Functional Analysis Of H3K27 Methyltransferase In Rice

Chromatin is the basis of epigenetics. Chromatin structure is dynamic due to various epigenetic modifications. Histone modification is one of the modifications of chromatin which can alter chromatin structure to regulate gene expression. Trimethylation of histone H3 lysine 27 (H3K27me3), one of most studied histone modifications, represses gene expression by inducing chromatin condensation. E(z) gene is one of the key subunits of Polycomb Repressive Complex2 (PRC2) which is highly conserved in plants and animals is required for H3K27me3 on target genes. There exist direct or indirect interactions between different chromatin modifications, the underlining mechanism is not clear. In this thesis we studied rice E(z) genes SDG711 and SDG718 for their roles in genome-wide H3K27me3 and plant development and their implication in DNA methylation.Our data show that SDG711 and SDG718 display different expression patterns in different organs revealed by different methods, and also demonstrate that the genes have different response to day length. To study the function of the two genes, we produced over-expression and RNAi plants of SDG711 and RNAi of SDG718. The effect of the transgenic genes on histone methylation was analyzed by Western blotting with histones isolated from leaves of the transgenic plants. The analysis revealed that compared to wild type the levels of H3K27me2/3 were lower in SDG711 RNAi lines, but higher in the SDG711 gain-of-function mutant and over-expression plants, suggesting that SDG711 was required for the overall H3K27me2/3 in rice.Over-expression and down-regulation of SDG711 respectively repressed and promoted flowering in LD, but had no effect in SD. By contrast, down-regulation of SDG718 had no effect in LD but delayed flowering in SD. SDG711 and SDG718 repressed OsLF (a repressor of Hdl) respectively in LD and SD, leading to a higher expression of Hdl thus late flowering in LD and early flowering in SD. SDG711 was also found to be involved in the repression of Ehdl in LD. SDG711 was shown to directly target to OsLF and Ehdl loci to mediate H3K27me3 and gene repression. The function of the rice E(z) genes in LD repression and SD promotion of flowering suggests that PRC2-mediated epigenetic repression of gene expression is involved in the accurate photoperiod control of rice flowering.We also found that over-expression of SDG711 can increase the number of spikelets per panicle and the number of primary branches. Mutant of JMJ703, a histone H3K4me3 demethylase, displays the same phenotype as that of SDG711-RNAi plants. To study the roles of SDG711 and JMJ703 in panicle developmental process, we produced SDG711 RNAi in jmj703. SDG711 RNAi in jmj 703 produces a more severe panicle phenotype. Analysis of gene expression revealed that the expression level of OsCKX2, encoding cytokinin oxidase, is significantly changed by during panicle development. The expression tendency of OsCKX2 opposite to that of JMJ703 and SDG711, Up- and down-regulation of SDG711 and JMJ703 affect the panicle development by affects cytokinin accumulation in the panicle meristem. ChIP analysis of OsCKX2 in SDG711 arid JMJ703 transgenic plants showed that SDG711 and JMJ703 directly targeted to OsCKX2 loci to mediate H3K27me3 increase and H3K4me3 decrease and gene repression. We also found that OsCKX2 have different expression levels in the leaves and young panicle also associated with H3K27me3 and H3K4me3. Therefore, we hypothesized that H3K27me3 and H3K4me3 antagonistically regulate OsCKX2 to regulate rice developmental process.In addition, we have shown that SDG711 interacts with DNA methyltransferase by Yeast two-hybrid, suggesting that H3K27me3 and DNA methylation may interact with each other to regulate gene expression. ChIP analysis of genome-wide H3K27me3 and bisulfate-sequencing analysis of genome-wide DNA methylation combined with RNA-seq of SDG711 transgenic plants are being analyzed to study the function of SDG711 in chromatin modification and gene expression.