Project description:In eukaryotic cells, nucleo-cytoplasmic trafficking of macromolecules across the nuclear envelope is an essential process that ensures a rapid exchange of different cellular components, including proteins and RNAs, between the nucleus and cytoplasm. The significance of the nucleo-cytoplasmic trafficking of chromatin regulators in regulating DNA methylation and gene silencing is not well understood. Here, using a genetic screen, we identified XPO1A, one of the nuclear export receptors in Arabidopsis, as an anti-silencing factor that protects transgenes from transcriptional silencing. Loss-of-function of XPO1A leads to locus-specific DNA hypermethylation at transgene promoters and some endogenous loci. We found that XPO1A directly interacts with histone deacetylase HDA6 in vivo and the xpo1a mutation causes increased nuclear retention of HDA6 protein, resulting in reduced histone acetylation and enhanced transgene silencing. Our results revealed a new mechanism of epigenetic regulation through the modulation of XPO1A-dependent nucleo-cytoplasm partitioning of a chromatin regulator.
Project description:The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The AUXIN RESPONSE FACTOR (ARF) family of transcription factors regulates auxin-responsive gene expression and exhibit nuclear localization in regions of high auxin responsiveness. Here we show that activating ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. The intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, thus providing a mechanism for cellular competence for auxin signaling.
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:We report the application ofr high-throughput profiling of total transcript in WT, hda6, ldl1/2 and hda6/ldl1/2. The total RNA were extracted and analysed by NGS to identify the differential expressed genes between WT with hda6, ldl1/2 and hda6/ldl1/2.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of H3Ac in 14 days old WT, hda6, ldl1/2 and hda6/ldl1/2 arabidopsis. By obtaining sequence from chromatin immunoprecipitated DNA, we mapped genome-wide H3AC levels of 14 days old WT, hda6, ldl1/2 and hda6/ldl1/2 arabidopsis. ChIP was performed using anti-H3K9K14Ac antibody (Millipore; 06-599), and ChIP DNA were analyzed by Illumina HiSeq 2500.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of H3K4me2 in 14 days old WT, hda6, ldl1/2 and hda6/ldl1/2 arabidopsis. By obtaining sequence from chromatin immunoprecipitated DNA, we mapped genome-wide H3K4me2 levels of 14 days old WT, hda6, ldl1/2 and hda6/ldl1/2 arabidopsis. ChIP was performed using anti-H3K4me2 antibody (diagenode; C15410035), and ChIP DNA were analyzed by Illumina HiSeq 2500.
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:To analyze the effect of Exportin-5 expression on the MEF cells in cell cycle re-entry phase, we have employed whole genome microarray expression profiling on the MEF cells in cell cycle re-entry phase with and without down regulation of Exportin-5 gene. Mouse MEF cells were transfected with 60nM of siRNA targeting either Exportin-5 or negative control, and incubated for 12 hours. After incubation, cells were starved with DMEM containing 0.2% FCS for 48 hours and re-fed with DMEM containing 20%FCS for 24 hours, along with second siRNA transfection. Two independant expreiments were preformed.