RA诱导P19细胞神经分化中组蛋白去甲基酶Jmjd3对Mash1基因表达的调控作用
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摘要
表观遗传学是指在基因的DNA序列不发生改变的情况下,基因的表达水平与功能发生改变,并产生可遗传的表型组,主要包括DNA甲基化、组蛋白修饰、染色质重塑、RNA介导的调控。组蛋白修饰是表观遗传学中很重要的调控方式。包括组蛋白的甲基化、乙酰化、磷酸化、泛素化等,它对基因的激活和抑制起着重要作用。
Mashl是碱性螺旋-环-螺旋(basic helix-loop-helix, bHLH)家族的一个重要蛋白,在神经分化过程中起关键作用。我们以全反式维甲酸(all-trans retinoic acid, atRA,简称RA)诱导小鼠胚胎瘤(embryonal carcinoma, EC) P19细胞作为神经分化的模型,探讨了表观遗传学特别是组蛋白修饰对神经分化关键基因Mash1的调控机制。
1.RA诱导P19细胞神经分化模型的建立
用0.5μM RA诱导培养P19细胞,细胞逐渐开始聚集成团状,4天后,将细胞重新置于正常培养基中培养,细胞出现类似神经元状的形态,细胞开始出现神经元分化的趋势。Western实验发现神经分化标志基因β-Tubulin在RA诱导后表达逐渐增加,表明RA诱导P19神经分化模型初步建立。
2.神经分化关键基因Mashl表达的检测
我们使用实时荧光定量PCR和1Western检测发现Mashl mRNA和蛋白在RA诱导2天后表达水平开始明显增加。免疫荧光实验发现,Mashl蛋白在RA诱导后表达增加。利用染色质免疫沉淀(Chromatin immunoprecipitation, ChIP)的方法检测RNA聚合酶Ⅱ(RNAPolⅡ)在基因组中Mash1基因区域结合情况发现,RA诱导后RNAPolⅡ在Mash1启动子区结合增加,表明Mash1基因开始被激活。
3.RA诱导P19神经分化过程中Mash1基因启动子区核心组蛋白修饰变化
组蛋白共价修饰可动态调节染色质的结构,对基因转录具有重要的调控作用。核小体核心组蛋白N端尾部以及核心区的多个氨基酸残基可发生乙酰化、磷酸化、甲基化、泛素化、SUMO化、ADP核糖基化等翻译后修饰。这些修饰可以改变核小体中组蛋白与DNA或组蛋白与其他蛋白的相互作用,引起基因转录激活或关闭。
我们使用ChIP的方法检测了RA诱导后Mash1基因上游调控区以及编码区相应的组蛋白修饰状态变化。结果发现,在RA诱导后Mash1基因区域组蛋白H3K9和H3K14乙酰化水平逐渐上升,特别是在RA诱导第四天变化显著。组蛋白甲基化也是表观遗传的一个关键调控因素。组蛋白特异赖氨酸位点甲基化对于调控基因的转录有重要作用。ChIP结构显示：H3K9me3水平无明显变化；H3K4me3在诱导过程中逐步增加,在启动子近端区域和外显子区尤为明显；H3K27me3水平在诱导后逐渐降低,特别在第3天大幅下降；这些结果提示,组蛋白修饰在Mashl表达激活过程中发挥重要作用。
1.组蛋白赖氨酸H3K27去甲基化酶Jmjd3促进Mashl基因表达
组蛋白赖氨酸H3K27me3去甲基化酶Jmjd3在RA诱导后mRNA和蛋白表达水平增加。神经分化关键基因Mashl的H3K27me3修饰水平在RA诱导后明显下降。过表达Jmjd3能够促进Mashl表达增加,而抑制Jmjd3能使Mashl表达降低。同时,双荧光素酶实验证明Jmjd3能够促进Mashl启动子活性。利用ChIP技术,我们发现RA诱导后,Jmjd3在Mashl启动子上的结合增加。去甲基酶活性突变的Jmjd3不能促进Mashl表达和启动子活性。以上的结果证明,组蛋白赖氨酸去甲基化酶Jmjd3能够促进神经分化关键基因Mashl的表达并且这种作用依赖于其去甲基化酶的功能。
Epigenetics is the study of stable alternation in gene expression and function without changing DNA sequence and leading to inheritable phenotype, mainly including DNA methylation, histone modification, chromatin remodeling and non-coding RNA-mediated regulation. Histone modifications including histone methylation, acetylation, phosphorylation and ubiquitination play crucial roles in gene activation and inactivation.
Mashl, one of bHLH protein, play a crucial role in neuronal differentiation. To investigate the epigenetic regulation especially histone modification of Mash1 gene during neurogenesis, RA (all-trans retinoic acid)-induced P19 embryonal carcinoma cells were used as a neuronal differentiation model.
1. The establishment of RA-incduced neuronal differentiation of P19 cell model
P19 cells were exposed to 0.5μM RA, cells began to aggregate. After 4 days, P19 cells cultured in medium without RA for another 3 or 4 days to differentiate into neuron-like cells. Western blotting assay showed that neuronal marker geneβ-Tubulin increased gradually with RA induced. These results indicated that RA-incduced neural differentiation of P19 cells model was established.
2. Analysis of neuronal marker gene Mashl expression
Quantitative RT-PCR and Western blotting assay showed that Mashl greatly increased after 2 days of RA treatment at mRNA and protein levels. Immunofluoresense assay showed that Mashl increased after RA induction. Simultaneously, RNA polymeraseⅡ(RNA PolⅡ) was enriched on the promoter of Mashl analyzed by chromatin immunoprecipitation assay (ChIP). These results suggested that the expression of Mashl gene was activated by RA induction in P19 cells.
3. Alternation of histone modifications in Mash1 gene region with RA treatment in P19 cells
Covalent modifications of histones are key regulations of chromatin dynamics, and play pivotal roles in genes regulation. The N-terminal tail of core histones, as well as positions in the globular domain, can carry post-translational modifications such as acetylation, phosphorylation, ubiquitination, methylation, SUMOylation and ADP-ribosylation. Such modifications can alter DNA-histone interactions within and between nucleosomes and affect gene activation.
ChIP assays were performed to investigate changes of histone modifications in Mash1 gene region during RA treatment. It was found that acetylation of histone H3K9 and H3K14 significantly increased, especially in the 4th day. Methylation is another important modification of histones, which is considered pivotal in epigenetic regulation. We analysed the methylation of histone H3K9me3, H3K4me3 and H3K27me3 in P19 cells during RA treatment by ChIP assays. It was found that H3K9me3 had no significant change. H3K4me3 increased steadily, especially in the proximal promoter region and exon region of the gene. H3K27me3 decreased gradually especially after 3rd day of RA treatment. These results suggested histone modifications play privotal roles in Mashl activation.
4. Histone lysine H3K27 demethylase Jmjd3 enhanced Mashl expression.
The mRNA and protein expression of Jmjd3, histone lysine H3K27 demethylase increased after RA treated while H3K27me3 level of Mashl, neuronal differentiation marker gene, decreased dramatically. Overexpression of Jmjd3 enhanced Mashl expression. Knockdown of Jmjd3 inhibited Mashl expression. Dual-luciferase reporter assay showed that Jmjd3 enhanced Mashl promoter activity. ChIP assay suggested remarkably elevated recruitment of Jmjd3 at the proximal promoter of Mash1 on the 3rd day of RA treatment led to an increased promoter activity of Mashl. Demethylase mutant of Jmjd3 abolished Mashl activation. These results suggested elevation of Jmjd3 expression and its demethylase activity was required to enhance the expression and promoter activity of Mashl in RA-induced neuronal differentiation of P19 cells. Demethylase activity of Jmjd3 was the prerequisite in RA induced Mashl expression.
Mashl是碱性螺旋-环-螺旋(basic helix-loop-helix, bHLH)家族的一个重要蛋白,在神经分化过程中起关键作用。我们以全反式维甲酸(all-trans retinoic acid, atRA,简称RA)诱导小鼠胚胎瘤(embryonal carcinoma, EC) P19细胞作为神经分化的模型,探讨了表观遗传学特别是组蛋白修饰对神经分化关键基因Mash1的调控机制。
1.RA诱导P19细胞神经分化模型的建立
用0.5μM RA诱导培养P19细胞,细胞逐渐开始聚集成团状,4天后,将细胞重新置于正常培养基中培养,细胞出现类似神经元状的形态,细胞开始出现神经元分化的趋势。Western实验发现神经分化标志基因β-Tubulin在RA诱导后表达逐渐增加,表明RA诱导P19神经分化模型初步建立。
2.神经分化关键基因Mashl表达的检测
我们使用实时荧光定量PCR和1Western检测发现Mashl mRNA和蛋白在RA诱导2天后表达水平开始明显增加。免疫荧光实验发现,Mashl蛋白在RA诱导后表达增加。利用染色质免疫沉淀(Chromatin immunoprecipitation, ChIP)的方法检测RNA聚合酶Ⅱ(RNAPolⅡ)在基因组中Mash1基因区域结合情况发现,RA诱导后RNAPolⅡ在Mash1启动子区结合增加,表明Mash1基因开始被激活。
3.RA诱导P19神经分化过程中Mash1基因启动子区核心组蛋白修饰变化
组蛋白共价修饰可动态调节染色质的结构,对基因转录具有重要的调控作用。核小体核心组蛋白N端尾部以及核心区的多个氨基酸残基可发生乙酰化、磷酸化、甲基化、泛素化、SUMO化、ADP核糖基化等翻译后修饰。这些修饰可以改变核小体中组蛋白与DNA或组蛋白与其他蛋白的相互作用,引起基因转录激活或关闭。
我们使用ChIP的方法检测了RA诱导后Mash1基因上游调控区以及编码区相应的组蛋白修饰状态变化。结果发现,在RA诱导后Mash1基因区域组蛋白H3K9和H3K14乙酰化水平逐渐上升,特别是在RA诱导第四天变化显著。组蛋白甲基化也是表观遗传的一个关键调控因素。组蛋白特异赖氨酸位点甲基化对于调控基因的转录有重要作用。ChIP结构显示：H3K9me3水平无明显变化；H3K4me3在诱导过程中逐步增加,在启动子近端区域和外显子区尤为明显；H3K27me3水平在诱导后逐渐降低,特别在第3天大幅下降；这些结果提示,组蛋白修饰在Mashl表达激活过程中发挥重要作用。
1.组蛋白赖氨酸H3K27去甲基化酶Jmjd3促进Mashl基因表达
组蛋白赖氨酸H3K27me3去甲基化酶Jmjd3在RA诱导后mRNA和蛋白表达水平增加。神经分化关键基因Mashl的H3K27me3修饰水平在RA诱导后明显下降。过表达Jmjd3能够促进Mashl表达增加,而抑制Jmjd3能使Mashl表达降低。同时,双荧光素酶实验证明Jmjd3能够促进Mashl启动子活性。利用ChIP技术,我们发现RA诱导后,Jmjd3在Mashl启动子上的结合增加。去甲基酶活性突变的Jmjd3不能促进Mashl表达和启动子活性。以上的结果证明,组蛋白赖氨酸去甲基化酶Jmjd3能够促进神经分化关键基因Mashl的表达并且这种作用依赖于其去甲基化酶的功能。
Epigenetics is the study of stable alternation in gene expression and function without changing DNA sequence and leading to inheritable phenotype, mainly including DNA methylation, histone modification, chromatin remodeling and non-coding RNA-mediated regulation. Histone modifications including histone methylation, acetylation, phosphorylation and ubiquitination play crucial roles in gene activation and inactivation.
Mashl, one of bHLH protein, play a crucial role in neuronal differentiation. To investigate the epigenetic regulation especially histone modification of Mash1 gene during neurogenesis, RA (all-trans retinoic acid)-induced P19 embryonal carcinoma cells were used as a neuronal differentiation model.
1. The establishment of RA-incduced neuronal differentiation of P19 cell model
P19 cells were exposed to 0.5μM RA, cells began to aggregate. After 4 days, P19 cells cultured in medium without RA for another 3 or 4 days to differentiate into neuron-like cells. Western blotting assay showed that neuronal marker geneβ-Tubulin increased gradually with RA induced. These results indicated that RA-incduced neural differentiation of P19 cells model was established.
2. Analysis of neuronal marker gene Mashl expression
Quantitative RT-PCR and Western blotting assay showed that Mashl greatly increased after 2 days of RA treatment at mRNA and protein levels. Immunofluoresense assay showed that Mashl increased after RA induction. Simultaneously, RNA polymeraseⅡ(RNA PolⅡ) was enriched on the promoter of Mashl analyzed by chromatin immunoprecipitation assay (ChIP). These results suggested that the expression of Mashl gene was activated by RA induction in P19 cells.
3. Alternation of histone modifications in Mash1 gene region with RA treatment in P19 cells
Covalent modifications of histones are key regulations of chromatin dynamics, and play pivotal roles in genes regulation. The N-terminal tail of core histones, as well as positions in the globular domain, can carry post-translational modifications such as acetylation, phosphorylation, ubiquitination, methylation, SUMOylation and ADP-ribosylation. Such modifications can alter DNA-histone interactions within and between nucleosomes and affect gene activation.
ChIP assays were performed to investigate changes of histone modifications in Mash1 gene region during RA treatment. It was found that acetylation of histone H3K9 and H3K14 significantly increased, especially in the 4th day. Methylation is another important modification of histones, which is considered pivotal in epigenetic regulation. We analysed the methylation of histone H3K9me3, H3K4me3 and H3K27me3 in P19 cells during RA treatment by ChIP assays. It was found that H3K9me3 had no significant change. H3K4me3 increased steadily, especially in the proximal promoter region and exon region of the gene. H3K27me3 decreased gradually especially after 3rd day of RA treatment. These results suggested histone modifications play privotal roles in Mashl activation.
4. Histone lysine H3K27 demethylase Jmjd3 enhanced Mashl expression.
The mRNA and protein expression of Jmjd3, histone lysine H3K27 demethylase increased after RA treated while H3K27me3 level of Mashl, neuronal differentiation marker gene, decreased dramatically. Overexpression of Jmjd3 enhanced Mashl expression. Knockdown of Jmjd3 inhibited Mashl expression. Dual-luciferase reporter assay showed that Jmjd3 enhanced Mashl promoter activity. ChIP assay suggested remarkably elevated recruitment of Jmjd3 at the proximal promoter of Mash1 on the 3rd day of RA treatment led to an increased promoter activity of Mashl. Demethylase mutant of Jmjd3 abolished Mashl activation. These results suggested elevation of Jmjd3 expression and its demethylase activity was required to enhance the expression and promoter activity of Mashl in RA-induced neuronal differentiation of P19 cells. Demethylase activity of Jmjd3 was the prerequisite in RA induced Mashl expression.
引文
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1. Martin C, Zhang Y. The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 2005,6:838849
2. Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA et al. Histone demethylation mediated by the nuclear amine oxidase homo log LSD1. Cell 2004,119:941953
3. Tsukada Y, Fang J, Erdjument-Bromage H, Warren ME, Borchers CH, Tempst P, Zhang Y. Histone demethylation by a family of JmjC domain-containing proteins. Nature 2006,439:811816
4. Stavropoulos P, Blobel G, Hoelz A. Crystal structure and mechanism of human lysine-specific demethylase-1. Nat Struct Mol Biol 2006,13:626632
5. Lee MG, Wynder C, Bochar DA, Hakimi MA, Cooch N, Shiekhattar R. Functional interplay between histone demethylase and deacetylase enzymes. Mol Cell Biol 2006,26:63956402
6. Holbert MA, Marmorstein R. Structure and activity of enzymes that remove histone modifications. Curr Opin Struct Biol 2005,15:673680
7. Metzger E, Wissmann M, Yin N, Muller JM, Schneider R, Peters AH, Gunther T et al. LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 2005,437:436439
8. Di Stefano, L.Ji, J. Y.Moon, N. S.Herr, A Dyson, N. Mutation of Drosophila Lsdl disrupts H3-K4 methylation, resulting in tissue-specific defects during development Curr Biol,2007,17:808-12
9. Rudolph, T.Yonezawa, M.Schmidt, A.Jenuwein, T.Reuter, G. Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3.Mol Cell,2007,26:103-15
10. Lan, F.Zaratiegui, M.Villen, Shi, Y.S. pombe LSD1 homologs regulate heterochromatin propagation and euchromatic gene transcription. Mol Cell,2007,26:89-101
11. Clissold, P. M.Ponting, C. P. JmjC:cupin metalloenzyme-like domains in jumonji, hairless and phospholipase A2beta. Trends Biochem Sci.2001,26:7-9
12. Ayoub, N.Noma, K.Isaac, S.Kahan, T.Grewal, S. I.Cohen, A. A novel jmjC domain protein modulates heterochromatization in fission yeast. Mol Cell Biol.2003,23:4356-70
13. Gray, S. G.Iglesias, A. H.Kokkotou, E.Dangond, F. Functional characterization of JMJD2A, a histone deacetylase-and retinoblastoma-binding protein. J Biol Chem. 2005,280:28507-18
14. Laumonnier, F.Holbert, S.Hamel, B. C.Briault, S. Mutations in PHF8 are associated with X linked mental retardation and cleft lip/cleft palate.J Med Genet.2005,42:780-6
15. Metzger, E.Wissmann, M.Schule, R. Histone demethylation and androgen-dependent transcription. Curr Opin Genet Dev.2006,16:513-7
16. Yamane, K.Toumazou, C.Tsukada, Y.Erdjument-Bromage, H.Tempst, P.Wong, J.Zhang, Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor.cell,125:483-95
17. Lee, N.Zhang, J.Klose, R. J.Erdjument-Bromage, H.Tempst, P.Jones, R. S.Zhang, Y. The trithorax-group protein Lid is a histonc H3 trimethyl-Lys4 demethylase. Nat Struct Mol Biol,2007,1545-9993
18. Xiang, Y.Zhu, Z.A. P.Chen, C. D. JARID1B is a histone H3 lysine 4 demethylase up-regulated in prostate cancer. Proc Natl Acad Sci U S A,2007,104:19226-31
19. Lockman K, Taylor JM, Mack CP. The histone demethylase, Jmjdla, interacts with the myocardin factors to regulate SMC differentiation marker gene expression. Circ Res.2007 Dec 7;101(12):e115-23. Epub 2007 Nov 8
20. Okada Y, Scott G, Ray MK, Mishina Y, Zhang Y. Histone demethylase JHDM2A is critical for Tnpl and Prml transcription and spermatogenesis. Nature. 2007 Nov 1;450(7166):119-23. Epub 2007 Oct 17
21. Loh YH, Zhang W, Chen X, George J, Ng HH.Jmjdla and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells. Genes Dev.2007 Oct 15;21(20):2545-57.
22. Jepsen K, Solum D, Zhou T, McEvilly RJ, Kim HJ, Glass CK, Hermanson O, Rosenfeld MG. SMRT-mediated repression of an H3K27 demethylase in progression from neural stem cell to neuron.。 Nature.2007 Nov 15;450(7168):415-9. Epub 2007 Oct 10.
23. Smith, E. R. Lee, M. G.Winter, B.Drosophila UTX is a histone H3 Lys27 demethylase that colocalizes with the elongating form of RNA polymerase Ⅱ Mol Cell Biol,2008,28,3