Project description:HDA6 is a RPD3-like histone deacetylase. In Arabidopsis, it mediates transgene and some endogenous target transcriptional gene silencing (TGS) via histone deacetylation and DNA methylation. Here, we characterized two hda6 mutant alleles that were recovered as second-site suppressors of the DNA demethylation mutant ros1-1. Although both alleles derepressed 35S::NPTII and RD29A::LUC in the ros1-1 background, they had distinct effects on the expression of these two transgenes. In accordance to expression profiles of two transgenes, the alleles have distinct opposite methylation profiles on two reporter gene promoters. Furthermore, both alleles could interact in vitro and in vivo with the DNA methyltransferase1 with differential interactive strength and patterns. Although these alleles accumulated different levels of repressive/active histone marks, DNA methylation but not histone modifications in the two transgene promoters was found to correlate with the level of derepression of the reporter genes between the two had6 alleles. Our study reveals that mutations in different domains of HDA6 convey different epigenetic status that in turn controls the expression of the transgenes as well as some endogenous loci.
Project description:DNA cytosine methylation is a hallmark of epigenetic gene silencing. DNA demethylation requires ROS1, a bifunctional DNA glycosylase/lyases and open chromatin status mediated by IDM1. HDA6 is a RDP3-like histone deacetylase and was confirmed to mediate DNA methylation. In previous screening for ros1 suppressor, we identified two hda6 mutants reverting ros1-caused hypermethylation at RD29A and 35S promoters respectively, indicating the antagonization of DNA methylation between HDA6 and ROS1. To learn antagonized effects between HDA6 and ROS1 at DNA cytosine methylation genome-wide, we performed whole genome bisulfite sequencing to search antagonized targets of HDA6 and ROS1 and their specific targets to evaluate their roles on DNA methylation. To evaluate HDA6’s roles in sRNA biogenesis and nucleosome positioning, we also performed small RNA sequencing and genome-wide mapping of nucleosome positioning of C24, ros1, hda6-9 and hda6-10. Our results indicate that around 43% ros1-caused CG hypermethylation, 74.5% and 84.5% ros1-caused CHG and CHH hypermethylation were reverted by the two hda6 alleles in the ros1 background respectively. These results indicate that most of ROS1-demethylated targets are also HDA6-mediated DNA methylated targets. In addition, we observed that HDA6-affected DNA methylation targets are far more than ROS1-demethylation targets at CHG and CHH context, but not at CG context. sRNA analysis showed that HDA6 inhibits LTR/Gypsy and TE gene 24nt siRNA accumulation, while promotes RC/Helitron 24nt siRNA accumulation. Our Mono-nucleosome positioning data further showed that the two hda6 alleles have striking difference on nucleosome positioning, hda6-10 obviously have different nucleosome positioning patterns with hda6-9. Our results indicate HDA6 not only mediates overall DNA methylation at CG, CHG and CHH context, and antagonizes with ROS1 mediated DNA demethylation, but also regulates nucleosome positioning and small RNA accumulation at some genome specific regions.
Project description:We carried out a microarray experiment by using Affymetrix Arabidopsis ATH1 Genechips in order to identify genes that show reduced expression in ros1-1 plants. 2 samples examined: C24 wild-type and ros1-1 mutant (in C24 background).
Project description:To understand the role of Arabidopsis histone deacetylase HDA6 in plant cold acclimation, we have employed transcriptional profiling of the hda6 mutant and its parental line under cold and control conditions to identify genes differentially expressed in the hda6 mutant under cold and control conditions. Aligent’s Whole Arabidopsis Gene Expression Microarray (G2519F, V4, 4x44K) were used.
Project description:To understand the role of Arabidopsis histone deacetylase HDA6 in drought tolerance, we have employed transcriptional profiling of the hda6 mutant and its parental line under drought and control conditions to identify genes differentially expressed in the hda6 mutant under drought and control conditions. Aligent's Whole Arabidopsis Gene Expression Microarray (Agilent-015059, G2519F, V3, 4x44K) was used.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of HDA6 in 14 days old arabidopsis. By obtaining sequence from chromatin immunoprecipitated DNA, we generated genome-wide HDA6-binding maps of 14 days old arabidopsis. To reveal bound genes by HDA6, chimeric protein HDA6-GFP was expressed under HDA6 promoter in hda6 (HDA6pro:HDA6:GFP/ hda6). ChIP was performed using anti-GFP antibody (ab290; ABCAM), and ChIP DNA were analyzed by Illumina HiSeq 2500.
Project description:DNA methylation is an important epigenetic modification involved in many biological processes, and active DNA demethylation plays critical roles in regulating expression of genes and anti-silencing of transgenes. In this study, we isolated mutations in one arabidopsis gene, ROS5, which causes the silencing of transgenic 35S-NPTII because of DNA hypermethylation, but no effect on transgenic RD29A-LUC. ROS5 encodes an atypical small heat shock protein. ROS5 can physically interact with IDM1 and is required for preventing DNA hypermethylation of some endogenous genes that are also regualated by IDM1 and ROS1. We propose that ROS5 may regulate active DNA demethylation by interacting with IDM1, thereby creating a friendly chromatin environment that facilitates the binding of ROS1 to erase DNA methylation.
Project description:This study used the mutants nrpe1-11 and ros1-4 to assess the impact of RNA-directed DNA methylation (RdDM) and ROS1 on the Arabidopsis thaliana (ecotype Col-0) methylome.