Molecular Mechanisms Of Crwn3 Involvement In Ros1-mediated Dna Demethylation Process In Arabidopsis

DNA cytosine methylation at carbon 5 of the pyrimidine ring[5-methylcytosine(5-meC)]controlled by methyltransferases is an important epigenetic mark,which has important effects on endosperm gene imprinting,transposition of transposable element,transcriptional gene silencing,mRNA processing and so on.In contrast to DNA methylation pathway,there exists a DNA demethylation process which counteracts the effects of DNA methylation.So far,DNA demethylation is found to play important roles in activating transcriptional gene silencing and chromatin decondensation.In Arabidopsis,it is generally accepted that DNA demethylation is mediated by DNA glycosylase/lyase-mediated base excision repair pathway,and a subfamily of DNA glycosylases,including ROS1(Repressor of Silencing 1),DME(DEMETER),DML2(DEMETER-LIKE protein 2)and DML3(DEMETER-LIKE protein 3),have been demonstrated to serve as DNA demethylases.The main roles of ROS1 in the process of DNA demethylation are to remove excessive DNA methylation caused by RNA-dependent DNA methylation(RdDM)pathway at particular loci,which therefore counteract gene silencing.Recent studies have demonstrated that for efficient demethylation a number of protein factors are required for creating a favorable chromatin environment to facilitate binding of ROS1 to target loci and the subsequent demethylation.To identify more protein factors associated with ROS1's functions,we obtained co-expressed genes of ROS1,and the corresponding T-DNA insertional mutants of such genes were used to screen for candidate mutants which showed similar hypermethylation levels to rosl at Chr2:10591372-10592250 region by using chop-PCR method(methylation-sensitive enzyme digestion-PCR).As a consequence,a mutant referred to as crwn3(CROWDED NUCLEI 3)was recovered.Further detailed investigations revealed that the functions of CRWN3 and ROS1 had quite a bit in common,and it is very likely that ROS1 serves to demthylate target loci with the help of CRWN3,which eventually contributes to decondensation of chromatin.The main results obtained are listed as follows:1.CRWN3 mutation leads to obvious elevation of genome-wide DNA methylation levelIt was previously reported that CRWN family consists of 4 members,and CRWN1 as well as CRWN4 were discovered to determine the size of nucleus;therefore,mutation of either of the two genes resulted in smaller nucleus.In order to know if the 4 CRWN proteins are all involved in DNA demethylation process,we obtained T-DNA insertional mutants of each gene.With the aim of creating double mutants,we systematically crossed these 4 mutants with each other and got 3 double mutants,i.e.crwn1 crwn2,crwn2 crwn3 and crwn3 crwn4;crwn3 crwn1 double mutant was obtained by creating mutation in CRWN1 by CRISPR/CAS9 technique in the crwn3 genetic background(herein referred as to crwn3 crwn1CR).With the exception of semi-dwarf phenotype of the crwn3 crwn1CR,the other 3 double mutants had no visible developmental defects.Chop-PCR assays demonstrated that only crwn3 out of the 4 single mutants exhibited increased DNA methylation at a few examined loci,suggesting that CRWN3 might have been evolved to participate in ROS1-mediated DNA demthylation process.RT-PCR results showed that in crwn3 mutant the expression levels of four genes associated with DNA demethylation,i.e.ROS1,ROS3,IDM1 and IDM2,remain unchanged,indicating that the effects of crwn3 on DNA methylation did not result from downregulation of these 4 genes.Whole-genome bisulfite sequencing results revealed that in crwn3 mutant there were 2557 hypermethylated genomic loci as compared with wild-type Col-0,anmong which 1082 loci overlapped with the hypermethylated loci in ros1-4,suggestive of functional connections of CRWN3 and ROS1.Introduction of nrpdla and rdml mutations into crwn3 background almost erased all tested hypermethylation,demonstrating that CRWN3 at least in part targeted RdDM loci.To our surprise,when crwn3 was introduced into C24-LUC transgenic line(in Col-0 background),the LUC luminescence and Kanamycin resistance remained unchanged relative to the C24-LUC,possibly indicating that CRWN3,unlike ROS1,has no capability of antisilencing.2.The relationships between CRWN3 and ROS1To better understand the relationships between CRWN3 and ROS1,we generated a crwn3 ros1 double mutant.Chop-PCR results indicated that DNA methylation level at Chrl:13,477,554-13,477,896 region was higher in the double mutant than that in either crwn3 or ros1 single mutant.Whole-genome bisulfite sequencing data revealed that there were a large number of hypermethylated loci in the double mutant exhibiting higher methylation levels than in each single mutant,among which methylation of many loci in the double mutant spreaded beyond the initiator sequences,whereas the total number of hypermethylated loci was almost unchanged,suggesting that the main functions of CRWN3 are to help ROS1 prevent methylation spreading to adjacent sequences.Given that CRWN3 is co-expressed with ROS1,we speculate that they may mutually regulate or interact with each other.RT-PCR results failed to detect expression changes of ROS1,ROS3,IDM1 and IDM2,indicating that crwn3 mutation does not alter expression levels of such genes.Yeast two hybrid(Y2H)assays demonstrated a physical interaction between CRWN3 and ROS1,and an in-depth analysis found that the N terminus of CRWN3(CRWN3-N)strongly interacted with the N terminus of ROS1(ROS1-N).Furthermore,BiFC and LCI experiments again corroborated such interaction.Taken together,these results provide a clear demonstration that CRWN3 presumably forms a protein complex with ROS1 in vivo through protein-protein interaction,and fulfills a function in DNA demethylation.3.CRWN3 and ROS1 may jointly contribute to chromain decondensation during seed germination.Previous studies revealed that ROS1 is able to actively decrease methylation levels of 5S rDNA chromatin regions and therefore facilliate their decondensation at the beginning of seed germination.Similar to ROS1,our Southern blot results as well indicated that methylation levels of 5S rDNA in crwn3 single and crwn3 ros1 double mutants hardly changed.Fluorescence in situ hybridization(FISH)experiments demonstrated that the decondensation of 5 S and 45 S rDNA regions in crwn3 ros1 double mutant at the onset of germination was obviously impaired compared to in wild-type Col-0,strongly implicating that both CRWN3 and ROS1 act collectively to promote decondensation of 5S and 45S rDNA regions.Our chromatin immunoprecipitation(ChIP)assays demonstrated that the promoter regions of 5S and 45S rDNA in the crwn3 ros1 double mutant displayed increased H3K9me2,which may be the result of higher DNA methylation plus impaired chromatin decondensation.Another ChIP experiment revealed that binding of ROS1 on its target genomic loci was actually weakened in the crwn3 background,suggesting that CRWN3 is required for ROSl's targeting to some extent.ABSCISIC ACID-INSENSITIVE 3(ABI3)was reported to enhance chromatin condensation during seed maturation,and our Y2H assays discovered that CRWN3 also physically interacted with ABI3.We subsequently examined expression levels of ABI3,CRWN3 and ROS1 in wild-type Col-0 at the beginning of seed germination.It is noticeable that ABI3 expression declined from 2nd day following germination onwards,while ROS1 expression rapidly increased and peaked at 4th day;by comparison,CRWN3 expression gradually increased and then falled at 3 rd day.Further investigations discovered the fact that the basal expression level of ABI3 in crwn3 ros1 double mutant at the onset of germination significantly higher than in wild-type Col-0 as well as the two single mutants,and it seems to be decreased at a slower rate with the prolonged period of time.All these results demonstrated that CRWN3 and ROS1 very likely operate together to antagonize the effects of ABI3,which in the end contributes to the chromatin decondensation.Surpringly,crwn3 ros1 double mutant clearly exhibited reduced germination speed under normal growth condition;however,exogenous application of ABA strongly inhibited its germination relative to Col-0,crwn3 and ros1 single mutants,indicative of elevated ABA content in such a double mutant.To prove this speculation,we made use of liquid chromatograph mass spectrometer(LC-MS)technique to detect the ABA contents of the four genotypes,and found that it is obviously higher in the double mutant than in the other 3 genotypes,suggesting that mutations of both CRWN3 and ROS1 seem to contribute to increase of ABA content of seeds in an as yet unknown manner.Altogether,our studies on the involvement of CRWN3 in ROS1-mediated DNA demethylation pathway discovered that CRWN3 presumably forms a protein complex with ROS1 in vivo to prevent methylation spreading from the initiator sequences to adjacent sequences.In addition,this protein complex was also found to play an important part in demethylating 5S and 45 S rDNA regions,which accordingly facilitates their decondensation at the onset of seed germination.All these results help increase our understanding of the mechanisms for DNA demethylation mediated by ROS1.