In order to address if ORCA interacts with the machinery that causes the establishment of heterochromatin, we used a candidate approach to investigate the interaction of ORCA with individual H3K9 KMTs that catalyze H3K9 repressive modifications. We observed robust interaction of endogenous ORCA with endogenous G9a and Suv39H1 (Figure 1Aa,Ab, Figure 1—figure supplement 1Aa,Ab). 1.31% of total G9a was found to be in a complex with ORCA. Quantitation was based on the amount of G9a immunoprecipitated with ORCA (based on 100% efficiency of ORCA IP, Figure 1—figure supplement 1B) (n = 7). Similarly, 1.44% of total Suv39H1 was in a complex with ORCA (n = 4). Note that only about 0.2% of the endogenous H3K9 KMTs co-purified with Suv39H1 (Fritsch et al., 2010). Co-immunoprecipitation (co-IP) using T7-tagged ORCA and Flag-tagged H3K9 KMTs revealed interaction of ORCA with G9a, GLP, and Suv39H1, all enzymes involved in the establishment of heterochromatin (Figure 1Ba,Bb). In addition, we carried out IP from cell lines stably expressing Flag-tagged-G9a or GLP. IP from nuclear extracts using Flag antibody to determine the association of endogenous ORCA with the KMTs. ORCA along with Orc2 and MCMs was found to interact with the KMTs (Figure 1—figure supplement 1C). However, ORCA did not associate with the arginine methyltransferase PRMT5 (Figure 1—figure supplement 1D), showing the specificity of the interactions.

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In order to show functional co-recruitment of ORCA and the H3K9 KMTs, we used an in vivo cell system (CLTon) that uses a 200 copy transgene array-containing lac operator repeats stably integrated into human osteosarcoma (U2OS) cells as a single heterochromatic locus that can be visualized by Cherry-lac repressor (LacI). Furthermore, transcriptional activation using doxycycline (DOX) causes the decondensation of the locus (Janicki et al., 2004; Shen et al., 2010). We tethered the triple fusion proteins of YFP-LacI-KMTs to the heterochromatic locus and examined if these enzymes could recruit ORCA to the locus. This approach corroborated the interaction of ORCA with G9a (Figure 1C).

We next examined whether ORCA and H3K9 KMTs (G9a and Suv39H1) assembly requires intact DNA. Co-IPs from cells expressing G9a and ORCA or Suv39H1 and ORCA were carried out in the presence or absence of ethidium bromide (EtBr). EtBr selectively inhibits DNA-dependent protein interactions (Lai and Herr, 1992). ORCA continued to show interaction with G9a as well as Suv39H1 even in the presence of EtBr (Figure 1Da,Db), indicating that these interactions were DNA-independent. The DNA-independent interactions were also corroborated by co-IP experiments in the presence of the nuclease benzonase (data not shown). Furthermore, the interaction of ORCA with G9a as well as Suv39H1 was direct and independent of DNA, as evident by the direct interaction of purified ORCA with G9a/Suv39H1 proteins (Figure 1Ea,Eb).

Recent studies have demonstrated that in addition to Suv39H1, G9a/GLP may participate in the establishment of pericentric heterochromatin (Vassen et al., 2006; Dong et al., 2008; Kondo et al., 2008). Since ORCA is enriched at heterochromatic regions, we carried out detailed functional characterization of the interaction of ORCA with Suv39H1 and also with G9a in order to dissect the biological relevance of these associations.

To map the interaction domains of ORCA with G9a and Suv39H1, we generated several truncation mutants of ORCA (Figure 2Aa), G9a (Figure 2Ab), and Suv39H1 (Figure 2Ac). Using co-IP experiments, we observe that the WD repeats of ORCA (truncation mutants 128–647 and 270–647 aa) interacted with G9a (Figure 2—figure supplement 1Aa) and Suv39H1 (Figure 2—figure supplement 1Ab). We found that the deletion of any one of the WD domains in ORCA resulted in loss of binding to heterochromatin consistent with the fact that the intact β-propeller structure of WD is crucial to maintain its functionality. We also observed that the leucine-rich repeats (LRR)-containing fragment of ORCA (1–127 aa), but not the one containing the linker (1–270 aa), interacted with G9a (Figure 2—figure supplement 1Aa) but not with Suv39H1 (Figure 2—figure supplement 1Ab). Co-IP experiments demonstrated that the ankyrin repeat (619–965 aa) of G9a (Figure 2Ba) and the SET domain (151–412 aa) of Suv39H1 were necessary for interaction with ORCA (Figure 2Bb).

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To address if the chromatin context affected the interaction between ORCA and G9a, we used the CLTon cells and examined the interaction of YFP-LacI-fused full-length and various truncation mutants of ORCA tethered to the heterochromatic locus, with full-length or truncation mutants of G9a (Figure 2Ca–Db, Figure 2—figure supplement 1Ba,Bb). The WD domain of ORCA was able to recruit CFP-G9a (Figure 2Ca,Cb) corroborating our IP results (Figure 2—figure supplement 1Aa). Interestingly, when CFP-LacI-ORCA was co-transfected with YFP-G9a mutants (Figure 2—figure supplement 1Ba), not only did the mutant YFP-G9a-1-618, which lacks the ankyrin repeats, show significantly reduced interaction but also YFP-G9a-1-965, which has an intact ankyrin repeat but lacks the SET domain, showed significantly reduced association with the locus (Figure 2—figure supplement 1Ba,Bb). We next addressed if the interaction of ORCA with G9a at heterochromatic regions required the catalytic domain of G9a in addition to its ankyrin repeats. YFP-LacI-G9a triple fusion protein was found to be enzymatically active as is evident by the accumulation of H3K9me2 at the CLTon locus upon the tethering of G9a full-length construct (Figure 2—figure supplement 1C). Tethering of YFP-LacI-G9a-ΔSET or YFP-LacI-G9a-H1166K (a point mutant, which abolishes the catalytic ability of G9a) to the locus (Figure 2Da) failed to recruit CFP-ORCA (Figure 2Da,Db). However, co-IP experiments demonstrated that T7-ORCA could interact with GFP-G9a-ΔSET (Figure 2—figure supplement 1D). These data suggest that while the ankyrin repeat of G9a is sufficient for the association with ORCA (Figure 2Ba), the interaction of ORCA and G9a at the heterochromatin also requires the methylating ability of G9a. Similarly, the interaction between ORCA and Suv39H1 requires the SET catalytic domain (Figure 2Bb).

Since we observed the interaction between ORCA and the H3K9 KMTs at the heterochromatic locus, we next asked if the interaction occurred in a chromatin context-dependent manner. We tethered ORCA to the CLTon locus and examined the recruitment of G9a upon induction of transcription from the decondensed locus (Figure 2Ea). In the absence of DOX, ∼80% of cells showed CFP-G9a recruitment to the locus when YFP-LacI-ORCA was tethered (Figure 2Ea,Eb). Upon transcriptional activation, there was a striking reduction (∼10%) in CFP-G9a association in the YFP-LacI-ORCA-tethered decondensed locus (Figure 2Ea,Eb). ORCA-tethered decondensed locus, in addition to G9a, also failed to recruit HP1α, but contained Cdk9, a component of the pTEFB kinase complex, which is part of the transcription elongation complex (Figure 2—figure supplement 1E) Two components of the ORC, Orc2, and Orc3 that require each other for their stability associate with one another at both the condensed and the open chromatin (Figure 2—figure supplement 1F). These results indicate that while ORCA can interact directly with G9a and Suv39H1 (Figure 1Ea,Eb), the interactions could be dependent on or regulated by chromatin within the cells. In a cellular milieu, the fraction of ORCA directly interacting with G9a or Suv39H1, independent of chromatin, is likely to be a very small pool, and therefore, too weak to be detected at the CLTon locus.

Our earlier work demonstrated the existence of a subset of multiple H3K9 KMTs in a single complex and functional cooperation between these molecules to regulate heterochromatin function and gene expression (Fritsch et al., 2010). Since ORCA interacts with different H3K9 KMTs, we investigated if ORCA is an integral component of this multi-KMT complex.

For this purpose, we employed the process of SiMPull analysis (Figure 3Aa,Ab) (Jain et al., 2011). This method is extremely sensitive and is a tour de force to examine protein complexes and also to accurately calculate the stoichiometry of proteins within the complexes (Shen et al., 2012). This approach obviates the need for ensemble experiments that require IPs with large quantities of cell lysates. Our initial estimates predicted that three grams of Flag-HA-Suv39H1-expressing Hela-S3 cell pellet, which is ∼3 billion cells, is required for a single glycerol gradient sedimentation to obtain other H3K9 KMT signals detectable by Western blotting (Fritsch et al., 2010). However, a relatively higher sensitivity can be achieved by the SiMPull approach by using only a million cells. We first measured the stoichiometry of ORCA bound ORC and H3K9 KMTs, respectively. We used cells co-expressing T7-ORCA and YFP-ORC1 (Figure 3Ba–Bd) or T7-ORCA and YFP-KMTs to perform SiMPull (Figure 3Ca–Dd). ORCA complexes containing YFP-ORC1 or YFP-KMTs were visualized as isolated fluorescent spots by single-molecule total internal reflection fluorescence (TIRF) microscopy (Figure 3Bb,Cb,Db). Pulldown by a control antibody (anti-HA) showed very low non-specific level of fluorescence, thereby demonstrating the high specificity of SiMPull assay. Individual fluorescence spots showed single- or multi-step decreases in fluorescence intensity corresponding to photobleaching of individual YFP molecules in a single complex (a representative schematic of the photobleaching analysis is shown in Figure 3Aa). After photobleaching analysis of many co-immunoprecipitated YFP-ORC1, YFP-G9a, and YFP-Suv39H1, we found that primarily one molecule each of ORC1, G9a, and Suv39H1 interacts with a single molecule of ORCA (Figure 3Bd,Cd,Dd). In addition, we also observed that in a small population, ORCA associates with two molecules of G9a (Figure 3Cd,Ce), suggesting that the ORCA-interacting-G9a may also be present as a homodimer.

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Next, we investigated whether ORCA bound to KMTs also contains ORC by performing SiMPull using biotin-conjugated anti-T7 antibody with cells co-transfected with YFP-ORC1, mCherry-G9a, and T7-ORCA (Figure 3Ea). To mimic endogenous expression levels of these proteins, we first systematically titrated the levels of plasmid transfected to obtain an expression of the candidate protein that is similar to endogenous levels (Figure 3—figure supplement 1Aa). Based on this analysis, we transfected 2 × 10⁶ cells with 100 ng of each plasmid and then carried out SiMPull (Figure 3—figure supplement 1Aa, lane3). Complexes of T7-ORCA that contain YFP-Orc1 were detected in the green imaging channel and those containing mCherry-G9a were detected in the red imaging channel (Figure 3Eb,Ec). After overlaying the two channels, 39 ± 5% of YFP-ORC1 molecules colocalized with mCherry-G9a, indicating that all three proteins, ORC, ORCA, and G9a are found in a substantial fraction of single complexes (Figure 3Ec). SimPull from cells that were transfected with higher concentration of plasmid showed similar extent of co-localization (Figure 3—figure supplement 1Ab,Ac). The results are consistent with our earlier study showing that ORCA is protected from ubiquitin-mediated proteolysis when bound to ORC and as a result is always associated with ORC (Shen and Prasanth, 2012). Next, we tested whether multiple H3K9 KMTs exist in a single complex with ORCA using triple transfections of T7-ORCA, YFP-Suv39H1, and mCherry-G9a in U2OS cells (Figure 3Fa,Fc, plasmid titrations: Figure 3—figure supplement 1Ba, lane3 used for the experiment). Interestingly, we could observe ∼55 ± 7% of YFP-Suv39H1 colocalized with mCherry-G9a, (Figure 3Fc). Similar results were obtained with higher concentration of plasmid transfection: (Figure 3—figure supplement 1Bb,Bc), suggesting the existence of a significant amount of ORCA-G9a-Suv39H1 complex. The true degree of cohabitation may be even higher because the fluorescent proteins may not all mature into active chromophores. This leads to dark molecules and appearance of either only green or only red spots even though both the KMTs are present in a complex. In addition, unequal expression of the transfected KMTs or the presence of endogenous KMTs in the complexes may also lead to a reduction in cohabitation detected by SiMPull. Finally, only a subset of G9a and Suv39H1 may exist as a single complex with ORCA (similar to reported data [Fritsch et al., 2010]). Elucidation of three different proteins in a single complex is one of the promised capabilities of SiMPull (Jain et al., 2011), and the data we present here constitute one of the first demonstrations of such a capability.

In order to corroborate our SimPull observations on the existence of ORC-ORCA-H3K9 KMTs and G9a-ORCA-Suv39H1 in a single complex, we utilized sequential IPs. We carried out triple transfections of T7-ORCA, HA-ORC1, and Flag-G9a in U2OS cells, followed by immunoprecipitation of HA-ORC1. Following HA peptide elution, the eluate was used for T7-Ab immunoprecipitation. T7-ORCA was immunoprecipitated, and a robust co-IP of Flag-G9a was detected (Figure 3—figure supplement 1C). This further confirmed the existence of ORC-ORCA-H3K9 KMTs in a single complex. Similarly, we performed triple transfections of T7-ORCA, HA-G9a, and Flag-Suv39H1 in U2OS cells followed by immunoprecipitation of HA-G9a. Following HA peptide elution, the eluate was used for T7-Ab immunoprecipitation. T7-ORCA was immunoprecipitated, and a robust co-IP of Flag-Suv39H1 was detected (Figure 3—figure supplement 1D), further confirming the existence of multiple H3K9 KMTs in a single complex with ORCA. The exogenous expression of Suv39H1 did not affect the association of G9a and ORCA; similarly, the exogenous expression of G9a did not compromise the association of Suv39H1 and ORCA (Figure 1—figure supplement 1Aa,Ab).

Previous studies indicated that ORCA along with ORC associates with heterochromatic regions (Prasanth et al., 2010; Shen et al., 2010) and also specifically binds to repressive histone and DNA marks (Vermeulen et al., 2010). Since ORCA interacts with both H3K9me2 and H3K9me3-catalyzing enzymes, we examined the direct binding of ORCA to these marks. We performed peptide pull-downs with N-terminal peptides of histone H3 with the K9 differentially modified with acetylation or mono-, di-, or tri-methylation (Figure 4—figure supplement 1A). Iodoalkyl agarose-conjugated peptides were incubated with purified His-tagged-ORCA. We found that ORCA displayed increased interaction with mono-, di-, and tri-methylated H3K9 compared to unmodified or K9-acetylated H3 peptides (Figure 4—figure supplement 1A).

To get a more quantitative estimation of the affinity of ORCA for differentially methylated H3K9, we have employed SiMPull for the first time as a potential substitute for isothermal calorimetry. Biotinylated histone H3 N-terminal tails were immobilized on passivated quartz slides followed by passing lysates expressing full-length YFP-ORCA or the fragment 1–127 aa, which contains only the LRR and lacks WD domain necessary for binding to methylated histones (Figure 4Aa). The level of YFP-ORCA expression was quantitated by a direct anti-GFP pull-down with both the lysates (Figure 4—figure supplement 1Ba) and analyses of the average number of molecules pulled down (Figure 4—figure supplement 1Bb,Bc). The lysates were then diluted so that the expression of the YFP-tagged proteins is nearly equal and passed through the flow chambers containing the immobilized peptides. Analysis of the average number of molecules pulled down by the peptides revealed that ORCA has the highest affinity for H3K9me3 followed by for me2 and me1 (Figure 4Ab,Ac). YFP-ORCA 1–127 aa showed a low-basal binding to all the peptides corroborating the necessity of WD domain of ORCA for specifically recognizing methylated histones.

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To further determine whether ORCA is required for the establishment of these histone marks on chromatin, depletion of ORCA (siRNA-mediated knockdowns [KDs]) was carried out both in cancerous cells (U2OS) and diploid fibroblasts (WI-38), and the total levels of H3K9me2 and H3K9me3 were analyzed by immunoblotting (IB) (Figure 4Ba,Bb). In both the cell lines, upon depletion of ORCA, the levels of H3K9me2 decreased while H3K9me3 remained unchanged at the resolution of Western blotting (Figure 4Ba,Bb).

To determine the involvement of ORCA in the genome-wide status of H3K9 methylation, we conducted H3K9me3 ChIP-sequencing upon ORCA depletion. We observed a significant decrease in H3K9me3 ChIP-seq signal upon ORCA KD (Figure 5A,B). Around 18% of the detected peaks showed highly significant (more than fivefold decrease) changes in H3K9 tri-methylation in cells lacking ORCA (Figure 5B, Supplementary file 1 and total H3K9 in Figure 5—figure supplement 1Aa). Interestingly, several regions did not show significant change in H3K9me3 association upon ORCA KD (Figure 5—figure supplement 1Ab). Since ORCA associates with heterochromatin, we specifically analyzed the number of reads of satellite repeats in H3K9me3 ChIP and found that there was a significant reduction of this mark at these regions of the genome upon ORCA KD (Figure 5Ca). Furthermore, H3K9me3 showed less association both with the telomeric (TAR1) and centromeric (SST1) repetitive DNA in cells lacking ORCA (Figure 5Cb,Cc).

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Our attempts on H3K9me2 ChIP-seq did not succeed because of the technical challenge associated with sequencing the broad H3K9me2 peaks. Similar problems with H3K9me2 ChIP-seq have been previously reported by other studies (Yuan et al., 2009). As an alternate, regions that showed significant reduction of H3K9me3 in the ChIP-seq experiment (as evident by the wiggle plots; Figure 5Da–Dd and Figure 5—figure supplement 1Ba,Bb) were chosen for H3K9me2 ChIP-qPCR (quantitative PCR) validation (Supplementary file 2). These regions also consistently showed a significant reduction of H3K9me2, corroborating the decrease seen in Western blotting (Figure 5—figure supplement 1Ca,Cb). C-FOS, a gene that does not associate with these repressive histone marks, was used as a negative control (Figure 5—figure supplement 1Bc–Cb). To determine whether the H3K9-targets are directly regulated by ORCA, we conducted ChIP using HA antibody in HA-ORCA expressing stable cell line. This allowed us to address if ORCA is associated with H3K9-occupied genomic sites. We observed a strong enrichment of ORCA at the H3K9-enriched loci (Figure 5Ea), while ORCA binding to C-FOS, a region devoid of H3K9, was comparable to that of IgG (Figure 5Eb).

To understand the mechanism of reduction of H3K9 methyl marks upon ORCA depletion, we first determined whether the protein stability or chromatin association of G9a and Suv39H1 was altered upon ORCA loss. Our data revealed that the total cellular levels of G9a and Suv39H1 were not reduced upon ORCA KD (Figure 5—figure supplement 1D). We next addressed if the loading of these KMTs onto chromatin is impaired upon ORCA KD. To investigate this, we performed G9a and Suv39H1 ChIP upon ORCA KD and analyzed the association of these enzymes to the loci that show H3K9me2 and H3K9me3 reductions. Suv39H1 showed a decrease at these loci (Figure 5Fa,Fb), indicating that the loading of Suv39H1 to these regions is reduced upon ORCA depletion. G9a association with these regions showed either no alteration or an increase at some regions (Figure 5—figure supplement 1Ea,Eb), indicating that the reduction in H3K9me2 that was observed was possibly due to impaired catalytic activity of G9a.

Next, we addressed if ORCA facilitates the assembly of the multimeric H3K9 KMT megacomplex. To address this, the association between the components of the KMT megacomplex, namely G9a and Suv39H1, was evaluated in cells that were treated with control or ORCA siRNAs. Flag-G9a and HA-Suv39H1 were expressed, and HA IP was carried out in control and ORCA-depleted (ORCA KD) cells. ORCA KD showed close to 50% reduction of Suv39H1 that co-immunoprecipitated with G9a (Figure 6Aa,Ab). This observation suggested that the stability of the KMT complex requires ORCA. We used SiMPull to obtain an accurate quantitative estimate of the reduction (Figure 6Ba–Bc). YFP-Suv39H1 and mCherry-G9a were expressed in cells depleted of ORCA. GFP pull-down was carried out, and the number of mCherry-G9a molecules associated with Suv39H1 was calculated (Figure 6Bb,Bc). ORCA KD led to ∼50% reduction in the complexes containing YFP-Suv39H1 and mCherry-G9a (note, 24 ± 3% mCherry-G9a pulled down by YFP-Suv39H1 in control vs 15 ± 1% in ORCA knock-down cells; Figure 6Bc). These results support the argument that ORCA acts as a scaffold protein that is necessary for stabilizing a subset of the complexes containing multiple H3K9 KMTs.

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To further confirm the role of ORCA as a scaffold protein, we addressed if over-expressing ORCA leads to any increase in G9a and Suv39H1-containing complexes. We performed triple transfections of YFP-Suv39H1, mCherry-G9a, and T7-ORCA. YFP-Suv39H1 SimPull was carried out, and the number of mCherry-G9a molecules pulled down was analyzed as a percentage of YFP-Suv39H1 pull-down (Figure 6Ca,Cc). The presence of full-length T7-ORCA showed 25 ± 1% association between Suv39H1 and G9A. The mutant T7-ORCA (1–270) that does not interact with either G9a or Suv39H1, when expressed along with G9a and Suv39H1 showed 14 ± 3% of mCherry-G9a in complex with YFP-Suv39H1. By contrast, the other T7-ORCA mutant (128–647) that interacts with both G9a and Suv39H1 stabilized mCherry-G9a and YFP-Suv39H1 complexes in the cell (29 ± 6%; Figure 6Cb,Cc).

These results collectively indicated that ORCA, by acting as a scaffold protein, stabilizes the association of multiple KMTs in a single complex. In the absence of ORCA, the integrity of this complex is compromised, leading to the reduction in the KMT-associated enzymatic activity and a subsequent reduction of H3K9me2 and H3K9me3 patterns on chromatin.

In general, chromatin at the nuclear periphery is significantly enriched with H3K9me2, whereas H3K9me3 is preferentially enriched around nucleolus (Yokochi et al., 2009). Typically, both of these regions replicate late during S-phase indicating that in general repressive histone marks-containing differentially condensed chromatin influences replication timing and chromatin positioning (Julienne et al., 2013). Therefore, we investigated whether the changes in H3K9me2 and H3K9me3 deposition in specific chromatin regions, upon ORCA depletion, also influences their replication timing. We depleted ORCA in U2OS cells and then synchronized the cells so as to analyze the spatio-temporal regulation of replication during S-phase (Figure 7A). Samples were collected at 4, 8, 12 hr post release from aphidicolin arrest with BrdU pulse-labeling prior to sample collection. This was followed by immunofluorescence to score for cells in early, mid, and late S-phase of cell cycle (Figure 7—figure supplement 1A). At 8 hr and 12 hr time points post-aphidicolin release, ORCA depletion caused dramatic reduction in cells showing late replication patterns (Figure 7B). BrdU (Bromodeoxyuridine)-PI flow cytometry results showed a significant reduction in BrdU incorporation in ORCA-depleted cells without significant changes in the early S-phase (Figure 7C). To determine if the changes in late replication pattern are a reflection of changes in the heterochromatin organization, we examined the replication timing of regions that showed a reduction in H3K9me2 and H3K9me3 upon ORCA KD. Initial analysis of the available repli-seq data set from various human cell lines in UCSC Genome Browser and ENCODE consortium revealed that the replication timing of large domains remains the same across cell lines. We, therefore, compared the H3K9me3 ChIP-seq data set to the HeLa repli-seq data set (Figure 7D). HeLa-S3 G1b and HeLa-S3 S1 are deep sequencing data sets for late G1 and early S replicating regions in HeLa-S3 cells (Hansen et al., 2010). The chromosomal regions that are replicating at these two stages are shown in black (early) and late (gray) along the length of the chromosome (Figure 7D and Figure 7—figure supplement 1B).

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Using the data set mentioned above, we examined the replication timing of the regions that showed reduction in H3K9me3 by ChIP-seq. On chromosome 19, the total H3K9me3 peaks in the control sample and the regions, which show greater than fivefold decrease in H3K9me3 upon ORCA depletion are represented as black bars above the HeLa-S3 G1b and HeLa-S3 S1 tracks. Upon ORCA depletion, most of the affected H3K9me3 peaks resided in late-replicating domains (Figure 7D and Figure 7—figure supplement 1B). This coupled with the loss of late-replication patterns by BrdU IF in ORCA-depleted cells made us hypothesize that ORCA could also regulate the replication of late-replicating regions.

Loss of ORCA could be causing either changes in replication timing of late-replicating regions or the complete loss of replication of these regions. To investigate these possibilities, we conducted BrdU ChIP in control and ORCA-depleted U2OS cells (Figure 7—figure supplement 1Ca–Cc). We depleted ORCA and arrested the cells using aphidicolin. The cells were then released into S-phase, pulse-labeled with BrdU, and analyzed by BrdU ChIP at different time points post-release (0, 4, 8, and 12 hr). The replication timing of various loci that showed significant reduction in H3K9me2 and me3 (Figure 5D and Figure 5—figure supplement 1Ca) was assessed by quantitative PCR. We observed changes in replication timing of these loci (Figure 7—figure supplement 1Cb,Cc shows two representative loci CELSR3 and FAM20A) upon loss of ORCA. For example, in control cells a significant population of CELSR3 locus replicates in late S (12 hr post release) as evident by the BrdU ChIP signal at 12 hr. Upon ORCA KD, there is a significant increase in the population of the locus replicating during early S (4-hr time point) and a concomitant reduction in BrdU ChIP signal at mid and late S (8 and 12 hr time points) (Figure 7—figure supplement 1Cb). The replication timing of C-FOS, a region that serves as a control showing no changes in H3K9me2 and me3 upon loss of ORCA, remains unaffected (Figure 7—figure supplement 1Ca), suggesting that the replication timing changes observed in ORCA-depleted cells may not be because of the direct role of ORCA/ORC in establishing the pre-replicative complex.

We have previously demonstrated that ORCA plays a key role in replication initiation (Shen et al., 2012). We addressed whether the observed defects in heterochromatin organization and replication patterning in cells lacking ORCA are due to defects in preRC assembly or reflect the more direct role of ORCA in heterochromatin organization. While it is well-known that ORC (along with ORCA) associates with heterochromatic regions in post-replicated cells in metazoans (Prasanth et al., 2004; Shen et al., 2010), its direct role in heterochromatin organization vs heterochromatin replication licensing has remained to be understood.

In order to understand ORCA's role in chromatin organization and if it is independent of its role in preRC function, we wanted to deplete ORCA after the establishment of pre-replication complex (post-G1 phase) but before DNA synthesis began. Depletion of a protein within a short, specific time window by RNA interference is challenging because even if the mRNA levels are dramatically reduced, the protein levels could persist for significantly longer times. This necessitates the use of a strategy that utilizes post-translation degradation process for reducing proteins levels efficiently. To achieve this, we utilized a commercially available Proteotuner kit (Clonetech). Briefly, an siRNA resistant version of T7-ORCA (T7-ORCA-siRNA NTV: non-targetable version) was tagged with a destruction signal or DD (destabilization domain) tag, a destabilization domain of the FKBP protein (Figure 8A; [Banaszynski et al., 2006]). This signal is recognized by the proteosomal machinery and results in the rapid degradation of the exogenous ORCA. In the presence of a ligand, Shield1, the DD tag is masked by the binding of Shield1, thereby preventing the degradation of the exogenous ORCA protein.

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In order to determine whether DD-T7-ORCA-siRNA NTV is functional and can substitute for endogenous ORCA, we examined whether it could efficiently rescue ORC levels on chromatin upon depletion of endogenous ORCA (Shen et al., 2010). We transfected DD-T7-ORCA-siRNA NTV into U2OS along with siRNA to KD endogenous ORCA. Following two rounds of siRNA treatment (48 hr) in the presence of DD-T7-ORCA-siRNA NTV (Figure 8B), we examined the loading of ORC2 on chromatin and compared it to ORC2 loading in control and ORCA-depleted cells. We observed that while ORC2 loading was affected upon ORCA depletion (levels of ORC2 decrease in the chromatin fraction with concomitant increase in the supernatant fraction), the expression of DD-T7-ORCA-siRNA NTV construct rescued this phenotype by restoring the levels of ORC2 on chromatin to an extent comparable to that of the control (Figure 8C). In addition, we also carried out immunoprecipitation of DD-T7-ORCA and found that it efficiently interacts with endogenous ORC2 (Figure 8—figure supplement 1A), further confirming that DD-T7-ORCA is functional.

To determine if the role of ORCA in heterochromatin organization is independent of its role in preRC assembly, we transfected U2OS cells with DD-T7-ORCA-siRNA NTV. We then depleted endogenous ORCA by using ORCA siRNA, while the levels of exogenous DD-T7-ORCA-siRNA NTV were maintained by growing the cells in the presence of Shield1. We synchronized the cells at early S by using aphidicolin and then degraded exogenous DD-T7-ORCA at early S by removing shield from the medium. Removal of Shield1 resulted in the loss of exogenous ORCA (in addition to endogenous ORCA that was removed by siRNA treatment [Figure 8D]). The cells were then allowed to progress through S-phase and chromatin organization and replication were examined at different time points during S-phase. Specific depletion of ORCA only in post-G1 cells also resulted in reduction in the H3K9me2 levels (Figure 8D). This demonstrates that the heterochromatin is disorganized in the absence of ORCA in post-G1 cells.

In these cells, we examined the replication patterning by BrdU immunofluorescence. We observed a decrease in cells showing mid- and late- patterns of DNA replication and a concomitant increase in cells showing early patterns (Figure 8Eb), similar to our previous observations (Figure 7B). In cells lacking ORCA, a large number of cells showing mid-replication patterns showed BrdU staining preferentially at perinucleolar regions (Figure 8Ea, Figure 8—figure supplement 1B). Furthermore, we observed that H3K9me3 and HP1α were mislocalized and formed perinucleolar rings in cells lacking ORCA (Figure 8Fa,Fb, Figure 8—figure supplement 1C). Such localization was reminiscent of HP1α localization in Orc1- and Orc5-depleted cells (Prasanth et al., 2010). Moreover, both control and ORCA-depleted cells progressed through S-phase at comparable rates (Figure 8—figure supplement 1D) indicating that the observed defects in HP1α localization and BrdU incorporation upon loss of ORCA are due to defects in heterochromatin and not due to indirect effects of defects in S-phase progression. Based on the results, we propose that the observed defects in heterochromatin organization in ORCA-depleted post-G1 cells are independent of its known functions in preRC assembly.

Human G9a (hG9a) full-length and mutant clones were obtained using PCR using pCDNA3 Flag G9a plasmid provided very kindly by Dr Martin Walsh. The mutants were cloned into pCGN, pEYFP, pECFP and pEmCherry vectors (Clonetech, Mountain View, CA), and pEYFP-LacI vector. pEGFP G9a full-length and ∆SET constructs were also kindly provided by Dr Walsh. Mouse G9a (mG9a) full-length was amplified from pSV2 YFP mG9a (Dr David Spector's lab) (Janicki et al., 2004). pEYFP LacI mG9a ∆SET and H1166K constructs were cloned by amplification from respective pSG5 HA mG9a constructs kindly provided by Dr Michael Stallcup. Flag GLP was kindly provided by Dr Dan Levy.

Human Suv39H1 full-length and mutant clones were obtained using PCR using Flag-Suv39H1 plasmid provided very kindly by Dr Rama Natarajan (Villeneuve et al., 2008). The mutants were cloned into pCGN vector and pEYFP vector (Clonetech, Mountain View, CA). pSV2-YFP-mSuv39H1 has been described previously (Janicki et al., 2004).

Myc-EZH2 was kindly provided by Dr Francois Fuks, Free University Brussels. EZH2 was PCR amplified and cloned into pEYFP-LacI vector.

Flag Suv420H1.1 and H2 constructs were kindly provided by Dr Craig Mizzen.

pEGFP-LacI vector was a kind gift from Dr Miroslav Dundr (Kaiser et al., 2008) and used for making pEYFP-LacI vector. T7-ORCA mutants, pEYFP and CFP ORCA, pECFP-LacI and pECFP-LacI-ORCA have been described previously (Shen et al., 2010). YFP-LacI-Orc2 was cloned by amplifying and inserting Orc2 into pEYFP-LacI vector. YFP-Orc1 construct has been described previously (Hemerly et al., 2009).

T7-ORCA-siRNA NTV was cloned into pPTuner IRES2 (Clonetech, Mountain View, CA) vector.

All the cloned constructs were confirmed by sequencing and used for immunoprecipitation and/or cell biological experiments.

U2OS cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing high glucose and supplemented with 10% fetal bovine serum (FBS—HyClone GE, Pittsburg, PA). WI38 cells were also grown in the same medium but supplemented with non-essential amino acids. Hela suspension cells (Hela-XLP GLP) were grown in DMEM supplemented with 5% fetal calf serum and penicillin-streptomycin. U2OS-2-6-3 CLTon cells were grown in DMEM with 10% Tet system approved FBS (Clonetech, Mountain View, CA).

For arresting cells at G1/S, aphidicolin (stock 10 mg/ml) was added to the cells at a final concentration of 5 μg/ml for 12 hr. Cells were then washed three times with PBS and released into S-phase with medium (DMEM + 10%FBS) lacking aphidicolin. Cells were then collected at 4, 8, and 12 hr post-G1/S block for immunofluorescence and flow cytometry analysis.

siRNA transfection for ORCA depletion: cells were grown to 30% confluency and siRNA against ORCA or control luciferease gene (Shen et al., 2010) was delivered into the cells at a final concentration of 100 nM using Lipofectamine RNAimax (Invitrogen, Carlsbad, CA). The siRNAs were delivered twice at the gap of 24 hr, and the cells were collected 24 hr after the second round of transfection for subsequent analysis.

U2OS cells were transfected with of DD-T7-ORCA-siRNA NTV construct along with siRNA against ORCA. 5 hr later Shield1 (0.5 μM) was added to the medium. 24 hr after the first round of KD, a second round of ORCA siRNA treatment was carried out in the presence of Shield1. Samples were collected 24 hr later for chromatin fractionation to examine ORC loading.

As described above, DD-T7-ORCA-si NTV (500 ng plasmid was transfected) was expressed in U2OS cells grown on coverslips in the presence of Shield1 (0.25 μM). This was followed by the addition of fresh medium containing aphidicolin (5 μg/ml) + Shield1 (0.25 μM). 14 hr post-aphidicolin block, cells were washed thrice with PBS containing aphidicolin, with or without Shield1, respectively. This was followed by performing control and ORCA depletions in Opti-MEM containing aphidicolin, with or without Shield1. The control and ORCA-depleted cells were washed with PBS containing or lacking Shield1, respectively. Then, the cells were released into S-phase using medium with or without Shield1 for control and ORCA-depleted cells followed by late S sample collection 12 hr later for Western blotting and immunofluorescence analysis.

His ORCA, G9a, and Suv39H1 viruses were generated by using pFastBac HT-B-His-tagged-ORCA, G9a, and Suv39H1, respectively (Shen et al., 2012) (by following Bac-to-Bac baculovirus expression system—Invitrogen). Virus production was carried out in Sf9 cells with viruses collected 72 hr post-infection (multiplicity of infection 5 to 10). Protein expression was carried out in Hi5 cells by collecting cells 65 hr post-infection. Nuclei were collected by using Hypotonic lysis buffer (20 mM potassium phosphate buffer pH 7.5, 5 mM KCl, 1.5 mM MgCl₂, and 5 mM b-mercaptoethanol) making nuclear extracts in PK50 buffer (20 mM potassium phosphate buffer pH 7.5, 50 mM KCl, 0.02% NP-40, 10% glycerol, 5 mM b-mercaptoethanol with protease and phosphatase inhibitors) (Siddiqui and Stillman, 2007). 45% ammonium sulfate precipitation was carried out followed by reconstitution of His-ORCA, G9a, and Suv39H1 in PK50 buffer.

Cells were fixed with 2% formaldehyde in phosphate buffered saline (PBS, pH 7.4) for 15 min in room temperature (RT) followed by permeabilization with 0.5% Triton X-100 in PBS for 7 min on ice or pre-extracted before fixing with 0.5% Triton X-100 in Cytoskeletal buffer (CSK: 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl₂, 10 mM PIPES at pH 6.8) for 5 min on ice. Blocking was then done for 30 min with 1% Normal goat serum (NGS) in PBS. Primary antibody incubation was then carried out for 1 hr in a humidified chamber followed by secondary antibody incubation for 25 min. The cells were then stained with DAPI (4',6-Diamidino-2-Phenylindole) and mounted using VECTASHIELD (Vector Laboratories Inc., Burlingame, CA). The following antibodies were used for immunofluorescence: BrdU (1:500; mAb BU-33, Sigma, St. Louis, MO), Lamin (1:750), H3K9me2 (1:100; 07-212, Millipore, Billerica, MA), H3K9me3 (1:200, Millipore 07-523), HP1α (1:100, Millipore 3584).

BrdU immunofluorescence: after primary and secondary antibody incubation for lamin immunofluorescence, cells were fixed with 2% formaldehyde solution in PBS. This was followed by acid denaturation of DNA using 4 N HCl for 25 min at RT. Three washes with PBS and two washes with PBS-NGS followed. This was followed by incubation of the cells with anti-BrdU antibody followed by secondary antibody incubation and mounting.

Zeiss Axioimager z1 fluorescence microscope (Carl Zeiss Inc., Jena, Germany) equipped with chroma filters (Chroma technology, Bellows Falls, CA) was used for observing the cells and statistics. Axiovision software (Zeiss) was used for digital imaging using Hamamatsu ORCA cooled CCD camera. Cells were also examined on the Delta vision optical sectioning deconvolution instrument (Applied precision, Pittsburgh, PA) on an Olympus microscope.

For co-IP with transiently transfected HKMTs and ORCA, co-transfections were carried out in U2OS cells. Cells were lysed, 24 hr post-transfection, in IP buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM DTT (Dithiothreitol) supplemented with the protease and phosphatase inhibitors). After pre-clearing with Gammabind Sepharose beads for 1 hr, the lysates were incubated with appropriate antibody-conjugated agarose overnight. The beads were washed in the same IP buffer and finally denatured by the addition of Laemmli buffer. The complex was analyzed by Western blotting.

For immunoprecipitations and IB the following antibodies were used anti-GFP (1:500; Covance, Princeton, NJ), anti-Flag M2 (1:500, Sigma), anti-HA 12CA5 (1:100) and anti-T7 (1:5000; Novagen, Billerica, MA), anti-ORCA pAb 2854-1 AP (1:500), anti-G9a (1:500, Sigma), anti-Suv39h1 (1:200, Cell Signaling, Danvers, MA), anti-ORC2 pAb 205-6 (1:1000), anti-Geminin (1:1000, Santa Cruz, Dallas, TX), anti-MCM3 TB3 (1:750), anti-α-tubulin (1:10,000, Sigma–Aldrich), anti-H3K9me2 (1:200, Millipore 07-212), anti-H3K9me3 (1:500, Millipore 07-523), anti-SF2 AK96 (1:750), anti-PCNA mAb PC10 (1:750) antibodies.

For IP in the presence of EtBr, lysates were made with IP buffer described above followed by addition of EtBr (stock 10 mg/ml, working 50 μg/ml). After pulldown, beads were washed with IP buffer supplemented with 80 μg/ml of EtBr. For Benzonase treatment, cells (grown in 6-cm plates) were lysed for 10 min in IP buffer (50 mM HEPES pH 7.9, 10% glycerol, 200 mM NaCl, 0.1% Triton X-100,1 mM MgCl₂) supplemented with protease and phosphatase inhibitors. 1000 U of Benzonase (Sigma) was then added followed by nutation at RT for 20 min. EDTA (final concentration 5 mM) was added to stop the reaction followed by centrifugation at 12,500 rpm, 5 min at 4°C. The supernatant was used for subsequent antibody incubation.

The nuclear extraction protocol has been described previously (Robin et al., 2007; Fritsch et al., 2010). We carried out HA immunoprecipitation in HeLa cells stably expressing Flag-HA-GLP and Flag-HA-G9a stable by retroviral transduction. First, nuclear extract was made using an equivalent of 20 g of dry cell pellet, which approximately corresponds to 3 billion cells. Cells were resuspended in hypotonic buffer (10 mM Tris pH 7.6, 1.5 mM MgCl₂, 10 mM KCl) with the volume of hypotonic buffer being equal to the packed volume of cells. The suspension was then dounced 20 times using tight pestle followed by adding one third the packed volume of sucrose buffer (20 mM Tris pH 7.6, 15 mM KCl, 60 mM NaCl, 0.34 M Sucrose, 0.15 mM Spermine, 0.5 mM Spermidine). Then, centrifugation was carried out (9000 rpm, 7 min). The supernatant was discarded, and the nuclei were resuspended in low salt buffer (20 mM Tris pH 7.6, 25% glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA, 20 mM NaCl). This was followed by adding high salt buffer (20 mM Tris pH 7.6, 25% glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA, 900 mM NaCl) dropwise while vortexing to make the final salt concentration 300 mM. After incubation on ice for 30 min, one third the volume of sucrose buffer was added followed by centrifugation at 1000 rpm, 10 min, 4°C. The supernatant is the nuclear soluble fraction. The pellet (chromatin bound fraction) was resuspended in sucrose buffer and CaCl₂ (final concentration 1 mM) was added. The sample was then preheated for 1 min at 37°C and MNase was added to a final concentration of 0.0025 U/μl. The sample was then incubated for exactly 12 min at 37°C followed by placing the tubes on ice. 0.5 M EDTA was added at a final concentration of approximately 3.6 μM. The samples were then sonicated (Bioruptor, high amplitude 5cycles: each cycle 1 min ON, 1 min OFF). Protease and phosphatase inhibitors were added, and the samples (nuclear soluble and chromatin bound fractions) were ultracentifuged at 40,000 rpm for 30 min. The supernatants were transferred to a fresh tube. Tagged-H3K9 HMTs complexes were then purified by immunoprecipitation using anti-FLAG antibody immobilized on agarose beads (cat# A2220, Sigma). After elution with the FLAG peptide (Ansynth, The Netherlands), the bound complexes containing nucleosomes were further affinity purified on anti-HA antibody-conjugated agarose (cat# A2095, Sigma) and eluted with the HA peptide (Ansynth, The Netherlands). The samples were then analyzed by IB.

Cells (HeLa for gel filtration and U2OS for SiMPull) were lysed in hypotonic buffer (10 mM HEPES-NaOH pH 7.9, 10 mM KCl, 2 mM MgCl₂, 0.34 M Sucrose, 10% glycerol, 0.1% Triton X-100) supplemented with 1 mM DTT, protease and phosphatase inhibitors. The lysate was incubated at 4°C for 5 min after which nuclei were collected by centrifuging at 1500×g for 5 min. The pellet was then resuspended in nuclear extraction buffer (10 mM HEPES-NaOH pH 7.9, 2 mM MgCl₂, 1 mM EGTA, 25% glycerol, 350 mM NaCl, 0.1% Triton X-100, and 1 mM DTT) supplemented with protease and phosphatase inhibitors. The supernatant was collected by centrifugation at 12,000 rpm for 5 min. The lysate was then used for co-localization studies by SiMPull.

Baculovirally purified His-ORCA and His-G9s/His-Suv39H1 were diluted using PK 50 buffer and incubated together for 2 hr at 4°C in the presence of ORCA antibody or pre-bleed. ORCA containing complexes were then pulled down followed by washes with PK 150 buffer (20 mM potassium phosphate buffer pH 7.5, 150 mM KCl, 0.5% NP-40, 10% glycerol, 5 mM b-mercaptoethanol with protease and phosphatase inhibitors). The samples were finally denatured by the addition of Laemmli buffer. The complexes were analyzed by Western blotting.

U2OS cells were grown in 6-cm plates to approximately 50% confluency followed by incorporation of BrdU (stock 10 mM; working 50 μM ) for 1 hr at 37°C. Cells were then harvested at 3500 rpm, 15 min followed by washing with 1% BSA (Bovine serum albumin) in PBS (Phosphate buffered saline) (pH 7.4). The cells were then resuspended in 0.9% NaCl (final cell density: 2 × 10⁶ cells/ml). The cells were then fixed by adding chilled 100% ethanol to a final concentration of 50% (left overnight at −20°C). After spinning down the fixed cells, DNA was denatured by resuspending in 2 N HCl + 0.5% Triton X—100 and incubating for 30 min at RT. The cells were then pelleted and resuspended in 0.1 M Sodium tetraborate pH 8.5. This was followed by centrifugation at 3500 rpm, 15 min at 4°C followed by resuspending the cells in PBS + 1% BSA + 0.5% tween 20. Anti-BrdU FITC antibody (1ug Ab/10⁶ cells) was added and the cells were incubated at RT for 1 hr. 1 ml of PBS + 1% BSA + 0.5% Tween 20 was added after that followed by spinning down the cells and proceeding with RNase A treatment and PI staining as described in the previous section.

Human Histone H3 (amino acids 1–15) peptides were synthesized (Biomer technology, Pleasanton, CA) with a Cysteine at the N terminus. The K9 (Lysine at position 9) of the peptides was unmodified, acetylated, mono-, di-, or tri-methylated. The peptides were dissolved in water, quantitated using reverse phase chromatography, lyophilized and stored at −20°C as 1-mg aliquots.

For coupling the peptides to SulphoLink Coupling Resin (Thermo Scientific, Waltham, MA), the peptides were reduced first. For this, 1 mg of each peptide was dissolved in 3 ml of coupling buffer (50 mM Tris pH 8.7, 5 mM EDTA, final pH adjusted to 8.5) supplemented with TCEP HCl (Thermo Scientific) and allowed to incubate at RT for 1 hr. 2 ml of the beads was washed with 5 ml coupling buffer (three 10 min washes) and resuspended in 1 ml of coupling buffer. 3 ml of the reduced peptides was added to the slurry followed by mixing immediately to distribute the peptide throughout the slurry. The mixture was incubated overnight at RT with gentle mixing. The beads were then spun down (2000 rpm, 2 min), washed with 6 ml of coupling buffer (three 5 min washes), resuspended in 5 ml coupling buffer + 1 ml L-Cysteine HCl. The mixture was incubated overnight at RT. The beads were then spun down and washed with 5 ml of 1 M NaCl (three 5 min washes). This was followed by washing two 5 min washes with 5 ml of storage solution (0.05% NaN₃ in water) and final suspension in 5 ml of storage solution and storage at 4°C.

50 μl packed volume of beads (300 μl of bead slurry) was washed with PK 150 buffer (20 mM potassium phosphate buffer pH 7.5, 150 mM KCl, 0.02% NP-40, 10% glycerol, 5 mM β-mercaptoethanol) and incubated with baculovirally purified His-ORCA for 2 hr at RT. This was followed with five washes with PK 150 buffer. The beads were then resuspended in Laemmli buffer and analyzed by Western blotting.

SiMPull experiments were carried out in flow chambers prepared on quartz microscope slides, which were passivated with methoxy-polyethylene glycol (mPEG) doped with 1% biotin-PEG (Lysan Bio, Inc, Arab, Al) (Jain et al., 2011). Appropriate biotinylated antibody was immobilized on PEG passivated surfaces at approximately 20 nM concentration for 20 min after coating the flow chambers with 0.2 mg/ml NeutrAvidin for 5 min. Antibodies were immobilized on NeutrAvidin (Thermo)-coated flow chambers either by incubating with biotinylated T7 antibody (Novagen) for 10 min. RIPA buffer lysed samples were then incubated in the chamber for 20 min and washed twice with the buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl 0.1 mg/ml BSA). Single-molecule data were acquired by a prism-type TIRF microscope and analyzed using scripts written in Matlab. For ORCA-Orc1; ORCA-G9a; ORCA-Suv39H1 SiMPull analysis, lysates were made from the cells transiently transfected with T7-ORCA with YFP-Orc1, YFP-G9a, or YFP-Suv39H1, respectively. For multimeric complex assembly analysis using SiMPull, cells were transfected with T7-ORCA, YFP-Orc1, and mCherry-G9a or T7-ORCA, YFP-Suv39H1, and mCherry-G9a.

For peptide pulldown experiments, biotinylated peptides were immobilized instead of antibodies. Cells lysed in RIPA buffer or nuclear extracts (depending on the experiment) were then incubated in the flow chamber for 20 min followed by wash with T300 buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1 mg/ml bovine serum albumin [BSA]). Single molecules were visualized by prism-type TIRF microscope and analyzed using Matlab scripts (https://github.com/vasuagg/SiMPull_Analysis). Cell lysate was appropriately diluted in T300 buffer to obtain optimal single molecule density on the surface.

Single molecule data were acquired as the average number of YFP or mCherry fluorescent molecules per imaging area (5000 μm²) as shown in the histograms. The error bars represent standard deviation of the mean values from 20 imaging areas. Number of fluorescence photobleaching steps was determined for each YFP-tagged molecule and accumulated to obtain the stoichiometry of the complex. Colocalization percentage between YFP and mCherry was calculated as the number of coaligned molecules of one fluorescent molecule with respect to the fluorescent molecules found in lower density on the surface. This was needed since the number of YFP and mCherry tagged proteins was not pulled down to the same extent due to their independent interaction with ORCA. Colocalization criterion was set at 2 pixels, which correspond to a diffraction limited spot (∼300 nm) for our TIRF setup. Error bars represent standard deviation of the mean values obtained from three independent experiments. For pulled down experiments performed using H3K9 peptides, the expression level of YFP-WT ORCA and YFP-mutant-ORCA was compared in the beginning by performing a direct pulldown by anti-GFP. The peptides pulldown was then performed at appropriate lysate dilution such that protein expression was same for WT (Wild type) and mutant ORCA.

H3K9me2 and me3 ChIPs: formaldehyde (Sigma) was added to culture medium to a final concentration of 1%. Crosslinking was allowed to proceed for 10 min at RT and stopped by the addition of glycine at a final concentration of 0.125 M. Fixed cells were washed and harvested with PBS. Chromatin was prepared by two subsequent extraction steps (10 min at 4°C) with Buffer 1 (50 mM Hepes/KOH pH 7.5; 140 mM NaCl; 1 mM EDTA; 10% glycerol; 0.5% NP-40; 0.25% Triton) and Buffer 2 (200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 10 mM Tris pH 8). Nuclei were then pelleted by centrifugation, resuspended in Buffer 3 (50 mM Tris pH 8; 0.1% SDS; 1% NP-40; 0.1% Na-Deoxycholate; 10 mM EDTA; 150 mM NaCl) and subjected to sonication with Bioruptor Power-up (Diagenode, Denville, NJ) yielding genomic DNA fragments with a bulk size of 150–300 bp. Chromatin was precleared with Protein A/G ultralink beads (53,133, Pierce, Grand Island, NY) for 2 hr at 4°C and immunoprecipitation with the specific antibodies carried out overnight at 4°C. Immune complexes were recovered by adding pre-blocked protein A/G ultralink beads and incubated for 2 hr at RT. Beads were washed twice with low salt buffer (0.1% SDS; 1% Triton; 2 mM EDTA; 20 mM Tris pH 8; 150 mM NaCl), twice with high salt buffer (0.1% SDS; 1% Triton; 2 mM EDTA; 20 mM Tris pH 8; 500 mM NaCl), once with LiCl wash buffer (10 mM Tris pH 8.0; 1% Na-deoxycholate; 1% NP-40, 250 mM LiCl; 1 mM EDTA), and twice with TE + 50 mM NaCl. Beads were eluted in TE + 1% SDS at 65°C, and cross-link was reversed O/N at 65°C. The eluted material was phenol/chloroform extracted and ethanol precipitated. DNA was resuspended in water and q-PCR performed using PowerSYBR Green PCR Master mix (Applied Biosystems, Pittsburgh, PA) and analyzed on a 7300 PCR System (Applied Biosystems). ChIP-qPCR results were represented as percentage (%) of IP/input signal (% input). HA-ORCA ChIPs were carried out using HA-ORCA stable cell lines in U2OS using a similar protocol with the following modifications. All the washing steps after immune complexes pulldown were done once followed by two washes with TE. Beads were eluted with 1% SDS + 0.1 M NaHCO3 at 65°C, and cross-link was reversed O/N at 65°C. The eluted material was purified using Qiagen gel purification kit and qPCR carried out.

G9a and Suv39H1 ChIPs were carried out using double-crosslinking protocols. The first crosslinking was carried out using disuccinimidyl glutarate (DSG) (Santa Cruz; stock 50 mM DSG in DMSO) and the second crosslinking using formaldehyde. U2OS cells were grown in 10-cm plates to 80% confluency, washed twice with PBS (pH 7.4). Freshly made crosslinking solution (2 mM DSG + 1 mM MgCl₂ in PBS-pH 8.0) was added for 45 min at RT. The cells were then washed twice with PBS (pH 7.4) and 10 ml of freshly made crosslinking solution (1% formaldehyde, 15 mM NaCl, 150 μM EDTA, 75 μM EGTA, 15 μM HEPES pH 7.9) was added for 10 min at RT. Then 3 ml of freshly made 1 M Glycine was added for 5 min at RT followed by two cold washes with PBS (pH 7.4). The cells were then pelleted in PBS (supplemented with protease inhibitors) followed by lysis with 300 μl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.0). The lysate was then subjected to sonication with Bioruptor Power-up (Diagenode). Chromatin was precleared with Dynabeads protein G (Life Technologies, Grand Island, NY) for 2 hr at 4°C and immunoprecipitation with the specific antibodies carried out overnight at 4°C. Immune complexes were recovered by adding pre-blocked Dynabeads (1 mg/ml BSA, 0.4 mg/ml salmon sperm DNA) and incubated for 2 hr at 4°C. Beads were washed once with low salt buffer (0.1% SDS; 1% Triton; 2 mM EDTA; 20 mM Tris pH 8; 150 mM NaCl), once with high salt buffer (0.1% SDS; 1% Triton; 2 mM EDTA; 20 mM Tris pH 8; 500 mM NaCl), once with LiCl wash buffer (10 mM Tris pH 8.0; 1% Na-deoxycholate; 1% NP-40, 250 mM LiCl; 1 mM EDTA), and twice with TE (10 mM Tris pH 8.0, 1 mM EDTA). Beads were eluted in elution buffer (1% SDS, 0.1 M sodium bicarbonate in water) at 65°C twice, 10 min each. The eluates were pooled (250 μl), NaCl added (final concentration 0.2 M) and cross-link was reversed O/N at 65°C. The eluted material was Rnase A treated (10 μg/ml, 1 hr at 37°C) followed by proteinase K treatment (4 μl 0.5 M EDTA, 8 μl 1 M Tris pH 6.9, 1 μl proteinase K 20 mg/ml) at 42°C for 2 hr. DNA was purified using QIAquick PCR purification kit (Qiagen) and q-PCR performed SYBR Green PCR Master mix and analyzed on a 7300 PCR System (Applied Biosystems). ChIP-qPCR results were represented as percentage (%) of IP/input signal (% input).

Two rounds of ORCA KD were carried out, 24 hr apart. The cells were then arrested using aphidicolin for 12 hr followed by release into S-phase and samples were collected 0, 4, 8, and 12 hr post release for BrdU ChIP. Prior to each time point collection, cells were pulsed for 2 hr with BrdU (10 μM).

Cells for each time point were then lysed with 300 μl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.0). The lysates were subjected to sonication with Bioruptor Power-up (Diagenode). 100 μl sheared chromatin aliquots were then placed on 95°C heat block for 10 min. This was followed by snap chilling the samples for 10 min. The samples were then diluted, precleared and processed further in a manner identical to the ChIP protocol described in the previous section.

qPCRs were carried out with purified DNA of input, BrdU ChIP, and mouse IgG ChIP samples obtained at 0, 4, 8, and 12 hr post-aphidicolin release time points. The qPCR signals of BrdU and mouse IgG samples were calculated as percent input values. Fold enrichment of BrdU ChIP over mouse IgG ChIP was calculated for each time point.

H3K9me2 and me3 ChIP were performed as above. Five to fifteen nanograms of ChIP DNA or un-enriched whole cell extract (Input) were prepared for sequencing on an Illumina HiSeq2000. Libraries were constructed with the Truseq DNA sample prep kit V2 (Illumina, San Diego, CA) with the following modifications: 10 ng of ChIP DNA were used as input material. DNA fragments were blunt-ended, 3′-end A-tailed and ligated to indexed TruSeq adaptors. The adaptors were diluted in a ratio of 1:20 to adjust for the input amount of DNA. Indexed adaptors allow for sequencing of multiple samples on the same lane (multiplexing). The adaptor-ligated ChIP DNAs were individually size selected on a 2% agarose gel (Ex-Gel, Life Technologies, CA) to obtain the ligated fragments 300–800 bp in length. Size-selected DNAs were amplified by PCR to selectively enrich for those fragments that have adapters on both ends. Amplification was carried out for 15 cycles with the Kapa HiFi polymerase (Kapa Biosystems, Woburn, MA) to reduce the likeliness of multiple identical reads due to preferential amplification. The final libraries were quantitated by qPCR on an ABI 7900, to allow for accurate quantitation and maximization of number of clusters in the flowcell. Final amplified libraries were also run on Agilent bioanalyzer DNA 7500 LabChips (Agilent, Santa Clara, CA) to determine the average fragment size and to confirm the presence of DNA of the expected size range.

The libraries were pooled and loaded onto a lane of an 8-lane flowcell for cluster formation and sequenced on an Illumina HiSeq2000. The libraries were sequenced from one end of the molecules to a total read length of 100 nt. The raw .bcl files were converted into demultiplexed compressed. fastq files using CASAVA 1.8.2.

The complete ChIP-seq data are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68129.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank members of the Prasanth laboratory for discussions and suggestions. We thank Drs M Stallcup, D Levy, R Natarajan, F Fuks, M Dundr, A Hernandez, R Donthu, C Fields, D Spector, and B Stillman for providing reagents and suggestions. We would like to thank Drs D Forsthoefel, S Ceman and D Rivier for critical reading of the paper. This work was supported by NSF-CMMB-IGERT and F31 (CA180616) NIH fellowship to SG; ACS (RSG11-174-01RMC) and NIH (1RO1GM088252) award to KVP and NSF career (1243372) and NIH (1RO1GM099669) awards to SGP. TH is an investigator with Howard Hughes Medical Institute. Work in SA group was supported by the: Agence Nationale de la Recherche (H3K9-methylome and EPILIS grants), Association Française contre les Myopathies (AFM); Fondation ARC; GEFLUC; Institut National du Cancer (INCa, grant 2012-1-PLBIO). JP was recipient of PhD fellowships from the MESR and Fondation ARC. The authors declare no competing financial interests.

© 2015, Giri et al.

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