Cre-mediated DNA Methylation Inhibits LoxP Recombination And Silences LoxP-sandwiched Genes

Although the cultivation of genetically modified(GM)crops has been increasing and brings certain social and economic benefits,the great value of this technology is far from being fully realized.Public concern addressing potential risks in ecology and food safety of transgenes,such as antibiotic,insecticidal and herbicide-resistance genes,has been raising,and thus limits the further commercialization of GM plants.How to use GM resources while avoid their potential risks is a key problem.Site-specific DNA recombinases can directly catalyze the cleavage,strand exchange,and ligation of DNA fragments at defined recognition sites.Depending on the orientation and location of the two recognition sites,DNA recombination reactions can produce deletion,integration,inversion,and translocation of targeted fragments.Cre/loxP is one of the most widely used and well studied recombination systems.Although this system is frequently used for conditional gene deletion in animals,the application of the system for gene deletion in plants is limited due to the low recombination efficiency.The mechanism underlying the poor gene-deletion performance of the recombinase system in plants remains unknown.Transgenes existing in reproductive tissues such as pollen and seeds constitute the main source of potential safety hazard.Germline promoter-controlled recombinase system can make it possible to delete transgenes from reproductive tissues to produce transgene-free pollen and seeds,while retain the genes in vegetative tissues to fufil their functions such as insect resistance and herbicide resistance.To achieve the goal of"generating non-transgenic products from transgenic plants",efficient recombinase system and its precise control are two prerequisites for the success.In this thesis,a series of promoters were screened and modified in tobacco,and the factors affecting the efficiency of site-specific DNA recombination reactions were investigated.It was demonstrated that:(1)No gene product(mRNA and protein)is detected in the offspring of some transgenic tobaccos in which Cre/loxP gene-deletion system is under the control of early flower-specific promoters;however,the target gene is actually retained.(2)loxP site and target gene are hypermethylated upon Cre action,resulting in inhibition of gene deletion and silence of the gene.(3)CG methylation in recombinase binding elements of loxP site has no effect on gene deletion;instead,CHH methylation in the crossover region contributes to the inhibition of loxP recombination,leading to a poor gene-deletion performance in plants where the occurrence of non-CG methylation is common.(4)Cre-mediated DNA methylation occurs strictly at the entire floxed region in an siRNA-independent manner,thus silencing the floxed genes.The main results are as follows:1.Screening and engineering germline promotersTwelve germline promoters from different species were evaluated for their ablility to direct the gene deletion system in terms of precision and efficiency.Using the negative rate of GFP fluorescence signal in the T₁ tobacco as an indicator to estimate the efficiency of the gene deletion system,deletion rates of the system under the control of various promoters differed significantly,from 2.7 to 100%.100%efficiency was achieved in the systems dericted by three tobacco early flower-specific promoters,NtAGIP2,NtAP1La,and NtAP1Lb1.GFP fluorescence observation of the T₀inflorescences showed that the GFP signal disappeared from the stamens and carpels of the NtAGIP2::Cre tobaccos and from the entire flowers of the NtAP1Lb1::Cre tobaccos.The tissues where the GFP signal disappeared were consistent with the tissues where the GUS reporter gene under the control of these two promoters expressed,indicating that the promoters can accurately and efficiently direct the expression of Cre/loxP system.To increase the activity of the germline promoters,the carpel-and stamen-specific promoter NtAGIP1 was modified.Results showed that:(?)Adding the 35S enhancer to NtAGIP1 promoter,the activity of the engineered promoter 35SNtAGIP1 extended from the center of the flower to the entire infloescence but did not cross the boundary of the reproductive organs into the vegetative tissues.GUS activity examination showed that the activity of 35SNtAGIP1 promoter was significantly improved compared to that of NtAGIP1.Consistent with this,when using this promoter to control Cre/loxP system,GFP fluorescence signal disappeared from the entire infloerescence in the T₀35SNtAGIP1::Cre tobacco.The statistics of GFP fluorescence elimination efficiency of offspring showed that 35SNtAGIP1 displayed 100%efficiency,while the maximum efficiency of NtAGIP1 was 91.2%.Serial deletion of the NtAGI-1 and chromatin immunoprecipitation(Ch IP)assay further revealed that a 100-bp fragment that contains a conserved GAGA factor binding motif could inhibit the activity of the 35S enhancer in vegetative tissues,by mediating histone H3 lysine 27 trimethylation(H3K27me3)modification,leading to enhanced promoter activity while retaining flower tissue specificity.(?)Fusing AP1 with NtAGIP1 promoter,the new promoter lost the activity in flower primordia displaying by AP1 promoter and activity in carpel-and stamen-primordia showing by NtAGIP1 promoter.Correspondingly,the efficiency of GFP fluorescence elimmiation directed by the new promoter was greatly decreased.This suggests that simply fusing AP1 with NtAGIP is unable to conbine the activity features of the two promoters together,suggesting an antagonist relationship between them.2.The expression of Cre results mostly in gene silencing rather than gene deletionNtAGIP2::Cre and NtAP1Lb1::Cre lines that showed 100%GFP fluorescence elimination rate were chosen for further examinations.RT-PCR and Western blot assay could not detect GFP mRNA and GFP protein in the NtAGIP2::Cre T₁ plants.However,to our surprise,genomic PCR assay showed that the gene did exist in most of the offspring.That is to say the bona fide gene-deletion efficiency of the three NtAGIP2::Cre lines was 0%,0%,and 14.6%,respectively.Similarly,the GFP DNA deletion efficiency of the three NtAP1Lb1::Cre was only 5.0%,29.2%,and 0.0%,instead of the expected 100%.A pair of primers was designed to amplify the floxed region.The results indicate that DNA recombination did not occur at all and the floxed DNA was still retained in the original position of the genome,excluding the possibility of the existence of circular DNA or reintegrating of floxed DNA into other positions in the genome.DNA sequencing confirmed that there was no mutation taking place either in the loxP sequence or in the 35S promoter that controls GFP expression.These results indicate that the GFP gene was silenced upon Cre action,rather than actually deleted.3.DNA methylation in crossover region of loxP sites contributes to the inhibition of loxP recombinationThere are two CG contexts and three CHH contexts(based on the one strand)in loxP site.Bisulfite sequencing showed that the CG and CHH contexts of loxP site were highly methylated in the GFP-negative carpels and stamens of the NtAGIP2::Cre tobaccos,while the methylation signal was undetectable in the sepals in which GFP was in active.These results suggested a connection between DNA methylation of loxP sequence and the poor deletion rate.Treating 35S::Cre×loxP-35S::GFP-loxP seeds with DNA methylation inhibitor 5-Aza C,the GFP gene-deletion rate was 81.9±6.4%,showing a 63-folds increase compared with the mock treatment(1.3±2.3%).In plants,de novo DNA methylation in all sequence contexts(CG,CHG,and CHH)is carried out by DOMAINS REARRANGED METHYLTRANSFERASE(DRM).An interference siRNA was designed to down-regulate DRM homologous genes in tobacco,and the siRNA cassette was delievere into the NtAGIP2::Cre line via retransformation.Like their transgenic acceptor material(NtAGIP2::Cre),three knockdown lines displayed100%GFP negative rate in the offspring seedlings.The NtDRM transcript level in the three lines was decreased over 80%.PCR assay showed that the GFP gene deletion efficiency was 52.8%,48.6%,and 50.0%,showing a 2.2-,1.9-,and 2.0-fold increase over the control(16.7%).Both chemical and genetic experiments indicate that interfering DNA methylation can effectively improve true gene deletion efficiency.To further study the mechanism of the inhibitory effect of DNA methylation on the site-specific DNA recombination,CG contexts of loxP site was methylated in vitro.Cre-mediated DNA recombination in vitro experiments showed that CG context methylation alone had no effect on gene deletion.In contrast,when CHH contexts in loxP site were also methylated,loxP recombination was abolished.The two CG sequences locate in the region has been shown to be important for the recombinase binding,while the two CHH sequences locate in the crossover region corresponding to DNA cleavage and reunion,suggesting a key role for methylation of the crossover region in DNA recombination inhibition.The recognition site RS of the R/RS recombination system has a CG context in the crossover region.Recombination experiments in E.coli showed that the methylation of the crossover region-located CG sequence inhibited the R recombinase-mediated DNA deletion reaction.These results indicate that DNA methylation,no matter CG or non-CG methylation,at crossover region of recognition sites contributes to the inhibition of site-specific DNA recombination.4.Cre-mediated DNA methylation within floxed region silences the genes and the methylation is independent of siRNA biosynthesisBisulfite sequencing showed that the p35S promoter was highly methylated in the carpels and stamens(GFP-negative)of the NtAGIP2::Cre tobaccos,while had almost no methyl-cytosine signal in the sepals(GFP-positive).The high level of DNA methylation could be inherited by the T₁ plants.Consistently,treating T₁NtAGIP2::Cre seeds with DNA methylation inhibitor 5-Aza C,the expression of the GFP gene was restored.Scanning the DNA methylation level in the T-DNA showed that in the carpels and stamens(GFP silenced)of the NtAGIP2::Cre tobacco,the whole floxed region was highly methylated,while in other part of the T-DNA region outside the floxed region,no signigicant methylation signal was detected.In contrast,for sepals(GFP expressing),no obvious methyl-cytosine signal was detected in the entire T-DNA region,except a small fragment at the 3'end of the NtAGIP2 promoter.In plants,small interferingRNAs(siRNAs)are required for the deposition of DNA methylation at target loci.Deep sequencing showed that in both GFP silenced and expressing tissues,no obvious siRNA transcript corresponding to the T-DNA was detected,except to the small fragment at the3'end of the NtAGIP2 promoter.Bioinformatic analysis showed that this particular small fragment has a large number of homologues in the tobacco genome,which may be the source of the matching siRNAs and the cause of siRNA-dependent DNA methylation of this sequence;but it has nothing to do with Cre expression.These data indicate that Cre-mediated DNA methylation of floxed region is independent of siRNAs.To further verify the characteristics of Cre-mediated DNA methylation,p NOS::Npt II cassette was relocated from outside of the floxed region to the loxP sandwich.As expected,the Npt II gene was also hypermethylated and silenced with an siRNA-independent manner,leading to loss of kanamycin resistance in the offspring.The results confirm that as long as floxed,siRNA-independent DNA methylation and gene silence will occur upon Cre expression.In summary,this thesis finds:Cre-mediated DNA methylation can cover the entire floxed region;methylation of loxP sites inhibits DNA recombination,and methylation of sequences between loxP sites results in silence of all floxed genes.This study reveals the mechanism for the poor gene deletion of the Cre/loxP recombinase system in plants,providing a theoretical basis for improving the efficiency of the recombinase system.Under the control of efficient flower-specific promoters,this Cre-mediated methylation can cause high level(100%)gene silence within large fragment sequence(>12kb),indicating a promising tool for epigenetic regulating the expression of multiple genes.In addition,the finding that siRNA biogenesis may not be required by Cre-mediated DNA methylation inspires an siRNA-independent model for the recombinase-mediated DNA methylation.