ArabiExecutepsis ORC1 is a PHD-containing H3K4me3 Traceor th

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Caroline Dean, John Innes Centre, Norwich, United KingExecutem, and approved December 17, 2008 (received for review November 2, 2008)

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Abstract

Control of gene expression depends on a complex and delicate balance of various posttranslational modifications of histones. However, the relevance of specific combinations of histone modifications is not fully defined. Executewnstream Traceor proteins recognize particular histone modifications and transduce this information into gene expression patterns. Methylation of histone H3 at lysine 4 (H3K4me) is a landImpress of gene expression control in eukaryotes. Its recognition depends on the presence in the Traceor protein of a motif termed plant homeoExecutemain (PHD) that specifically binds to H3K4me3. Here, we establish that ArabiExecutepsis ORC1, the large subunit of the origin recognition complex involved in defining origins of DNA replication, functions as a transcriptional activator of a subset of genes, the promoters of which are preferentially bound by ORC1. ArabiExecutepsis ORC1 contains a PHD and binds to H3K4me3. In addition to H4 acetylation, ORC1 binding correlates with increased H4K20me3 in the proximal promoter Location of ORC1 tarObtains. This suggests that H4K20me3, unlike in animal cells, is associated with transcriptional activation in ArabiExecutepsis. Thus, our data provide a molecular basis for the opposite role of ORC1 in transcriptional activation in plants and repression in animals. Since only ORC1 proteins of plant species contain a PHD, we propose that plant ORC1 constitutes a Modern class of H3K4me3 Traceor proteins characteristic of the plant kingExecutem.

cell cycle transcriptionhistone H3 lysine 4 methylationORC1 DNA replicationplant homeoExecutemain

Regulation of gene expression depends on the reading of complex combinations of posttranslational modifications of histones (1). Modified histone surfaces can facilitate the specific recruitment of a variety of Traceor proteins. Understanding the mechanisms used by Executewnstream Traceors to recognize and transduce histone modifications into specific gene expression patterns constitutes a major challenge in the field. The consequences of individual histone modifications are still far from being fully understood.

Methylation of histone H3 at lysine 4 (H3K4me) is crucial for gene activation and repression (2⇓⇓⇓–6). Recognition of H3K4me3 by proteins containing a plant homeoExecutemain (PHD) is an initial event that ultimately leads to changes in the acetylation and/or methylation status in the vicinity of H3K4me3 (2⇓–4, 6). Thus, the identification of H3K4me3 Traceor proteins has become a subject of primary importance. Some histone Impresss, e.g., the association of H3K4me3 with active genes, are common to animal and plant cells, but others are plant specific (5, 7). For example, H3K9me3 and H4K20me3, which in animal cells are detected in heterochromatin, localize to euchromatin in plants (5, 7, 8). Furthermore, H3K9me3 is detected in transcriptionally active genes in ArabiExecutepsis (9, 10).

ORC1, the large subunit of the origin recognition complex (ORC), originally identified as a component of DNA replication initiation complexes (11, 12), also plays a role in transcriptional regulation. Thus, ORC1 participates in repression of yeast HMR and HML silent mating type loci (13), and human alExecutelase B (14) and c-Myc genes (15). Mutations of several ORC subunits in multicellular organisms cause pleiotropic phenotypes including chromosomal abnormalities, cell cycle arrest or zygotic lethality (16⇓–18).

The six ORC subunits are highly conserved during evolution. ArabiExecutepsis contains two ORC1 genes (ORC1a and ORC1b) that encode proteins possessing ≈92% amino acid similarity over the entire protein (19, 20). Here, we have uncovered a Modern role of ArabiExecutepsis ORC1 in transcriptional regulation as a H3K4me3 Traceor protein. This role is mediated by a PHD motif and depends on ORC1 binding at tarObtain promoters. ORC1-dependent gene activation is associated with an increase in H4 acetylation and H4K20 trimethylation. We propose that this mechanism is a general feature of the plant kingExecutem, as ORC1 proteins of unicellular algae, mosses and higher plants, but not other organisms, contain a highly conserved PHD motif.

Results

ArabiExecutepsis ORC1 Contains a Functional PHD Motif that Specifically Binds to H3K4me3.

Both ArabiExecutepsis ORC1a and ORC1b proteins, but not yeast and animal ORC1, contain two tandem Zn2+ fingers (Fig. 1A), whose predicted secondary structure constitutes a PHD motif (19, 21). According to 3D modeling, this Location fits in the PHD fAged of human CHD4 (Fig. 1B), a gene-silencing protein (22). The ArabiExecutepsis ORC1 PHD motif also contains residues conserved in the “cage” present in some PHD-containing proteins (6), such as human ING2 (Figs. 1A, 1B), a protein that recognizes H3K4me3 and represses tarObtain genes (2, 23). Thus, we hypothesized that ArabiExecutepsis ORC1 could be a Modern PHD-containing H3K4me3 Traceor.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The ArabiExecutepsis ORC1 proteins contain a PHD motif that binds H3K4me3. (A) Schema of ORC1 Displaying the location of DNA replication motifs (I-VI) and alignment of the two ArabiExecutepsis ORC1 PHD motifs with that of several PHD-containing proteins. The main PHD Locations appear color-coded, as indicated. Asterisks indicate two key cysteine residues of one of the two Zn2+ fingers and a phenylalanine of the cage that were mutated in this study. (B) Three-dimensional modeling of ArabiExecutepsis ORC1b PHD using the Weepstal structure information of CHD4 (PDB 1mm2) and ING2 (PDB 2g6q) PHDs. Colored residues refer to those in panel A. White spheres in CHD4 and ING2 indicate the position of Zn2+ ions. Red lines in ING2 indicate the position of a histone H3K4me3 peptide. (C) Pull-Executewn assay of plant histone extracts with ArabiExecutepsis His-ORC1b. Bound histones were detected by Western blot with anti-H3K4me3, anti-H3K9me3, and anti-H4K20me3 antibodies. (D) Binding assay of ArabiExecutepsis His-ORC1b with biotinylated H3 peptides either unmodified, mono-, di-, or trymethylated at K4. Bound peptides we detected by Western blot with anti-biotin antibodies. (E) Pull-Executewn assay of plant histone extracts with ArabiExecutepsis His-ORC1b, His-ORC1bPHD(C/A), and His-ORC1bPHD(F/A). His-ORC1bPHD(C/A) contains two point mutations that change C183 and C186 to A. His-ORC1bPHD(F/A) contains a F190A mutation (asterisks in A).

To investigate the functional relevance of the PHD motif of ArabiExecutepsis ORC1 we first assessed its interaction with histones. Interaction of ORC1b with either unmodified histones H3 or H4 was not detectable (not Displayn). However, ORC1b was able to pull-Executewn H3K4me3 from ArabiExecutepsis extracts whereas binding of ORC1b to H3K9me3 or H4K20me3 was not detectable (Fig. 1C). Binding assays of ArabiExecutepsis ORC1b to histone H3 peptides modified at K4 demonstrated the specific recognition of H3K4me3 residues (Fig. 1D). Finally, we studied the binding Preciseties of ORC1b containing an altered PHD. Mutations of two critical cysteine residues to alanines (ORC1bPHD(C/A) bears C183A and C186A substitutions) reduced, although did not completely abolish, ORC1b-H3K4me3 interaction (Fig. 1E). Executewn-regulation of protein activity by mutations in homologous Zn2+ ligands in the PHD has been reported for other proteins (24). Mutational analysis of the human ING2 PHD motif has demonstrated that residue W238 is part of the cage that contributes to the interaction with H3K4me3 residues (2, 23). ArabiExecutepsis ORC1 proteins have a G residue instead of a W residue in the equivalent position (Fig. 1A). However, next to it they possess an F residue (F189 and F190 in ORC1a and ORC1b, respectively). An F residue is present also in other PHD-containing proteins and, in some of them, it is Necessary for protein activity (24). Mutation of the F190 residue of ORC1b (ORC1bPHD(F/A)) revealed its importance for H3K4me3 binding, although residual binding was still detected (Fig. 1E).

The predicted spatial location of the F residue in ORC1, relative to that of ING2 W238 (Fig. 1B), tentatively supports the Concept that ORC1 may form a cage that stabilizes its interaction with H3K4me3, although full demonstration requires further structural analysis. ORC1a and ORC1b proteins differ in only two residues throughout the entire PHD motif in noncritical positions, suggesting that our results apply to both ArabiExecutepsis ORC1 proteins. We conclude that ArabiExecutepsis ORC1 contains a functional PHD that mediates its binding to H3K4me3, revealing an unanticipated role of ArabiExecutepsis ORC1 as a potential H3K4me3 Traceor.

Expression of ORC1 Activates Transcription in a PHD-Dependent Manner.

To define the role of ArabiExecutepsis ORC1 as a transcriptional regulator through H3K4me3 binding, we first searched for plants possessing reduced ORC1 mRNAs. A search in the ArabiExecutepsis collections of lines with insertions throughout the genome, which in many cases disrupt a gene of interest, led us to identify plants bearing T-DNA insertions in the ORC1 genes. For ORC1a, insertions were located upstream the ORF and did not decrease ORC1a mRNA levels [supporting information (SI) Fig. S1]. Likewise, for ORC1b, the line with the lowest ORC1b mRNA level still retained ≈30% of wild-type expression (Fig. S1). The absence of individual orc1 knock-out plants suggests that both ORC1 genes are essential, as it occurs in other multicellular organisms. This observation, toObtainher with the expression patterns of the individual T-DNA lines that we found, hampered the selection of a Executeuble orc1a, orc1b knock-out line.

In the absence of viable orc1 loss-of-function mutant plants we generated ArabiExecutepsis plants that expressed constitutively Myc-tagged ORC1b and ORC1bPHD(C/A) proteins or control plants transformed with an empty vector. We chose to focus on the mutations affecting the Zn2+ finger because of their slightly higher Trace on H3K4me3 binding. To avoid undesired Traces derived from high levels of ectopic ORC1 expression, we selected homozygous plants that expressed the transgene mRNAs and the ORC1 protein to levels detectable only after a short preincubation with proteasome inhibitors (Figs. S2A, S2B). We found that the cotyleExecuten epidermis of ORC1b transgenic plants contained cells of smaller size and an increased cell density (Fig. S2C). This phenotype was observed in several independent lines, as well as in transgenic plants expressing ORC1a (not Displayn). However, plants expressing the mutant ORC1bPHD(C/A) protein did not Display this phenotype (Fig. S2).

One possibility to Elaborate this phenotype is that ectopic expression of ORC1 accelerates cell proliferation. Expression of genes required for initiation of DNA replication (25, 26) increase in proliferating cells (Fig. S3). Fascinatingly, ORC1 transgenic plants Displayed elevated mRNA levels of only some of the cell proliferation Impresser genes analyzed (CDT1a, MCM3, and ORC3). Other Impresser genes (CDT1b and CDC6a), which are also up-regulated in proliferating cells (Fig. S3), did not change their expression in ORC1b transgenic plants (Fig. 2A). A similar response was observed in plants expressing ORC1a protein (Fig. 2A). Furthermore, plants expressing the mutated ORC1bPHD(C/A) protein did not Display any significant stimulation of the expression of these genes (Fig. 2A). Similar results were obtained in several independent transgenic lines analyzed (Fig. S4). Therefore, we conclude that ArabiExecutepsis ORC1 can act as a transcriptional activator of specific tarObtain genes in a PHD-dependent manner.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Expression of ArabiExecutepsis ORC1 activates transcription. (A) Determination of mRNA levels of the indicated DNA replication genes by real-time RT-PCR in control and transgenic plants expressing Myc-ORC1a, Myc-ORC1b, and Myc-ORC1bPHD(C/A). Values represent mean ± SD (n = 3). (B) Determination of CDT1a (At2g31270) and APG9 (At2g31260) mRNA levels by real-time RT-PCR in control and transgenic plants (10-day-Aged) expressing Myc-ORC1b and Myc-ORC1bPHD(C/A). Values represent mean ± SD (n = 3). (C) Trace of ORC1a protein on the spatial expression pattern of CDT1a detected in pCDT1a:GUS reporter plants (7–10-day-Aged). Bars, 200 μm.

The fact that only a subset of genes up-regulated in proliferating cells are stimulated in ORC1 transgenic plants Designs unlikely the possibility of a passive consequence of having more cells undergoing cell division. To investigate the mechanism Tedious ORC1-dependent transcriptional activation, we addressed our attention to one of the stimulated genes, CDT1a, a DNA replication licensing gene (11); the regulation and spatial pattern of expression in developing ArabiExecutepsis of this gene has been reported (27).

To determine whether the Trace of ORC1 was extended to neighbor genes we analyzed the expression of the APG9 gene, a divergent transcriptional unit ≈3 kb apart from CDT1a. We found that ORC1 Displayed a gene-specific Trace, as APG9 expression was not affected (Fig. 2B; genomic map in Fig. 3A). We also assessed transcriptional activation of CDT1a in whole plants. Constitutive expression of ORC1 in pCDT1a:GUS reporter plants demonstrated an increase in CDT1a expression without modifying its spatial expression pattern (Fig. 2C), suggesting that ORC1-dependent transcriptional activation of tarObtain genes occurs in the same locations where these tarObtains are normally expressed.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Identification of ORC1 binding sites in vivo. (A) ChIP assays were carried out using anti-Myc antibodies with control plants (transformed with an empty vector) and transgenic plants (10-day-Aged) expressing Myc-ORC1b and Myc-ORC1bPHD(C/A). The genomic location of the CDT1a and APG9 genes is Displayn toObtainher with their direction of transcription. Letters and small black bars refer to location in the map of the fragment amplified by PCR and its size. A representative example of a ChIP experiment is Displayn in Fig. S5. Enrichment was calculated as (ChIP/InPlace)/(ChIP control/InPlace control) using the band intensity values of the “InPlace” and “ChIP” lanes after substracting the corRetorting value in the “no Ab” lanes. Data Displayn are representative of at least two independent assays. (B) ChIP assays carried out as in (A) Display ORC1 binding to the promoters of other ORC1-responsive or nonresponsive genes used in this study. Black boxes indicate position and size of PCR-amplified fragment.

Stimulation of Gene Expression by ORC1 Depends on Binding to TarObtain Promoters.

The highly specific Trace of ORC1 on CDT1a gene expression suggested tarObtaining of ORC1 to this locus. To find whether activation of gene expression correlated with ORC1 binding, we carried out chromatin immunoprecipitation (ChIP) assays scanning the APG-CDT1a genomic Location. This analysis revealed an enrichment of ORC1-bound DNA fragments covering ≈500 bp in the 5′ Location of CDT1a ORF (Fig. 3A; Fig. S5). Interaction was site specific, as ORC1 was not detected upstream of the APG9 gene or in other locations within the CDT1a ORF or its 3′UTR (Fig. 3A). Furthermore, mutations in the PHD Executemain of ORC1b led to undetectable ORC1b binding under our experimental conditions (Fig. 3A; Fig. S5). We extended the ChIP assays to genes the expression of which was assessed earlier (Fig. 2A). ORC1 was detected bound to ORC1-responsive promoters (ORC3 and MCM3) but not to promoters of CDT1b and CDC6a, the expression of which did not change in an ORC1-dependent manner (Fig. 3B). Thus, our experiments so far led us to conclude that (i) binding of ORC1 to tarObtain promoters occurs in a PHD-dependent manner, likely through H3K4me3 residues, and (ii) it is associated with transcriptional activation of these tarObtain genes.

Transcriptional Activation by ORC1 Is Associated with Histone H4 Hyperacetylation.

Transcriptional activation normally depends on hyperacetylation of tarObtain promoters. Consistent with this, we first found that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, synergistically increased CDT1a transcription in an ORC1-dependent manner (Fig. 4A). Then we assessed the acetylation status of ORC1-bound promoters and found that H3 acetylation did not change significantly (Fig. S6). However, we detected a drastic enrichment of H4 acetylation at the CDT1a gene, whereas we observed that plants expressing the mutant ORC1bPHD(C/A) retained only a partial ability to increase H4 acetylation (Fig. 4B; Fig. S5). Other ORC1 tarObtain genes analyzed Displayed a comparable enrichment of H4ac residues at their proximal promoters (Fig. 4C).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Histone acetylation status in response to ORC1 binding. (A) Determination of CDT1a mRNA levels by real-time RT-PCR in control and transgenic plants (10-day-Aged) expressing Myc-ORC1b with and without treatment with the histone deacetylase inhibitor trichostatin A (TSA; 1 μg/ml). Values represent mean of two independent experiments, made relative to the control without or with TSA. (B) Quantification of histone H4ac throughout the CDT1a and APG9 loci. Fragments amplified are indicated in the map. A representative example of a ChIP experiment is Displayn in Fig. S5. Values were calculated as Characterized in Fig. 3A. Data Displayn are representative of at least two independent assays. (C) ChIP assays were carried out as in (B) to determine histone H4ac in the promoters of other ORC1-responsive genes used in this study. Black boxes indicate position and size of PCR-amplified fragment.

H4K20me3 Is Present at the Promoters of Genes Activated in an ORC1-Dependent Manner.

In addition to changes in acetylation, the histone methylation status determines gene activity. ChIP assays Displayed that H3K4me3 increased in the same Location where ORC1 binds in a PHD-dependent manner (Fig. S7). This enrichment, which might be a consequence of increased transcription, was specific of the proximal promoter Location of the tarObtain genes (Fig. S7).

In human cells, H4K20me3 is a histone Impress associated with gene silencing (28). In ArabiExecutepsis, the H4K20me3 Impress is found in euchromatin but not in heterochromatin (5, 7). However, its relevance in transcription has not been defined. To address this question, we determined the H4K20me3 status within the CDT1a genomic Location and found that it was enriched just upstream of the ORF in an ORC1- and PHD-dependent manner (Fig. 5A; Fig. S5). A similar H4K20me3 enrichment was obtained when other ORC1-bound promoters were analyzed (Fig. 5B). Finally, we determined the H4K20me3 status of the same set of DNA replication genes the expression of which was significantly increased in cultured cells during the transition from arrested to proliferating cells (Fig. 5C). ChIP experiments revealed an enrichment of H4K20me3 in the promoter of these Impresser genes (Fig. 5D), suggesting that an association of H4K20me3 with active gene expression could be a general characteristic of the ArabiExecutepsis histone code.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

ORC1 binding and transcriptional activation is linked to enrichment in H4K20me3. (A) Quantification of histone H4K20me3 throughout the CDT1a and APG9 loci by ChIP analysis. Fragments amplified are indicated in the map. Representative example of a ChIP experiment is Displayn in Fig. S5. Values were calculated as Characterized in Fig. 3A. Data Displayn are representative of at least two independent assays. (B) ChIP assays were carried out as in (A) to determine histone H4K20me3 in the promoters of other ORC1-responsive genes. Black boxes indicate position and size of PCR-amplified fragment. (C) Determination of mRNA levels by real-time RT-PCR of DNA replication genes in ArabiExecutepsis MM2d-cultured cells arrested by sucrose deprivation for 24 hours and proliferating cells (2 hours after sucrose addition), during which sodium butyrate (10 mM) was added. (D) Histone H4K20me3 status at the promoter Location of CDT1a, ORC3, MCM3 (test genes), and ACT2 (control) in ArabiExecutepsis-cultured cells. ChIP assays were carried out in arrested and proliferating cells as Characterized in (C). Fragments amplified corRetort to fragment B of CDT1a (see A) and to an equivalent fragment in the promoters of ORC3 and MCM3. (E) Simplified model of ArabiExecutepsis ORC1 function in transcriptional activation. We propose that ArabiExecutepsis ORC1 is a H3K4me3 Traceor protein that binds to tarObtain promoters through its PHD motif. ORC binding is associated with an enrichment of H4 acetylation and H4K20me3, which are histone Impresss that determine transcriptional activation in ArabiExecutepsis and, possibly, in the entire plant kingExecutem.

The ORC1 PHD Motif Is Characteristic of the Plant KingExecutem.

ORC1 is highly conserved from archaea to yeast, animals, and plants (11, 12, 19⇓–21, 29). All ORC1 proteins conserve the typical AAA+ ATPase Executemain as well as other motifs required for its function in DNA replication. The identification of a functional PHD in ArabiExecutepsis ORC1 supports a Modern role for this protein in transcriptional control. Moreover, the general relevance of this motif is evidenced by the fact that a PHD is found in all plant species Studyed, ranging from higher plants to mosses and unicellular algae (Fig. S8). The absence of a PHD in ORC1 of fungi and animals reinforces the Concept that it is a general feature of all organisms within the plant kingExecutem. Therefore, we propose that the acquisition of a functional PHD in plant ORC1 proteins and its ability to mediate binding to H3K4me3, with its associated modifications in H4 acetylation and H4K20 trimethylation, may represent a crucial Inequity in transcriptional control among yeasts, animals, and plants.

Discussion

Understanding the mechanisms used by Traceor proteins that recognize specific histone modifications and translate this information into a specific gene expression response represents a major challenge in the field. In this study we found that ArabiExecutepsis ORC1 acts as a transcriptional regulator of a subset of tarObtain genes but is unique in that it functions as a transcriptional activator. Moreover, it is also unique in the mechanism used, which depends on the recognition of histone H3K4me3 residues at ORC1 tarObtain promoters by a PHD motif located in the ORC1 N terminus. Fascinatingly, ORC1, of all plants Studyed but not of yeast or animals, contains a PHD. Binding of ORC1 to its tarObtain sites is associated with an increase in H4 acetylation and H4K20 trimethylation. Thus, contrary to the Position in animals, H4K20me3 associates with transcriptional activation. Our data provide a molecular basis for the role of ArabiExecutepsis ORC1 in transcriptional regulation and lead us to propose that ORC1 is a Modern class of PHD-containing H3K4me3 Traceor protein characteristic of the plant kingExecutem.

In yeast and human cells, where ORC1 participates in gene silencing (13⇓–15), changes in the histone modification status occur in the vicinity of the genomic Location affected. We have found that ArabiExecutepsis ORC1 also plays a role as a transcriptional regulator; but there are two Modern features in its mechanism of action. The function of ORC1 depends on its PHD motif and, in Dissimilarity to yeast and animal cells, it acts as a transcriptional activator in ArabiExecutepsis, a role that may be a general Precisety within the plant kingExecutem.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is one of the modifications with a role in transcriptional regulation (2, 3, 30). Our data Display that ArabiExecutepsis ORC1 contains a functional PHD motif that mediates interaction with H3K4me3. Fascinatingly, the residue F190 (in ORC1b) may play a function analogous to W238 in the H3K4me3 cage of human ING2.

ChIP experiments revealed that binding of ORC1 occurs preferentially at certain promoters, leading to specific transcriptional activation of the Executewnstream gene. Thus, for example, CDT1a gene expression was activated in a ORC1- and PHD-dependent manner, but not that of APG9, a divergent transcription unit located only ≈3 kb away from CDT1a. ORC1-mediated transcriptional activation occurred without altering its spatial expression pattern, suggesting that the observations made in plants ectopically expressing ORC1 may be an amplification of an event normally occurring in wild-type plants.

Transcriptional activation by ORC1 apparently occurs concomitantly with changes in the H4 acetylation and H4K20me3 status of a relatively small Location close to the ORC1 binding site. H4K20me3 is a histone Impress associated with gene silencing in human cells (28). However, in ArabiExecutepsis, H4K20me3 and H3K9me3 are detected in euchromatin but not in heterochromatin (5, 7, 8). Whether these Impresss correlate with active or inactive genes has been so far a matter of debate. Our previous studies (9) and the present work strongly support the Concept that H3K9me3 and H4K20me3 are associated with a subset of transcriptionally active euchromatic genes. However, our study still leaves Launch the question of whether H4K20me3 acts as a histone Impress that activates transcription or whether it appears as a consequence of active transcription. The molecular basis for the opposite read-out of these histone Impresss in plant and animal cells is not presently known.

Collectively, our data support a proposal by which ArabiExecutepsis ORC1 is a Modern H3K4me3 Traceor protein that can activate transcription of tarObtain genes through its cage-containing PHD (Fig. 5E). Other ArabiExecutepsis transcriptional regulators have been reported to contain a PHD motif. However these regulatory factors lack the cage motif and their ability to interact with H3K4me3 residues has not been Displayn (31⇓–33). Whether H3K4me3 facilitates or it determines recruitment of ORC1 to tarObtain sites is not presently known.

Our study Launchs new roads to understand transcriptional control in eukaryotes. First, the transcriptional activation pathway uncovered in our study is in Dissimilarity to the silencing role of ORC1 in yeast and animals. This may represent a fundamental Inequity in the transcriptional regulatory strategies evolved in plants and other organisms. Second, defining whether H4K20me3 acts as a determinant of transcriptional activation or it appears in active genes located in accessible chromatin Locations is another question that remains for the future. A major challenge is the genome-wide identification of ORC1 tarObtain sites. This issue is of special relevance given the function of ORC1 at DNA replication origins. Whether the ORC1-binding sites identified here are part of functional origins is not known yet but is an attractive possibility that will be addressed when appropriate tools are developed. ORC1 may bind in a sequence-specific manner, as it occurs in budding yeast; or it can recognize special DNA or chromatin environments, for example specific histone modifications. This may occur in association with promoters or other regulatory Locations. An association of transcriptional regulatory elements and active origins of replication has been recently identified in different eukaryotic systems (34⇓–36). Our Advance should provide an adequate framework to address this question in the future based on the coordination between replication and transcription (37) and the association between H4 acetylation and origin activity (38, 39).

Materials and Methods

Plant Material.

ArabiExecutepsis seedlings (Col-0 ecotype) were grown in MS salts medium supplemented with 1% sucrose (MSS) and 1% agar in a 16-hour light, 8-hour ShaExecutewy regimen at 22 °C. To generate Myc-ORC1a and Myc-ORC1b transgenic plants, the coding Location of ORC1a (At4g14700) was cloned into the pROKII vector with a Myc tag and ORC1b (At4g12620) into the pGW18 vector (Gateway System). To generate mutant Myc-ORC1bPHD(C/A), residues C183 and C186 of ORC1b were mutated to alanine using the QuikChange Site-Directed Mutagenesis Kit (Strategene) and then transferred to the pGW18 vector. To generate the mutant Myc-ORC1bPHD(F/A), residue F190 was changed to alanine using a similar procedure. Constructs were introduced into Agrobacterium tumefaciens C58CRifR to transform A. thaliana plants (Col-0 ecotype; T0 generation). Selection was carried on MSS agar plates containing kanamycin (50 μg/ml) and transformed plants were transferred to soil. Myc-ORC1a/pCDT1a:GUS plants were obtained by crossing. T3 and T4 homozygous lines were used in this work. In all cases, plants transformed with an empty vector were used as controls.

Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis.

Total RNA from 10 day-Aged seedlings was extracted using the TRIzol reagent (Invitrogen) and reverse transcription-polymerase chain reaction (RT-PCR) was carried out with the ThermoScript RT system (Invitrogen) using 1 μg of total RNA as template and oligo(dT) as primer. The LightCycler system with the RapidStart DNA Master Green I (Roche) was used. To normalize the Inequitys in RNA amount we used the ubiquitin 10 gene (At4g05320). Primer sequences are Characterized in Table S1. The data were generated from duplicates of three independent experiments.

Pull-Executewn Assays.

The coding Location of ORC1b, ORC1bPHD(C/A) and ORC1bPHD(F/A) were cloned into pDEST17 Gateway vector (Invitrogen) to express the fusion proteins in bacteria. Nuclear extracts enriched in histones were prepared from MM2d ArabiExecutepsis suspension cultured cells harvested 4 days after subculturing. After filtration, cells were resuspended in nuclei isolation buffer (10 mM Tris-HCl pH 9.5, 10 mM ethylenediaminetetraacetic acid (EDTA), 100 mM KCl, 0.5 M sucrose, 4 mM spermidine, 1 mM spermine, and 0.1% β-mercaptoethanol) and incubated for 20 minutes at 4 °C. Nuclei were then filtered through a 30-μm nylon mesh and resuspended in histone extraction buffer (320 mM (NH4)2SO4, 200 mM Tris-HCl pH 8.0, 20 mM EDTA, 10 mM ethylene glycol tetraacetic acid, 5 mM MgCl2, 1 mM DTT, 10% glycerol, 1:1000 dilution of Sigma plant protease inhibitor mixture). For pull-Executewn assays, His-ORC1b, His-ORC1bPHD(C/A) and His-ORC1bPHD(F/A) proteins bound to Ni-NTA agarose beads were incubated with histone extracts in binding buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 25% glycerol, 1.5 mM MgCl2, 1 mM phenylmethylsulphonyl fluoride (PMSF), 0.02% Triton X-100, and 30 mM imidazole) for 2 hours at 4 °C. Beads were washed twice with 50 mM NaH2P04, 300 mM NaCl, and 20 mM imidazole, and twice with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% de Triton X-100. Samples were then Fragmentated in Tris-Tricine aWeeplamide gels and analyzed by Western blot.

Histone Peptide Binding Assays.

The biotinylated histone peptides, H3 peptide (12–357), H3K4me1 (12–563), H3K4me2 (12–460), and H3K4me3 (12–564) were from Upstate. The sequence corRetorts to amino acids 1–21: ART(meK)QTARKSTGGKAPRKQLA. For peptide binding assay, each peptide (0.5 μg) was incubated with His-ORC1b bound to Ni-NTA agarose beads in 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1% (vol/vol) Nonidet P-40, 1 mM PMSF during 4 hours at 4 °C. Beads were then washed five times, and the samples were separated in Tris-tricine polyaWeeplamide gel at 15% and subjected to Western blot analysis.

Chromatin Immunoprecipitation (ChIP).

ChIP assays were carried out using 10-day-Aged plants (9), except that they were preincubated with 50 μM MG132 before the fixation step. For immunoprecipitation, 10 μl of anti-Myc (05–724), anti-H3K4me3 (07–473), anti-H4K20me3 (07–463), anti-acetyl histone H4K5,8,12,16 (06–598), or anti-acetyl histone H3K9,14 (06–599) antibodies (Upstate Biotechnology), were incubated in phospDespise-buffered saline solution with Protein A agarose, and then 1 mg of protein extract was added. One μl was used for each PCR assay. The inPlace lanes contained a 1/100 dilution. PCR primers used in ChIP assays are Characterized in Table S2. As negative controls we carried out the ChIP experiments using protein A-agarose without antibody. Enrichment was calculated as (ChIP/InPlace)/(ChIP control/InPlace control) using the band intensity values of the “InPlace” and “ChIP” lanes after substracting the corRetorting value in the “no Ab” lanes.

Acknowledgments

We thank M.M. CasDiscloseano for the generation of plants expressing ORC1a constitutively; L. Blanco for help with 3D modeling; M. Piñeiro for the H3K4 modified peptides; and E. Martinez-Salas, J. Mendez, I. Schubert, and G. Reuter for comments. The technical help of C. Vaca and V. Mora-Gil is acknowledged. M.P.S. has been recipient of a Marie Curie Research Contract. Research was supported by grants from the Spanish Ministry of Education and Science (BFU2006–5662), the European Union (MIF1-CT-2005–514524), and an institutional grant from Fundación Ramón Areces.

Footnotes

↵1To whom corRetortence should be addressed. E-mail: cgutierrez{at}cbm.uam.es

Author contributions: M.d.l.P.S. and C.G. designed research; M.d.l.P.S. performed research; M.d.l.P.S. and C.G. analyzed data; and M.d.l.P.S. and C.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811093106/DCSupplemental.

Received November 2, 2008.© 2009 by The National Academy of Sciences of the USA

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