Folate deficiency induced H2A ubiquitination to lead to downregulated expression of genes involved in neural tube defects
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Neural tube defects (NTDs) are common congenital malformations resulting in failure of the neural tube closure during early embryonic development. Although it is known that maternal folate deficiency increases the risk of NTDs, the mechanism remains elusive.
Herein, we report that histone H2A monoubiquitination (H2AK119ub1) plays a role in neural tube closure. We found that the folate antagonist methotrexate induced H2AK119ub1 in mouse embryonic stem cells. We demonstrated that an increase in H2AK119ub1 downregulated expression of the neural tube closure-related genes Cdx2, Nes, Pax6, and Gata4 in mouse embryonic stem cells under folate deficiency conditions. We also determined that the E3 ligase Mdm2 was responsible for the methotrexate-induced increase in H2AK119ub1 and downregulation of neural tube closure-related genes. Surprisingly, we found that Mdm2 is required for MTX-induced H2A ubiquitination and is recruited to the sites of DSB, which is dependent on DNA damage signaling kinase ATM. Furthermore, folic acid supplementation restored H2AK119ub1 binding to neural tube closure-related genes. Downregulation of these genes was also observed in both brain tissue of mouse and human NTD cases, and high levels of H2AK119ub1 were found in the corresponding NTDs samples with their maternal serum folate under low levels. Pearson correlation analysis showed a significant negative correlation between expression of the neural precursor genes and H2AK119ub1.
Our results indicate that folate deficiency contributes to the onset of NTDs by altering H2AK119ub1 and subsequently affecting expression of neural tube closure-related genes. This may be a potential risk factor for NTDs in response to folate deficiency.
KeywordsNeural tube defect Histone ubiquitination Folate antagonist methotrexate Mouse double minute 2 homolog (Mdm2, MDM2) Neural tube closure-related genes
ataxia telangiectasia mutated
ATM- and Rad3-related
differentially expressed genes
DNA-dependent protein kinase
DNA double-strand breaks
mouse embryonic carcinoma
Kyoto Encyclopedia of Genes and Genomes
H2A monoubiquitylation at lysine119
half inhibition concentration
mouse embryo stem cells
neural tube defects
neural tube closure
polycomb protein complexes
upstream binding factor
Neural tube defects (NTDs) are severe malformations that originate during early embryonic development and result in failure of the neural tube closure. Both genetic and environmental factors contribute to NTDs . Maternal folate is one of the best-studied dietary factors closely related to neural tube closure (NTC). Worldwide fortification of food with folic acid has resulted in a significant reduction in the prevalence of NTDs . Particular attention has been paid to carbon metabolism, especially the finding of maternal supplementation with folic acid has reduced the risk of NTDs. Environmental factors may influence NTC through a direct effect on the biology of embryonic metabolism. Deficiency of specific folates at the cellular level may be responsible for NTDs as a result of disturbed bioavailability of folates. Folate one-carbon metabolism comprises a complex network of interlinked reactions that mediates transfer of 1-carbon groups to several biosynthetic processes  and it focuses particularly on the requirement for methylation reactions in neural tube closure. Abnormal thymidylate and purine biosynthesis have been identified in mouse NTD models and in a proportion of NTD cases [4, 5, 6]. Current data suggest that folate deficits likely represent only a moderate fraction of NTD risk, and the mechanisms underlying human NTDs remain elusive [7, 8]. In the prevention and treatment of NTDs with folic acid, anti-folate drugs used during pregnancy had deleterious effects and increased the incidence of NTDs and other congenital defects . As an inhibitor of dihydrofolate reductase, methotrexate (MTX) prevents the conversion of folate to its active form and inhibits nucleotide biosynthesis . Previous studies have shown that MTX inhibits neuronal differentiation in neural stem cells and that folate supplementation attenuates this effect .
There is growing awareness that gene–gene and gene–environment interactions are involved in the occurrence of NTDs [12, 13, 14]. More than 200 genes are known to cause NTDs in mice, and human NTDs are even more complicated, being multifactorial and polygenic [15, 16]. However, only few of the NTD-associated genes identified in mice are confirmed to be involved in humans , and genetic polymorphisms account for only a small proportion of cases. In fact, multiple NTD-causing genes arrange in a particular pathway in NTDs . Recently, potential causative factors for human NTDs have involved deregulation of gene expression through epigenetic mechanisms . Mouse NTD models have indicated that histone modifications might control the expression of patterning molecules that are essential for NTC. Since histone-modifying enzymes consume key metabolites, it is thought that they interpret the metabolic state of a given cell by altering chromatin modification patterns. Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) take part in NTC . The catalysis by PRC1 of H2A monoubiquitination at lysine119 (H2AK119ub1) is a critical component in mediating Polycomb group (PcG) silencing in developmental lineage genes to promote stemness [20, 21]. Histone demethylase UTX is required for NTC in PRC2-mediated H3K27 trimethylation (H3K27me3) . H2AK119ub1 is emerging as a prominent contributor to genetic and molecular etiology in neurodevelopmental disorders . Notably, mouse NTD models have shown that regulation of ubiquitination plays a vital role during NTC . Recent studies have reported that Mdm2 (mouse double minute 2 homolog) is physically associated with EZH2 on chromatin, enhancing H3K27me3 and H2AK119ub1 at its target genes. Removal of Mdm2 stimulates Ring1B/RNF2 and further induces cell dysdifferentiation. Folate-mediated 1-carbon metabolism is related to epigenetic modifications. It affects DNA methylation and is associated with NTDs. This metabolism also controls the fate of neural precursor cell during development and differentiation of embryonic stem cells (ESCs) [25, 26, 27]. This is consistent with the highly dynamic yet coordinated cellular and molecular programs that drive changes in morphology, patterning, proliferation, and differentiation during NTC. The observation of cranial NTDs among knock-out acetyltransferase p300 or Gcn5 suggested that activity of histone acetylase is essential for NTC [28, 29].
On the basis of findings that epigenetic regulators play key roles in mouse NTC and may be affected by folic acid [30, 31, 32], it is likely that genome-wide analyses will reveal epigenomic changes associated with NTDs. In animal models, advanced technologies allow researchers to evaluate the transcriptional profile of specific cell types and correlate this with changes in DNA methylation, histone marks, and higher order chromatin states. Future studies incorporating information on folic acid status should help to define potential folate-mediated changes, with a particular focus on the genes known to be necessary for NTC. Relation between previous reports showed that DNA hypomethylation of long interspersed nucleotide elements and risk of NTDs. The folate pathway is particularly important because altered patterns of histone modification regulate gene function and stability, in turn leading to congenital malformations. It has been demonstrated that increased H3K27me2 in cultured neural crest cells and neural tube explants from splotch (Pax3) mutant embryos resulted in development of NTDs . This finding showed that folate rescued H3K27me2 and was associated with Hes1 and Neurog2 promoters, thereby affecting gene transcription. However, to date only a few studies have addressed the possible role of folate level on the relationship between histone ubiquitination and ESC differentiation in ongoing occurrence of NTDs. This raises the possibility that the relationship between folic acid supplementation or folate 1-carbon metabolism and the risk of NTDs may be mediated in part through their effects on methylation.
Folate deficiency facilitates recruitment of upstream binding factor to hotspots of DNA double-strand breaks (DSBs)  of ribosomal (r)RNA genes and promotes their transcription. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were shown to be located exclusively within rRNA gene units, representing an H3K4me1 hallmark. Enrichment of H3K4me1 at the hotspots of DSB regions enhances the recruitment of upstream binding factor to rRNA genes, resulting in an increase in transcription of rRNA genes . Our previous study indicated that folate deficiency attenuated H3K79me2, affecting its regulate activation of target genes, some of which were known to be associated with NTDs, and interrupting early embryo development. Our results suggested that higher levels of homocysteine (Hcy) contribute to the onset of NTDs through upregulation of histone H3K79Hcy, leading to abnormal expression of selected NTC-related genes. Since folate levels contribute to the accumulation of DSBs in the genome, the effects of folate metabolism disorder on the relationship between DSBs in H2AK119ub1 and its transcriptional regulation in NTC genes are unknown.
In this study, we showed that the folate antagonist MTX induced H2AK119ub1 in mouse ESCs. We detected that an increase in H2AK119ub1 level downregulated the expression of NTC-related genes, including Cdx2, Nes, Pax6, and Gata4 in cells under folate deficient conditions. We also demonstrated that the E3 ligase Mdm2 is responsible for the MTX-induced increase in H2AK119ub1 and downregulated NTC-related genes. Surprisingly, Mdm2 is required for MTX-induced H2A ubiquitination and is recruited to the sites of DSB, which is dependent on DNA damage signaling kinase ATM. Furthermore, folic acid supplementation results in restored H2AK119ub binding to NTC-related genes. In addition, downregulation of NTC-related genes was observed in both brain tissue of mouse and human NTD cases, and low levels of folate found in the corresponding maternal serum samples. Our results provide strong evidence that folate deficiency induces aberrant H2AK119ub1, which is linked to abnormal expression of NTC-related genes and subsequently NTDs. This research further extends our understanding of aberrant epigenetic modification of NTC-related genes in NTDs.
Decreased expression of neural precursor marker genes in MTX-treated mouse ESCs
To identify the alterations in gene expression profile accompanied by MTX-induced changes, we performed RNA-seq on mESCs treated with 1 μM MTX. By comparing libraries from control and MTX-induced mESC samples, we identified a significant number of differentially expressed genes, including 1642 upregulated and 672 downregulated genes (Fig. 1b; Additional file 1: Table S1). Further analysis with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that these differentially expressed genes were enriched in GO terms related to multiple biological processes, molecular functions, and signaling pathways of MTX-treated cells (Additional file 2: Figure S1A), and KEGG pathways (Additional file 2: Figure S1B and Additional file 3: Table S2). Unsupervised clustering analysis revealed numerous differentially regulated genes under MTX treatment, with enrichment of gene sets involved in development and morphogenesis (Fig. 1c and Additional file 2: Figure S1C). Overall, 829 differentially expressed genes had > twofold changes (723 upregulated and 126 downregulated) following MTX treatment (Additional file 4: Table S3). Of the 42 genes strongly downregulated under MTX treatment, especially NTC-related genes associated with neural precursor stage (Fig. 1d). To determine the role of MTX in the neural plate stage in ESC, we determined the effect of MTX treatment on mESC using real-time quantitative PCR (RT-qPCR). The RT-qPCR analysis revealed that several NTC-related genes were downregulated in MTX-treated mESC, including neuronal filament genes Cdx2 and Nes and genes encoding the pro-neuronal transcription factors Pax6 and Gata4, consistent with RNA-seq data (Fig. 1e). Taken together, our data suggest that the MTX treatment of mESC resulted in changes in gene expression pattern and decreased expression of neural precursor marker genes.
MTX-induced H2AK119ub1 binding to NTC-related genes in mESCs
Mdm2 is required for regulation of H2AK119ub1 on NTC-related genes under MTX treatment
Mdm2 to DNA damage sites and contributes to MTX-induced H2AK119ub1
Supplementation with folinic acid attenuates H2AK119ub1 induced by MTX and affects H2AK119ub binding to NTC-related genes
MTX caused induced levels of H2AK119ub1 in the NTD mice
Aberrant H2AK119ub1 with decreased expression in low-folate NTD fetuses
Additional information of the human normal and NTD samples
Folate level (ng/mg)
A protective effect of folic acid on the occurrence and recurrence of NTDs has been clearly demonstrated. Abnormal 1-carbon metabolism has been implicated in the occurrence of NTDs. Maternal folate deficiency during pregnancy is associated with an increased risk of NTDs. However, the mechanisms underlying the effect of folic acid on NTDs remain to be elucidated. It seems that folate deficiency alone is insufficient to cause NTDs but can give rise to offspring with NTDs when combined with multiple genetic and environmental factors. We showed that increased H2AK119ub1 on neural precursor genes in mESCs under folate deficiency interfered with cell differentiation. Supplementation with folinic acid rescued potential proliferation in neural precursor marker genes in MTX-treated mESCs. It is possible that H2AK119ub1 in folate-mediated rescue affects transcription of neural precursor marker genes during the early developmental stage. Moreover, we found that decreased expression of NTC-related genes was accompanied by aberrant H2AK119 expression in human fetuses NTDs under folate deficiency.
Neurogenesis is regulated by genetic and epigenetic mechanisms. Genetic, non-genetic, epigenetic, and environmental factors all contribute to the prevalence of NTDs. Environmental factors may influence NTC through a direct effect on metabolism. Much evidence has shown that folate 1-carbon metabolism nutrients may affect epigenetic regulation, thereby influencing susceptibility to NTDs through altered gene expression. A causal link between histone methylation and nutritional status has also been demonstrated, where folate and methionine deficiency are associated with reduced histone methylation and can lead to changes in gene expression. In the present study, we explored pathways from the 1-carbon metabolism intermediate Hcy to the onset of NTDs based on several key observations, including the demonstrated increase in Hcy levels in women who give birth to infants with NTDs, the histone modifications KHcy and H3K79Hcy, and the well-established altered expression of NTC genes in NTDs. Our previous studies found that folate deficiency affected DNA breakage through histone H3K4me1, leading to regulation of transcription of rRNA genes during the early stage of development and offering new insights into the etiology of NTDs. In this study, we showed that folate deficiency affected H2AK119ub1. Our data showed that H2AK119ub1 was increased in the cranial neural tube of embryos with MTX-induced NTDs compared with wild-type controls, suggest that upregulation of H2AK119ub1 was involved in open NTDs (Figs. 6c, 7b). We also found that increased H2AK119ub1 altered expression of NTC genes in folate-deficient NTD samples (Fig. 7). The PcG proteins form large complexes that repress gene expression through H3K27me3 and H2AK119ub1. These histone modifications are essential in postnatal development and stem cell maintenance in the mouse . We focused on H2AK119ub1 because PcG proteins mediate suppression of PcG target genes, mammalian body patterning, posterior development, and possibly identity maintenance in embryonic stem cells. Folate deficiency leads to delayed embryonic development and growth retardation in early embryonic progression . Recent studies have shown that an epigenetic component of folate-mediated rescue could affect cell proliferation, including transcription of Hes1 and other genes involved in early development, and thereby rescue posterior neural tube development. Indeed, epigenetic mechanisms likely govern all aspects of the central nervous system, including neuronal gene expression , especially in cell differentiation during neuro-embryogenesis.
To select potential epigenetically altered genes, we focused on the neural precursor marker genes Cdx2, Nes, Pax6, and Gata4. Recent reports have shown that H2AK119ub1 alters the activities of these genes. Cdx2 and Nes are responsible for neuronal filament growth, Pax6 is critical in pro-neuronal transcription, and Gata4 is involved in maintenance of stem cell characteristics and sensory neurogenesis. In our study, inhibition of folate metabolism was specifically targeted to inactivate these selected NTC-related genes (CDX2, NES, PAX6, and GATA4) in low-folate fetuses (Fig. 7d). The upregulation by H2AK119ub1 of these NTC-related genes could be a molecular regulatory event involved in NTC. This study suggested that H2AK119ub1 repressed NTC-related genes in MTX-treated mESC (Fig. 2h). Folinic acid supplementation may attenuate H2AK119ub1 by decreasing levels of Mdm2 (Fig. 5a). Subsequently decreased Mdm2-repressed chromatin marks on the NTC-related genes at its promoter regions, resulting in increased expression of NTC-related genes.
Reports indicate that inadequate proliferation is associated with occurrence of NTDs [50, 51]. In mice, over 200 mutant genes are involved in open NTDs, but much less are known about genetic causation of human NTDs. The nested expression patterns of Pax6 demarcate the forebrain at the neural plate stage. Abnormal expression of Pax6 results in severe NTDs, in which the neural tube remains open from the midbrain to lower spine. The intermediate filament protein nestin is an important marker of multipotent neural stem cells. When H2AK119ub1 functioned on NTC-related genes PAX6 and NES, it progressed neural tube closure in an amphibian embryo. Folate, especially of maternal folic acid supplementation, reduces the risk of non-folic-acid-resistant NTDs. In our study, we demonstrated folate metabolism inhibition on transcription neural precursor-specific genes during early embryonic development in mESC. The occurrence of isolated NTDs at the cranial or caudal level in humans and different mouse models suggests the possible involvement of region-specific mechanisms, depending on expression of different genes; all stages have ubiquitous requirements. In mice, NTDs can be modeled by exposure to certain reagents. Our results suggest that the neural precursor-specific genes are involved in different phenotypes of NTDs. Thus, in different tissues, NTD phenotypes may be associated with prevention of neuronal differentiation. Together, these data indicated that H2AK119ub regulated NTC-related genes at its promoter regions under folate deficiency may result in NTDs.
Mdm2 forms an active heterodimer that catalyzes H2AK119ub1 and H3K27me3 by the physical association of Mdm2 and PRC2. Mdm2 regulates PRC2 target genes by promoting positive feedback between H3K27me3 and H2AK119ub1, which explains why H2AK119ub1 increases with increased Mdm2 expression with MTX treatment . Our previous research identified a role of MTX in changes in DNA DSBs in mESC. Furthermore, spontaneous DSBs in mammalian cells under folate deficiency have been located to H3K4me1 . H2AK119ub1 is induced locally at the sites of damaged DNA. We reported here on a new function of H2AK119ub1 in DNA damage response. Under MTX treatment, H2AK119ub1 colocalized with ATM and γH2AX. Mdm2 undergoes specific modifications in response to replicative stress of DNA damage and is associated with damage response factors such as the Mre11-Rad50-Nbs1 (MRN) complex [46, 47]. We demonstrated MTX-induced increased expression of Mdm2 in mESCs (Fig. 2a) and found that Mdm2 was bound to NTC-related genes under folate deficiency conditions (Fig. 3e). Downregulation of H2AK119ub1 attenuated expression of NTC-related gene when Mdm2 was depleted (Fig. 3f). Folate plays an important role in neuroplasticity and maintenance of neuronal integrity.
Our analyses provide novel insight into neural precursor marker genes and potential regulation of H2A modification under abnormal folate metabolism. We identified an association of H2AK119ub1 with NTC-related genes during early embryonic development, suggesting a dynamic interplay of Mdm2 in fine-tuning stem cell proliferation and differentiation. Dysregulation of H2AK119 in NTDs was involved in downregulation of NTC-related genes under folate deficiency. A better understanding of H2AK119ub1 may improve identification and diagnosis of NTDs. These results showed that abnormal histone ubiquitination regulates neural precursor marker genes in NTDs, and it provides evidence of regulation of histone ubiquitination followed by folinic acid supplementation. Our study may indicate one mechanism by which folate may affect human neural tube disorders.
Neural tube defects are common congenital malformations resulting from the failure of NTC during early embryonic development. Maternal folate level is one of the best-studied dietary factors relative to NTC. We showed that H2A monoubiquitination (H2AK119ub1) plays a role in NTC. We found that the folate antagonist MTX induced H2AK119ub1 in a mouse NTD model and in mESC. Using ChIP-seq and RNA-seq assays, we demonstrated that an increase in H2AK119ub1 level downregulated the expression of the NTC-related genes Cdx2, Nes, Pax6, and Gata4 in mESCs under conditions of folate deficiency. We also showed that the E3 ligase Mdm2 was responsible for the MTX-induced increase in H2AK119ub1 and downregulated NTC-related genes. Furthermore, folic acid supplementation restored H2AK119ub binding to NTC-related genes. Downregulation of NTC-related genes was observed in brain tissues of fetuses with NTDs, and the maternal serum were all under low folate condition, along with aberrant H2AK119ub1 levels. This evidence suggests that folate deficiency contributes to the onset of NTDs by altering H2AK119ub1 and subsequently affecting expression of NTC-related genes. This may be a potential risk factor for NTDs in response to folate deficiency.
In this study, we showed that the folate antagonist MTX induced H2AK119ub1 in mouse ESCs. We detected that an increase in H2AK119ub1 level downregulated the expression of NTC-related genes, including Cdx2, Nes, Pax6, and Gata4 in cells under folate deficiency conditions. We also demonstrated that the E3 ligase Mdm2 is responsible for the MTX-induced increase in H2AK119ub1 and downregulated NTC-related genes. Surprisingly, Mdm2 is required for MTX-induced H2A ubiquitination and is recruited to the sites of DSB, which is dependent on DNA damage signaling kinase ATM. Furthermore, folic acid supplementation results in restored H2AK119ub binding to NTC-related genes. In addition, downregulation of NTC-related genes was observed in both brain tissue of mouse and human NTD cases, and low levels of folate and higher levels of H2AK119ub1 were found in the corresponding maternal serum samples. Our results provide strong evidence that folate deficiency induces aberrant H2AK119ub1, which is linked to abnormal expression of NTC-related genes and subsequently NTDs. This research further extends our understanding of aberrant epigenetic modification of NTC-related genes in NTDs.
C57BL/6 mice (6–8 weeks, 18–20 g), were provided by Jinmyung animal laboratory, and feed under SPF (Specific Pathogen Free) growing condition, approved facility on a 12-h light/dark cycle. Male and female C57BL/6 mice were fed on low folate diet for more than 4 weeks. Sexually matured individuals mated overnight; vaginal plug was detected at 8:00 am in the following morning, which designated as E0.5 days. NTDs mouse models were induced by intraperitoneal injection with 1.5 mg/kg (body weight) of MTX (Sigma, USA) on E7.5. On E13.5, pregnant mice were euthanized and killed by cervical dislocation. Embryos were dissected and placed in ice-cold tubes, DEPC-treated PBS. All procedures involving animal handling were in compliance with institutional guidelines on the care of experimental animals.
Cell culture and differentiation assays
Mouse embryonic stem cells (mESCs) Sv/129 were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 0.1 mM β-mercaptoethanol (Invitrogen, Carlsbad, USA), 0.1 mM non-essential amino acids (Invitrogen, Carlsbad, USA), 0.1 mM glutamate (Invitrogen, Carlsbad, USA), 15% fetal bovine serum (Gibco, USA), and 1000 U/ml mouse leukemia inhibitory factor (Millipore, Billerica, USA), cultured in culture dishes coated with 0.2% gelatin (Invitrogen, Carlsbad, USA). Cells were placed in humidified incubator with 37 °C, 5% CO2 passaged every 2–3 days. F9 cells were cultured in Dulbecco’s modified Eagle medium with 10% FBS. mESCs were suspended to form embryoid body (EB), with 20% serum. mESCs were treated with a concentration gradient of MTX for 12, 24, 48 h. Cells were incubated at 37 °C/5% CO2 and passaged every 2 days.
SiRNA Mdm2: 1-GGAACAAGAGACUCUGGUUTT; 2-AACCAGAGUCUCUUGUUCCTT.
The blots were incubated with the primary antibody, mouse anti- H2AK119ub monoclonal antibody (1:1500, CST, USA) and mouse anti-H2A monoclonal antibody (1:1,500,000, CST, USA) overnight at 4 °C, and then incubated with secondary anti-rabbit HRP-conjugated antibody (1:5000, CST, 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.
RNA-Seq and data analysis
Total RNA was extracted from cells with Trizol (Invitrogen) according to the manufacturer’s protocol and purified by mRNA enrichment. mRNA poly(A) tails then enriched with oligodT magnetic bead. rRNAs were removed. rRNA hybridized with DNA probe. RNaseH chose to digest DNA/RNA hybridization chain. DNase-digested DNA probe resulted in purified RNA. The cDNA libraries were constructed for each pooled sample with VAHTSTM Total RNA-seq (H/M/R) according to the manufacturer’s instructions. Final cDNA libraries were created by PCR purification and enrichment, then quantified by Agilent2200. The tagged cDNA libraries were pooled in equal ratio and used for 150 bp paired-end sequencing in a single lane of Illumina HiSeq Xten by NovelBio Corp. Laboratory. NovelBrain Cloud Analysis platform was used to analyse high-throughput sequencing data. All RNA-Seq reads were mapped to mouse genome-using TopHat2. Transcript abundance was quantified using Cufflinks and annotations from Ensembl release 70, and FPKM (fragments/kb of transcript/million fragments mapped) values were calculated. To minimize dispersion effect by low-FPKM values, all the FPKM values were modified by addition of 0.1 in log2 transformation. Gene ontology analysis for biological process of the selected genes was performed using Partek Genomic Suite (Ryoka systems).
ChIP-Seq and data analysis
Before CHIP-sequencing, samples were qualified by Agilent2200. Sample fragments and concentration have to be 100-500 bp and greater or equal to 1 ng/μl. 107 mESC cells were used as ChIP samples. ChIP was done with Simple ChIP Enzymatic chromatin IP kit (9003 s) 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 NovaSeq 6000. DNA libraries were prepared by NEB Ultra DNA library prep kit under Illumina guide. All ChIP-Seq reads were mapped to the mouse genome (mm9) using Bowtie2 with default parameters. Genomic profiles were generated using IGVtools and were viewed in Integrative Genomics Viewer (IGV). Peaks of H2AK119ub1 ChIP-Seq signals on genome were determined using MACS2 with false-discovery rate as 0.05. Each associated gene for the peaks was determined using Entrez gene annotation with in-house computer program, in which Ash1 l-target genes were defined as genes containing H2AK119ub1-peaks around transcription start site (TSS) within + /24 kb. Datasets for reads/kb/million mapped (RPKM) values of H2AK119ub1 in coding regions of each gene were normalized to 75th percentile.
Chromatin immunoprecipitation (ChIP) analysis
ChIP assays were performed using the SimpleChIP Enzymatic chromatinIP system (Cell Signaling, California, USA) following the manufacturer’s protocols. Chromatin was prepared, sonicated to DNA segments between 100 and 500 bp and then immunoprecipitated with Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb (CST, USA). The immunoprecipitated DNA was analyzed by RT-qPCR, which were performed using QuantStudio 7 Flex with SYBR Green detection. The primers used for ChIP assays were shown in Additional file 8: Table S7. Normal Rabbit IgG is used as negative controls in immunoprecipitations. The following equation was used to calculate percent input = 2% × 2^ (CT) 2% input sample − (CT) IP sample).
Total RNA was extracted using the Trizol reagent (Invitrogen, USA), first-strand synthesis was done with RevertAid First Strand cDNA Synthesis Kit (ABM, Canada). Maxima SYBR Green/ROX qPCR Master Mix (ABM, Canada) was used for RT-qPCR and the procedure was as follows: enzyme activation was done at (95 °C, 10 min) × 1 cycle; denature at (95 °C, 35 s) × 35 cycles; annealing at (60 °C, 60 s) x 35 cycles; melting curve at (95 °C, 15 s; 60 °C, 60 s; 95 °C, 15 s; 60 °C 15 s) × 1 cycles; primers were shown in Additional file 8: Table S7.
For detection of subcellular localization by immunofluorescence, cells were fixed in methanol under − 20° and permeabilized in 0.2% Triton X-100 (PBS) for 20 min, cells were incubated with the indicated Mdm2, H2AK119ub, γH2AX, and pATM antibodies (dilution 1:50; CST, Santa Cruze) for 16 h at 4 °C, followed by incubation with Alexa Fluor-conjugated secondary antibody (dilution 1:200; CST, USA) for 1 h at 25 °C. The nuclei were stained with DAPI (Sigma), and images were visualized with a Zeiss LSM 510 Meta inverted confocal microscope. Colocalization values were calculated from FV10-ASW 3.0 Viewer of Mdm2, H2AK119ub, gH2AX, and ATM.
The NanoString nCounter was used to detect the number of transcripts in human brain tissues. Total RNA was extracted following the manufacturer’s instructions (miRNeasy Mini Kit, Qiagen) and gene specific probes were designed by the manufacturer (NanoString Technologies). Hybridizations were carried out according to the nCounter Element 24-plex 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. Gene expression data were filtered using quality control (QC) criteria according to the manufacturer’s recommendations. Raw counts of QC-passed samples were normalized using three reference genes as internal controls (GAPDH, CLTC and GUSB). All QC and normalization procedures were performed using nSolver Analysis Software v2.0; all data were log2-transformed before further analysis. The Student’s t test was used to compare normalized expression values between normal and NTDs.
All the experiments were repeated independently at least twice, and data are presented as mean ± SD. Statistical significance was determined using the Student’s t test. A P value of < 0.05 was considered to be statistically significant and is presented as *P < 0.05 or **P < 0.01.
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 8 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.
We thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
TZ conceived the project and performed the project planning. PP performed the main experimental work. XYC performed human histone modification analysis. JY participated in mouse culture. PP and SW designed the experiments, analyzed the data and wrote the manuscript. All the authors read and approved the final manuscript.
We are grateful to all participating hospitals for their assistance in sample collection and recording of clinical information. This work was supported by CAMS initiative for Innovative Medicine (CAMS-I2M-1-008), The National Natural Science Foundation Projects (31571324), The National Natural Science Foundation Projects (81771584), Beijing municipal program of medical research (Grant No. 2016-04).
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The authors declare that they have no competing interests.
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