Methyl-CpG-binding domain 9 (MBD9) is required for H2A.Z incorporation into chromatin at a subset of H2A.Z-enriched regions in the Arabidopsis genome
[['Nucleosomes , the fundamental units of chromatin that consist of ~147 bp of DNA wrapped around a histone octamer , efficiently condense large eukaryotic DNA molecules inside the nucleus .', 'At the same time , nucleosomes present a physical barrier that restricts the access of DNA-binding proteins to regulatory sequences .', 'This physical constraint imposed by nucleosomes on DNA can be modulated to expose or occlude regulatory DNA sequences , and is thereby used as a mechanism to control processes such as transcription that rely on sequence-specific DNA binding proteins .', 'Thus , enzymatic complexes that can remodel chromatin structure by manipulating the position and/or composition of nucleosomes are essential for proper transcriptional regulation and the execution of key developmental programs .', 'All chromatin-remodeling complexes ( CRCs ) contain a DNA-dependent ATPase catalytic subunit that belongs to the SNF2 family of DNA helicases , along with one or more associated subunits [1 , 2] .', 'There are four major subfamilies of CRCs: SWI/SNF , INO80 , ISWI , and CHD , all of which use the energy of ATP to either slide , evict , or displace nucleosomes , or to replace the canonical histones within nucleosomes with histone variants .', 'One member of the INO80 CRC subfamily is the SWI2/SNF2-related 1 ( SWR1 ) chromatin remodeler , a multisubunit protein complex required for incorporation of the H2A variant , H2A . Z , into chromatin [3 , 4] .', 'H2A . Z is a highly conserved histone variant found in all eukaryotes that plays important roles in regulating a variety of cellular processes , including transcriptional activation and repression , maintenance of genome stability and DNA repair , telomere silencing , and prevention of heterochromatin spreading [5–12] .', 'Although loss of H2A . Z is not lethal in yeast [2 , 13] , H2A . Z is essential for viability in other organisms such as Tetrahymena [14] , Drosophila [15 , 16] , and mice [17] .', 'Interestingly , H2A . Z-deficient plants are viable but display many developmental abnormalities such as early flowering , reduced plant size , altered leaf morphology , and reduced fertility [18–20] .', 'The SWR1 complex that mediates H2A . Z incorporation into chromatin was first described in yeast and is composed of 13 subunits , including Swr1 , the catalytic and scaffolding subunit of the complex [3 , 4 , 21 , 22] .', 'In mammals , the functional and structural homolog of yeast SWR1 complex is the SRCAP ( SNF2-related CREB-binding protein activator protein ) complex .', 'This complex is composed of 11 of the same subunits found in yeast SWR1 and is likewise able to exchange H2A/H2B dimers for H2A . Z/H2B dimers in nucleosomes [23–26] .', 'Intriguingly , higher eukaryotes possess an additional multi-subunit complex , 60 kDa Tat-interactive protein ( TIP60 ) , that has histone acetyltransferase ( HAT ) activity and can also mediate the deposition of H2A . Z into nucleosomes [27–30] .', 'Furthermore , the yeast Nucleosome Acetyltransferase of histone 4 ( NuA4 ) complex , which is structurally related to SWR1 through the sharing of four subunits [31–35] , was shown to regulate the incorporation of H2A . Z into chromatin cooperatively with the SWR1 complex [34] .', 'Many homologs of yeast SWR1 and animal SRCAP complex subunits have been identified in Arabidopsis thaliana including ACTIN RELATED PROTEIN 6 ( ARP6 ) , SWR1 COMPLEX SUBUNIT 2 ( SWC2 ) , SWC6 , PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 ( PIE1 ) , and three H2A . Z paralogs: HTA8 , HTA9 , and HTA11 .', 'Numerous genetic and biochemical experiments suggest that the SWR1 complex is conserved in Arabidopsis .', 'For example , it has been recently shown that Arabidopsis SWC4 protein directly interacts with SWC6 and YAF9a , two known components of the SWR1 complex [36] .', 'Additionally , protein interaction experiments have demonstrated that PIE1 interacts directly with ARP6 , SWC6 , HTA8 , HTA9 , and HTA11 [18 , 20 , 37 , 38] , suggesting that PIE1 may serve as the catalytic and scaffolding subunit of an Arabidopsis SWR1-like complex .', 'Furthermore , functional characterizations of PIE1 , ARP6 and SWC6 have revealed that mutations in these genes have similar pleiotropic effects on Arabidopsis development , including a loss of apical dominance , curly and serrated rosette leaves , early flowering due to reduced expression of FLOWERING LOCUS C ( FLC ) , altered petal number , and reduced fertility [18 , 37–43] .', 'Interestingly , genetic experiments revealed that the pie1 null phenotypes are more severe than those of arp6 , swc6 , hta9;hta11 , or hta8;hta9;hta11 ( h2a . z near-null ) plants [18 , 19 , 37–43] .', 'The more dramatic phenotypes in pie1 plants suggest that PIE1 has additional functions outside of H2A . Z deposition by SWR1 , as previously proposed [5 , 6 , 18 , 19] .', 'A recent report also showed that mutant plants null for pie1 and h2az exhibited early developmental arrest , dying shortly after germination [19] , further supporting the notion that PIE1 has H2A . Z-independent functions in Arabidopsis .', 'On the other hand , genetic analyses of pie1;swc6 double mutant plants revealed that they had a phenotype indistinguishable from pie1 single mutants [38] , and arp6;swc6 plants displayed the same defects as either arp6 or swc6 single mutant plants [18 , 37] .', 'These results further support the idea that PIE1 , ARP6 , and SWC6 act in the same genetic pathway and/or are the components of the same protein complex , but that PIE1 has additional functions .', 'Despite the strong genetic and biochemical evidence that Arabidopsis contains many conserved subunits homologous to the components of the yeast SWR1 complex and mammalian SRCAP , the plant SWR1 complex has not been successfully isolated and characterized .', 'Recently , Bieluszewski and colleagues used Arabidopsis SWC4 and ARP4 proteins , two subunits shared between yeast SWR1 and NuA4 , as well as mammalian SRCAP and TIP60 complexes , as baits to affinity purify their interacting partners from Arabidopsis cell suspension cultures [44] .', 'These studies identified most of the subunits normally found in the SWR1 and NuA4 complexes , but it was not possible to determine whether this collection of proteins represented a single large complex or multiple complexes .', 'Overall , it is not yet clear whether plants possess separate SWR1 and NuA4 complexes , SWR1 and TIP60-like complexes , or all three complexes [44] .', 'The main goal of our study was to purify the Arabidopsis SWR1 complex in order to identify all of its components .', 'To achieve this , we used the ARP6 protein , a subunit unique to SWR1 in other organisms , as bait in Tandem Affinity Purification ( TAP ) experiments .', 'We performed three independent TAP experiments to isolate and identify ARP6-associated proteins .', 'We identified all 11 conserved subunits found in yeast SWR1 and mammalian SRCAP complexes , demonstrating that Arabidopsis contains a bona-fide functional and structural homolog of these complexes .', 'In addition , we identified several unexpected proteins that associated with ARP6 , including the plant homeodomain ( PHD ) - and Bromo domain-containing protein Methyl CpG-BINDING DOMAIN 9 ( MBD9 ) , and the PHD domain-containing proteins ALFIN-LIKE 5 ( AL5 ) , AL6 , and AL7 .', 'Association of these proteins with the SWR1 complex is consistent with the results of a recent related study that used ARP6-MYC or -FLAG tag epitope purification followed by mass spectrometry to identify ARP6-interacting proteins ( Potok et al . 2019 , bioRxiv 10 . 1101/657296 ) .', 'Genetic analyses revealed that mbd9 mutants showed phenotypic similarities to arp6 mutants , so we further explored a possible role for MBD9 in regulating H2A . Z incorporation into chromatin .', 'We found that MBD9 is required for H2A . Z incorporation at a subset of the sites that normally harbor H2A . Z nucleosomes , and that these MBD9-dependent H2A . Z sites have distinct chromatin features .', 'Furthermore , MBD9 strongly interacted with acetylated histone H4 peptides , H3 peptides mono- or dimethylated at lysine 4 , as well as H3 dimethylated at arginines 2 or 8 .', 'We also found that MBD9 is not a core subunit of the Arabidopsis SWR1 complex and that double mutant arp6;mbd9 plants exhibited much more severe phenotypes than single arp6 or mbd9 mutants .', 'These results collectively suggest that MBD9 targets the SWR1 complex to a subset of genomic loci but also has important functions beyond H2A . Z deposition .'], ['To isolate the Arabidopsis SWR1 complex , we decided to use ARP6 protein as bait in Tandem Affinity Purification ( TAP ) experiments because ARP6 is exclusively found in the SWR1 complex in other organisms and is not shared by any other known CRCs [4 , 18 , 34] .', 'For our purification experiments we used a GSrhino TAP-tag , which consists of two protein G domains , a tandem repeat of rhinovirus 3C protease cleavage site , and the streptavidin-binding peptide .', 'This tag has been successfully used to purify several plant nuclear complexes , including SWI/SNF type CRCs [45] .', 'Furthermore , the use of this tag provides high yield of purified proteins and specificity of purification .', 'In addition , a list of 760 proteins that nonspecifically bind to this tag or the associated purification beads has been assembled from data on 543 GS-based TAP experiments [45] .', 'We fused the GSrhino TAP-tag to either the N-terminal end ( N-TAP-ARP6 ) or the C-terminal end ( C-TAP-ARP6 ) of the genomic ARP6 coding sequence , containing the endogenous ARP6 promoter , and introduced the constructs into arp6-1 mutant plants to test for the ability of each transgene to complement a null arp6 allele .', 'Using western blotting , we first detected the presence of the 67 kDa ARP6-TAP-tag fusion protein specifically in plants homozygous for the transgene and not in arp6-1 or wild-type ( WT ) plants ( Fig 1A ) .', 'Next , we assessed the ability of the transgenes to rescue the morphological defects of arp6-1 plants .', 'When grown in parallel , the transgenic plants appeared almost indistinguishable from WT plants , with more compacted , non-serrated rosette leaves as compared to arp6-1 mutants ( Fig 1B ) .', 'The N-TAP-ARP6 and , to a lesser degree , the C-TAP-ARP6 transgenes are able to rescue the early flowering phenotype of arp6-1 plants ( Fig 1C ) as evident by the significantly higher number of rosette leaves at the time of flowering in wild type and transgenic plants compared to arp6-1 plants ( Fig 1D ) .', 'Finally , all transgenic plants showed full complementation for the loss of apical dominance and fertility defects of arp6-1 mutant plants ( Fig 1E and 1F ) .', 'Overall , we conclude that the N-TAP-ARP6 and C-TAP-ARP6 transgenes are fully functional and thus suitable for affinity purification .', 'Since the N-TAP-ARP6 and C-TAP-ARP6 transgenes were fully functional , we proceeded with the affinity purification experiments using two independent N-TAP-ARP6 transgenic lines ( N-TAP 11–4 and N-TAP 1–2 ) and one C-TAP-ARP6 line ( C-TAP 10–2 ) .', 'We followed the protocol described by Van Leene and colleagues to purify and elute the ARP6-TAP-tag interacting proteins and identified the eluted proteins by liquid chromatography coupled with tandem mass spectrometry ( LC-MS/MS ) [45] .', 'All eluted proteins detected in our three TAP-tag experiments are listed in S1 Table .', 'Using the database of non-specific binders of the GSrhino TAP-tag , we eliminated many proteins from this list as false positives and compiled a list of ARP6-interacting proteins .', 'Among these proteins , we identified ARP6 , SWC2 , SWC4 , SWC6 , PIE1 , RuvB1 , RuvB2 , ACTIN1 , ARP4 , YAF9a , and H2A . Z proteins as Arabidopsis homologs of all 11 conserved subunits found in both yeast SWR1 and mammalian SRCAP complexes ( Table 1 ) .', 'While we were able to detect HTA9 or HTA11 in all of our TAP-tag experiments , we never detected HTA8 , the third member of the H2A . Z family .', 'This finding is perhaps not surprising considering the fact that HTA8 has the lowest expression in all tissues when compared to the other H2A . Z family members in Arabidopsis ( S1A Fig ) .', 'We also did not detect the YAF9b protein ( Table 1 ) even though Arabidopsis YAF9a and YAF9b have been previously shown to act redundantly and are required for proper FLC expression [44 , 46 , 47] .', 'In addition to H2A . Z , we identified three H2B histones in our TAP experiments: HTB2 , HTB4 , and HTB9 ( Table 1 ) .', 'Since H2A . Z histones are deposited into nucleosomes as H2A . Z/H2B dimers , we sought to investigate whether specific H2A . Z proteins might have preferential H2B partners .', 'If this is true , we would expect to see synchronized expression of specific H2A . Z/H2B pairs in various Arabidopsis tissues .', 'Using publicly available microarray expression data [48] for the two H2A . Z and three H2B histones that we identified , we observed that HTA11 and HTB2 had highly similar expression profiles across tissues ( S1B Fig ) , while the expression of HTA9 matched very well with HTB4 expression and , to a slightly lesser degree , with HTB9 expression ( S1C and S1D Fig ) .', 'These results indicate that the Arabidopsis H2A . Z histones may have preferential H2B partners when deposited as dimers into nucleosomes .', 'Interestingly , in addition to known subunits of the SWR1 complex , we also reproducibly identified several other nuclear proteins as being associated with the SWR1 complex ( Table 1 ) .', 'These include MBD9 , a protein with a methyl-CpG-binding domain and various chromatin-binding domains [49–51] , TRA1A and TRA1B , two subunits in the SPT module of an Arabidopsis SAGA complex [52] that are also homologs of the yeast NuA4 subunit Tra1 and the mammalian TIP60 subunit TRRAP [34 , 44] , and three members of a plant-specific Alfin1-like family ( AL5 , AL6 , and AL7 ) best known for their regulation of abiotic stress responses in Arabidopsis and ability to bind di- and tri-methylated lysine 4 of histone H3 ( H3K4me2/3 ) [53 , 54] .', 'MBD9 , a methyl-CpG-binding domain-containing protein , was identified in all three TAP-tag experiments as an ARP6-interacting partner ( Table 1 ) .', 'Previous studies have shown that mbd9 mutants flowered significantly earlier than WT plants , due to reduced FLC expression , and produced more inflorescence branches when compared to WT plants [50] , which are phenotypes also found in arp6 mutants [40] .', 'We discovered that , in addition to the above-mentioned defects , mbd9 plants have serrated rosette leaves and a significantly increased number of flowers with extra petals ( S2 Fig ) , which are phenotypes also associated with the loss of ARP6 [18 , 39 , 40] .', 'Furthermore , examination of the previously reported MBD9 enrichment pattern at the FLC locus revealed that MBD9 occupied the two FLC regions previously shown to also have the highest H2A . Z enrichment in WT plants [41 , 51] .', 'Given that arp6 and mbd9 plants have similar phenotypes , that H2A . Z and MBD9 appear to occupy the same sites at the FLC locus , and that MBD9 co-purified with ARP6 in our TAP-tag experiments , we investigated whether MBD9 plays a role in the incorporation of H2A . Z into chromatin .', 'We performed three biological replicates of chromatin immunoprecipitation coupled with high-throughput sequencing ( ChIP-seq ) using an H2A . Z antibody [41] on WT , arp6-1 , and mbd9-1 seedlings .', 'At least 8 million high-quality , nuclear reads were aligned to the TAIR10 genome for each replicate , resulting in thousands of reproducible H2A . Z peaks for each genotype ( S3A Fig ) .', 'Spearman correlation analysis indicated a high degree of overlap among H2A . Z ChIP replicates for a given genotype with the exception of mbd9-1 replicate 3 , which correlated moderately with other two mbd9 replicates ( S3B Fig ) .', 'The average H2A . Z enrichment profile ( the average of all three scaled replicates ) across all gene bodies in WT plants showed the highest enrichment of H2A . Z just after the transcription start site , with decreasing enrichment toward the 3’ end , as expected ( Fig 2A ) .', 'The pattern of H2A . Z enrichment across genes in arp6-1 showed a similar profile but with extremely reduced enrichment , while mbd9-1 plants had an intermediate level of H2A . Z enrichment between WT and arp6-1 ( Fig 2A ) .', 'To analyze all of the regions normally enriched for H2A . Z , we identified peaks of H2A . Z enrichment that were present in at least two of the three H2A . Z ChIP-seq replicates in WT plants , and we then examined H2A . Z levels at these same sites in arp6-1 and mbd9-1 mutants .', 'As observed for gene bodies , the 7039 sites reproducibly enriched for H2A . Z in WT were nearly depleted of H2A . Z in arp6-1 , while there was an intermediate H2A . Z depletion in mbd9-1 plants ( Fig 2B ) .', 'Western blotting for H2A . Z on acid-extracted nuclear proteins isolated from WT , arp6-1 , and mbd9-1 showed a decrease in H2A . Z levels in both arp6-1 and mbd9-1 , suggesting that the lack of chromatin incorporation may lead to H2A . Z degradation ( S4A and S4B Fig ) .', 'To test whether the observed reduction of H2A . Z incorporation might be due to either SWR1 components or H2A . Z genes being misregulated in mbd9 plants , we performed qRT-PCR experiments to measure the expression of PIE1 , ARP6 , MBD9 , SWC4 , SWC6 , and YAF9a , as well as HTA8 , HTA9 , and HTA11 genes , in WT , arp6-1 , and mbd9-1 plants .', 'We found that the expression of these genes was not substantially altered and , therefore , unlikely to account for the observed depletion of H2A . Z levels in mbd9-1 plants ( S5 Fig ) .', 'Using RNA-seq , a related study has also demonstrated that the expression of SWR1 subunit genes was largely unaffected in mbd9-3 plants ( Potok et al . 2019 , bioRxiv 10 . 1101/657296 ) .', 'Overall , these results indicate that MBD9 is indeed required for proper H2A . Z incorporation into chromatin .', 'To further confirm our results with respect to the role of MBD9 in H2A . Z deposition , we performed ChIP-qPCR experiments using WT , arp6-1 , mbd9-1 , and two additional mbd9 T-DNA alleles , mbd9-2 and mbd9-3 [50] .', 'We first assayed H2A . Z abundance at two distinct regions of the FLC gene: the first and last exon ( regions 2 and 9 , respectively , as described in [41] ) .', 'Regions 2 and 9 are the sites on the FLC gene where H2A . Z is most highly enriched in WT plants , and that enrichment is lost in arp6-1 mutant plants ( S6A Fig , [41] ) .', 'We found that in plants homozygous for any of the three mbd9 alleles , the amount of H2A . Z at FLC regions 2 and 9 was reduced at least 2-fold when compared to WT plants ( S6A Fig ) , indicating that MBD9 contributes to H2A . Z deposition at the FLC gene .', 'We also measured the H2A . Z abundance at ASK11 and At4 , two phosphate starvation response genes previously shown to have H2A . Z deposited in their chromatin [55] .', 'We discovered that in mbd9 plants these genes were depleted of H2A . Z to similar levels as in arp6-1 plants when compared to WT ( S6B Fig ) .', 'Taken together , our results indicate that MBD9 is required for proper H2A . Z levels at multiple Arabidopsis genomic loci and is , therefore , functionally related to the SWR1 complex .', 'As MBD9 was previously reported to have histone acetyltransferase ( HAT ) activity in vitro and was found to associate with acetylated H4 [51] , we also examined the global levels of histone H4 N-terminal acetylation ( H4Ac ) in WT , mbd9-1 , and arp6-1 plants .', 'At least 5 million high-quality , nuclear reads were aligned to the TAIR10 genome for each replicate , resulting in thousands of reproducible , H4Ac peaks for each genotype ( S3A Fig ) .', 'Spearman correlation analysis indicated a high degree of overlap among H4Ac ChIP replicates for a given genotype with the exception of mbd9-1 replicate 3 , which correlated moderately with other two mbd9-1 replicates due to having the lowest levels of H4Ac among the replicates ( S3C and S7 Figs ) .', 'If MBD9 is responsible for global acetylation of H4 , as previously reported [51] , we would expect to see a dramatic reduction of acetylated H4 in mbd9-1 mutants compared to WT .', 'However , we found that the average genome-wide distribution of acetylated H4 was only modestly reduced in mbd9-1 plants , while arp6-1 plants had H4Ac levels similar to WT when examined across all gene bodies or at all sites enriched for acetylated H4 in WT plants ( Fig 2C and 2D ) .', 'Western blot analysis using H4Ac antibodies on acid-extracted nuclear proteins from WT , arp6-1 , and mbd9-1 plants was consistent with the ChIP-seq findings that mbd9-1 plants have moderately reduced levels of H4Ac when compared to WT ( S4A and S4C Fig ) .', 'Collectively , our ChIP-seq results demonstrate a major role for MBD9 in maintaining proper H2A . Z levels .', 'MBD9 may also have a minor role in the acetylation of H4 , but this is likely to be indirect given that the protein does not contain an identifiable acetyl CoA-binding or acetyltransferase domain .', 'The intermediate loss of H2A . Z in mbd9-1 plants compared with arp6-1 plants ( Fig 2A and 2B ) suggested that MBD9 may be required for incorporation of H2A . Z at only a subset of H2A . Z-enriched sites or it may be required for complete H2A . Z deposition at all sites .', 'To identify genomic regions that require MBD9 for H2A . Z incorporation into chromatin , we quantified the normalized H2A . Z ChIP-seq read abundance from WT , arp6-1 , and mbd9-1 mutant plants across all of the H2A . Z-enriched regions that were reproducibly identified in the WT replicates .', 'We performed DESeq analysis [56] to quantitatively compare WT to mbd9-1 and WT to arp6-1 H2A . Z levels at each site ( S2 Table ) .', 'H2A . Z levels were significantly depleted in arp6-1 at nearly all of the H2A . Z sites , as expected for a mutation that disrupts the SWR1 complex ( Fig 3A ) .', 'In contrast , out of the 7039 H2A . Z-enriched sites , we identified only 1391 sites that had reduced H2A . Z by at least 1 . 5-fold ( log2 fold change of at least 0 . 6 with an adjusted p value ≤ 0 . 05 ) in mbd9-1 compared to WT ( Fig 3B ) .', 'To further examine the nature of the H2A . Z deposition defect in mbd9 mutants , we visualized H2A . Z enrichment and distribution across the 1391 sites that lose H2A . Z in mbd9-1 , which we refer to as MBD9-dependent H2A . Z sites .', 'For comparison , we selected a similarly sized set of MBD9-independent H2A . Z sites ( 1505 sites with an average fold difference of less than 1 . 19 between WT and mbd9-1 , which is an absolute log2 fold change of less than 0 . 25 ) .', 'This analysis revealed a drastic reduction in H2A . Z occupancy at each of the MBD9-dependent H2A . Z sites when comparing WT and mbd9-1 , but with maintenance of the same overall pattern of occupancy ( Fig 3C ) .', 'The level of H2A . Z in mbd9-1 at MBD9-dependent sites was depleted to levels similar to arp6-1 .', 'In contrast , the MBD9-independent H2A . Z sites showed equivalent profiles and occupancy levels between WT and mbd9-1 , but still showed reduced H2A . Z in arp6-1 ( Fig 3C ) .', 'To further validate these results , we chose three MBD9-dependent and three MBD9-independent H2A . Z sites and performed qPCR experiments using the same H2A . Z ChIP material from WT and mbd9-1 plants previously utilized for ChIP-qPCR analysis of FLC , ASK11 , and At4 genes .', 'We found that all three MBD9-dependent loci showed statistically significant depletion of H2A . Z in mbd9-1 when compared to WT ( S8A and S8B Fig ) , while none of the three MBD9-independent sites had significantly different levels of H2A . Z between mbd9-1 and WT plants ( S8C and S8D Fig ) .', 'Thus , we conclude that MBD9 is required for proper H2A . Z deposition at a subset of the sites that this histone variant normally occupies .', 'In order to understand why MBD9 is required for H2A . Z deposition at certain sites and not others , we first analyzed the genomic distribution of MBD9-dependent H2A . Z sites compared to the MBD9-independent H2A . Z sites .', 'We found that the two sets are distributed similarly across the genome , with more than 80% of each set of coordinates localizing within genic regions ( S9A Fig ) .', 'Both MBD9-dependent and MBD9-independent H2A . Z sites were found primarily at the 5’ end of protein-coding gene ( PCG ) bodies , with the MBD9-dependent sites found slightly more upstream of the MBD9-independent sites ( S9B and S9C Fig ) .', 'PCGs nearest to the MBD9-dependent sites had reduced H2A . Z levels throughout the entire gene body in mbd9-1 and arp6-1 plants , whereas PCGs nearest to the MBD9-independent sites had similar gene body H2A . Z levels in WT and mbd9-1 , but still had reduced H2A . Z levels in arp6-1 ( S9C Fig ) .', 'For the 1322 PCGs found nearest to the MBD9-dependent H2A . Z sites ( S2 Table ) there was no significantly overrepresented Gene Ontology ( GO ) terms identified using either of the two different GO analysis tools ( see methods ) , indicating that MBD9-mediated deposition of H2A . Z is not associated with functionally-related gene sets or a particular cellular pathway .', 'We also examined various histone modification profiles at the two types of sites using publicly available ChIP-seq data from WT plants in order to discern any differences between H2A . Z sites that require MBD9 and those that do not .', 'Interestingly , we found that in WT plants the average level of histone H3 lysine 9 acetylation ( H3K9Ac ) is higher at MBD9-dependent H2A . Z sites than it is at the sites that do not require MBD9 ( Fig 4A ) .', 'However , no significant differences were found in the average enrichment of histone H3 lysine 18 acetylation or H3 lysine 27 trimethylation ( H3K18Ac and H3K27me3 , respectively ) between the two types of loci ( Fig 4B and 4C ) .', 'On the other hand , we did observe less abundant enrichment of dimethylated lysine 9 of histone H3 ( H3K9me2 ) at MBD9-dependent H2A . Z sites ( Fig 4D ) .', 'The same pattern was observed for H3 trimethylation at lysine 4 or lysine 36 ( H3K4me3 and H3K36me3 , respectively ) , with consistently lower levels of each mark at MBD9-dependent H2A . Z sites ( Fig 4E and 4F ) .', 'To better understand the differences in chromatin profiles between the two sets of loci , ten different histone marks ( H2A . Z , H4Ac , H3K27me3 , H2AK121Ub , H3K9me2 , H3K9Ac , H3K18Ac , H3K27Ac , H3K4me3 , and H3K36me3 ) were plotted around a 2 kb window centered on the H2A . Z peak of either the MBD9-dependent or MBD9-independent sites ( S10A Fig ) .', 'The heatmaps were clustered into 4 k-means clusters and three similar sets of chromatin states were observed in both MBD9-dependent and MBD9-independent loci .', 'These include euchromatic regions ( S10B Fig ) , H3K27me3-rich sites likely regulated by PRC2 ( S10C Fig ) , and sites that have H2A . Z but are relatively depleted of the other examined histone modifications .', 'The observed average differences in specific histone marks between the two sets of loci ( Fig', '4 ) appear to be driven by increased levels of H3K9Ac at MBD9-dependent euchromatic regions ( S10B Fig ) , decreased levels of H3K9me2 and H3K4me3 in MBD9-dependent PRC2-regulated regions ( S10C Fig ) , as well as reductions in H3K9me2 and H3K36me3 at the MBD9-dependent regions relatively depleted of most histone modifications ( S10D Fig ) .', 'Given the predominant gene-body localization of H2A . Z , the differences in histone modification levels between MBD9-dependent and -independent sites could simply reflect differences in expression levels of the underlying genes .', 'However , using publically available RNA-seq data , we found that genes nearest to the sites in each category span a wide range of expression levels and are not significantly different from one another in terms of steady-state transcript levels ( unpaired t-test , p < 0 . 05 , S11 Fig ) .', 'Thus , MBD9 may specifically recognize or be repelled by specific chromatin features , which could help guide the SWR1 complex to specific DNA sites .', 'To examine which histone marks MBD9 may directly recognize , we performed a histone peptide array assay using the full-length MBD9 protein ( S12 Fig ) .', 'We found that MBD9 most strongly interacted with histone H4 peptides that were acetylated at lysine ( K ) residues 12 , 16 , and 20 , or acetylated at K12 and K16 while being di- or trimethylated at K20 ( Fig 5 ) .', 'The protein also bound to H4 peptides containing both K16Ac and K20Ac , or K12Ac and K16Ac along with K20me1 .', 'This affinity of MBD9 for acetylated H4 is consistent with a previous report [51] .', 'Interestingly , MBD9 interacted with H3 peptides that were mono- or dimethylated at K4 , but did not appear to bind the H3 peptide that was trimethylated at K4 .', 'This lack of affinity for H3K4me3 could explain the relative depletion of this mark at MBD9-dependent H2A . Z sites ( Fig 4E ) .', 'Other H3 peptides that showed significant binding of MBD9 include those dimethylated at arginines 2 and/or 8 ( Fig 5 ) .', 'Collectively , these results suggest that MBD9 recognizes distinct combinations of histone modifications that are likely to influence its chromatin binding characteristics .', 'To determine whether the MBD9 protein , with an estimated molecular mass of 240 kDa , is an integral component of the Arabidopsis SWR1 complex ( Fig 6A ) we performed size-exclusion chromatography ( SEC ) experiments on protein extracts from WT and mbd9-1 plants , followed by western blotting for ARP6 .', 'This allowed us to define the native size of the complex and to determine whether this size changes in the absence of MBD9 , as would be expected if this protein were a stoichiometric component of the SWR1 complex ( Fig 6B ) , as previously demonstrated for the PIE1 subunit [41] .', 'Using an ARP6 monoclonal antibody [40] , we detected ARP6 protein in its native form as a part of a multi-subunit complex with a molecular mass of ~800 kDa ( Fig 6C ) .', 'When the SEC experiments were performed on mbd9-1 extracts , the ARP6 peak did not significantly shift and the estimated molecular mass of the native ARP6 complex in mbd9-1 plants was ~775 kDa ( Fig 6C ) in two biological replicates .', 'These results strongly suggest that MBD9 is not a core component of the ARP6-containing SWR1 complex , but most likely interacts with it in a more transient manner .', 'Alternatively , MBD9 may be a stable component of a minor subset of SWR1 complexes .', 'Collectively , we discovered that MBD9 is required for proper H2A . Z deposition at many sites , but is not stably associated with the SWR1 complex .', 'To investigate genetic interactions between MBD9 and ARP6 , we generated arp6-1;mbd9-1 double mutant plants .', 'We have shown that single arp6-1 and mbd9-1 mutant plants have similar phenotypic defects ( S2 Fig ) and that both ARP6 and MBD9 regulate H2A . Z incorporation into chromatin ( Fig 2A and 2B ) .', 'If these two proteins are subunits of the same complex or function exclusively in the same genetic pathway then double mutant plants should be phenotypically indistinguishable from single mutants , as previously shown for arp6;swc6 plants [18 , 37] .', 'Instead , we observed that the double mutants displayed much more severe defects ( dwarf stature , deformed leaves , and drastically reduced fertility ) than the individual single mutants throughout development ( Fig 7 ) .', 'Importantly , these phenotypes in the double mutant plants reverted back to those of each single mutant by introducing either the genomic ARP6 or MBD9 constructs into the double mutants ( S13 Fig ) , indicating that these defects were truly the result of simultaneous loss of ARP6 and MBD9 functions .', 'To investigate whether the more severe phenotype in arp6-1;mbd9-1 plants is caused by further reduction of H2A . Z incorporation into chromatin , we performed a ChIP-qPCR experiment using H2A . Z antibody on WT , arp6-1 , mbd9-1 , and arp6-1;mbd9-1 plants at FLC regions 2 and 9 .', 'We found that double mutant plants had similar levels of H2A . Z depletion at FLC when compared to arp6-1 ( S14 Fig ) .', 'Collectively , our findings support the idea that MBD9 is not a core subunit of the SWR1 complex and suggest that this protein has additional functions outside of H2A . Z incorporation [57] .'], ['Previous studies provided important evidence suggesting that Arabidopsis contains a SWR1-like complex that mediates incorporation of H2AZ into chromatin [6 , 18 , 2
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['The SWR1 chromatin remodeling complex , which deposits the histone variant H2A . Z into nucleosomes , has been well characterized in yeast and animals , but its composition in plants has remained uncertain .', 'We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 ( ARP6 ) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana .', 'We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes , demonstrating that this complex is conserved in plants .', 'We also identified several additional proteins not previously associated with SWR1 , including Methyl-CpG-BINDING DOMAIN 9 ( MBD9 ) and three members of the Alfin1-like protein family , all of which have been shown to bind modified histone tails .', 'Since mbd9 mutant plants were phenotypically similar to arp6 mutants , we explored a potential role for MBD9 in H2A . Z deposition .', 'We found that MBD9 is required for proper H2A . Z incorporation at thousands of discrete sites , which represent a subset of the genomic regions normally enriched with H2A . Z .', 'We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides , as well as those carrying mono- or dimethylated H3 lysine 4 , or dimethylated H3 arginine 2 or 8 .', 'Considering that MBD9-dependent H2A . Z sites show a distinct histone modification profile , we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A . Z deposition .', 'Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions .', 'The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex , along with the synergistic phenotype of arp6;mbd9 double mutants , suggests that MBD9 also has important roles beyond H2A . Z deposition .']
['The histone H2A variant , H2A . Z , is found in all known eukaryotes and plays important roles in transcriptional regulation .', 'H2A . Z is selectively incorporated into nucleosomes within many genes by the activity of a conserved ATP-dependent chromatin remodeling complex in yeast , insects , and mammals .', 'Whether this complex exists in the same form in plants , and how the complex is targeted to specific genomic locations have remained open questions .', 'In this study we demonstrate that plants do indeed utilize a complex analogous to those of fungi and animals to deposit H2A . Z , and we also identify several new proteins that interact with this complex .', 'We found that one such interactor , Methyl-CpG-BINDING DOMAIN 9 ( MBD9 ) , is required for H2A . Z incorporation at thousands of genomic sites that share a distinct histone modification profile .', 'The histone binding properties of MBD9 suggest that it may guide H2A . Z deposition to specific sites by interacting with modified nucleosomes and with the H2A . Z deposition complex .', 'We hypothesize that this represents a general paradigm for the targeting of H2A . Z to specific sites .']
['biotechnology', 'engineering and technology', 'brassica', 'dna-binding proteins', 'fungi', 'plant science', 'model organisms', 'genetically modified plants', 'experimental organism systems', 'epigenetics', 'molecular biology techniques', 'plants', 'chromatin', 'genetic engineering', 'arabidopsis thaliana', 'research and analysis methods', 'bioengineering', 'genetically modified organisms', 'artificial gene amplification and extension', 'animal studies', 'chromosome biology', 'proteins', 'gene expression', 'histones', 'molecular biology', 'nucleosomes', 'yeast', 'biochemistry', 'eukaryota', 'plant and algal models', 'cell biology', 'polymerase chain reaction', 'genetics', 'biology and life sciences', 'plant biotechnology', 'organisms']