion is located in the brain, while the spina bifida lesion is in the
spinal cord. In this study, we collected brain tissue from human
fetuses with different NTDs; therefore, it is not surprising that HOX
genes were expressed differently in the brain in the different NTDs
phenotypes. This result indicated the NTDs are not one disease, but
many; therefore, it is better to explore the etiology according to the
NTDs phenotype. A limitation of our study was that only a few human
cases were collected; more human cases are needed to analyze the
correlation of H3K27me3 levels with HOX gene expression in human NTDs.
Conclusions
In conclusion, environmental factors have a profound influence on
neural tube closure. Our study showed that epigenetic modifications of
H3K27me3 could cause abnormal Hox gene expression after exposure to a
detrimental environment factor, such as RA, which may significantly
contribute to development and etiology of NTDs. The present study
provided novel insight into the synergistic function of transcriptional
dysregulation and epigenetic modifications. It may be possible to
identify novel measures to prevent this devastating birth defect. These
results provide evidence supporting the hypothesis that deregulation of
Hox gene expression through epigenetic mechanisms may be associated
with human NTDs. Our findings have broad implications for the
mechanisms underlying epigenetic memory which is currently under
investigation.
Methods
Animals
C57BL/6 mice (44007200007011, 9–10 weeks, 18–23 g) were purchased from
Guangdong Medical Laboratory Animal Center (Guangzhou, China), and
housed in SPF cage, approved facility on a 12-h light/dark cycle. NTDs
mouse embryos were induced by gavage with 28 mg/kg of RA (Sigma, USA,
dissolved in sesame oil) on E7.5. On E8.5, E9.5 and E10.5, pregnant
mice were euthanized by cervical dislocation and the cranial neural
tissue of embryos was collected according to the previous study
[[159]37]. All procedures involving animal handling were approved by
the Animal Research Ethics Board of Guangdong medical laboratory animal
center in China, and were in compliance with institutional guidelines
on the care of experimental animals.
Embryonic stem cell culture and RA treatment
Sv/129 mouse embryonic stem cells (ESCs), were obtained from Xuanwu
Hospital (Beijing, China), and maintained in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco, USA) supplemented with 0.1 mM
β-mercaptoethanol (Invitrogen, Carlsbad, USA), non-essential amino
acids (Invitrogen, Carlsbad, USA), 2 mM glutamate (Invitrogen,
Carlsbad, USA), 15% fetal bovine serum (Gibco, USA), and 1000 U/ml
leukemia inhibitory factor (Millipore, Billerica, USA), cultured in the
culture dishes coated with 0.2% gelatin (Invitrogen, Carlsbad, USA).
Cells were incubated at 37 °C/5% CO[2] and passaged every 2 days. ESCs
were treated with 1 μM RA for 24 h [[160]38].
Human samples collection
Normal and NTDs case subjects were obtained from Linxian and Liulin
counties, located in the north of Shanxi Province of China. The
enrolled pregnant women were diagnosed by trained local clinicians
using ultrasonography and then registered in a database. 39
NTDs-affected fetuses and 39 age-matched controls were collected, and
the neural tissue samples were used in the following experiments. The
sample information was described in Additional file [161]9: Table S4.
The Committee of Medical Ethics in the Capital Institute of Pediatrics
(Beijing, China) approved this study (SHERLLM2014002). Written informed
consent was obtained from the parents on behalf of the fetuses.
RNA extraction, cDNA library construction and Illumina sequencing
Total RNA was extracted from mouse embryo cranial neural tissue tissues
using the Trizol method (Invitrogen, USA). RNA concentration and
quality were assessed by Agilent 2100 Bioanalyzer. The cDNA library was
constructed using a TruSeq RNA Sample Preparation Kit (RS-122-2101,
Illumina, USA) according to the manufacturer’s protocols. And then, the
library could be sequenced using PE91 + 8+91 of Illumina HiSeq™ 2000.
After quality of control (QC), the clean reads were obtained and have
been deposited into NCBI SRA (accession numbers: SRP070626).
Bioinformatic analysis of RNA-seq and Hox genes screening
The gene expression level is calculated by using RPKM method [[162]39]
(reads per kilobase transcriptome per million mapped reads), and the
unsupervised clustering analysis was performed using R. The
differentially expressed genes (DEGs) were identified by the
combination of P value < 0.05, FDR < 0.001 and the absolute value of
log[2] ratio > 1, which expression patterns were analyzed by Cluster
3.0 [[163]40] and Java Treeview. The Gene Ontology (GO) and pathway
enrichment analysis were used to identify the significantly enriched
functional classification, signaling and metabolic pathways of DEGs,
which were performed based on Gene Ontology Database
([164]http://www.geneontology.org/) [[165]41] and KEGG pathway database
([166]http://www.genome.jp/kegg/) [[167]42]. GO terms were identified
to be significantly enriched when corrected P value < 0.05. Pathways
were identified to be significantly enriched when FDR < 0.05 meantime.
The DEGs were selected by taking intersection among E8.5, E9.5 and
E10.5, and the important Hox genes were screened by fold change > 20 at
E9.5 and E10.5 meantime.
RT-qPCR
To validate the RNA-seq findings, we prepared new mouse cranial neural
tissue of E10.5, and some DEGs were selected and performed with
RT-qPCR. In addition, we also validated the important Hox genes
expression in Sv/129 mouse ESCs treated with 1 μM RA. Total RNA was
extracted using the Trizol method (Ambion, USA), first-strand synthesis
was done with RevertAid First Strand cDNA Synthesis Kit (Thermo, USA).
Maxima SYBR Green/ROX qPCR Master Mix (Thermo, USA) were used for qPCR
and the procedure was as follows: (50 °C, 2 min) × 1 cycle; (95 °C,
10 min) × 1 cycle; (95 °C, 15 s; 60 °C, 30 s; 72 °C, 30 s) × 40 cycles;
collect fluorescence at 72 °C. Primer sequences were shown in
Additional file [168]10: Table S5.
Protein extraction
Core histone proteins of cells were extracted using acid extraction
[[169]43]. Briefly, the brain tissue was first homogenized in lysis
buffer [10 ml solution containing 10 mM Tris–HCl with pH 8.0, 1 mM KCl,
1.5 mM MgCl[2] and 1 mM dithiothreitol (DTT)] and chilled on ice. 5% of
protease inhibitors were added immediately before lysis of cells,
chilled on ice for 30 min, and nuclei were isolated by centrifugation
(1500g for 5 min). For the preparation of histones, nuclei were
incubated with four volumes of 0.2 M sulfuric acid (H[2]SO[4]) for
overnight at 4 °C. The supernatant was precipitated with 33%
trichloroacetic acid (final concentration) and followed by
centrifugation (12,000g for 5 min at 4 °C). The obtained pellet was
washed with cold acetone and subsequently dissolved in distilled water.
Core histone proteins of mouse and human brain samples were extracted
using EpiQuik™ Total Histone Extraction kit (EPIGENTEK, Farmingdale,
NY) according to the manufacturer’s protocols. Nucleoprotein extraction
was extracted from cells or mouse and human brain samples using
Nucleoprotein Extraction Kit (Sangon Biotech, China) according to the
manufacturer’s protocols.
Western blotting
5 μg of histone was separated on a 12% SDS-PAGE for H3K27me3 and H3
detection. The blots were incubated with the primary antibody, mouse
anti- H3K27me3 (1:800, Abcam, Cambridge, UK), anti-H3K27me2,
anti-H3K27me1, anti- Suz12 monoclonal antibody (1: 500, Cell Signaling
Technology, USA), and mouse anti-H3 monoclonal antibody (1:1,500,000,
Abcam, Cambridge, UK) overnight at 4 °C, and then incubated with
secondary anti-mouse HRP conjugated antibody (1:5000, Santa, USA) for
1 h at room temperature. The blots were developed with SuperSignal West
Pico Chemiluminescence Substrate (Thermo, USA) and quantitated on
densitometer (Bio-Rad, Universal HoodII, USA) using Quantity One
software.
Immunofluorescence
Hep-G2 cells were maintained in DMEM (Gibco, USA) supplemented with 10%
fetal bovine serum (Gibco, USA), treated with 1 μM RA for 24 h. The
primary antibodies used for immunofluorescence staining were the mouse
monoclonal anti-H3K27me3 antibody (1: 200, Abcam, UK).
Immunohistochemistry
Embryos (E10.5) were fixed in 4% paraformaldehyde overnight and
processed to generate 5-μm paraffin sections. Immunohistochemistry was
performed on transverse sections according to the method previously
described [[170]44]. The primary antibodies used were the mouse
monoclonal anti-H3K27me3 antibody (1: 200, Abcam, UK). The area
percentage of the H3K27me3 positive was analyzed by Image J software.
ChIP-Seq and data analysis
Cranial neural tissue of pooled mouse embryos on E10.5 were collected
and performed by ChIP-seq. ChIP was done with Simple ChIP Enzymatic
chromatinIP kit (9003s) from Cell Signaling Technology and chromatin
was sheared to an average DNA fragment size of 100–300 bp. ChIP-Seq
libraries were prepared according to Illumina protocols. Sequencing was
done with Illumina HiSeq platform. After quality of control (QC), the
clean reads were obtained and have been deposited into NCBI SRA
(Accession Numbers: SRP193168). The clean data were mapped to the mouse
genome (mm9) using SOAPaligner/SOAP2 (Version: 2.21t), and no more than
two mismatches are allowed in the alignment. Peaks of H3K27me3 ChIP-Seq
signals on genome were determined using MACS2 with false-discovery rate
as 0.05. Tracks of H3K27me3 ChIP-seq were viewed by UCSC Genome Browser
for mm9.
Chromatin immunoprecipitation (ChIP) analysis
ChIP assays were performed using the SimpleChIP Enzymatic chromatin IP
system (Cell Signaling, California, USA) following the manufacturer’s
protocols. Chromatin was prepared, sonicated to DNA segments between
200 and 1000 bp and then immunoprecipitated with anti-H3K27me3,
anti-UTX (Abcam, Cambridge, UK) and anti-H3K27me2 (Cell Signaling
Technology, USA). The immunoprecipitated DNA was analyzed by qPCR,
which were performed using QuantStudio 7 Flex with SYBR Green
detection. The primers used for ChIP assays were shown in Additional
file [171]11: Table S6 [[172]45–[173]53]. Mouse IgG antibodies were
used as negative controls in the immunoprecipitations. The following
equation was used to calculate percent input = 2% × 2^(CT) 2% input
sample − (CT) IP sample).
Human mRNA detection
NanoString nCounter system was used to analyze the 10 HOX mRNA
expression level of degradative brain tissues from human fetus samples.
The RNA was extracted using miRNeasy Mini Kit (Qiagen, Germany).
Hybridizations were performed according to the NanoString miRGE Assay
Manual. Approximately 100 ng of each RNA sample was mixed with 20 μl of
nCounter Reporter probes in hybridization buffer and 5 μl of nCounter
Capture probes for a total reaction volume of 30 μl. The hybridizations
were incubated at 65 °C for approximately 16 h, then eluted and
immobilized in the cartridge for data collection, which was performed
on the nCounter Digital Analyzer. The counts were analyzed by log[2]
transformation using nSolver Analysis Software 2.5, and normalized by
housekeeping genes such as GAPDH, CLCT, GUSB, HPRT1 and PKG1 genes.
UTX activity detection
UTX activity was detected according to the manufacturer’s protocols of
Epigenase JMJD3/UTX Demethylase activity/inhibition assay kit
(Epigentek, Farmingdale, NY) using 10 μg of nuclear extracts. The UTX
activity was calculated as the following formula: UTX activity
(OD/min/mg) = sample OD − Blank OD/(protein amount (μg) × min) × 1000.
UTX inhibitor
Sv/129 ESCs were treated with 30 nM of UTX inhibitor GSK J4 sc-391114
(Santa Cruz, USA) for 6 h according to the manufacturer’s protocol.
Suz12 siRNA and overexpression of transfection
Mouse F9 cells were maintained in DMEM (Gibco, USA) supplemented with
10% fetal bovine serum (Gibco, USA). siRNA was delivered to cells using
Lipofectamine 2000 according to the manufacturer’s instruction. siRNAs
specific for Suz12 5′-AAGCTGTTACCAAGCTCCGTG-3′ and a nonspecific siRNA
5′-TTCTCCGAACGTGTCACGT-3′ were designed and synthesized (Sangon
Biotech, China), and the latter was transfected as negative control.
Suz12 expression plasmid was purchase from Origene. After transfection
for 24 h, F9 cells were treated with 1 μM RA for 24 h, and then
harvested for further analysis.
Statistical analysis
Statistical analysis was performed using SPSS software, version 22.0
(SPSS, Inc., Chicago, IL, USA). Data were expressed as the mean ± SD,
Student’s t test or ANOVA analysis was performed. And Pearson’s
correlation analysis was used to analyze the association between HOXs
expression and H3K27me3 level. P < 0.05 was considered statistically
significant.
Ethics statement
All animals were handled in strict accordance with the “Guide for the
Care and Use of Laboratory Animals” and the “Principles for the
Utilization and Care of Vertebrate Animals”, and all animal work was
approved by Institutional Animal Care and Use Committee (IACUC) at the
Beijing Institute of Radiation Medicine. The study using clinical
samples including 39 paired human NTDs and matched normal tissues were
approved by department of Lvliang area of Shanxi Province in northern
China. Informed consent was obtained from all subjects or their
relatives. Human samples were collected and analyzed in accordance with
Capital Institute of Pediatrics approval. The Ethics Board of Capital
Institute of Pediatrics approved the study protocol. All animal
experiments were conducted in compliance with the guidelines of the
Institute for Laboratory Animal Research, Capital Institute of
Pediatrics.
Supplementary information
[174]13072_2019_318_MOESM1_ESM.pdf^ (556.2KB, pdf)
Additional file 1: Figure S1. Bioinformatic analysis of RNA-seq. A.
Morphology of mouse NTDs embryos induced by RA. (a). Normal mouse
embryo. (b). Mouse embryo showed growth retardation, neural tube close
incompletely. Arrow indicates unclosed neural tube. (c). Mouse embryo
showed anencephaly, enlarged heart and ventricular chambers, and short
tail. Arrow indicates hindbrain, heart and tail respectively. B.
Unsupervised hierarchical clustering plot of genes detected in mouse
embryo cranial neural tissue. C. GO functional classification of DEGs.
Blue represents cellular component, red represents molecular function,
and green represents biological process. D. KEGG pathway analysis of
DEGs. E. Protein-protein interaction (PPI) network of 196 genes
analyzed by STRING database.
[175]13072_2019_318_MOESM2_ESM.doc^ (137.5KB, doc)
Additional file 2: Table S1. Enriched GO terms of DEGs in
Con-E8.5-vs-RA-E8.5, Con-E9.5-vs-RA-E9.5 and Con-E10.5-vs-RA-E10.5
comparisons.
[176]13072_2019_318_MOESM3_ESM.docx^ (27.1KB, docx)
Additional file 3: Table S2. Enriched KEGG pathways of DEGs in
Con-E8.5-vs-RA-E8.5, Con-E9.5-vs-RA-E9.5 and Con-E10.5-vs-RA-E10.5
comparisons.
[177]13072_2019_318_MOESM4_ESM.pdf^ (81.2KB, pdf)
Additional file 4: Figure S2. H3K27me3/2/1 analysis in mouse NTD
embryos of E10.5. A. Relative protein expression of H3K27me3, H3K27me2
and H3K27me1 in mouse NTDs embryo. Data are shown as the mean (SD; n=
3). *P < 0.05. B. Peak statistics of mouse embryos used for ChIP-seq.
C. GO analysis of differential peak related gene.
[178]13072_2019_318_MOESM5_ESM.xlsx^ (75.4KB, xlsx)
Additional file 5: Table S3. Differential peaks related genes of mouse
NTDs embryos of E10.5.
[179]13072_2019_318_MOESM6_ESM.pdf^ (221.7KB, pdf)
Additional file 6: Figure S3. H3K27me3/2/1 analysis in RA-induced ESCs.
A. Relative protein expression of H3K27me3, H3K27me2 and H3K27me1in
RA-induced ESCs. Data are shown as the mean (SD; n= 3). *P < 0.05. B.
ChIP assays of H3K27me2 were performed using F9 cells treated with 1 μM
RA for 24 h. Mouse IgG was used as control. Enrichment of Hox gene
promoters was measured by qPCR.
[180]13072_2019_318_MOESM7_ESM.pdf^ (109.9KB, pdf)
Additional file 7: Figure S4. Suz12 and Ezh2 decreased in RA-induced
mouse NTDs and ESCs. A. RNA-seq analysis showed Suz12 expression in
cranial neural tissue of RA-induced mouse NTDs embryos from E8.5 to
E10.5. B. RNA-seq analysis showed Ezh2 expression in cranial neural
tissue of RA-induced mouse NTDs embryos from E8.5 to E10.5. C. Ezh2
level in cranial neural tissue of RA-induced mouse NTDs was measured by
RT-qPCR and Western blotting. Actb and Gapdh were used as a loading
control respectively. Data are shown as the mean (SD; n= 4). *P < 0.05.
D. Ezh2 level in mouse ESCs treated with RA was measured by RT-qPCR and
Western blotting. Actb and Gapdh were used as loading control
respectively. Data are shown as the mean (SD; n= 3). *P < 0.05. E.
Relative protein expression of H3K27me3 after overexpression of Suz12
in RA-induced F9 cells. Data are shown as the mean (SD; n= 3).
Different letters represent the difference had statistic significance,
P < 0.05.
[181]13072_2019_318_MOESM8_ESM.pdf^ (397.6KB, pdf)
Additional file 8: Figure S5. HOX gene expression in human spinal
bifida, hydrocephaly and encephalocele. A. HOX genes were detected with
NanoString in the spinal cord from human spinal bifida and normal
cases. Data are shown as the mean (SD; n= 10). P < 0.05 indicates
statistical significance. B. HOX genes were detected with NanoString in
the brain tissues from human hydrocephaly and normal cases. Data are
shown as the mean (SD; n= 10). P < 0.05 indicates statistical
significance. C. HOX genes were detected with NanoString in the brain
tissues from human encephalocele and normal cases. Data are shown as
the mean (SD; n= 9). P < 0.05 indicates statistical significance. D.
Whole genome sequencing of 100 human NTDs samples. E, F. Variant
distribution allele frequency of 10 HOX genes in 100 human NTDs
samples. G. The number of variants by minor allele frequency (MAF) in
100 human NTDs samples. H, I. Variant rate of 10 HOX genes in 100 human
NTDs samples.
[182]13072_2019_318_MOESM9_ESM.docx^ (19.5KB, docx)
Additional file 9: Table S4. Information of human NTDs samples. The
human cases with anencephaly, spinal bifida, hydrocephaly and
encephalocele involved in this study. Gender, gestational age, sample
type and NTDs phenotype are listed.
[183]13072_2019_318_MOESM10_ESM.docx^ (25.3KB, docx)
Additional file 10: Table S5. RT-qPCR primer sequences. All
oligonucleotides were synthesized by Sangon Biotech.
[184]13072_2019_318_MOESM11_ESM.docx^ (16.3KB, docx)
Additional file 11: Table S6. ChIP-qPCR primer sequences. All
oligonucleotides were synthesized by Sangon Biotech.
Acknowledgements