玉米多梳蛋白MEZ1调控组蛋白修饰和DNA甲基化的研究
发布时间：2019-03-16 18:52
【摘要】：多梳家族蛋白(polycomb group proteins)是首先在果蝇中发现的一大类染色质调节因子。PRC2(polycomb repressive complex 2)是其中最典型的一类复合体,在动物和植物中是高度保守的。对PRC2的研究在动物中最为广泛,自2000年以来在拟南芥中也有很多报道,但是对其在玉米生长发育中所发挥的作用却是了解甚少。本研究利用反向遗传学,通过Crispr-Cas9敲除玉米PRC2核心组分ME探究MEZ1对玉米籽粒发育的影响。我们发现,MEZ1是一个突变后有表型的母本表达的印记基因。携带母本allele的mez1/MEZ1在籽粒发育早期出现胚和胚乳的发育滞后,但是最终能够恢复到野生型的水平,暗示MEZ1可能具有类似于拟南芥ME4的自我调控(autoregulation)的机制。纯合的mez1/mez1的籽粒小、败育。结合生物化学手段,我们首次证实了玉米MEZ1和FIE1处在一个约700 kDa的PRC2复合体中。在mez1/mmez1纯合突变体中,胚乳中的H3K27me3水平几乎完全丧失,证实了 MEZ1是一个有功能的H3K27me3甲基转移酶。虽然籽粒中还同时表达MEZ1的两个旁系同源物MEZ2和MEZ3,但是它们不能弥补MEZ1缺失带来的影响,表明MEZ1在胚乳中发挥的作用是特异的。对转录组数据分析发现MEZ1调控了胚乳细胞化、胁迫响应、激素响应和模式形成(patterning)等过程;印记基因中的 PEG(paternally expressed imprinted genes)受 MEZ1 影响最大。在 BETL中特异表达的基因,受到MEZ1的间接调控。MEZ1可能是上游调控BETL特异表达基因的通用因子。H3K27me3的ChIP-seq结果表明,MEZ1-PRC2的靶基因表达量低,且具有组织表达特异性。靶基因富集在基因转录调控、细胞壁相关、胁迫响应、脂肪酸代谢和氮代谢。同时一些家族的的转录因子基因也有富集。在胚乳发育过程中起着重要调控作用的MADS家族基因也是MEZ1的偏好靶点。全基因组亚硫酸氢盐测序(BS-seq)发现与野生型相比,虽然整体上基因区的DNA甲基化水平升高幅度小,但是受MEZ1调控的转录因子和PEG的甲基化水平变化表现明显。MEZ1可能是CHH island的上游调控因子。MEZ1主要影响的是TE上的DNA甲基化。mez1中TE上CG甲基化水平降低,而CHG和CHH甲基化水平升高。MEZ1可能起着抑制TE内部CHH甲基化的作用。综上,我们的研究揭示了 MEZ1在玉米籽粒发育中不可或缺的作用。
[Abstract]:(polycomb group proteins) is a major chromatin regulatory factor first found in Drosophila melanogaster. PRC2 (polycomb repressive complex 2 is one of the most typical complexes, which is highly conserved in animals and plants. The study of PRC2 is the most widespread in animals and has been reported in Arabidopsis since 2000. However, little is known about its role in the growth and development of maize. In this study, reverse genetics was used to explore the effect of MEZ1 on maize kernel development by Crispr-Cas9 knockout of ME, the core component of maize PRC2. We found that MEZ1 is a mutant imprinted gene expressed by the maternal parent. The development of embryo and endosperm of mez1/MEZ1 with maternal allele appeared lag in early grain development, but finally recovered to the level of wild type, suggesting that MEZ1 may have the mechanism of self-regulation (autoregulation) similar to Arabidopsis ME4. The seeds of homozygous mez1/mez1 were small and aborted. Combined with biochemical methods, we confirmed for the first time that maize MEZ1 and FIE1 are in a PRC2 complex of about 700 kDa. In the homozygous mutant of mez1/mmez1, the level of H3K27me3 in endosperm was almost completely lost, which confirmed that MEZ1 was a functional H3K27me3 methyltransferase. Although MEZ2 and MEZ3, two side-line homologues of MEZ1, were expressed simultaneously in grains, they could not make up for the effect of MEZ1 deletion, which indicated that MEZ1 played a special role in endosperm. Analysis of transcriptional data showed that MEZ1 regulated endosperm cellulation, stress response, hormone response and pattern formation of (patterning), and PEG (paternally expressed imprinted genes) in imprinted genes was most affected by MEZ1. The gene specifically expressed in BETL is indirectly regulated by MEZ1. MEZ1 may be a general factor for upstream regulation of BETL-specific expression gene. ChIP-seq results of H3K27me3 show that the target gene expression of MEZ1-PRC2 is low and has tissue-specific expression. Target genes are enriched in gene transcription regulation, cell wall correlation, stress response, fatty acid metabolism and nitrogen metabolism. At the same time, some family transcription factor genes are also enriched. MADS family genes, which play an important role in endosperm development, are also the preferred targets of MEZ1. Genome-wide bisulfite sequencing (BS-seq) found that although the overall DNA methylation level in the gene region was slightly higher than that in the wild type, MEZ1 may be the upstream regulatory factor of CHH island. The main effect of MEZ1 is DNA methylation on TE, and the decrease of CG methylation level on TE in MEZ1, but the change of transcription factor and methylation level of PEG regulated by MEZ1 is obvious. Mez-1 may play an important role in inhibiting CHH methylation in TE. CHG and CHH methylation levels are increased. In summary, our research reveals that MEZ1 plays an indispensable role in maize kernel development.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S513
本文编号：2441876
[Abstract]:(polycomb group proteins) is a major chromatin regulatory factor first found in Drosophila melanogaster. PRC2 (polycomb repressive complex 2 is one of the most typical complexes, which is highly conserved in animals and plants. The study of PRC2 is the most widespread in animals and has been reported in Arabidopsis since 2000. However, little is known about its role in the growth and development of maize. In this study, reverse genetics was used to explore the effect of MEZ1 on maize kernel development by Crispr-Cas9 knockout of ME, the core component of maize PRC2. We found that MEZ1 is a mutant imprinted gene expressed by the maternal parent. The development of embryo and endosperm of mez1/MEZ1 with maternal allele appeared lag in early grain development, but finally recovered to the level of wild type, suggesting that MEZ1 may have the mechanism of self-regulation (autoregulation) similar to Arabidopsis ME4. The seeds of homozygous mez1/mez1 were small and aborted. Combined with biochemical methods, we confirmed for the first time that maize MEZ1 and FIE1 are in a PRC2 complex of about 700 kDa. In the homozygous mutant of mez1/mmez1, the level of H3K27me3 in endosperm was almost completely lost, which confirmed that MEZ1 was a functional H3K27me3 methyltransferase. Although MEZ2 and MEZ3, two side-line homologues of MEZ1, were expressed simultaneously in grains, they could not make up for the effect of MEZ1 deletion, which indicated that MEZ1 played a special role in endosperm. Analysis of transcriptional data showed that MEZ1 regulated endosperm cellulation, stress response, hormone response and pattern formation of (patterning), and PEG (paternally expressed imprinted genes) in imprinted genes was most affected by MEZ1. The gene specifically expressed in BETL is indirectly regulated by MEZ1. MEZ1 may be a general factor for upstream regulation of BETL-specific expression gene. ChIP-seq results of H3K27me3 show that the target gene expression of MEZ1-PRC2 is low and has tissue-specific expression. Target genes are enriched in gene transcription regulation, cell wall correlation, stress response, fatty acid metabolism and nitrogen metabolism. At the same time, some family transcription factor genes are also enriched. MADS family genes, which play an important role in endosperm development, are also the preferred targets of MEZ1. Genome-wide bisulfite sequencing (BS-seq) found that although the overall DNA methylation level in the gene region was slightly higher than that in the wild type, MEZ1 may be the upstream regulatory factor of CHH island. The main effect of MEZ1 is DNA methylation on TE, and the decrease of CG methylation level on TE in MEZ1, but the change of transcription factor and methylation level of PEG regulated by MEZ1 is obvious. Mez-1 may play an important role in inhibiting CHH methylation in TE. CHG and CHH methylation levels are increased. In summary, our research reveals that MEZ1 plays an indispensable role in maize kernel development.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S513
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1 宋宁;玉米多梳蛋白MEZ1调控组蛋白修饰和DNA甲基化的研究[D];中国农业大学;2017年
，本文编号：2441876
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