Fuctional Analysis Of Chromatin Regulators In Rice

Chromatin regulators are classified into two groups:the first group are chromatin modification factors including DNA methyltransferases and histone modification enzymes. The second group are the other factors related to epigenetic regulation except the first group such as proteins that can recognize specific chromatin modification sites or chromatin remodeling factors. But in rice a few are reported on the function of chromatin regulators especially the second group. So characterization of these proteins is critical to elucidate the molecular mechanism of epigenetic regulation in rice.This study mainly investigated the function of CHD (Chromodomain Helicase DNA-binding protein), LHP1 (LIKE HETEROCHROMATIN PROTEIN 1) and HDAC11 (histone deacetylase 11) and explored their roles in epigenetic regulation by clone, transformation, yeast two hybrid and other tools. The results are displayed below:1. Phylogenetic analyses of rice SNF2 proteins were conducted and three rice CHD genes were identified.2. We constructed RNAi vectors of CHD genes and transformed them into rice to get transgenic plants. The gene expressions of some of these transgenic plants were tested. Moreover, we found out the mutants of CHD genes and performed genotype and gene expression analyses.3. The expression of CHD1 was comparatively high in leaves especially young leaves in which CHD1 expression was several dozen times than that in others. Even in mature leaves the expression of CHD1 was much higher than that in other organs.4. The RNAi transgenic plants and mutants of CHD1 exhibited the similar phenotype: dwarf, thinner culm, narrower leaves including vegetative leaves and flag leaves, bleached upper surface of leaf, narrower seeds, smaller and shorter roots when planted in the field.5. The number of several kinds of tissues and cells in chdl were declined including big and small vascular bundles, bulliform cells and mesophyll cells. Cell numbers were also decreased in culm but both cell number and size were not affected in shoot apical meristem.Moreover, the number of trichomes on chdl leaf surface were dramatically increased and the content of chlorophyll in chdl leaf were deceased.6. In chdl WEE1 expression was increased but only in leaf when the mutants were planted in the field and not in the other tisues or organs such as shoot, seedling, root. WEE1 expression was contrary to that of CHD1, which was relatively low in leaves especially in mature leaves compared to the other tissues or organs.7. In rice the expression of WEE1 was induced by MMS treatment. And this induction only occurred in leaves. Contrarily, CHD1 expression was down-regulated by MMS treatment.8. chdl plants were hypersensitive to MMS treatment. The expression level of WEE1 was not significantly different between wild type and chdl plants when they were treated with MMS in the same concentration.9. The whole genome micoarray analysis indicated that amounts, of genes related to stress were induced to different extent in chdl.10. Histone modifications such as H3K4 trimethylation, H3K27 trimethylation and H3, H4 acetylation were not affected in chdl. CHD1 mutation and MMS treatment did not affect H3K27 trimethylation at WEE1.11. PHD domain of CHD1 could bind to TrimeH3K27 in vitro and two tadem chromodoamin could also bind to histones but with no specific hisotne, modification binding sites.12. RNAi transgenic plants of LHP1 were constructed but no visible phenotype was observed in positve plants. Rice LHP1 had splicing isoforms and were distinct in different rice subspecies. LHP1 was ubiquitiously expressed in different rice organs. Yeast two hybrid assay showed that chromoshadow domain of CHD1 could form homozygous dimer.13. We find out a rice-specific gene which was interacted with HDAC11 by yeast two hybrid screening. This gene didn't have conserved domain and no homolog was found out in other species. This gene showed polymorphism between indica and japonica. Some nucleotide changes led to amino acids changes and some did not. Deletion of thymine (22) in indica allele led to the pre-stop. N-terminal of this protein was not involved in the interaction between this protein and HDAC11. It expressed in all tested organs similar to that of HDAC11.All those results demonstrated that rice CHDl participated in DNA damage signal transduction. When DNA was damaged, the signal would down-regulate CHD1 expression and then was transduced to WEE1 resulting in the activation of WEE1 expression which delayed or stopped cell cycle process. So conclusion could be drawn that epigenetic regulation may play a role in cell cycle regulation.