In order to generate chromatin fibers that contain HP1, we have expressed Drosophila
HP1a (HP1) in bacteria and purified it to homogeneity over four consecutive columns (74
) (Fig. ). Throughout the present article we will refer to this recombinant HP1a protein as HP1 unless stated otherwise. The purified HP1 dimerizes and interacts specifically with peptides that resemble the H3 N terminus dimethylated at K9 (Fig. ). For the chromatin binding studies, we assembled recombinant Drosophila
) onto DNA fragments containing 11 repeats of the 5S nucleosome positioning sequence using salt dialysis (9
). The level of assembly was tested by micrococcal nuclease digestion (Fig. , right panel). The DNA fragments were asymmetrically labeled with biotin and immobilized using streptavidin-coupled paramagnetic beads. Fully assembled arrays were coupled and used for binding assays after washing with a buffer containing 100 mM salt. The addition of the highly purified HP1 dimer at a 4:1 molar ratio of HP1/nucleosome to the immobilized chromatin fiber resulted in only weak binding (Fig. , lane 2) of HP1. This is consistent with previous observations that report binding of HP1 only at HP1-to-nucleosome ratios of more than 500:1 (74
). In contrast to HP1, the linker histone H1 binds very efficiently to chromatin fibers even at a molar ratio of 2:1 (Fig. , lane 3). From these experiments we concluded that HP1 requires high-affinity docking sites in order to bind with a recognizable strength to chromosomal arrays.
FIG. 2. Generation of H3K9-methylated chromatin. (A) To the left is a Coomassie blue-stained SDS-15% PAA gel of reconstituted unmodified (lanes 1 and 2) and H3K9-methylated (lanes 3 and 4) octamers. Displayed in the lower panel is a MALDI-TOF analysis of H3 peptide (more ...)
One of the best-characterized binding sites for HP1 in vivo is an H3 molecule that is methylated at K9 (40
). The enzyme responsible for creating the site in a living cell is the histone methyltransferase SU(VAR)3-9, which interacts with HP1 and has been suggested to create an autoregulatory loop that helps in maintaining the methylated state of heterochromatin (60
). We wanted to generate a high-affinity binding site for HP1 by reconstituting chromatin using in vitro-methylated recombinant histones. To do this we used recombinant SU(VAR)3-9 (20
) to methylate a mixture of four recombinant expressed core histones that were reconstituted into octamers (44
). Subsequently, the recombinant SU(VAR)3-9 as well as the cofactors S
-adenosylmethionine and S
-adenosylhomocysteine were separated from the histone octamer using a cation exchange resin (70
). Only histone preparations that contained no detectable SU(VAR)3-9 protein (as measured by Western blotting) were used for subsequent experiments. The purified histones were analyzed by mass spectrometry, which showed that more than 85% of all H3 molecules were methylated at K9, with more than 80% carrying two or three methyl groups (Fig. , right panel and MALDI-TOF spectrum). No other lysine in H3, H2A, H2B, or H4 was found to be methylated, and no SU(VAR)3-9 was detectable in the purified histones (data not shown). The highly methylated histone octamers were then used to assemble chromatin fibers as described above. Micrococcal nuclease digestion showed that the methylated chromatin has a similar spacing and sensitivity toward the nuclease (Fig. , right panel, compare lanes 2 to 4 and 5 to 7). However, despite the high content of methylated H3, recombinant HP1 showed only a weak binding that was independent of histone methylation and was even weaker than its affinity to free DNA (Fig. ). From these experiments we concluded that HP1 either binds to methylated histones before assembly of chromatin or it requires additional factors for the binding to its substrate.
In addition to its interaction with the methylated H3 tail, HP1 has also been shown to interact with core residues of H3 and H1 (51
), which are buried within chromatin, suggesting that HP1 may bind to H3 before assembly. In order to test this hypothesis, we had to use a different assembly method, as we reasoned that the HP1 binding would not sustain the high salt concentration during the salt assembly reaction. Therefore, we used a S150 chromatin assembly extract from early Drosophila
) that allowed us to assemble chromatin at lower salt concentrations (less than 100 mM) (Fig. ). However, even though recombinant HP1 was added at the same time as the assembly extract, we could detect only a weak association of HP1 with the assembled chromatin (Fig. , lane 3). As we have previously shown that histones from early Drosophila
embryos contain less than 5% H3K9 methylation (7
), we added either unmodified or in vitro-methylated histones to the extract before the assembly reaction (Fig. , lanes 4 and 5). The addition of exogenous histones led to a slight decrease in sensitivity towards MNase (Fig. , compare lanes 2, 3, and 4 with lanes 6, 7, 8, 10, 11, and 12). However, we could not observe any difference in nucleosomal repeat length when supplementing the S150 with either unmodified or H3K9Me octamers. MS analysis of the chromatin after assembly showed that it contained K9-methylated chromatin only when the in vitro-methylated histones were added (data not shown), indicating that the exogenously added histones are incorporated by the assembly extract. Under these conditions, HP1 bound to chromatin arrays where methylated octamers were added before the assembly reaction but only weakly interacted with chromatin to which unmodified histones were added (Fig. , compare lanes 4 and 5). A quantification of this experiment is shown in Fig. . There is 10 times more HP1 bound to H3K9Me chromatin than to unmodified chromatin. As we assembled chromatin using a heterogeneous extract we could not directly conclude from these experiments whether HP1 bound to the methylated H3 before the assembly or whether the binding was enhanced by the action of accessory factors. To distinguish between these two possibilities, we assembled chromatin from unmodified or methylated histones by salt dialysis and added S150 extract together with recombinant HP1 after the assembly reaction. It turned out that the S150 extract was able to facilitate HP1 binding to methylated chromatin even at concentrations that were not sufficient to assemble nucleosomes in vitro (Fig. ). As chromatin assembly is ATP dependent, we wondered whether the loading process required ATP hydrolysis. The addition of ATP stimulated binding of HP1 to the nucleosomal array irrespective of its methylation state (Fig. , compare lanes 4 and 6). The stimulation of HP1 binding to the methylated chromatin, however, was not dependent on ATP hydrolysis (Fig. , compare lanes 5 and 7). A quantification of this HP1 binding experiment is shown in Fig. . In the presence of an assembly extract HP1 is bound more than 11 times better to H3K9Me chromatin compared to unmodified chromatin. From these results we reckoned that the assembly extract does indeed contain factors that facilitated HP1 binding to the methylated H3 tail and that can assist HP1 binding to assembled chromatin. The presence of ATP in the reaction moderately stimulates the affinity of HP1 to chromatin but does not increase the specific binding to methylated chromatin.
FIG. 3. Reconstitution of methylated chromatin using an S150 Drosophila assembly extract and HP1 binding. (A) Scheme of the assay. (B) Micrococcal digestion pattern of chromatin assembly reactions as described for panel C, without HP1 added. MNase digestions (more ...)
FIG. 4. HP1 is bound to salt-assembled chromatin in the presence of Drosophila assembly extract. (A) Scheme of the assay. (B) Salt-assembled unmodified or H3K9Me chromatin attached to paramagnetic beads was incubated for 1 h at 26°C with HP1, plus and (more ...)
As discussed above, HP1 has three domains, all of which are involved in HP1 function. The CD binds histone H3 methylated at K9, the hinge domain is important for DNA and RNA binding, and the CSD carries a protein-protein interaction domain. In order to get more insight into the nature of HP1 binding to methylated chromatin, we expressed and purified mutant HP1 proteins (Fig. ) and added them to a chromatin assembly reaction as shown in Fig. . As has been reported before (35
), a V26M mutation within the CD of HP1 prevented binding to a peptide containing methylated K9 (Fig. , lane 7). This mutation also resulted in a reduction of HP1 binding to the methylated chromatin (Fig. , lane 6). A point mutation of W to A at position 200 in the CSD of HP1 that has been shown to selectively interfere with the interaction between HP1 and associated proteins (8
) also resulted in a loss of HP1 binding to methylated chromatin (Fig. , lane 7), despite its ability to interact with the methylated peptide (Fig. , lane 10). A quantification of this experiment is shown in Fig. . These results pointed towards a protein-protein interaction rather than an HP1 DNA or HP1 RNA interaction playing a key role in the loading of HP1 to heterochromatin.
FIG. 5. Expression of HP1 mutant proteins and binding of these to H3K9Me chromatin during assembly. (A) Scheme of HP1 mutants generated. (B) Coomassie blue-stained 15% SDS-polyacrylamide gel of the purified HP1 proteins. MM, molecular mass. (C) Peptide pulldown (more ...)
Several chromatin-associated factors have been suggested to play a role in heterochromatin formation and its function (55
). The chromatin-remodeling factor ACF consisting of the ISWI ATPase and the regulatory ACF1 protein is very abundant in early Drosophila
embryos. A mutation in the gene ACF1
has been identified as a suppressor of position effect variegation, which places ACF1 in the same genetic pathway as HP1 (24
). Mammalian ACF1 has been shown to colocalize with HP1β in NIH 3T3 cells and is suggested to have a role in replication of heterochromatin (11
). As we have observed a strong impairment of chromatin binding of HP1 mutants that carry a mutation within the CSD, we first investigated whether recombinant ACF was able to interact directly with HP1 and whether this interaction was mediated by the CSD. We purified an ACF complex using a baculoviral system expressing a FLAG-tagged ACF1 protein together with untagged ISWI (Fig. ), and the immobilized complex was then incubated with various HP1 mutant proteins. We could detect binding of HP1 to the reconstituted ACF complex (Fig. ) as well as to the isolated ACF1 subunit (Fig. ). Consistent with previous findings showing that most heterotypic protein-protein interactions with HP1 are mediated by the CSD (67
), the ACF1-HP1 interaction was also mediated by this domain, as the point mutation within the CSD motif impaired the interaction (Fig. , compare lanes 3 and 6 with lane 9). The fact that the isolated ACF1 subunit is sufficient for the HP1 binding may explain the specific effect of an ACF1
mutation on heterochromatin formation (24
). In order to map the interaction domain within ACF1 that is responsible for the HP1 interaction, we performed GST pulldown experiments using GST-HP1 and in vitro-translated ACF1 fragments (Fig. ). In these experiments we could detect binding of all fragments containing amino acids 202 to 468 (Fig. ).
FIG. 6. HP1 interacts with the ACF complex and ACF1. (A) Coomassie-stained SDS-8% PAA gel of FLAG affinity-purified recombinant ACF1 and ACF complex from Sf9 cells coinfected with FLAG-ACF1 in the presence or absence of untagged ISWI. MM, molecular mass. (B) (more ...)
The region responsible for ACF1 binding to HP1 contains the evolutionarily conserved DDT motif (14
), suggesting that this motif most likely represents an HP1 interaction domain. For Drosophila
ACF1, the region containing the DDT motif has been shown to be required for ISWI interaction (16
Another prominent factor that is known to interact with HP1 in vivo and which plays an important role in heterochromatin formation is the histone methyltransferase SU(VAR)3-9 (1
). We performed protein-protein interaction assays using different SU(VAR)3-9 or HP1 mutants in order to biochemically map the interaction regions for each protein. The N terminus of SU(VAR)3-9 was necessary for its association with HP1 (Fig. ), while the CSD of HP1 was required for SU(VAR)3-9 binding (Fig. ). This further demonstrated the importance of the HP1 CSD for protein-protein interaction (8
). The in vivo target loci of HP1 and SU(VAR)3-9 have been mapped in Drosophila
Kc cells (27
). HP1 and SU(VAR)3-9 colocalized at multiple sites, suggesting a possible targeting of HP1 by SU(VAR)3-9, but the fact that HP1 can also be found at other chromatin sites supports the idea that SU(VAR)3-9 binding is not the sole way of stabilizing HP1 binding to chromatin. We therefore tested whether the known SU(VAR) proteins ACF1 and SU(VAR)3-9 could facilitate HP1 binding to methylated chromatin in vitro. In order to do this, we used salt-assembled chromatin that contained either methylated or nonmethylated histones and added recombinant HP1 together with recombinant SU(VAR)3-9 or ACF (Fig. ). Consistent with the model that HP1 requires multiple binding sites for efficient chromatin binding, we observed an increased association of HP1 with methylated chromatin when ACF complex (Fig. , lane 5, and 8C [quantification]) or SU(VAR)3-9 (Fig. , lane 5, and 8E [quantification]) was present. This was not due to an intrinsically higher affinity of the auxiliary factors to methylated chromatin, as both bound efficiently to the chromatin fiber independently from its modification state (Fig. , compare lanes 4 and 5, and 8D, compare lanes 4 and 5). We also found that neither ATP nor HMTase activity was required for preferential binding of HP1 to methylated chromatin (Fig. , compare lanes 5 and 7).
FIG. 7. HP1 interacts with SU(VAR)3-9. (A) SU(VAR)3-9 constructs used for in vitro translation and GST constructs are shown at the top. The GST pulldown is shown at the bottom. SU(VAR)3-9 was detected by autoradiography. CHROMO SH., chromo shadow. (B) The upper (more ...)
FIG. 8. SU(VAR) 3-9 and ACF facilitate HP1 binding to H3K9Me chromatin. (A) Scheme of the assay. (B) Salt-assembled unmodified or H3K9Me chromatin bound to paramagnetic beads was incubated with HP1 in the presence or absence of ACF and ATP for 1 h at 26°C. (more ...)
This evidence suggests that only the protein-protein interaction served as a second binding site within chromatin and stabilized the interaction of HP1 and the methylated chromatin. This finding is further strengthened by the observation that a mutation in the CSD of HP1 (W200A) that no longer interacts with SU(VAR)3-9 has a reduced binding affinity towards methylated chromatin (Fig. , compare lanes 3 and 9).