Arabidopsis Homologs of Retinoblastoma-Associated Protein 46/48 Associate with a Histone Deacetylase to Act Redundantly in Chromatin Silencing
[['Cytosine DNA methylation is critical for stable silencing of transposable elements ( TE ) and repetitive sequences and for epigenetic regulation of endogenous gene expression in eukaryotes [1]–[3] .', 'DNA methylation is thought to play an ancestral role in the defense against invasive DNA elements to maintain genome stability and integrity [1]–[3] .', 'In the model plant Arabidopsis , cytosine methylation occurs in three different sequence contexts: CG , CHG and CHH .', 'CG and CHG methylation are heritably maintained respectively by DNA METHYLTRANSFERASE 1 ( MET1 ) and the plant-specific CHROMOMETHYLASE 3 ( CMT3 ) .', 'CHH methylation is dynamically maintained through de novo methylation by the DOMAINS-REARRANGED METHYLTRANSFERASE 2 ( DRM2 ) and the RdDM pathway [1] .', 'RdDM is a mechanism by which siRNAs direct de novo cytosine methylation in all sequence contexts of target DNA sequences ( complementary to the siRNAs ) .', 'In Arabidopsis , the plant-specific RNA polymerase Pol IV is thought to initiate silencing by generating single-stranded RNA transcripts that are subsequently converted to double-stranded RNAs ( dsRNAs ) by RNA-DEPENDENT RNA POLYMERASE 2 ( RDR2 ) .', 'dsRNAs are processed by DICER 3 ( DCL3 ) to produce 24-nt siRNAs , which are subsequently loaded to an ARGONAUTE 4 ( AGO4 ) -containing effector complex known as RISC ( for RNA-Induced Silencing Complex ) .', 'Through their interaction with long non-coding RNA transcripts from target loci , generated by the RNA polymerase Pol V , the loaded RISC complexes in association with DRM2 are targeted to RdDM target loci to establish cytosine methylation in CG , CHG and CHH contexts , leading to heterochromatin formation and transcriptional silencing [for reviews , see [2] , [4] .', 'siRNAs not only direct DNA methylation , but also trigger repressive histone modifications at RdDM target loci , including histone deacetylation , H3K9 dimethylation ( H3K9me2 ) and H3K27 monomethylation ( H3K27me1 ) .', 'Functional loss of the RISC component AGO4 causes a strong reduction in H3K9me2 at the endogenous RdDM target loci including transposable and repetitive elements [5] , [6] .', 'Furthermore , it has been shown that at RdDM target loci H3K27 monomethylation , a hallmark for silenced heterochromatin [7] , is reduced upon loss of Pol V or AGO4 activity [5] .', 'Together with DNA methylation , these repressive histone modifications establish a silenced heterochromatin state at RdDM target loci .', 'Histone modifications are involved in the control of DNA methylation .', 'For instance , the H3K9 methyltransferase KRYPTONITE ( KYP ) /SUVH4 and SUVH4 homologs including SUVH2 , SUVH5 , SUVH6 and SUVH9 , catalyze dimethylation of H3K9 , which is recognized and bound by CMT3 , leading to the maintenance of CHG methylation [1] , [8] .', 'Histone H3 lysine-4 ( H3K4 ) demethylation is also involved in DNA methylation .', 'Recent studies reveal that cytosine methylation is depleted in genomic regions with di- or tri-methylated H3K4 at a genome-wide level [9]; the H3K4 demethylase known as JMJ14/PKDM7B is required for H3K4 demethylation and CHG and CHH methylation at various RdDM target loci [10] .', 'The histone deacetylase HDA6 deacetylates lysines of core histones including H3 and H4 , and is required for cytosine methylation in transgenes and silenced rRNA genes [11]–[13] .', 'Multiple genetic screens have revealed that HDA6 is critical for transgene silencing [13] , [14] .', 'Loss of HDA6 activity causes a substantial decrease of symmetric cytosine methylation and a moderate reduction in asymmetric CHH methylation in an RdDM-silenced transgene promoter , leading to the transgene reactivation [13] .', 'In addition , disruption of HDA6 function gives rise to histone hyperacetylation and decreased CG and CHG methylation at silenced rRNA gene promoters [11] .', 'HDA6 plays a dual role in silencing of these loci: deacetylating core histones and mediating cytosine methylation [15] .', 'In this way , HDA6 and DNA methylation machinery are thought to work collaboratively to silence target loci .', 'The histone-binding proteins RbAp46 and RbAp48 are highly homologous WD40-repeat proteins and were first identified in mammalian cells as the tumor-suppressor Rb-binding proteins [16] .', 'Subsequent studies revealed that RbAp46/48 is an integral subunit of multiple chromatin-modifying or -assembly complexes [for a review , see [16]] .', 'RbAp46 forms a complex with the histone acetyltransferase called HAT1 that acetylates H4 , whereas RbAp48 is a subunit of the Chromatin Assembly Factor-1 ( CAF-1 ) complex that deposits nucleosomes .', 'Both RbAp46 and RbAp48 are components of several histone deacetylase ( HDAC ) co-repressor complexes such as the Sin3 complex , which deacetylate core histones to repress target gene expression .', 'In addition , RbAp46/48 is an integral subunit of the evolutionarily conserved Polycomb Repressive Complex 2 ( PRC2 ) -like complexes that catalyze H3K27 trimethylation ( H3K27me3 ) , resulting in transcriptional repression .', 'Recent studies have shown that RbAp46/48 functions as a histone ( H3–H4 dimer ) -binding protein [17] .', 'It is believed that the RbAp46/48-containing complexes interact with histone substrates via either RbAp46 or RbAp48 [17] .', 'RbAp46 and RbAp48 are evolutionarily conserved in animals and plants .', 'There are five homologs in Arabidopsis known as MSI1–MSI5 ( MSI for MULTICOPY SUPPRESSOR OF IRA1 ) [18] .', 'Biological functions of MSI1 and MSI4/FVE have been identified , whereas the functions of MSI2 , MSI3 and MSI5 are not known .', 'MSI1 is required for proper vegetative development and plays an essential role in gametophyte and seed development [19] , [20] .', 'The MSI1 protein is an integral subunit of the conserved Arabidopsis CAF-1 complex , and has also been found in several PRC2-like complexes [18] , [20] , [21] .', 'In addition , MSI1 directly interacts with the Arabidopsis Rb homolog ( RBR ) , a key cell-cycle regulator [22] , to repress MET1 expression in female gametogenesis , presumably resulting in a reduction in CG methylation [23] .', 'In addition to MSI1 , MSI4/FVE also interacts with a plant Rb homolog [24] , but the biological implication of this interaction is unclear .', 'FVE has been shown to repress expression of the central floral repressor FLC and several cold-responsive genes in Arabidopsis [24] , [25] .', 'FLC inhibits the transition from a vegetative to a reproductive phase ( i . e . flowering ) , and loss of FVE function causes FLC de-repression , resulting in late-flowering [24] , [25] .', 'Previous studies reveal that fve mutations cause increased levels of histone acetylation at FLC chromatin [24] , [26] , indicating that FVE may be involved in deacetylation of FLC chromatin to repress FLC expression .', 'However , recent studies show that loss of FVE function also gives rise to a strong reduction in PRC2-catalyzed H3K27me3 , a repressive chromatin mark , in FLC chromatin [27] .', 'Given that the human FVE homologs , RbAp46/48 , are subunits of multiple histone-modifying complexes , the mechanisms underlying FVE-mediated transcriptional repression/silencing remain elusive .', 'FLC plays a crucial role in flowering-time regulation in Arabidopsis and FLC expression is affected by a range of chromatin modifiers ( reviewed in refs 28 , 29 ) .', 'In most rapid-cycling ( i . e . early flowering ) Arabidopsis ecotypes , FLC expression is repressed or silenced by a group of proteins that mediate or trigger repressive histone modifications at the FLC locus , among which , in addition to FVE , are two conserved RNA 3′end-processing factors called CstF64 and CstF77 , RNA-binding proteins known as FCA and FPA , a putative H3K4 demethylase FLOWERING LOCUS D ( FLD ) , and a putative CLF ( for CURLY LEAF ) -containing PRC2-like complex [for reviews , see [28] , [29]] .', 'Furthermore , recent studies show that FLC antisense transcripts trigger FLC silencing [30] , [31] .', 'There are two groups of antisense transcripts resulting from alternative polyadenylation .', 'CstF64 and CstF77 function together with FCA and FPA to promote polyadenylation of FLC antisense transcripts at a proximal site , triggering FLC silencing [30] , [31] .', 'FLD activity is required for , and acts downstream FCA and FPA in this silencing mechanism [30] , [32] .', 'It is believed that the 3′ processing at the proximal polyadenylation site on FLC antisense transcripts leads to co-transcriptional decay of the antisense RNA downstream the proximal site , which may generate aberrant RNAs and trigger repressive histone modifications such as FLD-mediated H3K4 demethylation , and consequent silencing [30] .', 'FLC antisense transcript-triggered silencing is mechanistically different from the siRNA-triggered silencing of RdDM target loci , although both involve RNA molecules .', 'So far , no siRNAs targeting the FLC genomic coding region or 5′ promoter have been detected in Arabidopsis .', 'Consistent with this , knockout of siRNA-silencing pathway components such as Pol IV , Pol V , RDR2 or AGO4 has little effect on FLC silencing [33] .', 'In addition , cytosines in genomic FLC in most Arabidopsis ecotypes are not methylated [34] .', 'Thus , unlike RdDM-mediated silencing , cytosine methylation is not directly involved in FLC silencing .', 'However , both silencing mechanisms require repressive histone modifications such as histone deacetylation , H3K4 demethylation , and/or H3K9 and H3K27 methylation , and involve chromatin silencing .', 'In this study , we explored the role for FVE and MSI5 in chromatin silencing of various loci targeted by siRNAs or asRNAs .', 'We show that MSI5 acts in partial redundancy with FVE to silence FLC and endogenous RdDM target loci including FWA ( containing two tandem repeats ) , AtMu1 ( DNA transposon ) , AtSN1 ( retrotransposon ) and IG/LINE ( intergenic transcripts ) .', 'FVE and MSI5 associate with the histone deacetylase HDA6 to form HDAC complexes , and directly interact with the target loci , leading to histone deacetylation and transcriptional silencing .', 'Together , these results show that FVE and MSI5 play an important role in the chromatin silencing of various loci targeted by siRNAs or asRNAs in plants .'], ['FVE and MSI5 are Arabidopsis homologs of the human histone-binding RbAp46/48 [18] , [24] .', 'The amino acid sequence similarity between FVE and RbAp48 over the entire RbAp48 is 45% , and the similarity between MSI5 and RbAp48 over the entire RbAp48 is also 45% , whereas the identity between FVE and MSI5 over the entire MSI5 is 77% ( Figure S1 ) .', 'The high degree of sequence conservation between MSI5 and FVE suggests that these two proteins may have a similar biochemical function .', 'Previous studies have shown that FVE represses the floral transition in Arabidopsis [24] , [25] .', 'We sought to address the biological functions of MSI5 .', 'Two loss-of-function mutants of MSI5 carrying insertional T-DNAs were identified , in which the full-length transcription of MSI5 was severely disrupted ( Figure 1A and 1B ) .', 'Grown in long days ( LD; 16-hr light/8-hr dark ) , msi5-1 did not exhibit any visible phenotypes , whereas msi5-2 flowered slightly later than wild-type Col ( Figure 1C and 1D ) , as measured by the developmental criterion of the number of leaves formed prior to flowering , from the primary apical meristem .', 'In short days ( 8-hr light/16-hr dark ) , both mutants flowered moderately later than Col ( Figure 1E ) .', 'In both long and short days , msi5-2 flowered later than msi5-1 , indicating that msi5-2 is a strong allele .', 'We further confirmed that the moderate late-flowering of msi5-2 was indeed caused by the mutation in a complementation test in which the wild-type copy of MSI5 complemented the msi5-2 mutation ( Figure 1F ) .', 'To examine whether MSI5 acts redundantly with FVE to repress flowering , we introduced both msi5 alleles into fve mutants .', 'In LDs , both msi5-1;fve and msi5-2;fve flowered later than the late-flowering fve mutants ( Figure 1D ) .', 'Of note , msi5-2;fve flowered with 56 leaves on average which is much later than fve ( 34 leaves on average ) ( Figure 1D ) .', 'Hence , MSI5 functions redundantly with FVE to promote Arabidopsis flowering .', 'Vernalization ( an extended period of cold exposure ) promotes Arabidopsis flowering .', 'We examined the effect of cold treatment on the flowering times of msi5-1;fve .', 'The late flowering phenotypes of this double mutant were partially suppressed by 7-day cold treatment , and after 35 days of cold exposure , the mutant flowered similar to Col ( Figure 1G ) .', 'It is well known that vernalization largely represses FLC expression to accelerate flowering in Arabidopsis [28] , [29] .', 'These data indicate that the late-flowering phenotypes of msi5;fve is largely dependent on FLC and that the activities of MSI5 and FVE are not required for FLC repression by vernalization .', 'FVE has been shown to repress FLC expression [24] , [25] .', 'To examine whether the late flowering of msi5;fve was caused by FLC de-repression , we created an flc;msi5-2;fve triple mutant .', 'In long days , the triple mutant flowered much earlier than msi5-2;fve , but still moderately later than flc ( Figure 2A ) .', 'Hence , the late-flowering of fve;msi5-2 is partly dependent on FLC .', 'We further examined the flowering times of flc , fve;flc and flc;msi5-2;fve in short days , and found that the triple mutant flowered later than fve;flc and flc ( Figure 2B ) , suggesting that FVE and MSI5 may repress other floral repressors to promote flowering , in addition to FLC .', 'Besides FLC , Arabidopsis has five FLC homologs including FLOWERING LOCUS M ( FLM ) ( also known as MAF1 ) , and MAF2-MAF5 ( MAF for MADS BOX AFFECTING FLOWERING ) ; these genes moderately repress flowering [35]–[37] .', 'We quantified transcript levels of FLC and FLC homologs in Col , msi5 , fve , and msi5;fve seedlings .', 'FLC expression was slightly increased in msi5-2 compared to Col , whereas it remained unchanged in msi5-1 ( Figure 2C ) .', 'However , both msi5 alleles caused strong increases in FLC transcript levels in the fve background ( Figure 2C ) .', 'Furthermore , we found that in fve mutants both MAF4 and MAF5 were de-repressed , and this de-repression was enhanced upon loss of MSI5 function in the fve background , whereas MAF1 , MAF2 and MAF3 expression remained unchanged upon loss of FVE and MSI5 function ( Figure 2C ) .', 'Together , these data show that MSI5 acts redundantly with FVE to repress the expression of MAF4 and MAF5 , in addition to FLC , and promote the floral transition .', 'Recent genetic analyses have revealed that FVE is partly required for proper silencing of the RdDM target loci AtSN1 ( retrotransposon ) and AtMu1 ( DNA transposon ) , although the underlying mechanism is unknown [38] , [39] .', 'This prompted us first to explore whether FVE plays a broad role in silencing of the RdDM target loci including TEs and repetitive elements .', 'We examined the effect of loss of FVE function on the silencing of two other representative RdDM loci , FWA and IG/LINE .', 'FWA , encoding a homeodomain-containing transcription factor that can repress flowering , is sporophytically silenced by cytosine methylation in two sets of tandem repeats containing a sequence related to a SINE ( for Short Interspersed Nuclear Element ) retroelement located in the 5′ region of FWA [40] , [41] .', 'IG/LINE is a spurious intergenic transcript initiated from a flanking solo-LTR ( for Long Terminal Repeat ) that functions as a promoter [42] .', 'Upon loss of FVE function , FWA and IG/LINE were re-activated in the fve or fve;flc seedlings , respectively ( Figure 3A , 3B ) .', 'We asked whether MSI5 was required for silencing of RdDM target loci .', 'To this end , we first quantified the transcript levels of AtSN1 , AtMu1 and IG/LINE in msi5-2;flc and msi5-2;fve;flc seedlings .', 'Both msi5-2 and msi5-2;fve were introduced into the flc background to exclude the possibility that FLC de-repression may affect reactivation of these loci .', 'Loss of MSI5 function alone had little effect on silencing of these three loci; however , upon the combined loss of FVE and MSI5 function , all three loci were strongly re-activated to levels much higher than fve alone ( Figure 3B–3D ) .', 'Next , we measured FWA transcript levels in msi5 and msi5;fve seedlings [in the Col background; note that FLC upregulation does not affect FWA silencing [43]] .', 'FWA is fully silenced in the msi5 seedlings ( Figure 3A ) , but the msi5 mutations greatly enhanced FWA reactivation upon loss of FVE function ( Figure 3A ) , like the situation in the other three loci .', 'Together , these data suggest that MSI5 and FVE may play a broad role in silencing of transposable and repetitive elements in Arabidopsis genome , and that MSI5 functions redundantly with FVE to silence these elements .', 'These distinct four loci have a common feature , that is , their de novo silencing is established by the siRNA-triggered DNA methylation pathway [40] , [42] , [44] , [45] .', 'To test whether MSI5 and FVE were involved in silencing of TEs other than RdDM target loci , we examined the transcript levels of Ta3 in msi5 and/or fve mutant seedlings ( in the flc background ) , which is a pericentromeric TE that is silenced independently of siRNAs [46] .', 'Loss of MSI5 and/or FVE function did not cause Ta3 reactivation ( Figure 3E ) .', 'These data indicate that MSI5 and FVE may only be required for the silencing of RdDM-targeted TEs and repetitive elements .', 'FWA , AtMu1 and IG/LINE are silenced by cytosine methylation [40]–[42] , [44] .', 'We sought to determine whether MSI5 and FVE are required for cytosine methylation in these loci .', 'Using bisulfite sequencing , we examined cytosine methylation at the tandem repeats ( TRs ) , terminal inverted repeats ( TIRs ) and solo-LTR , respectively , in FWA , AtMu1 and IG/LINE in msi5 and/or fve mutant seedlings ( note that these repeats generate siRNAs ) ( Figure 4A ) .', 'At the FWA locus , CG methylation was slightly reduced , but a strong reduction in CHG and CHH methylation was observed , in msi5-2;fve compared to wildtype ( mCHG: 14% in WT , but 4% in msi5-2;fve; mCHH: 7% in WT , but 2% in msi5-2;fve ) ; neither CHG nor CHH methylation was affected in msi5-2 , whereas upon loss of FVE function CHG and CHH methylation was moderately reduced ( Figure 4B ) .', 'At AtMu1 , CG methylation was not affected , but CHG methylation was greatly reduced in msi5-2;fve ( in the flc background ) ; in addition , CHH methylation was moderately reduced upon loss of FVE and MSI5 function ( Figure 4C ) .', 'At solo-LTR , cytosine methylation in all contexts was reduced upon the combined loss of FVE and MSI5 function ( Figure 4D ) .', 'The reduction of non-CG methylation at the FWA , AtMu1 and solo-LTR loci was further confirmed using the methylation-sensitive restrictive endonucleases Fnu4HI or AluI ( Figure S2 ) .', 'Together , these results show that MSI5 and FVE primarily mediate CHH and CHG methylation at RdDM target loci .', 'Recent studies show that symmetric CHG methylation is largely maintained by CMT3 in concert with the H3K9 methyltransferase KYP , whereas CHH methylation cannot be maintained , but is de novo methylated by the RdDM pathway [1] .', 'Hence , we conclude that MSI5 and FVE are required for the de novo CHH methylation and maintenance of CHG methylation at the RdDM target loci .', 'Cytosine methylation at both AtMu1 TIRs and solo-LTR causes transcriptional silencing .', 'The reduction in cytosine methylation at the non-transcribed and siRNA-targeted regions ( TIRs and solo-LTR ) upon loss of FVE and MSI5 function , suggests that these two genes silence RdDM target loci partly by mediating DNA methylation in these loci .', 'Both FVE and MSI5 are required for de novo CHH methylation at the endogenous RdDM target loci .', 'We sought to examine whether they could be involved in de novo cytosine methylation in all sequence contexts on previously unmethylated sequences using an FWA transgene assay .', 'When an unmethylated FWA transgene is introduced into Arabidopsis genome , siRNAs from the endogenous FWA are able to target the transgene and through the RdDM pathway direct de novo cytosine methylation in all sequence contexts , leading to its silencing [40] , [47] .', 'Otherwise , ectopic FWA expression would give rise to a late-flowering phenotype [47] , [48] .', 'We introduced an FWA transgene [47] , [48] into flc and flc;msi5-2;fve mutant backgrounds .', 'Consistent with our previous finding that FWA transgene is de novo silenced in the flc background [43] , T1 transformants of the flc background flowered only slightly later than flc ( Figure 5A ) .', 'By contrast , T1 transformants of the flc;msi5-2;fve mutant flowered much later than the non-transformed control ( Figure 5A ) .', 'Hence , MSI5 and FVE are required for de-novo silencing of the incoming FWA transgene .', 'We further examined the methylation state of FWA transgene in the flc and flc;msi5-2;fve backgrounds by bisulphite sequencing , and observed that CG methylation ( a primary contributor for FWA silencing ) , was significantly reduced in the transgene upon the combined loss of FVE and MSI5 function ( Figure 5B ) , in contrast to the slight reduction in CG methylation of the endogenous FWA ( Figure 4B ) .', 'In addition , non-CG methylation of FWA transgene was also reduced in flc;msi5-2;fve compared to the flc background .', 'Together , these results show that FVE and MSI5 are required for the establishment of cytosine methylation in all sequence contexts of the newly introduced FWA transgene and thus de novo FWA silencing .', 'The functional redundancy of MSI5 with FVE raised the possibility that both genes could be expressed in the same tissues .', 'To test this , we examined the spatial expression patterns of MSI5 and FVE using translational fusions to the reporter gene β-GLUCURONIDASE ( GUS ) ; the constructs contained the promoter plus part of the protein-coding region of FVE or MSI5 .', 'In seedlings , both MSI5 and FVE were preferentially expressed in shoot apices , root tips and leaf vasculature ( Figure 6A–6D ) .', 'In the reproductive phase , both genes were mainly expressed in styles and the junctions of ovary and receptacle ( Figure 6E , 6F ) .', 'In general , FVE-GUS was expressed at a level higher than that of MSI5-GUS .', 'We confirmed that indeed , FVE transcript levels were much higher than those of MSI5 in both seedlings and floral buds ( Figure 6G ) .', 'This may partly explain why FVE plays a more dominant role in gene silencing than MSI5 does .', 'Given the high protein-sequence homology of MSI5 with FVE , the overlapping expression patterns of these two genes provide an explanation for the functional redundancy of MSI5 with FVE .', 'The mammalian homologs of MSI5 and FVE , RbAp46/48 , are subunits of several chromatin-modifying complexes involved in gene silencing such as HDAC co-repressor complexes; RbAp46/48 binds H3–H4 dimers and is thought to recognize and bind histone substrates in these complexes [17] .', 'Using a candidate-gene approach , we explored whether FVE and/or MSI5 could associate with HDA6 , an HDAC that has been shown to be involved in FLC repression , DNA methylation maintenance and gene silencing in Arabidopsis [11] , [13] , [49] .', 'Bimolecular fluorescence complementation ( BiFC ) [50] was employed to examine whether MSI5 and FVE could associate with HDA6 in plant cells .', 'A non-fluorescent N-terminal EYFP ( for Enhanced Yellow Fluorescent Protein ) fragment was fused to the full-length FVE and MSI5 individually , whereas a non-fluorescent C-terminal EYFP fragment was fused to the full-length HDA6 .', 'nEYFP-FVE and HDA6-cEYFP were simultaneously expressed in onion epidermal cells , and fluorescence was observed in the nuclei , reflecting the physical association of FVE with HDA6 in the nucleus ( Figure 7A ) .', 'Similarly , we also found that MSI5 associated with HDA6 in the nuclei of onion cells ( Figure 7B ) .', 'Next , we performed protein pull-down assays to confirm the association of HDA6 with FVE and MSI5 .', 'Transgenic lines ( T3 homozygotes ) expressing MSI5-YFP-HA ( in msi5-2 background ) or FVE-FLAG ( in fve background ) were created .', 'The MSI5 transgene was fully functional ( Figure S3A ) , whereas the FVE-FLAG was partially functional ( Figure S3B .', 'Total proteins were extracted from transgenic seedlings and mixed with the purified GST-HDA6 from E . coli .', 'HDA6 was able to pull down the MSI5 fusion and FVE-FLAG from the protein extracts ( Figure 7C–7D ) .', 'Thus , HDA6 can directly associate with FVE and MSI5 from Arabidopsis seedlings .', 'We further performed co-immunoprecipitation ( co-IP ) experiments to determine whether HDA6 is part of a complex with FVE or MSI5 in vivo .', 'First , we created transgenic lines expressing a functional HDA6-FLAG ( Figure S3C ) , and a line expressing a functional HA-FVE ( Figure S3D ) .', 'HDA6-FLAG-expressing plants were crossed to the HA-FVE line or the MSI5-YFP-HA line , and from the resulting F1 seedlings total proteins were extracted for co-IP analysis .', 'Indeed , we found that anti-FLAG ( recognizing HDA6-FLAG ) immunoprecitated the MSI5 fusion protein and HA-FVE from the seedlings ( Figure 7E , 7F ) .', 'Of note , we detected only a small portion of the HA-FVE protein in the HDA6-FLAG immunoprecipitates from the F1 seedlings ( note that no HA-FVE was immunoprecipitated from the seedlings expressing only HA-FVE ) ; this is most likely due to an unstable association of FVE with HDA6 .', 'Taken together , these results led us to infer that FVE or MSI5 forms an HDAC complex with HDA6 in Arabidopsis .', 'Consistent with the HDA6 association with FVE and MSI5 , recent studies show that HDA6 , like FVE and MSI5 , represses FLC , MAF4 and MAF5 expression to promote the floral transition [49] .', 'It was of interest therefore to determine whether HDA6 also silences endogenous RdDM target loci .', 'We measured transcript levels of FWA , AtMu1 , AtSN1 and IG/LINE in WT ( Col ) and hda6 seedlings .', 'Indeed , loss of HDA6 activity , like loss of MSI5 and FVE function , caused re-activation of all four loci ( Figure 8A–8C ) .', 'Thus , HDA6 , like MSI5 and FVE , is required for silencing of the RdDM target loci .', 'We further examined cytosine methylation state in the FWA , AtMu1 and solo-LTR loci in hda6 seedlings .', 'At FWA , loss of HDA6 function , like of loss of FVE and MSI5 function , caused a reduction in CHG and CHH methylation ( Figure 8D ) .', 'At solo-LTR , cytosine methylation in all sequence contexts was reduced in hda6 compared to wildtype ( Figure 8D ) , similar to the situation in the msi5-2;fve mutant ( Figure 4D ) .', 'In addition , at AtMu1 , upon loss of HDA6 function cytosine methylation in all sequence contexts was reduced ( Figure 8D ) .', 'Together , these results show that HDA6 , like MSI5 and FVE , is required for cytosine methylation at these RdDM target loci .', 'To investigate whether FVE could bind to the chromatin of genes that exhibit altered expression in fve mutants , we performed chromatin immunoprecipitation ( ChIP ) experiments using the HA-FVE line .', 'Using anti-HA antibodies , we immunoprecipitated DNA fragments from HA-FVE-expressing seedlings ( wild-type Col was used as a negative control ) , and quantified DNA fragments from FLC [the 5′ Intron I region that is essential for FLC silencing; see [26]] , AtMu1 ( the promoter region immediately downstream the 5′ TIR ) , solo-LTR and FWA ( a region in the silencing tandem repeats ) ( Figure 9A ) .', 'Compared with the control , the abundances of HA-FVE protein associated with FLC , solo-LTR and AtMu1 chromatin increased in the HA-FVE line ( Figure 9B ) .', 'In addition , FVE was strongly enriched in FWA ( Figure 9B ) .', 'Of note , the moderate enrichment of HA-FVE at AtMu1 and solo-LTR is likely due to that in the ChIP experiments , anti-HA may not bind effectively to the single HA epitope tag fused to FVE at these TE-containing loci .', 'Taken together , these data suggest that FVE directly interacts with FLC and the three RdDM loci .', 'FVE or MSI5 forms an HDAC complex with HDA6 and may mediate histone deacetylation for transcriptional silencing .', 'Hence , we examined H3K9 and K14 acetylation state in FVE and MSI5 targets in WT and msi5-2;fve seedlings .', 'H3K9K14 acetylation levels were moderately increased in the AtMu1 promoter region and solo-LTR , and were strongly elevated in the 5′ Intron I of FLC and FWA tandem repeats upon combined loss of FVE and MSI5 function ( Figure 9C ) , consistent with FVE enrichments at these regions .', 'Together , these data suggest that FVE and MSI5 are required for histone deacetylation at FLC and the three RdDM target loci .', 'Given the association of HDA6 with FVE and MSI5 , these two proteins may act as part of the HDA6-containing HDAC complexes to mediate deacetylation of target loci and silence their expression .'], ['FVE and MSI5 are required for cytosine methylation at three representative RdDM target loci .', 'These two proteins may mediate cytosine methylation partly via FVE/MSI5-HDA6 complex-catalyzed histone deacetylation .', 'HDA6-catalyzed histone deacetylation is known to be required for cytosine methylation in transgenes and endogenous rRNA genes .', 'It has been shown that silencing of a transgene promoter targeted by RdDM requires HDA6 [13] .', 'The absence of HDA6 activity causes a substantial decrease in symmetric CG and CHG methylation and a moderate decrease in CHH methylation in the promoter region , leading to reactivation of the silenced transgene [13] .', 'Recent studies have shown that HDA6 exhibits a complex interrelationship with cytosine methylation in silenced rRNA genes: loss of HDA6 activity leads to a decrease in symmetric CG and CHG methylation and de-repression of intergenic transcription resulting in overproduction of siRNAs and consequent increase in CHH methylation [11] .', 'Thus , HDA6 is not essentially required for CHH methylation at rRNA genes; however , this does not exclude that HDA6 is still partly involved in this methylation at loci other than rRNAs .', 'A very recent study has revealed that HDA6 is required for both CHH and CHG methylation at a few loci , and partly for CG methylation in some of these loci in Arabidopsis [51] .', 'We have found histone hyperacetylation , loss of cytosine methylation and transcriptional reactivation at the RdDM target loci FWA , AtMu1 and solo-LTR ( IG/LINE ) upon combined loss of MSI5 and FVE function .', 'This raises the possibility of that the loss of cytosine methylation might result from transcriptional activities at these loci .', 'However , the reasons below argue for that the loss of DNA methylation at least partly causes re-activation of these silent loci .', 'First , both TIRs and solo-LTR are non-transcribed regions .', 'Second , the examined regions with a loss of cytosine methylation including TRs in FWA , TIRs in AtMu1 and solo-LTR in IG/LINE have been shown to generate siRNAs that trigger DNA methylation at these regions leading to transcriptional silencing [40] , [42] , [44] , [45] .', 'In this study , we have revealed that MSI5 and FVE act to silence various loci targeted by siRNAs or asRNAs .', 'This raises the possibility of that these genes could be involved in the production of siRNAs and asRNAs for transcriptional silencing .', 'Recent studies show that loss of FVE function does not affect the production of neither asRNAs from the FLC locus nor the siRNAs from AtMu1 , solo-LTR and AtSN1 [30] , [38] .', 'We have measured the levels of Pol V-dependent silencing scaffold RNAs from solo-LTR and AtSN1 [5] , [45] , in mutant seedlings carrying a knockout allele of FVE and/or MSI5 , and observed that the loss of FVE and/or MSI5 function does not affect the production of these RNAs ( Figure S4 ) .', 'It has also been shown that HDA6 is not involved in siRNA production from the promoter region of a silencing-reporter transgene [13] .', 'Based on these findings , we infer that FVE/MSI5-HDA6 complexes act downstream of or in parallel to siRNA/asRNA production for transcriptional silencing of FLC and RdDM loci .', 'De novo CHH methylation and maintenance of symmetric CHG methylation at the endogenous RdDM loci are catalyzed by the DNA methyltransferases DRM2 and CMT3 , respectively , and MSI5/FVE-HDA6 complex-mediated histone deacetylation is expected to facilitate these cyt
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['RNA molecules such as small-interfering RNAs ( siRNAs ) and antisense RNAs ( asRNAs ) trigger chromatin silencing of target loci .', 'In the model plant Arabidopsis , RNA–triggered chromatin silencing involves repressive histone modifications such as histone deacetylation , histone H3 lysine-9 methylation , and H3 lysine-27 monomethylation .', 'Here , we report that two Arabidopsis homologs of the human histone-binding proteins Retinoblastoma-Associated Protein 46/48 ( RbAp46/48 ) , known as MSI4 ( or FVE ) and MSI5 , function in partial redundancy in chromatin silencing of various loci targeted by siRNAs or asRNAs .', 'We show that MSI5 acts in partial redundancy with FVE to silence FLOWERING LOCUS C ( FLC ) , which is a crucial floral repressor subject to asRNA–mediated silencing , FLC homologs , and other loci including transposable and repetitive elements which are targets of siRNA–directed DNA Methylation ( RdDM ) .', 'Both FVE and MSI5 associate with HISTONE DEACETYLASE 6 ( HDA6 ) to form complexes and directly interact with the target loci , leading to histone deacetylation and transcriptional silencing .', 'In addition , these two genes function in de novo CHH ( H\u200a=\u200aA , T , or C ) methylation and maintenance of symmetric cytosine methylation ( mainly CHG methylation ) at endogenous RdDM target loci , and they are also required for establishment of cytosine methylation in the previously unmethylated sequences directed by the RdDM pathway .', 'This reveals an important functional divergence of the plant RbAp46/48 relatives from animal counterparts .']
['Chromatin , made of histones and DNA , is often covalently modified in the nucleus , and modifications can regulate gene transcription .', 'RNA molecules such as small-interfering or silencing RNAs ( siRNAs ) and antisense RNAs ( asRNAs ) can trigger silencing of gene expression in eukaryotes .', 'We have found that in the flowering plant Arabidopsis , two homologous putative histone-binding proteins associate with a histone deacetylase and function in partial redundancy in chromatin-based silencing of various loci targeted by siRNAs or asRNAs .', 'They act in partial redundancy to silence a development-regulatory gene that controls the transition to flowering and whose silencing is triggered by asRNAs , and genomic loci containing transposable and repetitive elements whose silencing is triggered by siRNAs via the siRNA–directed DNA Methylation ( RdDM ) pathway .', 'In addition , these two genes function in maintenance of DNA methylation at RdDM loci and are also required for establishment of DNA methylation in the previously unmethylated sequences , revealing that histone modifications are partly required for DNA methylation .', 'Our findings implicate that RNA–triggered transcriptional silencing involves repressive histone modifications such as deacetylation at a target locus .']
['plant science', 'plant growth and development', 'plant biology', 'genetics', 'plant genetics', 'epigenetics', 'biology', 'dna modification', 'genetics and genomics', 'histone modification']