The Role Of SAC3B In Gene Antisilencing In Arabidopsis

Epigenetic silencing plays an important role in gene expression regulation,development processes,and stress responses in plants.Meanwhile,plant antisilencing factors can counteract the epigenetic silencing effects via different ways such as DNA de-methylation,histone modification and chromatin remodeling.To study the anti-silencing mechanism of plant,we adopt a screening system based on luciferase(LUC)reporter to find new factors involved in gene antisilencing using the model plant Arabidopsis.In this screening system,a construct containing d35S:LUC and d35S:NPTII was transformed into rdr6-11(RNA-dependent RNA polymerase 6-11)background.From the forward genetic screening based on luminescence intensity,we found a mutant(named P31)showing LUC and NPTII silencing phenotype.We propose that the responsible gene should function in antisilencing.In this thesis,we use this P31 mutant to study the antisilencing mechanism of the responsible gene,and found the following results:1.The gene responsible for the silencing phenotype in P31 mutant is SAC3B(At3g06290).Through map based cloning we found the mutation occurs within a mapping interval of 280K between 1800K-2087K on chromosome 3.Whole genome sequencing analysis found a point mutation in SAC3B gene(At3g06290)which ecodes a central component of mRNA export complex TREX-2.The point mutation from C(1321)to T changed the amino acid from Glu to a premature stop codon.The P31 silencing phenotype can be complemented by a wild type genomic SAC3B,confirming that SAC3B gene is the responsible gene for the P31 silencing phenotype.2.SAC3B mutation caused not only transgene silencing phenotype,but also morphological phenotypes including shorter root length,curly leaves and reduced height.A sac3b T-DNA insertion line sac3b-4 also exhibit similar morphological defects.Complementation by a wild type SAC3B gene rescued these morphological defects,confirming that SAC3B gene is also the responsible gene for these morphological defects.3.SAC3B mutation caused epigenetic modifications and transcription impairment of the reporter gene.A DNA methylation inhibitor 5-Aza-dC treatment recoverd the LUC expression in P31 mutant,and bisulfite sequencing result shows that the methylation level at d35S promoter bottom strand is increased.Chromatin imunoprecipitation(Chip)analysis data shows that in P31 mutant,dimethylation at histone H3K9 is increased while RNA polymerase Pol II occupancy is decreased at d35S promoter region,indicating that SAC3B mutation results in epigenetic silencing.4.SAC3B mutation does not cause genome-wide hypermethylation.Instead,it causes weak hypomethylation in CHH and CHG contents.Whole genome’ bisulfite sequencing analysis found that in P31 mutant,only 298 loci are hypermethylated while 425 loci are hypomethylated.These indicate that SAC3B is not a direct player in demethylation.5.Besides SAC3B,mutation of other mRNA export factors THP1 and NUP1 can also lead to transgene silencing.By introducing the d35S:LUC reporter gene into thpl-1 and nupl-1 mutant,we found that mutation of THP1(another component of TREX-2),and NUP1(Nucleoporin)can both lead to silencing of the reporter gene LUC and NPTII,indicating that mRNA export factors may have a role in antisilencing through similar mechanisms.6.SAC3B mutation caused R-loop stabilization in the promoter region of the reporter gene.By R-loop foot-printing experiment and DNA IP assay using an antibody which specifically recognizes DNA:RNA hybrid,we found that R-loop is accumulated in the d35S promoter region and the stability of R-loop is increased by SAC3B mutation by a factor of 4,indicating that R-loop stabilization is the reason for transgene silencing.7.We propose a mechanism model for gene silencing in the sac3b mutant:SAC3B mutation causes uncoupling of transcription and export,inducing R-loop stabilization,which acts as a "roadblock" to cause Pol II transcription impairment of the reporter genes.Meanwhile,the non-coding RNA retained in the R-loop structure may facilitate DNA methylation and heterochromatin formation,leading to stable epigenetic silencing.Above all,this study found at the first time that mutations of mRNA export factors can lead to epigenetic silencing in Arabidopsis,and proposed that R-loop stabilization by SAC3B mutation is the reason for the gene silencing.Thus,this study revealed a role of mRNA export factors in antisilencing,and provided new information to further understand the mechanism of epigenetic sil.encing and antisilencing.