Epigenetic regulation mechanism of folic acid deficiency inhibiting <italic style="font-style: italic">Brachyury</italic> expression and its effect on differentiation of embryoid bodies

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Epigenetic regulation mechanism of folic acid deficiency inhibiting Brachyury expression and its effect on differentiation of embryoid bodies

Basic Medicine | 更新时间：2026-02-13

- Epigenetic regulation mechanism of folic acid deficiency inhibiting Brachyury expression and its effect on differentiation of embryoid bodies
- Journal of Army Medical University Vol. 44, Issue 14, Pages: 1401-1409(2022)
- 作者机构：
  
  1.100020 北京，首都儿科研究所，儿童发育营养组学北京市重点实验室
  2.100026 北京，朝阳区妇幼保健院儿科
  3.266033 山东青岛，青岛市海慈医疗集团泌尿外科
- 作者简介：
- 基金信息：
  
  the National Natural Science Foundation for Young Scholars of China(82001555)
- DOI：10.16016/j.2097-0927.202201119
  CLC： R321.4;R345.638;R394.1
- Received：14 January 2022，
  
  Revised：2022-03-16，
  
  Published：30 July 2022
- 稿件说明：
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Shaoyan CHANG, Jiuye GUO, Yan CHEN, et al. Epigenetic regulation mechanism of folic acid deficiency inhibiting Brachyury expression and its effect on differentiation of embryoid bodies[J]. Journal of Army Medical University, 2022, 44(14): 1401-1409.
DOI：

Shaoyan CHANG, Jiuye GUO, Yan CHEN, et al. Epigenetic regulation mechanism of folic acid deficiency inhibiting Brachyury expression and its effect on differentiation of embryoid bodies[J]. Journal of Army Medical University, 2022, 44(14): 1401-1409. DOI： 10.16016/j.2097-0927.202201119.

摘要

目的

2

探讨叶酸缺乏抑制发育关键基因

Brachyury

表达的表观调控机制及对拟胚体(embryoid bodies，EBs)分化的影响。

方法

2

建立叶酸缺乏的小鼠胚胎干细胞(embryonic stem cell，ESCs)分化为EBs的细胞模型。根据叶酸浓度分为3组：正常组，叶酸缺乏组和叶酸补充组。采用massARRAY和RT-PCR分别检测EBs中

Brachyury

基因的甲基化和表达水平，明确叶酸缺乏对

Brachyury

基因表达的影响；RT-PCR和ChIP-qPCR分别检测DNA甲基化酶的表达和组蛋白甲基化修饰，探讨叶酸缺乏对

Brachyury

基因的表观调控机制；检测多潜能markers和三胚层markers的表达，探讨发育关键基因

Brachyury

表达异常对EBs分化的影响。

结果

2

① 在叶酸缺乏的ESCs分化为EBs模型中

Brachyury

表达显著降低，叶酸缺乏组的相对表达量为正常组的(0.19±0.08)倍，叶酸补充后上升至正常组的(0.62±0.14)倍。EBs中

Brachyury

基因启动子区甲基化

水平从正常组的(30.75±0.50)%升高至叶酸缺乏组的(37.93±1.24)%，补充叶酸后则降低至(31.88±2.67)%。②DNA从头甲基化酶

DNMT

3

b

表达升高，叶酸缺乏组其相对表达量为正常组的(1.59±0.31)倍。叶酸缺乏的EBs中H3K27me3修饰增加，正常组、叶酸缺乏组、叶酸补充组的相对富集程度分别为(0.22±0.03)、(0.32±0.02)、(0.24±0.01)。③在叶酸缺乏ESCs分化为EBs后，细胞形态发生改变：EBs小球体积变小，形态不规则；叶酸补充后EBs小球体积接近正常，呈规则的球体结构。④ESCs多潜能性markers包括

Nanog

和

Sox

2表达显著上调，其相对表达量在叶酸缺乏组的EBs中分别为正常组的(3.18±0.95)和(4.23±1.59)倍；叶酸缺乏的EBs中外胚层marker

Fgf

5和中胚层marker

Gsc

表达量显著下调，分别为正常组的(0.42±0.04)倍和(0.61±0.20)倍。

结论

2

叶酸缺乏通过DNA甲基化和组蛋白H3K27甲基化的双重表观调控作用，引起

Brachyury

基因的表达显著下调，最终导致拟胚体EBs的分化受阻。

Abstract

Objective

2

To investigate the epigenetic regulation mechanism of folic acid deficiency in inhibition of the expression of

Brachyury

a key developmental gene

and its effect on the differentiation of embryoid bodies (EBs).

Methods

2

Firstly

the cell model of folic acid-deficient embryonic stem cells (ESCs) differentiated into EBs was established. According to the folate level

they were divided into normal group

folate deficiency group and folate supplement group. MassARRAY system and RT-PCR were used to detect the methylation and expression level of

Brachyury

gene in EBs to clarify the effect of the deficiency on the expression. RT-PCR and chromatin immunoprecipitation (ChIP)-qPCR were used to detect the expression of DNA methylase and histone methylation modification

respectively to explore the epigenetic regulation mechanism of folic acid deficiency on

Brachyury

expression. At the same time

the expression levels of pluripotent markers and triploblastic markers were detected to explore the effect of abnormal expression of

Brachyury

on EBs differentiation.

Results

2

① In the differentiation of ESCs into EBs under folic acid deficie

ncy

the expression level of

Brachyury

was decreased significantly

only 0.19±0.08 times of that in the normal group

and folate supplementation increased the level to 0.62±0.14 times of that of the normal group. The methylation level of promoter region of

Brachyury

in EBs was increased from (30.75±0.5)% in the normal group to (37.93±1.24)% in the folic acid-deficient group

and decreased to (31.88±2.67)% after folic acid supplementation. ② The expression level of

DNMT

3

b

DNA de novo methylase

in folic acid-deficiency group was increased by 1.59±0.31 times higher than that of normal group. H3K27me3 modification was enhanced in the EBs with folate deficiency

and the relative enrichment level was 0.22±0.03 in the normal group

0.32±0.02 in the folate deficiency group

and 0.24±0.01 in the folate supplement group. ③After the differentiation of folic acid-deficient ESCs into EBs

the cell morphology was significantly changed with the smaller EBs spheres and irregular shape. Folic acid supplementation resulted in normal volume and regular shape of EBs spheres. ④ ESCs pluripotent markers

Nanog

and

Sox

2

were significantly up-regulated

and their relative levels in EBs of folate deficiency group was 3.18±0.95 and 4.23±1.59 times higher than those of normal group. The expression of ectodermal marker

Fgf

5 and mesodermal marker

Gsc

in folic acid-deficient EBs were significantly down-regulated

which was 0.42±0.04 times and 0.61±0.20 times in folic acid-deficient EBs when compared with those in normal EBs

respectively.

Conclusion

2

Through the dual epigenetic regulation of DNA methylation and histone H3K27 methylation

folic acid deficiency causes significant down-regulation of

Brachyury

and ultimately leads to the blocked differentiation of EBs.

关键词

Keywords

references

BEKDASH R A. Earlylife nutrition and mental health: the role of DNA methylation[J]. Nutrients, 2021, 13(9): 3111. DOI: 10.3390/nu13093111.

CHANG S Y, JING J, SHANGGUAN S F, et al . The effect of folic acid deficiency on Mest/Peg1 in neural tube defects[J ] . Int J Neurosci, 2021, 131(5): 468-477. DOI: 10.1080/00207454.2020.1750386.

LI B Y, CHANG S Y, LIU C, et al . Low maternal dietary folate alters retrotranspose by methylation regulation in intra-uterine growth retardation (IUGR) fetuses in a mouse model[J ] . Med Sci Monit, 2019, 25: 3354-3365. DOI: 10.12659/MSM.914292.

WANG L, CHANG S, GUAN J, et al . Tissue-specific methy-lation of long interspersed nucleotide element-1 of Homo sapiens (L1Hs) during human embryogenesis and roles in neural tube defects[J ] . Curr Mol Med, 2015, 15(5): 497-507. DOI: 10.2174/1566524015666150630130229.

ZHANG Q, XUE P, LI H L, et al . Histone modification mapping in human brain reveals aberrant expression of histone H3 lysine 79 dimethylation in neural tube defects[J ] . Neurobiol Dis, 2013, 54: 404-413. DOI: 10.1016/j.nbd.2013.01.014.

CHANG S Y, WANG L, GUAN Y Q, et al . Long interspersed nucleotide element-1 hypomethylation in folate-deficient mouse embryonic stem cells[J ] . J Cell Biochem, 2013, 114(7): 1549-1558. DOI: 10.1002/jcb.24496.

XU Q H, XIE W. Epigenome in early mammalian development: inheritance, reprogramming and establishment[J]. Trends Cell Biol, 2018, 28(3): 237-253. DOI: 10.1016/j.tcb.2017.10.008.

LADSTÄTTER S, TACHIBANA K. Genomic insights into chromatin reprogramming to totipotency in embryos[J]. J Cell Biol, 2019, 218(1): 70-82. DOI: 10.1083/jcb.201807044.

KISPERTA, KOSCHORZ B, HERRMANN B G. The T protein encoded by Brachyury is a tissue-specific transcription factor[J]. Embo J, 1995, 14(19): 4763-4772.

BRUCE A E E, WINKLBAUER R. Brachyury in the gastrula of basal vertebrates[J]. Mech Dev, 2020, 163: 103625. DOI: 10.1016/j.mod.2020.103625.

POSTMA A V, ALDERS M, SYLVA M, et al . Mutations in the T (brachyury) gene cause a novel syndrome consisting of sacral agenesis, abnormal ossification of the vertebral bodies and a persistent notochordal canal[J ] . J Med Genet, 2014, 51(2): 90-97. DOI: 10.1136/jmedgenet-2013-102001.

BARRESI V, IENI A, BRANCA G, et al . Brachyury: a diagnostic marker for the differential diagnosis of chordoma and hemangioblastoma versus neoplastic histological mimickers[J ] . Dis Markers, 2014, 2014: 514753. DOI: 10.1155/2014/514753.

SHARIFNIA T, WAWER M J, CHEN T, et al . Small-molecule targeting of brachyury transcription factor addiction in chordoma[J ] . Nat Med, 2019, 25(2): 292-300. DOI: 10.1038/s41591-018-0312-3.

CHANG S Y, LU X L, WANG S, et al . The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects[J ] . FASEB J, 2019, 33(4): 4688-4702. DOI: 10.1096/fj.201801536R.

YAMAKOSHI K, SHIMODA N. De novo DNA methylation at the CpG island of the zebrafishno tail gene[J]. genesis, 2003, 37(4): 195-202. DOI: 10.1002/gene.10245.

STANCHEVA I, EL-MAARRI O, WALTER J, et al . DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos[J ] . Dev Biol, 2002, 243(1): 155-165. DOI: 10.1006/dbio.2001.0560.

JACKSON M, KRASSOWSKA A, GILBERT N, et al . Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells[J ] . Mol Cell Biol, 2004, 24(20): 8862-8871. DOI: 10.1128/MCB.24.20.8862-8871.2004.

KUMAR D, CINGHU S, OLDFIELD A J, et al . Decoding the function of bivalent chromatin in development and cancer[J ] . Genome Res, 2021, 31(12): 2170-2184. DOI: 10.1101/gr.275736.121.

LIU X Y, WANG C F, LI U W Q, et al . Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos[J ] . Nature, 2016, 537(7621): 558-562. DOI: 10.1038/nature19362.

FOSTER C T, DOVEY O M, LEZINA L, et al . Lysine-specific demethylase 1 regulates the embryonic transcriptome and CoREST stability[J ] . Mol Cell Biol, 2010, 30(20): 4851-4863. DOI: 10.1128/MCB.00521-10.

SCHLESINGER Y, STRAUSSMAN R, KESHET I, et al . Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer[J ] . Nat Genet, 2007, 39(2): 232-236. DOI: 10.1038/ng1950.

CHARLTON J, DOWNING T L, SMITH Z D, et al . Global delay in nascent strand DNA methylation[J ] . Nat Struct Mol Biol, 2018, 25(4): 327-332. DOI: 10.1038/s41594-018-0046-4.

GALUPA R, HEARD E. X-chromosome inactivation: new insights intoCis and trans regulation[J]. Curr Opin Genet Dev, 2015, 31: 57-66. DOI: 10.1016/j.gde.2015.04.002.

BRINKMAN A B, GU H C, BARTELS S J J, et al . Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk[J ] . Genome Res, 2012, 22(6): 1128-1138. DOI: 10.1101/gr.133728.111.

QIU A Q, ZHANG H, KENNEDY B K, et al . Spatio-temporal correlates of gene expression and cortical morphology across lifespan and aging[J ] . NeuroImage, 2021, 224: 117426. DOI: 10.1016/j.neuroimage.2020.117426.

TONELOTTO V, TRAPANI V, BRETAUD S, et al . Spatio-temporal expression and distribution of collagen VI during zebrafish development[J ] . Sci Rep, 2019, 9(1): 19851. DOI: 10.1038/s41598-019-56445-4.

LOLAS M, VALENZUELA P D T, TJIAN R, et al . Charting Brachyury-mediated developmental pathways during early mouse embryogenesis[J ] . Proc Natl Acad Sci USA, 2014, 111(12): 4478-4483. DOI: 10.1073/pnas.1402612111.

GENTSCH G E, OWENS N D L, MARTIN S R, et al . In vivo T-box transcription factor profiling reveals joint regulation of embryonic neuromesodermal bipotency[J ] . Cell Rep, 2013, 4(6): 1185-1196. DOI: 10.1016/j.celrep.2013.08.012.

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