Isolation And Functional Characterization Of Rice Histone Deacetylase Gene

The term "epigenetics" defines heritable states of gene activity not encoded in the DNA sequence.Any given epigenetic state appears to be correlated with histone modifications,DNA methylation,and chromatin remodeling.Histone modification is an integral component of the epigenetic network in many eukaryotes.Research over the past few years has focused on yeast, animal,human and Arabidopsis.The study of epigentics in monocots,especially in rice lagged behind.The objective of this study is to isolate and characterize genes encoding histone modification enzyme,which are involved in the developmental regulation and epigenetic inheritance.The main results are as follows.1.A total of 19 HDAC genes were identified in rice genome.Of the 19 HDACs,14 belong to the RPD3/HDA1 superfamily,three belong to the HD2 family,and two belong to the SIR2 family.2.Sequence alignments and phylogenetic analysis were performed to elucidate the relationship of different members in the three HDAC families.3.A total of 10 genes were constructed for RNAi suppression,and 5 genes were constructed for over-expression.The constructs were used to transform an indica rice variety 'Minghui63'.4.This study focused on OsSRT1,a member of SIR2 family,which is NAD⁺-dependent histone deacetylase.We studied the expression profile of OsSRT1.The results showed that OsSRT1 was a widely expressed nuclear protein with higher levels in rapidly dividing tissues.5.OsSRT1 RNA interference induced an increase of histone H3K9(lysine-9 of H3) acetylation and a decrease of H3K9 dimethylation,leading to H₂O₂ production,DNA fragmentation,cell death,and lesions mimicking plant hypersensitive responses during incompatible interactions with pathogens.6.No particular visible or morphological phenotypic changes were observed in OsSRT1 over-expression plants.But over-expression of OsSRT1 gene in transgenic rice resulted in enhanced tolerance to oxidative stress compared to the wild type. 7.To determine more downstream OsSRT1-regulated genes and its regulation mechanism in OsSRT1 RNAi plants,we performed microarray analysis using the Affymetrix oligonucletide rice chip array.The results revealed that the transcription of many transposons and retrotransposons in addition to genes related to hypersensitive response and/or programmed cell death was activated.8.The results indicated that the analysis of GO classification in differential expressed genes showed that OsSRT1 actively regulated defense response,DNA repair,and phenylpropanoid metabolism,those of which always related to the stress-responsive and stress-related or PCD signaling pathways,respectively.9.To confirm the microarray data,we performed semiquantitative RT-PCR analysis of RNA isolated from OsSRT1 RNAi young leaves harvested at 7,11,and 21 days after germination to compare with wild-type and OsSRT1 overexpression plants.10.ChIP assays showed that OsSRT1 down-regulation induced histone H3K9 acetylation on the transposable elements and some of the hypersensitive response-related genes, suggesting that these genes may be among the primary targets of deacetylation regulated by OsSRT1.11.DNA methylation analysis suggested that down-regulation of OsSRT1 was likely to have a stochastic effect on DNA methylation.12.Narrow leaves were produced in OsHDT702 RNAi plants,and dwarf phenotype was observed in OsHDA710 RNAi plants,further analysis is needed.Our data together suggest that the rice SIR2-1ike gene is required for safeguard against genome instability and cell damage to ensure plant cell growth,but likely implicates different molecular mechanisms than yeast and animal homologs.