Functional Analysis Of Histone Methyltransferase And Deacetylase In Rice

Histone modifications, including histone methylation, acetylation, phosphorylation, ubiqutination and others, play pivotal roles in plant development and gene regulation. There are few reports on the molecular mechanism of epigenetic regulation in monocots, especially in rice. Therefore it is of great significance to study the function of histone modification gene from rice.This study mainly focused on the histone methyltransferase (HMTs) and histone deacetylase (HDACs). The function and the roles of these genes in epigenetic regulation were investigated by bioinformatics, cloning, transformation and other methods. Main results are shown as following:1.12 SUVHs (Suppressor of variegation 3-9 homologue) histone methyltransferases and 18 HDACs (Histone deacetylase) were identified from rice. Sequence alignment and phylogenetic analysis of SUVHs and HDACs were performed to elucidate the evolution relationship of members within these two families. Gene structure analysis revealed that SUVH proteins contained three conserved domain including YDG (named after 3 conserved amino acids)-SRA (SET-and Ring finger-associated) and pre-SET, SET (named after 3 genes from Drosophila). To study the expression profiles of the rice SUVHs and HDAC genes, a serial Affimetrix microarray analysis of transcripts from various organs at different developmental stages were performed (http://crep.ncpgr.cn). The results revealed that most of the rice SVUH genes showed a similar expression pattern, whereas their expression levels in same tissues or organs were different.2. Over-expression and RNAi vectors of SUVHs and HDACs genes were constructed and transformed into rice to get transgenic plants.3. RNA level analysis indicated that there were the increased expression of each target genes in over-expression and decreased expression in RNAi transgenic plants.4. Down-regulation of SDG713 and SDG728 (SUVHs) led to the lethal dwarf plant and reduced seed thickness, respectively. The dwarf phenotype and twist flag leaf was caused by reduced expression level of HDA704, which were not complemented by treating with gibberellin (GA3).5. The expression pattern of the SDG713-GFP fusion protein showed that SDG713 was a widely expressed nuclear protein with high expressions in root tip, young stem, pistil and others tissues.6. SDG714 RNA interference induced abnormal stigma development and led to the increased miR159 RNA level and decreased miR164 RNA level in rice (indica).7. Western Blot results revealed that SDG728 was required to regulate H3K9me3 and SDG704, SDG726 and SDG727 participated in the regulation of H3K9me3.8. The proteins of GST-SDG728 and GST-SDG714 which were expressed and purified from E.coli were used to study the methyltransferase activity in vitro. Results showed that SDG728 could methylate calf core histones, but not the synthesized non-methylated histones, whereas SDG714 showed a methyltransferase activity on both types of sbustrates. This observation indicated that SDG728 has a trimethyltransferase activity.9. The results of McrBC-PCR denmostrated that DNA methylations of Tos17 and a Ty1-copia like retrotransposon (Os08g03880) were affected by SDG728 and other members such as SDG703, SDG713 and SDG715.10. ChIP (Chromatin immunoprecipitation) data indicated that SDG728 was required for histone H3K9me3 on the Tosl 7 and Os08g03880 loci.All those data demonstrated that SDG728 was a histone trimethyltransferase in rice, which regulated the DNA methylation on the gene region of transposable elements by the changes of histone H3K9me3. The fact that SDG728 could repress the retrotransposons and maintain the genome stability indirectly, suggested its role in regulating the retrotransposon expression of SDG728. Other members of SUVHs and HDACs played important roles in histone modification and rice development.