| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online August 12, 2005; 10.1105/tpc.105.034165 © 2005 American Society of Plant Biologists
Monomethyl Histone H3 Lysine 4 as an Epigenetic Mark for Silenced Euchromatin in Chlamydomonas
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
|---|
; Loidl, 2004). Histone residues can be covalently altered by enzymatic complexes that include, among others, methylation, acetylation, phosphorylation, or ubiquitination activities (Fischle et al., 2003; Loidl, 2004), whereas the main postsynthetic DNA change usually involves cytosine methylation (Richards and Elgin, 2002; Tariq and Paszkowski, 2004). These modifications can affect chromatin states by directly modulating nucleosome structure and/or by creating binding surfaces for chromatin structural/regulatory factors (Richards and Elgin, 2002; Fischle et al., 2003).
Posttranslational histone modifications can function as epigenetic marks for either active or repressed chromatin (Kouzarides, 2002; Fischle et al., 2003; Lachner et al., 2003). In most eukaryotes, methylated histone H3 Lys 4 (H3K4) correlates with transcriptionally competent chromatin, whereas methylated H3K9 occurs preferentially in transcriptionally silent chromatin (Bernstein et al., 2002; Alvarez-Venegas et al., 2003; Fischle et al., 2003; Lachner et al., 2003; Schneider et al., 2004; Tariq and Paszkowski, 2004). However, methylatable Lys residues can exist in monomethylated, dimethylated, or trimethylated states, and recent evidence suggests that the degree of methylation has considerable influence on transcriptional stimulation or repression (Santos-Rosa et al., 2002; Tamaru et al., 2003). In addition, histone methylation likely cooperates with DNA methylation in maintaining heterochromatic domains (Richards and Elgin, 2002; Tariq and Paszkowski, 2004). Moreover, underscoring the complexity of these processes, the RNA interference (RNAi) machinery has recently been implicated in the establishment of silent heterochromatin (Matzke et al., 2004; Pal-Bhadra et al., 2004).
Methylation of most histone Lys residues is mediated by proteins containing a conserved SET domain, first identified in the Drosophila melanogaster chromatin factors Su(var)3-9, Enhancer-of-zeste, and Trithorax (Trx) (Rea et al., 2000). Trx displays histone methyltransferase (HMTase) activity specific for H3K4 (Smith et al., 2004), and complexes with similar enzymatic capacity participate in transcriptional activation in several eukaryotes (Roguev et al., 2001; Nakamura et al., 2002; Hughes et al., 2004). In Arabidopsis thaliana, ATX1, a Trx homolog, acts as an activator of homeotic genes (Alvarez-Venegas et al., 2003). However, there is also unexplained evidence linking the H3K4 methylation machinery to chromatin repression. In mammals, the H3K4 HMTase Mixed-Lineage Leukemia (MLL) is part of a supercomplex with both repressive and activating functions (Nakamura et al., 2002; Xia et al., 2003). Moreover, in Saccharomyces cerevisiae, subunits of a H3K4 methylating complex are required for silencing of certain chromosomal loci (Bryk et al., 2002; Carvin and Kladde, 2004).
We have previously characterized a Chlamydomonas reinhardtii mutant, Mut-11, defective in the transcriptional silencing of RbcS2:aadA:RbcS2 transgenes (conferring spectinomycin resistance), TOC1, a retrotransposon-like element, and Gulliver, a cut-and-paste DNA transposon (Jeong et al., 2002; Zhang et al., 2002). Mut11 encodes a WD40-repeat protein (Mut11p) homologous to S. cerevisiae Swd3 and human WDR5, conserved subunits of activating H3K4 HMTase complexes (Roguev et al., 2001, 2004; Hughes et al., 2004; Dou et al., 2005). WDR5 has also been implicated in the transcriptional repression mediated by the clock protein PERIOD1 (Brown et al., 2005). Here, we show that Mut11p copurifies with histone methylating activities. Deletion of Mut11 or RNAi-mediated suppression of Set1 (encoding a H3K4 methyltransferase) results in defects in H3K4 monomethylation and transcriptional derepression of certain genes, transgenes, and transposons. Our findings suggest that monomethyl H3K4 is associated with silenced euchromatin and that certain Trx-like complexes may function in gene repression.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
|---|
) as well as yeast two-hybrid screens. For affinity purification, the Mut11 coding sequence was fused to the TAP tag, placed under the control of a constitutive promoter, and transformed into Mut-11. Expression of Mut11-TAPp partly rescued the mutant phenotype, evidenced by resilencing of RbcS2:aadA:RbcS2 (Figure 1A). Mut11-TAPp–associated proteins were isolated by affinity purification, resolved by SDS-PAGE, and identified by tandem mass spectrometry (Figure 1B, Table 1). Three of the purified polypeptides were similar to HMTase complex subunits (Roguev et al., 2001, 2004; Hughes et al., 2004). The 83-kD protein (band 1) is related to yeast Swd1/human Rbbp5 (for Retinoblastoma binding protein 5), whereas the 42-kD polypeptide (band 8) is homologous to yeast Bre2/human Ash2L (for Absent, small or homeotic discs 2-like) (Figure 1B, Table 1). The protein represented by band 9, named Set4p, contains a plant homeodomain zinc finger and a C-terminal SET domain with similarity to the Trx class of HMTases (Figures 2C and 3) (Kouzarides, 2002). Control purifications using a TAP-tagged Ble fusion, expressed from an integrated transgene that confers bleomycin resistance, did not identify any of the Mut11-TAPp–associated proteins (Figure 1B).
|
|
|
|
) and regulate the formation of certain repressive complexes with histone deacetylase activity (Guenther et al., 2002). Thus, it is tempting to speculate that these chaperones are required for the proper folding of Mut11p and, possibly, its assembly into a protein complex(es). Intriguingly, a Mut11p-ß-glucuronidase fusion protein, although predominantly localized in the nucleus, can also be detected at lower abundance in the cytosol (Zhang et al., 2002), the likely subcellular location of a HSP70/90/CCT-mediated step.
In yeast two-hybrid screens with Mut11p as a bait, two types of clones were repeatedly isolated from two Chlamydomonas cDNA expression libraries (Figure 2A). One class of cDNAs encoded a SET domain–containing protein, named Set1p. Based on the similarity of aligned SET motifs (Figures 2B, 2C, and 3), Set1p, a predicted polypeptide of 1795 amino acids, is related to the Trx class of HMTases (Kouzarides, 2002). This protein is encoded by C_1710009 in the draft Chlamydomonas genome sequence (http://genome.jgi-psf.org/chlre2/chlre2.home.html). The second type of cDNAs coded for a polypeptide named QAp, for a long stretch of alternating Gln/Ala residues. QAp is encoded by C_410063 and corresponds to a predicted protein of 1472 amino acids containing a Leu zipper domain (Figure 2B), typical of DNA binding proteins. The QAp C-terminal region is weakly similar to S. cerevisiae Ssn6, which forms a global transcriptional corepressor with Tup1. Intriguingly, Mut11p, the interacting partner of QAp, has similarity to the carboxyl end of Tup1 (Zhang et al., 2002).
We have tried to verify the interaction between Mut11p and Set1p by coimmunoprecipitation assays and by in vivo protein cross-linking with formaldehyde (Rohila et al., 2004) prior to Mut11-TAPp complex purification. However, all of our attempts have been unsuccessful (data not shown). Thus, we cannot rule out that the yeast two-hybrid interaction between Mut11p and Set1p is artifactual. Alternatively, the failure to detect Set1p in association with Mut11-TAPp could be due to technical limitations, such as low affinity interactions, compounded by the low abundance of the proteins under study. Interestingly, in animals and fungi, the Mut11p, Ash2L, and Rbbp5 homologs are conserved components of virtually all affinity-purified Trx-like HMTase complexes, suggesting that they represent strongly interacting core subunits. By contrast, other proteins, including several HMTases, appear to associate more transiently and vary in different complexes depending on the purification conditions (Roguev et al., 2001, 2004; Nakamura et al., 2002; Goo et al., 2003; Wysocka et al., 2003; Hughes et al., 2004; Yokoyama et al., 2004; Popovic and Zeleznik-Le, 2005). Nevertheless, our findings, taken together, clearly support an association of Mut11p with conserved HMTase complex subunits.
The Mut11p Complex(es) Possesses Histone Methylating Activity and Recombinant Set1p Methylates H3K4
Given the relatedness of Mut11-TAPp copurifying polypeptides to H3K4 HMTase complex subunits, we examined whether the Mut11-TAPp complex(es) had HMTase activity. Purified proteins were incubated with core histones and the methyl donorS-adenosyl-L-(methyl-14C)methionine (14C-SAM). In this assay, the Mut11-TAPp complex(es) displayed several activities, methylating not only H3 but also H2A and H4 (Figure 4A), even though by affinity purification we had only identified Set4p interacting with Mut11-TAPp. By contrast, no HMTase activity was detected in association with the TAP-tagged Ble control. Since SET domain-containing HMTases are quite specific in terms of the histone and Lys residue that they modify, because of the knot-like structure of the active site (Lachner et al., 2003; Zhang et al., 2003; Collins et al., 2005), these observations suggest that Mut11p likely interacts with several HMTases, either as part of one supercomplex or, as described in mammals, of distinct complexes. Of the SET domain HMTases with known substrate specificity (Kouzarides, 2002; Lachner et al., 2003; Collins et al., 2005), Set4p is more similar to the Trx class but relatively weakly (Figures 2C and 3), and, thus far, we have not been able to detect any HMTase activity associated with a partly truncated recombinant protein (data not shown).
|
). HMTases containing Phe at the position corresponding to Y1768 of Set1p (Figure 2C, arrowhead) function preferentially as dimethyltransferases and/or trimethyltransferases, whereas those containing Tyr at this position work predominantly as monomethyltransferases and/or dimethyltransferases (Collins et al., 2005). The only known exception to this Phe/Tyr switch hypothesis is S. cerevisiae Set1 that has a Tyr residue at the comparable position and yet can monomethylate, dimethylate, and trimethylate H3K4 (Santos-Rosa et al., 2002; Boa et al., 2003; Collins et al., 2005). However, recent findings have indicated that an N-terminal RNA-recognition motif (RRM), perhaps regulated by interaction with additional proteins, is required for the H3K4 trimethylation activity of the yeast enzyme (Fingerman et al., 2005; Morillon et al., 2005; Schlichter and Cairns, 2005). In its absence or when mutated, S. cerevisiae Set1 behaves mostly as a monomethyltransferase and dimethyltransferase. A corresponding RRM domain is missing from both Chlamydomonas Set1p and Set4p (Figure 2B; data not shown). Thus, based on their active site sequences (Figure 2C), Set1p would be predicted to function as a monomethyltransferase and/or dimethyltransferase, while Set4p would work as a dimethyltransferase and/or trimethyltransferase.
Deletion of Mut11 Causes Global Changes in H3K4 Methylation
Because our results suggested that gene silencing in Chlamydomonas requires a H3K4 HMTase complex subunit, Mut11p, we examined whether the mutant strain showed defects in H3K4 methylation. In immunoblot analyses with specific antibodies, Mut-11 displayed almost undetectable levels of H3K4me1 but significantly increased amounts of H3K4me2, in comparison with the parental strain (Figure 4D). Although the anti-H3K4me3 antibody cross-reacted with several nonhistone proteins in Chlamydomonas, it revealed that Mut-11 was also defective in H3K4 trimethylation (Figure 4E). We also tested for additional alterations in histone methylation since, given the multiple HMTase activities associated with Mut11-TAPp, we expected that Mut-11 would be deficient in several modifications. However, using antibodies specific for H3K9me2 or H4K20me2, we were unable to detect a signal corresponding to histones H3 or H4, respectively (data not shown). This is in agreement with previous data indicating that Chlamydomonas may not contain significant amounts of dimethyl H3K9 (Waterborg et al., 1995). When we examined the global levels of H3K9me1, all strains behaved similarly to the wild type (Figure 4D).
In agreement with our findings in Chlamydomonas, recent experiments demonstrated that small interfering RNA (siRNA)-mediated downregulation of the mammalian Mut11p homolog, WDR5, results in defects in both H3K4me1 and H3K4me3, but not in H3K4me2 (Wysocka et al., 2005). Furthermore, WDR5 was reported to play a role in the transcriptional activation of homeotic Hox genes, coincidental with the conversion of H3K4me2 to H3K4me3 by an associated HMTase (Wysocka et al., 2005). However, the function of H3K4 monomethylation was not examined in this study. Interestingly, lack of WDR5 has also been implicated in defects in H3K9me2 and in gene repression mediated by the clock protein PERIOD1 (Brown et al., 2005). These apparently contradictory observations could be explained if WDR5 is a core component of distinct HMTase complexes with either activating or repressing function(s) (or of a supercomplex with distinctly regulated activities), and the observed outcome (activation or silencing) depends on the gene(s) examined (see Discussion). This may also be the case with Chlamydomonas Mut11p. Trimethylation of H3K4 has been strongly linked to transcriptional activity in virtually all examined eukaryotes (Lachner and Jenuwein, 2002; Schneider et al., 2004). Thus, we hypothesize that Mut11p and Set4p (based on its active site sequence) may be involved in H3K4me3 and gene activation (although this awaits further experimental support). However, since we have been exploring the role of Mut11p in gene silencing, we decided to examine the function of H3K4 monomethylation, perhaps performed by Set1p and/or an equivalent HMTase.
Suppression of Set1 Expression by RNAi Causes Gene Silencing Defects and a Global Deficiency in H3K4 Monomethylation
We tested whether RNAi-mediated Set1 suppression induced defects in gene repression similar to those of Mut-11, potentially supporting a role for this H3K4 HMTase in silencing as well as an in vivo interaction between Mut11p and Set1p. However, since Mut11p appears to associate with several HMTase activities in Chlamydomonas (Figure 4A), a defect in Mut11 may result in stronger phenotypic abnormalities than the lack of a single SET domain protein. Chlamydomonas 11-P[300], containing a silenced RbcS2:aadA:RbcS2 transgene, was transformed with an inverted repeat construct (Rohr et al., 2004) designed to produce double-stranded RNA homologous to Set1 (Set1-IR). We recovered several transformants displaying strong downregulation of Set1 expression (Figure 5A; data not shown). These Set1-IR strains grew on spectinomycin-containing media, albeit to a different extent than Mut-11, indicating transcriptional derepression of RbcS2:aadA:RbcS2 (Figure 5B; data not shown). Moreover, similar to our previous observations with Mut-11 (Zhang et al., 2002), Set1-IR strains also showed enhanced expression of the TOC1 retrotransposon-like element (Figure 5C), whose transcripts are heterogeneous in size and produce a smeary signal in RNA gel blot analyses (Jeong et al., 2002). Expression of Trxh2, encoding a cytosolic thioredoxin isoform that is typically transcribed at low levels (Sarkar et al., 2005), was also enhanced in Mut-11 and Set1-IR2 (Figure 7A). On the contrary, transcription of a highly active gene, encoding the 40S ribosomal protein S3 (Rps3), did not change in the RNAi strains or the mutant (Figure 7A).
|
|
; Wysocka et al., 2005). Consequently, Mut11p-dependent silencing in Chlamydomonas may be mediated by more than one histone modification, although thus far we have only detected alterations in H3K4 methylation in the mutant background (see Discussion).
Monomethylated H3K4 Is Associated with Transcriptionally Repressed Loci
To determine if changes in H3K4 monomethylation were directly involved in the transcriptional reactivation of TOC1, RbcS2:aadA:RbcS2, and Trxh2, we analyzed their chromatin environment by chromatin immunoprecipitation (ChIP) assays (Figures 6 and 7). Using primers specific for the promoter or transcribed sequences of the TOC1 transposon, we detected a strong decrease in monomethylated H3K4 associated with these elements in both Mut-11 and Set1-IR2 (Figure 6), as compared with the parental strain (11-P[300]) where TOC1 is repressed. Consistent with the global changes in histone H3 methylation (Figure 4D), we also found a dramatic enrichment in H3K4me2 associated with TOC1 in Mut-11. On the other hand, no differences in H3K4 dimethylation were observed between Set1-IR2 and 11-P[300] (Figure 6). Similar patterns of H3K4me1 and H3K4me2 were identified for the RbcS2:aadA:RbcS2 transgene (Figure 6) and the weakly expressed Trxh2 gene (Figure 7B). We also tested by ChIP whether H3K9me2 was associated with the chromatin of the silenced loci, since this epigenetic mark has been shown to correlate with transcriptional repression in a number of eukaryotes (Richards and Elgin, 2002; Tachibana et al., 2002; Tariq and Paszkowski, 2004). However, in agreement with our immunoblotting results (see above), we could not detect H3K9me2 associated with TOC1 in any of the strains (data not shown). Lastly, as the available antibodies for H3K4me3 exhibited cross-reactivity with additional (unknown) Chlamydomonas proteins (Figure 4E), we did not examine this histone modification by ChIP.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
|---|
, 2004; Nakamura et al., 2002; Hughes et al., 2004; Dou et al., 2005). The isolated Mut11-TAPp complex(es) displays several HMTase activities and includes at least one detectable SET domain–containing protein, Set4p. Moreover, deletion of Mut11 causes defects in the H3K4 monomethylation associated with derepressed transcription units and a corresponding increase in H3K4 dimethylation. The mutant strain also shows a global deficiency in H3K4 trimethylation. Although the Mut11p machinery could have an indirect effect on H3K4me1 and H3K4me3, perhaps by modifying other histone residues that stimulate the activity of a H3K4 HMTase(s), a direct role seems much more likely given the evolutionarily conserved function of Mut11p homologs as components of Trx-like complexes in fungi and animals (Roguev et al., 2001; Nakamura et al., 2002; Goo et al., 2003; Wysocka et al., 2003; Hughes et al., 2004; Yokoyama et al., 2004; Popovic and Zeleznik-Le, 2005). In addition, similar defects in H3K4me1 and H3K4me3 have recently been reported for siRNA-mediated suppression of WDR5 in mammalian cells (Wysocka et al., 2005). However, Set4p is unlikely to be the enzyme responsible for H3K4 monomethylation since, according to the Phe/Tyr switch model (Cheng et al., 2005; Collins et al., 2005), it would be expected to act predominantly as a dimethyltransferase and/or trimethyltransferase. As already mentioned, we speculate that Mut11p homologs may be components of HMTase complexes with either activating or silencing functions (see below). In this context, Set4p could conceivably be involved in H3K4 trimethylation and gene activation, or alternatively, it could modify other histone residue(s). We expect to address the Set4p substrate specificity in the future by the phenotypic and molecular characterization of strains showing RNAi-mediated suppression of Set4.
Several Trx-like HMTase complexes, which include the putative core subunits (Mut11p, Ash2L, and Rbbp5 homologs) but differ in the associated H3K4 methyltransferases, have been isolated from mammalian cells (Nakamura et al., 2002; Goo et al., 2003; Wysocka et al., 2003; Hughes et al., 2004; Yokoyama et al., 2004; Dou et al., 2005; Popovic and Zeleznik-Le, 2005). Likewise, the multiple activities displayed by affinity-purified Mut11-TAPp also suggest that Mut11p may associate with several methyltransferases. Indeed, it is tempting to speculate that a variable range of proteins, including potential transcription corepressors or activators and different HMTases, may interact with Mut11p homologs and confer specific histone-modifying activity(ies) to the resulting complexes. In yeast two-hybrid assays, Mut11p associates with Set1p, a Trx-like H3K4 HMTase. However, we have been unable to verify this interaction, despite repeated attempts by a variety of approaches. Thus, we cannot rule out that Set1p functions independently from Mut11p. Yet, it is also possible that the interaction between these proteins is relatively weak precluding its detection due to technical limitations. As already discussed, there is some precedence for this in the characterization of similar complexes from mammalian cells, where different components are isolated depending on the purification conditions (Nakamura et al., 2002; Hughes et al., 2004; Yokoyama et al., 2004; Popovic and Zeleznik-Le, 2005).
Recombinant Set1p methylates H3K4, and based on its active site sequence (Collins et al., 2005), it is expected to function predominantly as a monomethyltransferase and/or dimethyltransferase. Consistent with this interpretation, suppression of Set1 by RNAi resulted in reduction of monomethylated H3K4 associated with transcriptionally repressed loci as well as gene silencing defects. These phenotypes were similar to those of the Mut11 deletion mutant but of lower magnitude. The Set1-IR strains also failed to display the increase in H3K4me2 and the reduction in H3K4me3 observed in Mut-11. Thus, these results are ambiguous with respect to a potential interaction between Set1p and Mut11p. These proteins could be components of independent molecular machineries capable of modifying H3K4. Alternatively, they could work together, but Mut11p likely associates with additional HMTases, and its depletion results in further defects than those caused by Set1 suppression. Interestingly, deletion of Mut11 did not entirely abolish H3K4 monomethylation (Figures 6 and 7B), suggesting that other WD40-repeat containing proteins may partly compensate for the loss of Mut11p, a H3K4 monomethyltransferase normally associated with (or stimulated by) Mut11p may be able to act with reduced efficacy in its absence, and/or an alternative H3K4 monomethylating machinery is still functional in the Chlamydomonas mutant. Systematic suppression by RNAi of the putative H3K4 HMTases encoded in the Chlamydomonas genome (http://genome.jgi-psf.org/chlre2/chlre2.home.html) will help address some of these issues. However, independent of the specific molecular machinery(ies) responsible for H3K4 monomethylation, our results strongly suggest that this epigenetic mark is associated with transcriptionally repressed euchromatin in Chlamydomonas.
H3K4 Monomethylation and Euchromatin Silencing
In all eukaryotes examined to date, transcriptionally competent chromatin has been firmly linked to histone H3K4 dimethylation and/or trimethylation (Lachner and Jenuwein, 2002; Alvarez-Venegas et al., 2003; Schneider et al., 2004; Tariq and Paszkowski, 2004). Although the specific role(s) of these modifications remains poorly understood, S. cerevisiae H3K4me3 appears to be associated with the transition from transcript initiation to elongation and requires members of the Paf1 elongation complex, phosphorylation of Ser 5 of the C-terminal domain of RNA polymerase II, and ubiquitination of K123 on H2B (Briggs et al., 2002; Krogan et al., 2003; Ng et al., 2003; Morillon et al., 2005; Peters and Schubeler, 2005). Moreover, in metazoans, sites of H3K4me2/H3K4me3 correlate with the transcription starts of active genes, and the degree of modification correlates positively with the transcriptional rate (Schneider et al., 2004; Schubeler et al., 2004; Bernstein et al., 2005). However, in D. melanogaster, the H3K4 HMTase Trx is only required for transcriptional activation in the presence of repressive activity from Polycomb group proteins (Klymenko and Muller, 2004; Peters and Schubeler, 2005). This suggests that Trx-mediated H3K4 di/trimethylation functions as an antirepressor, as a mark that maintains the active state, rather than as a transcriptional activator (Peters and Schubeler, 2005). In this context, if repression is weakened, transcription can likely occur (although perhaps at relatively low rates) without an increase in H3K4me2/H3K4me3, as detected for some metazoan genes (Perkins et al., 2004; Schubeler et al., 2004) and suggested by our results with the Set1-IR strains. In more active genes, H3K4 di/trimethylation may facilitate higher transcription rates through the maintenance of greater chromatin accessibility (Nishioka et al., 2002; Zegerman et al., 2002; Santos-Rosa et al., 2003; Pray-Grant et al., 2005). Thus, H3K4me2/H3K4me3 may be part of a positive feedback loop facilitating transcription and providing a molecular memory of transcriptional activity (Gerber and Shilatifard, 2003; Ng et al., 2003). In contrast with this wealth of data supporting a correlation between H3K4 di/trimethylation and gene activity, the role of H3K4 monomethylation has remained virtually unexplored.
In Chlamydomonas, Mut11p and Set1p are required for H3K4 monomethylation and the maintenance of transcriptional repression of certain genes, transgenes, and transposons. The silenced transcription units that we have examined likely correspond to euchromatic domains. The TOC1 and Gulliver transposons are dispersed throughout the genome as single insertions in intergenic regions (http://genome.jgi-psf.org/chlre2/chlre2.home.html). These elements, as well as repressed single copy RbcS2:aadA:RbcS2 transgenes, lack the heavy DNA methylation typical of heterochromatic repeats (Cerutti et al., 1997; Jeong et al., 2002; Zhang et al., 2002), and silent transgenes do not show the restricted accessibility to nucleases typical of heterochromatin (Cerutti et al., 1997). Moreover, Mut11 and Set1 are necessary for the repression of some euchromatic genes, such as Trxh2. Interestingly, when grown under normal laboratory conditions, the majority of Chlamydomonas chromatin appears to be in a silenced state since only 
20% of histone H3 is dynamically multiacetylated (Waterborg, 1998), an indication of transcriptional activity (Zhang et al., 2004). Moreover, recent microarray experiments have demonstrated that 60% of the Chlamydomonas predicted genes are not expressed during flagellar regeneration (Stolc et al., 2005).
Under normal Chlamydomonas growth conditions, 81.2% of histone H3 is monomethylated at K4, and 15.6% shows K9 monomethylation (Waterborg et al., 1995). Consistent with our ChIP results, the extensive amount of H3K4me1 (equivalent to the silenced chromatin fraction) strongly indicates that this modification is associated with repressed chromatin. Moreover, our findings also suggest that monomethyl H3K4 is necessary for gene silencing since a decrease in H3K4me1 in the Set1-IR strains resulted in reactivation of transcriptionally repressed loci. In the Mut-11 mutant, we observed more dramatic changes, as loss of H3K4me1 was accompanied by a substantial increase in H3K4me2, reduction in H3K4me3, and more extensive defects in gene silencing (Jeong et al., 2002; Zhang et al., 2002). In this case, we speculate that lack or diminished H3, H2A, and/or H4 HMTase activities likely results in more marked deficiencies in repressive chromatin (although potential alterations in H2A and H4 methylation remain to be explored). In addition, if Mut11p is also a component of activating HMTase complexes (as suggested by the Mut-11 defect in H3K4me3), the mutant strain may also be deficient in the activation of certain genes. Substantial alterations in chromatin states were also observed in Arabidopsis after RNAi-mediated suppression of a Mut11 homolog (J. Xu, K. van Dijk, S. Sato, T. Clemente, and H. Cerutti, unpublished data), suggesting that at least some of the Mut11p-mediated processes have been evolutionarily conserved in the plant lineage.
Links between H3K4 methyltransferase subunits and gene repression have also been observed in yeast and mammals, but the actual mechanism has remained unexplained (Spada et al., 2005). In mammalian cells, the DNA methyltransferase Dnmt3a, involved in gene silencing, copurifies with both H3K9 and H3K4 HMTase activities (Fuks et al., 2003). Some mammalian H3K4 methyltransferases occur in supercomplexes containing transcriptional repressors such as Sin3 and NuRD (Nakamura et al., 2002; Wysocka et al., 2003; Xia et al., 2003). Moreover, knockdown of WDR5 has recently been demonstrated to result in several histone H3 methylation defects (including reduction in H3K4me1, H3K4me3, and H3K9me2) and alterations in both gene activation (Wysocka et al., 2005) and gene silencing (Brown et al., 2005). In budding yeast, loss of H3K4 HMTase subunits results in derepression of several genes (Carvin and Kladde, 2004) and defects in heterochromatic silencing (Bryk et al., 2002). In the latter case, H3K4 methylation has been postulated to play an indirect role in gene repression (Bernstein et al., 2002; van Leeuwen and Gottschling, 2002; Santos-Rosa et al., 2004). Indeed, H3K4me2 is depleted from silenced, heterochromatic domains and marks genome-wide active chromatin in S. cerevisiae (Bernstein et al., 2002). Therefore, loss of H3K4me2/H3K4me3 has been proposed to allow the nonspecific binding of silencing factors throughout the genome, resulting in reduced concentration of these proteins at normally repressed heterochromatic loci and corresponding defects in silencing (van Leeuwen and Gottschling, 2002; Santos-Rosa et al., 2004). However, this hypothesis cannot explain our results regarding euchromatic silencing in Chlamydomonas since monomethylated H3K4 is predominantly associated with repressed transcriptional units, and its loss correlates with gene reactivation, without a change (Set1-IR2 strain) or with an increase (Mut-11 strain) in H3K4me2. Conversely, H3K4me1 is barely detectable at constitutively active genes that, by comparison, show relatively high levels of H3K4me2.
The dual (activating and silencing) nature of the H3K4 methyl mark may be alternatively explained if monomethyl H3K4 is one of the epigenetic marks associated with euchromatic gene silencing, perhaps as part of a combinatorial set of posttranslational histone modifications (Fischle et al., 2003). In this respect, H3K4me1 has recently been found associated with the repressed MET16 gene in S. cerevisiae, and its levels drop rapidly upon gene reactivation, particularly at the promoter region, coincidental with the appearance of H3K4me3 (Morillon et al., 2005). Moreover, consistent with the presence of H3K4me1 on inactive genes, this modification is independent of H2B ubiquitination or Paf1, which are required for H3K4me2 and H3K4me3 (Morillon et al., 2005). Furthermore, in mouse embryonic stem cells, initiation of X chromosome inactivation correlates with a reduction in H3K4me2/H3K4me3, but no change in H3K4 monomethylation, suggesting that this mark is compatible with a state of transcriptional repression (Kohlmaier et al., 2004). In Chlamydomonas, as already described, loss of monomethyl H3K4 mimics some of the molecular defects caused in mammalian and plant cells by reduced H3K9me2, a well established epigenetic silencing mark (Richards and Elgin, 2002; Tachibana et al., 2002; Tariq and Paszkowski, 2004). Thus, our results suggest functional differentiation between dimethyl H3K4 (and most likely trimethyl H3K4, although we have been unable to study this modification by ChIP due to the nonspecific cross-reaction of available antibodies with nonhistone proteins) and monomethyl H3K4, with the latter correlating with transcriptional repression in euchromatic domains. We also hypothesize that in some eukaryotes there may be at least two kinds of Trx-like complexes containing Mut11p homologs: a repressive one that monomethylates H3K4 (and likely modifies other histone residues) and an activating one that trimethylates H3K4. In other eukaryotes, such as S. cerevisiae, a single complex may be able to perform monomethylation, dimethylation, or trimethylation of H3K4, but the degree of methylation is likely modulated by interacting proteins (Morillon et al., 2005).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
|---|
; Zhang et al., 2002). Strains 11-P[300] and Mut-11 have been described previously (Cerutti et al., 1997; Zhang et al., 2002). To suppress the expression of Set1 or Maa7 (encoding Trp synthase ß-subunit), we used an RNAi approach explained in detail elsewhere (Rohr et al., 2004). Briefly, 11-P[300] was transformed by the glass bead method (Cerutti et al., 1997; Zhang et al., 2002) with inverted repeat transgenes designed to produce hairpin double-stranded RNA and induce the degradation of homologous transcripts. For Set1, a 628-bp SmaI-HincII fragment spanning 60 bp of coding sequence and 568 bp of the 3' untranslated region (UTR) was inserted in sense and antisense orientation flanking a spacer sequence in vector Maa7/X IR (Rohr et al., 2004). The Maa7 construct, employed as a negative control in our experiments, has already been described (Rohr et al., 2004). To test for expression of the RbcS2:aadA:RbcS2 transgene, serially diluted cells were spotted onto selective (containing spectinomycin) and nonselective TAP medium and allowed to grow for 7 to 10 d (Cerutti et al., 1997; Zhang et al., 2002).
DNA and RNA Analyses
Standard procedures were used for DNA and RNA manipulations (Sambrook and Russell, 2001). For RT-PCR analyses, total cell RNA was isolated with TRI reagent, according to the manufacturer's directions (Molecular Research Center), treated with DNase-I (Ambion), and amplified as previously described (Rohr et al., 2004). Five-microliter aliquots of each RT-PCR reaction were resolved on 2.0% agarose gels and visualized by ethidium bromide staining. The following primer sets were used for specific amplifications: Set1, Set1-1 (5'-ACCGCAGCATATCCCCTTCA-3') and Set1-2 (5'-CCGTCCACAGCGAACATATAG-3'); Maa7, Maa7-F (5'-TGAACATCACTGCCCCTACTC-3') and Maa7-R (5'-CCCAGCGAGTTGTTGATCTTA-3'); Trxh2, Trxh2-1 (5'-TGTGCGCTAACTCGAACAAC-3') and Trxh2-2 (5'-CCTCAATAGCAGCGAGCAT-3'); Rps3, C_20102codL1, (5'-GAGATTATCATCCGCGCTACTC-3') and C_20102codR1 (5'-AAGACACCATGTAGCCATCCTT-3'); Mut9, Mut9-5 (5'-GCTGTACATCTCGTGCGTGT-3') and Mut9-2 (5'-ATGGCGGTCACGTAGAAGC-3'). Real-time RT-PCR analysis was performed on the Bio-Rad iCycler iQ using SYBR Green. PCR was performed in triplicate, and the experiments were repeated twice starting with independently isolated RNA samples. After each run, a melting curve was performed to ensure that no primer dimer contaminated the quantification. Relative mRNA levels were determined using the method developed by Pfaffl (2001).
Yeast Two-Hybrid Screens
We generated two Chlamydomonas cDNA libraries in pGADT7-Rec, using the Matchmaker Library Construction and Screening kit (BD Biosciences). These prey plasmids contain unidirectionally cloned cDNAs expressed as fusion proteins with the GAL4 activation domain. As bait, we used a fusion protein consisting of the Mut11 coding sequence (accession number AF443204) and the GAL4 DNA binding domain cloned into pGBKT7. The libraries were screened by interaction mating for activation of two GAL4 responsive selectable markers, HIS3 and ADE2, on medium lacking His and Ade. Isolated clones were retransformed, together with the bait, into the auxotrophic yeast strain AH109 and retested for the activation of HIS3, ADE2, as well as the GAL4-regulated MEL1 gene, encoding 
-galactosidase.
Protein Expression, Purification, and Identification by Mass Spectrometry
For the isolation of a Mut11p protein complex from Chlamydomonas, we fused the TAP tag (Rigaut et al., 1999), modified by Rohila et al. (2004), and the coding sequence of the bleomycin gene (ble) (Rohr et al., 2004) to the N-terminal end of Mut11p. This construct was placed under the control of RbcS2 regulatory sequences (Cerutti et al., 1997), allowing constitutive expression, and transformed into Mut-11. Transformants were selected on TAP medium containing 4 µg/mL of bleomycin and verified by DNA gel blot hybridization. Protein expression was examined by protein gel blotting with a rabbit anti-Ble antibody (Cayla) using standard procedures (Sambrook and Russell, 2001). To purify Mut11-TAPp–associated polypeptides, transgenic Mut-11(Mut11TAP) strains were grown to midlogarithmic phase in TAP medium containing 0.7 µg/mL of bleomycin and collected by centrifugation. For each experiment, 4 x 1010 cells were resuspended in IPP150 lysis buffer (Rigaut et al., 1999), containing 2 µL/mL of plant protease inhibitor cocktail (Sigma-Aldrich), and broken by two passages through a French-press at 5000 psi. Subsequent purification steps were performed as previously described by Rigaut et al. (1999). Isolated proteins were fractionated by SDS-PAGE, stained with Sypro Ruby (Bio-Rad), trypsin digested in gel, and identified by tandem mass spectrometry (Rohila et al., 2004). For the expression of recombinant proteins in Escherichia coli, we used the pET system, according to the manufacturer's protocol (Novagen). DNA fragments encoding the C-terminal 1512 to 1795 amino acids of Set1p (amplified by RT-PCR and verified by sequencing) and the full-length Mut11p coding sequence were subcloned in pET30a, expressed in Rosetta DE3, and purified on nickel-nitrilotriacetic acid agarose His binding columns following standard recommendations (Novagen).
HMTase Assays
The histone methylating activity of the affinity-purified Mut11-TAPp complex or of recombinant proteins, isolated from E. coli, was tested as described (Rea et al., 2000). Briefly, Mut-11(Mut11TAP) protein lysates were directly incubated with 200 µL Calmodulin-Sepharose (Stratagene). These beads were subsequently washed with 25 mL Calmodulin binding buffer (Rigaut et al., 1999) followed by 5 mL of methylase activity buffer (Rea et al., 2000). Proteins retained on the sepharose beads were incubated, in 40 µL of methylase activity buffer, with 250 nCi of the methyl donor 14C-SAM (Amersham) and 10 µg of Chlamydomonas core histones (Waterborg et al., 1995) or 10 µg of calf thymus histones (Roche) (Rea et al., 2000). After 60 min at 30°C, samples were resolved by SDS-PAGE on 15% gels, stained with Coomassie Brilliant Blue, and dried onto filter paper. The incorporated radioactivity was detected with a phosphor imager (Amersham). A similar protocol was used to examine the activity of purified recombinant proteins.
N-Terminal Edman Sequencing
Calf thymus histone H3 (Roche) was methylated as described above using 14C-SAM (Amersham) as the methyl donor. After separation by SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane (Millipore) and stained with Coomassie Brilliant Blue, and the histone band was excised and sequenced by sequential Edman degradation at the University of Nebraska Medical Center Protein Structure Core Facility. The residue containing the incorporated radiolabel was identified by scintillation counting of the amino acid fractions.
Protein Gel Blot Analysis
To examine the histone methylation status in vivo, 2.5 x 106 cells from each strain were pelleted and frozen in liquid nitrogen. Cell pellets were directly boiled in SDS gel loading buffer, and the total protein extracts were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes. Specific methylated states of histone H3 Lys residues were detected with antibodies against monomethyl H3K4 (Abcam, ab8895), dimethyl H3K4 (Upstate, 07-030), trimethyl H3K4 (Abcam, ab8580), monomethyl H3K9 (Upstate, 07-395), or dimethyl H3K9 (Upstate, 07-212). A modification-insensitive anti-H3 antibody (Abcam, ab1791) was used to adjust sample loading. The methylation status of histone H4 was examined with a rabbit antiserum that recognizes dimethylated H4 Lys 20 (Upstate, 07-367). Reacting proteins were detected by chemiluminescence with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Sigma-Aldrich).
ChIP Assays
To determine the methylation status of histone H3 at specific chromosomal loci, 2 x 107 TAP-grown cells were treated with 1% formaldehyde for 10 min followed by 0.1 M Gly for 5 min. Cells were then pelleted, washed, and frozen in liquid nitrogen. The cell pellet was resuspended in 2 mL ChIP lysis buffer (20 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 2 µL/mL of plant protease inhibitor cocktail [Sigma-Aldrich], 0.5 mM PMSF, and 2 mM benzamidine), and cells were lysed by two passages through a French press at 5000 psi. Chromatin was sheared by sonication to an average size of 500 bp (determined experimentally) and subjected to immunoprecipitation using a ChIP assay kit (Upstate). Immunoprecipitations were performed with antibodies against H3 (5 µL, Abcam ab1791), monomethylated H3K4 (20 µL, Abcam ab8895), dimethylated H3K9 (15 µL, Upstate 07-212), dimethylated H3K4 (5 µL, Upstate 07-030), or human IgG (2 µL at 6 µg/µL, Sigma-Aldrich I-2511). Immunoprecipitations, histone/DNA complex elutions, and reversions of cross-linking were performed according to the manufacturer's instructions (Upstate). DNA was recovered by phenol/chloroform extraction and ethanol precipitation and resuspended in 30 µL of TE buffer. Standard PCR analysis (Sambrook and Russell, 2001) was performed on 1:10, 1:20, and 1:50 dilutions of the input and antibody precipitated DNA (using samples precipitated with human IgG as negative controls). The amount of immunoprecipitated DNA used in each reaction was adjusted empirically based on the quantity needed to amplify equal amounts of each specific PCR fragment from the anti-H3 antibody samples. The number of PCR cycles was also optimized to show a linear relationship between input DNA and band intensity of the final products. PCR fragments were resolved by electrophoresis on 2% agarose gels, images acquired with the Bio-Rad Gel Doc system, and quantitation performed with the Quantity One software (Bio-Rad). The primer pairs used were as follows: aadA promoter (aadA-ProL, 5'-TGATGTTTGATGGGGTATTTGA-3'; aadA-ProR, 5'- GAGTCGATACTTCGGCGATAAC-3'); aadA coding region (aadA-CodL, 5'-TCTGGCTATCTTGCTGACAAAA-3'; aadA-CodR, 5'-TAGTGATCTCGCCTTTCACGTA-3'); Trxh2 3'UTR (Trxh2-1, 5'-TGTGCGCTAACTCGAACAAC-3'; Trxh2-2 5'-CCTCAATAGCAGCGAGCAT-3'); TOC1 LTR (TOC1-LTR-5', 5'-ATCGCAGCGCAGTGGAGAT-3'; TOC1-LTR-3', 5'-GTCCCGGCAGAGGGACGAT-3'); TOC1 internally transcribed sequence (TOC1-5', 5'-TTCGTTGTACATGCCTTTGC-3'; TOC1-3', 5'-TCACTGAGGGCGAAATATCC-3'); Rps3 promoter (C_20102proL1, 5'-AAGGGCGCTGCTAGTATAACCA-3'; C_20102proR1, 5'-CCTTTGTTCCCGAGAGAGAGAA-3').
Phylogenetic Analysis
SET domain sequences were aligned using ClustalX (Thompson et al., 1994) with default gap penalties (see Supplemental Table 1 online). An unrooted phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei, 1987) from the PHYLIP version 3.6b package (kindly distributed by J. Felsenstein, University of Washington, Seattle, WA).
Accession Numbers
Sequence data for this article can be found in the EMBL/GenBank data libraries under the following accession numbers: human EZH2, gi:21361095; D. melanogaster DmE(z), gi:29429136; D. melanogaster DmTRX, gi:12644002; human MLL, gi:5174569; human KIAA1076, gi:41149776; human KIAA0339, gi:27500172; S. cerevisiae ySet1, gi:6321911; A. thaliana ATXR7 (At5g42400), gi:30694058; D. melanogaster Ash1, gi:17737643; A. thaliana KYP (At5g13960), gi:30580520; human SUV39H1, gi:4507321; human G9a, gi:18375637; and Chlamydomonas Set3p, AY702654.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Online version contains Web-only data. 
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.034165.
Received May 9, 2005; Revision received July 14, 2005. accepted July 19, 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
|---|
Bernstein, B.E., Humphrey, E.L., Erlich, R.L., Schneider, R., Bouman, P., Liu, J.S., Kouzarides, T., and Schreiber, S.L. (2002). Methylation of histone H3 Lys 4 in coding regions of active genes. Proc. Natl. Acad. Sci. USA 99, 8695–8700.
Bernstein, B.E., Kamal, M., Lindblad-Toh, K., Bekiranov, S., Bailey, D.K., Huebert, D.J., McMahon, S., Karlsson, E.K., Kulbokas III, E.J., Gingeras, T.R., Schreiber, S.L., and Lander, E.S. (2005). Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120, 169–181.[CrossRef][Web of Science][Medline]
Boa, S., Coert, C., and Patterton, H.G. (2003). Saccharomyces cerevisiae Set1p is a methyltransferase specific for lysine 4 of histone H3 and is required for efficient gene expression. Yeast 20, 827–835.[CrossRef][Web of Science][Medline]
Briggs, S.D., Xiao, T., Sun, Z.-W., Caldwell, J.A., Shabanowitz, J., Hunt, D.F., Allis, C.D., and Strahl, B.D. (2002). Gene silencing: Trans-histone regulatory pathway in chromatin. Nature 418, 498.[CrossRef][Web of Science][Medline]
Brown, S.A., Ripperger, J., Kadener, S., Fleury-Olela, F., Vilbois, F., Rosbash, M., and Schibler, U. (2005). PERIOD1-associated proteins modulate the negative limb of the mammalian circadian oscillator. Science 308, 693–696.
Bryk, M., Briggs, S.D., Strahl, B.D., Curcio, M.J., Allis, C.D., and Winston, F. (2002). Evidence that Set1, a factor required for methylation of histone H3, regulates rDNA silencing in S. cerevisiae by a Sir2-independent mechanism. Curr. Biol. 12, 165–170.[CrossRef][Web of Science][Medline]
Carvin, C.D., and Kladde, M.P. (2004). Effectors of lysine 4 methylation of histone H3 in Saccharomyces cerevisiae are negative regulators of PHO5 and GAL1-10. J. Biol. Chem. 279, 33057–33062.
Cerutti, H., Johnson, A.M., Gillham, N.W., and Boynton, J.E. (1997). Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. Plant Cell 9, 925–945.
Cheng, X., Collins, R.E., and Zhang, X. (2005). Structural and sequence motifs of protein (histone) methylation enzymes. Annu. Rev. Biophys. Biomol. Struct. 34, 267–294.[CrossRef][Web of Science][Medline]
Collins, R.E., Tachibana, M., Tamaru, H., Smith, K.M., Jia, D., Zhang, X., Selker, E.U., Shinkai, Y., and Cheng, X. (2005). In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases. J. Biol. Chem. 280, 5563–5570.
Dou, Y., Milne, T.A., Tackett, A.J., Smith, E.R., Fukuda, A., Wysocka, J., Allis, C.D., Chait, B.T., Hess, J.L., and Roeder, R.G. (2005). Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF. Cell 121, 873–885.[CrossRef][Web of Science][Medline]
Fingerman, I.M., Wu, C.-L., Wilson, B.D., and Briggs, S.D. (2005). Global loss of Set1-mediated H3 Lys4 trimethylation is associated with silencing defects in Saccharomyces cerevisiae. J. Biol. Chem. 280, 28761–28765.
Fischle, W., Wang, Y., and Allis, C.D. (2003). Histone and chromatin cross-talk. Curr. Opin. Cell Biol. 15, 172–183.[CrossRef][Web of Science][Medline]
Fuks, F., Hurd, P.J., Deplus, R., and Kouzarides, T. (2003). The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res. 31, 2305–2312.
Gerber, M., and Shilatifard, A. (2003). Transcriptional elongation by RNA polymerase II and histone methylation. J. Biol. Chem. 278, 26303–26306.
Goo, Y.H., et al. (2003). Activating signal cointegrator 2 belongs to a novel steady-state complex that contains a subset of trithorax group proteins. Mol. Cell. Biol. 23, 140–149.
Guenther, M.G., Yu, J., Kao, G.D., Yen, T.J., and Lazar, M.A. (2002). Assembly of the SMRT-histone deacetylase 3 repression complex requires the TCP-1 ring complex. Genes Dev. 16, 3130–3135.
Hughes, C.M., et al. (2004). Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus. Mol. Cell 13, 587–597.[CrossRef][Web of Science][Medline]
Jeong, B.-r., Wu-Scharf, D., Zhang, C., and Cerutti, H. (2002). Suppressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements. Proc. Natl. Acad. Sci. USA 99, 1076–1081.
Klymenko, T., and Muller, J. (2004). The histone methyltransferase Trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins. EMBO Rep. 5, 373–377.[CrossRef][Web of Science][Medline]
Kohlmaier, A., Savarese, F., Lachner, M., Martens, J., Jenuwein, T., and Wutz, A. (2004). A chromosomal memory triggered by Xist regulates histone methylation in X inactivation. PLoS Biol 2, 991–1003.
Kouzarides, T. (2002). Histone methylation in transcriptional control. Curr. Opin. Genet. Dev. 12, 198–209.[CrossRef][Web of Science][Medline]
Krogan, N.J., Dover, J., Wood, A., Schneider, J., Heidt, J., Boateng, M.A., Dean, K., Ryan, O.W., Golshani, A., Johnston, M., Greenblatt, J.F., and Shilatifard, A. (2003). The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: Linking transcriptional elongation to histone methylation. Mol. Cell 11, 721–729.[CrossRef][Web of Science][Medline]
Lachner, M., and Jenuwein, T. (2002). The many faces of histone lysine methylation. Curr. Opin. Cell Biol. 14, 286–298.[CrossRef][Web of Science][Medline]
Lachner, M., O'Sullivan, R.J., and Jenuwein, T. (2003). An epigenetic road map for histone lysine methylation. J. Cell Sci. 116, 2117–2124.
Loidl, P. (2004). A plant dialect of the histone language. Trends Plant Sci. 9, 84–90.[CrossRef][Web of Science][Medline]
Manzur, K.L., Farooq, A., Zeng, L., Plotnikova, O., Koch, A.W., Sachchidanand, and Zhou, M.-M. (2003). A dimeric viral SET domain methyltransferase specific to Lys27 of histone H3. Nat. Struct. Biol. 10, 187–196.[CrossRef][Web of Science][Medline]
Matzke, M., Aufsatz, W., Kanno, T., Daxinger, L., Papp, I., Mette, M.F., and Matzke, A.J. (2004). Genetic analysis of RNA-mediated transcriptional gene silencing. Biochim. Biophys. Acta 1677, 129–141.[Medline]
Morillon, A., Karabetsou, N., Nair, A., and Mellor, J. (2005). Dynamic lysine methylation on histone H3 defines the regulatory phase of gene transcription. Mol. Cell 18, 723–734.[CrossRef][Web of Science][Medline]
Nakamura, T., Mori, T., Tada, S., Krajewski, W., Rozovskaia, T., Wassell, R., Dubois, G., Mazo, A., Croce, C.M., and Canaani, E. (2002). ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol. Cell 10, 1119–1128.[CrossRef][Web of Science][Medline]
Ng, H.H., Robert, F., Young, R.A., and Struhl, K. (2003). Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol. Cell 11, 709–719.[CrossRef][Web of Science][Medline]
Nishioka, K., Chuikov, S., Sarma, K., Erdjument-Bromage, H., Allis, C.D., Tempst, P., and Reinberg, D. (2002). Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev. 16, 479–489.
Pal-Bhadra, M., Leibovitch, B.A., Gandhi, S.G., Rao, M., Bhadra, U., Birchler, J.A., and Elgin, S.C. (2004). Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303, 669–672.
Perkins, E.J., Kee, B.L., and Ramsden, D.A. (2004). Histone 3 lysine 4 methylation during the pre-B to immature B-cell transition. Nucleic Acids Res. 32, 1942–1947.
Peters, A.H.F.M., and Schubeler, D. (2005). Methylation of histones: Playing memory with DNA. Curr. Opin. Cell Biol. 17, 230–238.[CrossRef][Web of Science][Medline]
Pfaffl, M.W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.
Popovic, R., and Zeleznik-Le, N.J. (2005). MLL: How complex does it get? J. Cell. Biochem. 95, 234–242.[CrossRef][Web of Science][Medline]
Pray-Grant, M.G., Daniel, J.A., Schieltz, D., Yates III, J.R., and Grant, P.A. (2005). Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation. Nature 433, 434–438.[CrossRef][Medline]
Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B.D., Sun, Z.W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C.P., Allis, C.D., and Jenuwein, T. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593–599.[CrossRef][Medline]
Richards, E.J., and Elgin, S.C. (2002). Epigenetic codes for heterochromatin formation and silencing: Rounding up the usual suspects. Cell 108, 489–500.[CrossRef][Web of Science][Medline]
Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032.[CrossRef][Web of Science][Medline]
Roguev, A., Schaft, D., Shevchenko, A., Pijnappel, W.W., Wilm, M., Aasland, R., and Stewart, A.F. (2001). The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4. EMBO J. 20, 7137–7148.[CrossRef][Web of Science][Medline]
Roguev, A., Shevchenko, A., Schaft, D., Thomas, H., and Stewart, A.F. (2004). A comparative analysis of an orthologous proteomic environment in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Mol. Cell. Proteomics 3, 125–132.
Rohila, J.S., Chen, M., Cerny, R., and Fromm, M.E. (2004). Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. Plant J. 38, 172–181.[CrossRef][Web of Science][Medline]
Rohr, J., Sarkar, N., Balenger, S., Jeong, B.R., and Cerutti, H. (2004). Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J. 40, 611–621.[CrossRef][Web of Science][Medline]
Saitou, N., and Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.[Abstract]
Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).
Santos-Rosa, H., Bannister, A.J., Dehe, P.M., Geli, V., and Kouzarides, T. (2004). Methylation of H3 lysine 4 at euchromatin promotes Sir3p association with heterochromatin. J. Biol. Chem. 279, 47506–47507.
Santos-Rosa, H., Schneider, R., Bannister, A.J., Sherriff, J., Bernstein, B.E., Emre, N.C., Schreiber, S.L., Mellor, J., and Kouzarides, T. (2002). Active genes are tri-methylated at K4 of histone H3. Nature 419, 407–411.[CrossRef][Medline]
Santos-Rosa, H., Schneider, R., Bernstein, B.E., Karabetsou, N., Morillon, A., Weise, C., Schreiber, S.L., Mellor, J., and Kouzarides, T. (2003). Methylation of histone H3 K4 mediates association of the Isw1p ATPase with chromatin. Mol. Cell 12, 1325–1332.[CrossRef][Web of Science][Medline]
Sarkar, N., Lemaire, S., Wu-Scharf, D., Issakidis-Bourguet, E., and Cerutti, H. (2005). Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage. Eukaryot. Cell 4, 262–273.
Schlichter, A., and Cairns, B.R. (2005). Histone trimethylation by Set1 is coordinated by the RRM, autoinhibitory, and catalytic domains. EMBO J. 24, 1222–1231.[CrossRef][Web of Science][Medline]
Schneider, R., Bannister, A., Myers, F., Thorne, A., Crane-Robinson, C., and Kouzarides, T. (2004). Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat. Cell Biol. 6, 73–77.[CrossRef][Web of Science][Medline]
Schubeler, D., MacAlpine, D.M., Scalzo, D., Wirbelauer, C., Kooperberg, C., van Leeuwen, F., Gottschling, D.E., O'Neill, L.P., Turner, B.M., Delrow, J., Bell, S.P., and Groudine, M. (2004). The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev. 18, 1263–1271.
Siegers, K., Bolter, B., Schwarz, J.P., Bottcher, U.M., Guha, S., and Hartl, F.U. (2003). TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes. EMBO J. 22, 5230–5240.[CrossRef][Web of Science][Medline]
Smith, S.T., Petruk, S., Sedkov, Y., Cho, E., Tillib, S., Canaani, E., and Mazo, A. (2004). Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex. Nat. Cell Biol. 6, 162–167.[CrossRef][Web of Science][Medline]
Spada, F., Vincent, M., and Thompson, E.M. (2005). Plasticity of histone modifications across the invertebrate to vertebrate transition: Histone H3 lysine 4 trimethylation in heterochromatin. Chromosome Res. 13, 57–72.[CrossRef][Web of Science][Medline]
Stolc, V., Samanta, M.P., Tongprasit, W., and Marshall, W.F. (2005). Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. Proc. Natl. Acad. Sci. USA 102, 3703–3707.
Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., Fukuda, M., Takeda, N., Niida, H., Kato, H., and Shinkai, Y. (2002). G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 16, 1779–1791.
Tamaru, H., Zhang, X., McMillen, D., Singh, P.B., Nakayama, J., Grewal, S.I., Allis, C.D., Cheng, X., and Selker, E.U. (2003). Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa. Nat. Genet. 34, 75–79.[CrossRef][Web of Science][Medline]
Tariq, M., and Paszkowski, J. (2004). DNA and histone methylation in plants. Trends Genet. 20, 244–251.[CrossRef][Web of Science][Medline]
Thompson, J., Higgins, D., and Gibson, T. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.
van Leeuwen, F., and Gottschling, D. (2002). Genome-wide histone modifications: Gaining specificity by preventing promiscuity. Curr. Opin. Cell Biol. 14, 756–762.[CrossRef][Web of Science][Medline]
Waterborg, J.H. (1998). Dynamics of histone acetylation in Chlamydomonas reinhardtii. J. Biol. Chem. 273, 27602–27609.
Waterborg, J.H., Robertson, A.J., Tatar, D.L., Borza, C.M., and Davie, J.R. (1995). Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation. Plant Physiol. 109, 393–407.[Abstract]
Wysocka, J., Myers, M.P., Laherty, C.D., Eisenman, R.N., and Herr, W. (2003). Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell-proliferation factor HCF-1. Genes Dev. 17, 896–911.
Wysocka, J., Swigut, T., Milne, T.A., Dou, Y., Zhang, X., Burlingame, A.L., Roeder, R.G., Brivanlou, A.H., and Allis, C.D. (2005). WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development. Cell 121, 859–872.[CrossRef][Web of Science][Medline]
Xia, Z.-B., Anderson, M., Diaz, M.O., and Zeleznik-Le, N.J. (2003). MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein. Proc. Natl. Acad. Sci. USA 100, 8342–8347.
Yokoyama, A., Wang, Z., Wysocka, J., Sanyal, M., Aufiero, D.J., Kitabayashi, I., Herr, W., and Cleary, M.L. (2004). Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol. Cell. Biol. 24, 5639–5649.
Zegerman, P., Canas, B., Pappin, D., and Kouzarides, T. (2002). Histone H3 lysine 4 methylation disrupts binding of nucleosome remodeling and deacetylase (NuRD) repressor complex. J. Biol. Chem. 277, 11621–11624.
Zhang, C., Wu-Scharf, D., Jeong, B.R., and Cerutti, H. (2002). A WD40-repeat containing protein, similar to a fungal co-repressor, is required for transcriptional gene silencing in Chlamydomonas. Plant J. 31, 25–36.[CrossRef][Web of Science][Medline]
Zhang, K., Siino, J.S., Jones, P.R., Yau, P.M., and Bradbury, E.M. (2004). A mass spectrometric "Western blot" to evaluate the correlations between histone methylation and histone acetylation. Proteomics 4, 3765–3775.[CrossRef][Web of Science][Medline]
Zhang, X., Yang, Z., Khan, S.I., Horton, J.R., Tamaru, H., Selker, E.U., and Cheng, X. (2003). Structural basis for the product specificity of histone lysine methyltransferases. Mol. Cell 12, 177–185.[CrossRef][Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  A. Patel, V. Dharmarajan, V. E. Vought, and M. S. Cosgrove On the Mechanism of Multiple Lysine Methylation by the Human Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex J. Biol. Chem., September 4, 2009; 284(36): 24242 - 24256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sebastian, P. Sreenivas, R. Sambasivan, S. Cheedipudi, P. Kandalla, G. K. Pavlath, and J. Dhawan MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation PNAS, March 24, 2009; 106(12): 4719 - 4724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yamasaki, H. Miyasaka, and T. Ohama Unstable RNAi Effects Through Epigenetic Silencing of an Inverted Repeat Transgene in Chlamydomonas reinhardtii Genetics, December 1, 2008; 180(4): 1927 - 1944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Casas-Mollano, J. Rohr, E.-J. Kim, E. Balassa, K. van Dijk, and H. Cerutti Diversification of the Core RNA Interference Machinery in Chlamydomonas reinhardtii and the Role of DCL1 in Transposon Silencing Genetics, May 1, 2008; 179(1): 69 - 81. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Casas-Mollano, B.-r. Jeong, J. Xu, H. Moriyama, and H. Cerutti The MUT9p kinase phosphorylates histone H3 threonine 3 and is necessary for heritable epigenetic silencing in Chlamydomonas PNAS, April 29, 2008; 105(17): 6486 - 6491. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Saleh, R. Alvarez-Venegas, M. Yilmaz, O. Le, G. Hou, M. Sadder, A. Al-Abdallat, Y. Xia, G. Lu, I. Ladunga, et al. The Highly Similar Arabidopsis Homologs of Trithorax ATX1 and ATX2 Encode Proteins with Divergent Biochemical Functions PLANT CELL, March 1, 2008; 20(3): 568 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Casas-Mollano, K. van Dijk, J. Eisenhart, and H. Cerutti SET3p monomethylates histone H3 on lysine 9 and is required for the silencing of tandemly repeated transgenes in Chlamydomonas Nucleic Acids Res., February 16, 2007; 35(3): 939 - 950. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | THE PLANT CELL | PLANT PHYSIOLOGY | |
|---|---|---|---|
; 5, CCT subunits 
, 
, and 
; 6, Mut11-TAPp; 7, CCT subunits 
, and 
; 8, Ash2L homolog; 9, Set4p. Lane 1 corresponds to a control purification with a TAP-tagged Ble protein (indicated by the asterisk).
