Functional Analysis Of Histone Modification Enzymes In Rice

Histone-modifying enzymes,including HDACs(Histone deacetylases),HMTs (Histone methyltransferases),and HDMs(Histone demethylases),play important roles in gene expression and development in animals as well as in plants.However,a few investigations of these enzymes have been reported in rice,the model species of monocots.This study deep analyzed the function of Histone deacetylase gene OsSRT1 and the Histone demethylase gene JMJ706 in rice development.The OsSRT1 belongs to Sirtuin subfamily of HDACs.Western blot analysis of its RNAi lines showed an increase of H3K9ace and a decrease of H3K9me2,while overexpressioning plants leads to a decrease of H3K9ace.The results supported OsSRT1 having histone deacetylase activity at H3K9 site in vivo,and also related to methylation at this site.In order to analyze the function of OsSRT1 in-depth,we produced its antibody successfully.The purifed antibodies were successfully used to ChIP assays,and the immunoprecipitated DNA were purified for high throughput deep-sequencing.LSD1 and JMJ family proteins are two classes of histone demethylation enzymes. Phylogenetic analysis of jmjC gene family showed that plant jmjC genes have both conserved and specific features compared with human homologues.JMJ706,a jmjC-domain-containing protein in rice,belongs to JMJD2 group,and encodes a heterochromatin-associated protein.Loss-of-function mutations of the JMJ706 gene lead to increased di- and trimethylations of H3K9 and affect the spikelet development.The spikelet mutant phenotype can be rescued by retransformating JMJ706 cDNA. Transgenic plants expressing an amiRNA of the JMJ706 showed a phenotype similarity to jmj6 mutants,mRNA expression level of 16 MADS-box genes and DH1 were analyzed by RT-PCR,and found OsMADS8 were induced,OsMADS47 and DH1 were reduced. ChIP-PCR showed the modification levels of H3K9me2 and H3K9me3 on the promoter and 5'regions of DH1 and OsMADS47 were more abundant in the mutants.Microarray analysis were performed to study the gene regulation mechanism of JMJ706.Compared the transcripts of two development stage of jmj6 mutant and the wild-type plants,we found:â‘ 60 genes were up-regulation at seedling stage,while 30 genes were down-regulation;â‘¡145 genes were up-regulation,97 genes were down-regulation at 3rd stage of panicle developmental;â‘¢there are many elements such as transposable element,DNA repeats,and small RNAs are found at chromosomal contexts of changed genes.We test the DNA methylation patterns of the up-regulated gene Os03g02470,and found symmetric DNA methylations(CG and CHG) were changed.Also we constructed two small RNA cDNA libraries for high throughput deep-sequencing.In order to analyze the functional relationship between HMTs and HDMs,we retransformed the amiRNAs of SUVH gene family members into jmj6 mutants.We observed the phenotype weaker in the retransgenic plants than in the jmj6 mutant.The real-time PCR results showed the expressions of amiRNA targeted genes were reduced in transgenic plants.