Methylated histones, histone-modifying and nucleosome-remodeling proteins remain associated with DNA during all stages of mitosis

Previously we developed a PLA-based Chromatin Association Assay (CAA) that detects close proximity of a protein to 5-ethynyl-2′-deoxyuridine (EdU)-labeled DNA in vivo [28]. Using CAA and other assays, we found that, in Drosophila embryos, major methylated histone forms H3K27me3 and H3K4me3 are replaced during replication with unmodified histone H3 [28]. Similarly, H3K4me1, H3K4me2, H3K9me3, H4R3me2, H3R17me2 and H3K27Ac were displaced during replication and were accumulated at nascent DNA with various delays after DNA replication [29]. These results suggested that modified histones are unlikely to carry epigenetic information through DNA replication.

Here, using CAA, we asked whether modified histones are associated with DNA during mitosis. PLA has a unimolar sensitivity (10−40 M, Olink) and CAA detects very close proximity (40 nm, Olink) of a protein to DNA. Therefore, compared with conventional IF, this method provides a direct and very sensitive approach to detect association of proteins with labeled DNA at all stages of mitosis. We used CAA in Drosophila embryos at nuclear divisions 14–16 to detect the association of proteins with DNA during mitosis. At this developmental stage, the cell cycles are very short (S phase is 50 min, G2 is from 30 to 150 min and M is about 10 min) and lack the G1 interphase. DNA was labeled with EdU for 30 min followed by a 2 h chase; this allowed EdU-labeled cells to enter into mitosis. EdU was subsequently conjugated to biotin, and antibodies to biotin and tested protein were then used in PLA to examine the proximity of a protein to labeled DNA as described previously [28, 29]. Following PLA, embryos were counterstained with antibody to histone H3 phosphorylated at serine 10 (H3S10-p, a specific marker for mitosis) to visualize mitotic stages and with antibody to biotin to detect EdU-labeled nuclei.

Since in these experiments the cell cycle is not synchronized, and DNA in each nucleus may be labeled with EdU for varying times and at different stages of S phase, some EdU-labeled nuclei may lack PLA signals. Therefore, to quantify potential changes in association of these proteins with DNA during mitosis, we compared the total number of EdU-labeled nuclei with the number of EdU-labeled nuclei positive for PLA signals on interphase and mitotic chromosomes. All tested modified histones and chromosomal proteins were detected with similar efficiency during interphase and mitosis (Figure 1). In control experiments, PLA signals between a mitotic marker H3S10-p and EdU-labeled DNA are detected at all stages of mitosis but were only occasionally detected in the interphase nuclei (Figures 1 and 2a and Table 1).

Figure 1 Comparison of association of chromosomal proteins and histones with DNA during interphase and mitosis. Statistical comparison of nuclei with positive proximity ligation assay (PLA) signals in interphase versus all stages of mitosis. All proteins/histones tested, except H3S10-p (*P<0.05 determined by two-tailed independent t-test), have similar association with DNA during all phases of mitosis. Note: For HP1 and OSA (presented in Figure 2c and d), we were unable to accurately quantify the percentages of PLA-positive nuclei in mitosis versus interphase nuclei because their antibodies are from the same species as antibody to the mitotic marker H3S10-p. Error bars represent s.d. Full size image

Figure 2 Modified histones, histone-modifying enzymes and histone-remodeling proteins are associated with DNA during mitosis. Proximity of the modified histones H3S10-p, H3K27me3, H3K4me3 (a), TRX, ASH1, SU(VAR)3–9 (b), ISWI, OSA, BRM (c) and HP1 (d) to DNA during interphase and all phases of mitosis was tested by Chromatin Association Assay. Proximity ligation assay (PLA), red; H3S10-p, blue; 5-ethynyl-2′-deoxyuridine (EdU), green. H3S10-p is a marker for mitosis and serves as a positive control. Note: As antibodies against HP1 and OSA are from the same species (mouse) as antibody to H3S10-p, H3S10-p marker was not used in these experiments. PLA, red; H3S10-p, blue; EdU, green. PLA signals only are shown in the bottom black and white panels. Full size image

Table 1 Number of PLA signals for chromosomal proteins and modified histones on labeled DNA during interphase and mitotic stages Full size table

The quantification of the results of these experiments for the interphase and mitotic phases is presented in Table 1 and images for representative groups of proteins are shown in Figure 2a–d. Our results suggest that major methylated histones forms, H3K27me3, H3K4me3, H3K9me3, H4R3me2 and H3R17me2, which are associated with EdU-labeled DNA in interphase, remain associated with DNA at all four stages of mitosis. These results are in contrast to the absence of these histone forms using CAA during early stages of DNA replication [28, 29] and suggest that methylated histones may have an epigenetic role during mitosis.

With the exception of the H3K4 HMT ASH1 and H3K9 HMT SU(VAR)3–9, all other tested histone-modifying proteins were detected in close proximity to the replication complex and on short nascent DNA [28, 29]. Here we found that TrxG proteins, lysine HMTs TRX, ASH1, and SETD1; the PcG proteins PHO, PC and lysine HMT E(Z); and suppressor of variegation H3K9 HMT SU(VAR)3–9, as well as the arginine HMTs DART1 and DART4, remain associated with DNA during all stages of mitosis (Figures 1 and 2b and Table 1). We found that the lysine demethylases LID, LSD1 and UTX, which counteract the activities of lysine HMTs, are also stably associated with DNA in mitosis. Similarly, other groups of histone-modifying enzymes, histone acetyltransferases CBP and PCAF and histone deacetylase HDAC1, were also found to be stably associated with mitotic DNA (Figure 1 and Table 1).

TrxG proteins BRM, OSA and ISWI are components of six major chromatin-remodeling complexes. Our previous CAA-based data suggested that these proteins are displaced from DNA during replication and are recruited back from 10 min to 2 h after replication [29]. In contrast, we found that all these proteins are associated with mitotic chromosomes during all stages of mitosis (Figures 1 and 2c and Table 1). Thus all tested histone-modifying enzymes and chromatin-remodeling complexes, including ASH1, SU(VAR)3–9, BRM, OSA and ISWI, that are dissociated from DNA during replication are stably associated with DNA during mitosis.

The number of protein–DNA foci decreases at late stages of mitosis

For most of the examined proteins, we detected a relatively low number of PLA signals on interphase and mitotic chromosomes; moreover, only a few signals are detected at later stages of mitosis. One explanation may be steric hindrance that leads to epitope masking, especially owing to condensation of chromatin in mitosis, thus reducing the number of PLA signals. We tested this by performing CAA experiments following heat denaturation of nuclei in formamide and found that this treatment did not increase the number of PLA signals for H3K27me3 at any stage of mitosis (Figure 3a). We concluded that a low number of DNA–protein signals cannot be explained by low accessibility of epitopes during mitosis. Another possibility is that there is a local jackpot effect for detecting epitope interactions on mitotic chromosomes. If this were the case, then all antibodies would detect the same, limited number of foci in our CAA assay. To examine this, we performed CAA experiments with a mixture of different antibodies to proteins known to be members of different protein complexes. As seen in Figure 3b, mixing antibodies against chromosomal proteins or modified histones in a single experiment leads to a significant increase in the number of PLA signals at each of the mitotic stages. Together, these results suggest that there are no technical limitations in detecting proteins through most of the regions of mitotic chromosomes by CAA.

Figure 3 Number of protein-DNA foci decreases at late stages of mitosis. (a) Comparison of the number of proximity ligation assay (PLA) signals in Chromatin Association Assay (CAA) assays for H3K27me3 and 5-ethynyl-2′-deoxyuridine (EdU)-labeled DNA between a regular (top) and a denatured (bottom) sample. (b) CAA assays of mitotic nuclei with the indicated mixtures of chromosomal proteins (top) and modified histones (bottom). In (a and b), PLA, red; H3S10-p, blue; EdU, green. PLA signals only are shown in the bottom black and white panels. (c) Analysis of CAA in mammalian SEM cells using super-resolution microscopy (SR-SIM). Top panels, SR-SIM of the CAA assays for H3K27me3 in SEM cells. Scale bars are indicated. Middle panels, PLA signals only. Bottom panels, magnified SR-SIM images of nuclei shown in the top panels (white outline). Full size image

The low number of PLA signals may also stem from the small sizes of nuclei of Drosophila embryos and the limit of resolution using conventional fluorescent microscopy. To improve the resolution of these experiments, we analyzed the association of H3K27me3 using super-resolution structured illumination microscopy (SR-SIM) [30] in mammalian SEM cells that have much larger nuclei than Drosophila. These assays yielded a significantly larger number of PLA signals in the prophase. However, despite much higher resolution of SR-SIM, we also detected a significant decrease in the number of PLA signals at later stages of mitosis (Figure 3c). We conclude that a low number of DNA–protein signals for a particular protein may reflect condensation of mitotic chromatin or some previously unrecognized features of mitotic chromosomes.

Loss of H3K27 trimethylation does not affect the association of PRC1 or PRC2 with DNA

It was proposed that H3K27me3 is essential for association of PcG PRC1 and PRC2 complexes following replication and mitosis [1, 31]. To determine whether the level of H3K27me3 affects the stable association of PRC2 and PRC1 subunits during mitosis, embryos were treated with the inhibitor of the only Drosophila H3K27me3 HMT E(Z), GSK343 [32, 33]. Treatment with GSK343 caused a significant decrease in the amount of nuclear H3K27me3 (Figure 4a). In control experiments, no difference in the amount of H3K4me3 was observed between treated and non-treated embryos, confirming the specificity of GSK343 for E(Z) (Figure 4a). We also detect a marked decrease in the number of PLA signals for DNA-associated H3K27me3 in both interphase and mitotic nuclei following GSK343 treatment (Figure 4b). The reduced H3K27me3 levels did not affect the number of PLA signals for PC and E(Z) (components of PRC1 and PRC2, respectively) in either mitotic or interphase nuclei (Figure 4b and c). Together, our results suggest that H3K27me3 is not essential for either recruitment or association of the PcG PRC1 and PRC2 complexes during mitosis.

Figure 4 Reduced amount of H3K27me3 does not affect the association of E(Z) or PC with DNA. Embryos were untreated or treated with 50 μM of the E(z) inhibitor GSK343. (a) Embryos were immunostained with antibodies to H3K27me3 or H3K4me3 in green and H3S10-p in red. Split channels are indicated. (b) Chromatin Association Assay was performed between H3K27me3 (top), E(z) (middle) or Pc (bottom) and biotin (5-ethynyl-2′-deoxyuridine (EdU)). Proximity ligation assay (PLA), red; H3S10-p, blue; EdU, green. White arrowheads indicate PLA-containing nuclei that are labeled with EdU and H3S10-p. Pair-wise and single split channels are indicated. (c) Quantification of the results in (b). A statistically significant difference (P-value<0.05) is indicated by an asterisk (*). Full size image

RNAs are associated with DNA during mitosis but are not essential for binding of PcG and TrxG proteins

It was proposed that ncRNAs recruit PcG proteins to DNA following mitosis. However, the fate of any RNA during mitosis is unknown [34]. To assess this experimentally, we used the newly developed RNA–DNA Interaction Assay (RDIA) [16]. DNA in embryos was pulse-labeled with EdU for 30 min, chased for 1 h 40 min and then RNA was labeled with 5-bromouridine (BrU) for 15 min. RNA–DNA proximity was then detected by PLA (see below and Materials and Methods). The specificity of this assay was demonstrated previously [16] and is illustrated by the fact that multiple PLA signals between RNA and DNA are detected only in nuclei that are labeled by EdU (Figure 5a). Moreover, treatment of embryos with RNase almost completely eliminates PLA signals, further suggesting that these signals are specific to labeled RNA (Figure 5a). We found that in Drosophila embryos bulk RNAs are associated with DNA during all stages of mitosis (Figure 5b), similar to human lymphoblast cells (GM22737) examined previously [16].

Figure 5 RNA is associated with DNA at all stages of mitosis. (a) Proximity of 5-bromouridine (BrU)-labeled RNA and 5-ethynyl-2′-deoxyuridine (EdU)-labeled DNA was assessed by RNA–DNA Interaction Assay (RDIA). Following labeling with BrU, embryos were either untreated (left) or treated (right) with RNase A. PLA, red; EdU, green; 4,6-diamidino-2-phenylindole (DAPI), blue. (b) Association of RNA with DNA at all mitotic stages was determined by RDIA. Black and white bottom panels show PLA signals only. PLA, red; EdU, green; H3S10-p, blue. Full size image

RNAs are unlikely to be essential for recruitment of chromosomal proteins to DNA as proposed previously (discussed in Steffen and Ringrose [34]), as our CAA results suggest that these proteins are not dissociated from DNA through all mitotic stages. Nevertheless, the presence of RNA at mitotic chromosomes may imply that RNAs have a role in stabilizing chromosomal proteins at DNA during this stage of the cell cycle. To test this possibility, we assessed association of the PcG proteins PC and E(Z) and the TrxG protein TRX following extensive treatment of embryos with RNase. This treatment led to complete elimination of the PLA signals between RNA and DNA (Figure 5a), suggesting that most RNAs were destroyed. However, RNase treatment had no detectable effect on association of PC, E(Z) and TRX at any mitotic stage (Figure 6). Together, these results suggest that while RNAs are associated with DNA during mitosis and may thus have a certain epigenetic role, they are unlikely to be essential for either recruitment or association of chromosomal proteins during this stage of the cell cycle.