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Previous Article | Next Article 
Molecular and Cellular Biology, November 2002, p. 7553-7561, Vol. 22, No. 21
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.21.7553-7561.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
and Steven Henikoff1,2*
Howard Hughes Medical Institute,2 Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 981091
Received 29 May 2002/ Returned for modification 18 July 2002/ Accepted 30 July 2002
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ABSTRACT |
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INTRODUCTION |
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Canonical nucleosomes consist of 146 bp of DNA wrapped around an octamer of two each of the core histones H2A, H2B, H3, and H4 and are the fundamental packaging unit of chromatin (45). The first step in chromatin assembly is deposition of an (H3.H4)2 tetramer, which by itself organizes 120 bp of DNA (8, 11, 24). Subsequent deposition of two H2A.H2B dimers completes a nucleosome core particle (45). CenH3 replaces canonical H3 in nucleosomes both in vivo and in vitro (1, 2, 5, 18, 25, 29, 33, 34, 35, 47). CenH3 and H3 do not heterodimerize in the same nucleosome (1, 3, 33) and are therefore probably found as separate tetramers with histone H4 before incorporation into chromatin. Maintenance of CenH3 chromatin depends upon targeting of (CenH3.H4)2 tetramers to centromeres, a process that is poorly understood.
CenH3s differ from canonical H3 in that they have diverged histone fold domains (HFDs), noncanonical N-terminal tails, and slightly longer loop I (LI) regions (12, 36). Differences between CenH3s and canonical H3 appear to specify centromeric targeting of CenH3s. Deletion analysis showed that the HFD and not the N-terminal tail of CENP-A targeted centromeres in a human cell line (33, 37). However, no single targeting element could be mapped by swapping amino acids between CENP-A and canonical H3 (33, 37). For Saccharomyces cerevisiae CenH3 (Cse4), targeting was not specifically assayed, although residues throughout the HFD were similarly required for centromere function (9, 16). Since these regions were predicted to contact the DNA, it was proposed that DNA specificity was important for centromeric targeting of CenH3 (9, 16, 33, 37). However, this proposal appears to be contradicted by the lack of DNA sequence conservation at centromeres (6, 12, 36) and by the existence of human neocentromeres, known to be devoid of the 
-satellite DNA found at canonical human centromeres (7, 18, 19, 30, 43).
Recent evolutionary studies of Drosophila and Arabidopsis CenH3s support a role for DNA specificity in localizing CenH3-containing tetramers (12, 21, 39). In Drosophila, both the N-terminal tail and LI of the Cid HFD were found to be evolving adaptively (21). LI is known to form a DNA-binding domain together with LII of H4 (20), suggesting that LI mediates DNA specificity in Cid-containing chromatin, presumably at the level of targeting of (Cid.H4)2 tetramers. Given the lack of mechanistic insight into the process of CenH3 targeting to centromeres, we have investigated the targeting determinants of Cid.
We reasoned that amino acid swaps between H3 and CenH3 did not reveal a targeting element because, although they are expected to have similar structures (20, 29, 47), they perform different functions and are therefore evolving under different selective constraints. H3-containing nucleosomes mediate essential processes that impact chromatin, such as transcription and gene silencing (15), whereas CenH3-containing nucleosomes are required for centromere function and maintenance. In addition, (H3.H4)2 tetramers are assembled onto the bulk of genomic DNA, whereas (CenH3.H4)2 tetramers only need to be incorporated into a small subset of the genomic DNA sequences found at their respective centromeres. These differences in DNA-binding requirements may be driving the rapid, adaptive evolution of CenH3s (12, 21, 39). In contrast, H3 is highly conserved. Furthermore, assembly of canonical H3 and CenH3s use different processes, with most H3 being deposited in a replication-coupled manner and CenH3 deposition occurring in a replication-independent manner throughout the cell cycle (1, 32). The interpretation of swap experiments between canonical H3 and CenH3 is further complicated by the possible dimerization of chimeric proteins with endogenous partners.
Here we examine CenH3 targeting by making swaps within a single lineage, Drosophila. Given the rapid evolution of Cid (12, 21) we predict that Cid from a diverged Drosophila species will not have the ability to target D. melanogaster centromeres. Such a diverged Cid would be a suitable recipient in swap experiments aimed at mapping targeting determinants. By using this approach, we have delineated a single targeting element, and it corresponds to LI. We show that LI is both necessary and sufficient for centromeric targeting and identify single amino acids within LI that are essential for targeting. (Cid.H4)2 tetramers may be targeted to centromeres by a LI mediated discrimination step during their assembly.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture and immunostaining. D. melanogaster Kc167 cells were cultured and transfected with 10 µg of DNA as described previously (13). For inducible constructs, HS was for 1 h at 37°C, 16 to 24 h after transfection. Recovery was for 2 to 6 h as indicated. For the Cid promoter, cells were processed 24 h after transfection. Immunostaining was carried out as described with an anti-Cid antibody and Texas red secondary antibody (13). The anti-Cid polyclonal antibody recognizes an 18-amino-acid peptide present in the N-terminal tail of D. melanogaster, D. simulans, and D. erecta Cid proteins (13). Images were analyzed using Delta Vision software (Applied Precision).
All transfections were done at least three times. Transfection efficiency varied from 50 to 90%. Data from transfections were only used if CidMel-GFP (full length or HFD) showed high transfection levels in a parallel experiment, thus controlling for transfection efficiency of different populations of Kc cells. Constructs that were scored as displaying no targeting had no detectable GFP at the centromere for at least 1,000 cells examined. For constructs scored as targeting centromeres, Cid-GFP expressed from either the Cid or HS promoters could typically be detected at the centromere in 250 to 800 cells out of every 1,000, with intensities up to 100-fold above background for the HS promoter. It should be noted that expression from the Cid promoter is cell cycle specific (13) and at low levels compared to the high expression level from the HS promoter. Although some proteins were targeted when expressed from the HS promoter but not from the Cid promoter, we never observed the converse, i.e., targeting when expressed from the Cid promoter but not from the HS promoter. The reproducible absence of green dots at the centromeres in all cells examined when a fusion protein was expressed from the Cid promoter is likely to reflect reduced targeting efficiency rather than variation in expression levels, because all open reading frames could be produced at high levels from the HS promoter irrespective of targeting, as evidenced by green fluorescence throughout the entire nucleus.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 h (1). Therefore, mitotic chromosomes seen 2 h after HS must have been in G2 phase at the time of the induction, whereas mitotic chromosomes seen 6 h after HS must have been from cells that were induced during S phase. A pulse of CidMel-GFP targeted it to centromeres both in S-phase and G2 cells (Fig. 2C and D). CidBip-GFP was not incorporated into chromatin in G2 phase cells (Fig. 2G) but could be visualized in euchromatin where the protein had been induced during S phase (Fig. 2H). We conclude that CidBip-GFP is competent for assembly into D. melanogaster chromosomes in S phase, but not into centromeres. CidMel must therefore contain a centromeric targeting element that CidBip does not. The HFD of CENP-A has been shown to target it to human centromeres (33, 37). We found that the HFD of CidMel similarly targets it to D. melanogaster centromeres (Fig. 3A). Just like the full-length CidBip, the HFD of CidBip did not localize to D. melanogaster centromeres (Fig. 3A). Furthermore, a chimera of the CidMel N-terminal tail and the CidBip HFD also did not localize to D. melanogaster centromeres, and conversely, a chimera of the CidBip N-terminal tail and CidMel HFD did localize (not shown). Therefore, targeting determinants map to the HFD. The HFD of CidBip has 59% amino acid identity to the CidMel HFD (Table 1) (22) with differences spread throughout the domain. To select candidate targeting determinants, we compared the HFDs of Cids that are localized to D. melanogaster centromeres (CidMel and CidLut) to the CidBip HFD that is not localized (Fig. 3B). We concentrated on amino acids that are unique to CidBip. Three regions of the CidMel HFD (N-terminal region [NR], LI, and C-terminal region [CR]) were chosen for swap experiments with the CidBip HFD. These regions encompass all but four of the amino acids that are unique to CidBip HFD compared to both CidMel and CidLut.
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LI of CidMel is 1 amino acid longer than that of CidBip and differs from it in 12 of the remaining 14 amino acids (Fig. 4A). To identify critical amino acids for centromere targeting of CidMel LI, we performed a mutational analysis. Mutations were introduced into the CidBip HFD with LI of CidMel (HFDBip LIMel), because this construct localizes to centromeres yet is not expected to dimerize with endogenous CidMel. Shorter segments of CidMel LI did not target Cid to D. melanogaster centromeres, suggesting that multiple contacts within LI are required for targeting (Fig. 4A; M1, M2, and M3 mutants). To identify key targeting residues, we introduced single-amino-acid scanning mutations to alanine or glycine throughout D. melanogaster LI (Fig. 4A, M4 to M18). The results are displayed together with a sequence Logos representation of Drosophila LI in Fig. 4B.
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A hypervariable region flanked by more-conserved amino acids may be a hallmark of LI in CenH3 histones in general. For example, CENP-A targeting is unaffected by replacement of the central four hypervariable amino acids (T79, R80, G81, and V82) by only two (KT) (33) Fig. 5A), whereas deletion of three central hypervariable amino acids of Cse4 (K172, D173, and Q174) similarly does not affect viability in a null background (9, 16) (Fig. 5A). In contrast, changing conserved residues causes severe effects: point mutation of W86 of CENP-A revealed a requirement for an aromatic amino acid for targeting, and mutation of the respective W178 in Cse4 to either Y, D, or E was lethal (16, 33) (Fig. 5A).
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All CenH3s have a longer LI region than that of canonical H3 (Fig. 5B). We found that shortening D. melanogaster LI by one amino acid compromised centromeric targeting of HFDBip LIMel (Fig. 4A, M23 and M25). Shortening LI by two or more amino acids abolished all targeting (Fig. 4A, M24 and M26), even when the deletion was within the hypervariable region where mutation of the same amino acids had no effect on targeting (Fig. 4A, compare M26 with M21 and M22). The added length of the LI peptide backbone compared to that of H3 appears to be necessary to ensure targeting, rather than any specific interactions of the acidic side chains of D166, D167, and E168. In contrast, the Cse4 LI was found to tolerate a three-amino-acid deletion (16). In this case, LI is not the primary targeting determinant, but rather Cse4 is targeted by CBF3 binding to its cognate centromeric DNA element, CDEIII (28), and so LI may be evolving under relaxed constraints. We conclude that the crucial targeting function of CidMel LI is mediated by several amino acids located at both ends of the loop and that a minimum length of LI appears to be required for targeting.
Consistent with our results for the full-length Cid proteins, the LI regions of CidSim and CidLut also targeted Cid to D. melanogaster centromeres when introduced into CidBip HFD (Fig. 4A, M28 and M29). The CidBip HFD with CidPse LI localized to D. melanogaster centromeres when it was expressed at levels high enough to appear in euchromatin too (Fig. 4B). Centromeric targeting correlates with the degree of conservation of the amino acids that are critical for centromeric targeting of CidMel LI (Fig. 4B [number of critical amino acids that are conserved is shown in parentheses]). CidPse LI contains four of the eight critical amino acids and weakly targets Cid to D. melanogaster centromeres. CidBip LI contains only one of the eight amino acids and fails to target Cid to D. melanogaster centromeres. It is interesting that the CidBip N-terminal tail has undergone a nine-amino-acid repeat expansion (22). We have shown that this expansion is significantly similar to DNA minor groove-binding motifs and have suggested that these and other minor groove binding repeats in the Cid N-terminal tail facilitate centromere-specific binding (22). CidBip may rely on its N-terminal tail for targeting to D. bipectinata centromeres. In this way, there would be a relaxed constraint on LI in the D. bipectinata lineage. We conclude that the LI region is a common targeting element of CidMel, CidSim, CidLut, and CidPse for D. melanogaster centromeres, whereas CidBip may have followed a different evolutionary trajectory and lost this function.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LI was found to be one of several regions of the HFD to be required for targeting and centromere function in previous swap experiments between CenH3s and canonical H3 (9, 16, 33). These experiments did not reveal a single targeting element, likely because of different selective constraints acting on canonical H3 and CenH3s. Canonical H3 did not target centromeres in swap experiments (33, 37), perhaps because centromeric targeting signals had to compete against regulatory elements in the highly conserved histone H3. Like CENP-A LI (33), LI of CidMel does not target centromeres when introduced into canonical H3, either in the presence or absence of the H3 N-terminal tail (data not shown). CidMel LI is also insufficient to target CenH3 heterologs (Cse4 or Caenorhabditis elegans HCP3) to D. melanogaster centromeres (data not shown). This is consistent with the possibility that CenH3s are derived from multiple independent lineages where they are evolving under different selective constraints. Indeed, CenH3 heterologs cannot functionally substitute for one another: Cse4, CENP-A, and HCP3 do not target centromeres when expressed in Drosophila cells (13), and chimeric CenH3s fail to rescue centromere defects in yeast (16).
Our mutational analysis identifies a framework of amino acids within LI that are essential for targeting. These more conserved amino acids are likely to play a general role in maintaining LI structure. In the crystal structure of the nucleosome, LI of H3 forms a DNA-binding domain together with LII of H4 (10, 20) (Fig. 5B). The arginine side chain of H3 is inserted into the minor groove of the DNA (Fig. 5B, R84 of H3), and hydrogen bonds are formed between DNA phosphate groups and protein main chain and side chains. There are three hydrogen bonds between H3 LI (R84, F85, and Q86) and LII of H4. L170 and R171 of CidMel are conserved with the equivalent residues in LI of H3 (L83 and R84) (Fig. 5B). Mutation of L170 in the same position of Cse4 LI (Fig. 5B; L176 of Cse4) is lethal when combined with another point mutation that is involved in contacting H4 in canonical nucleosomes (9). Mutation of L170 in Cid LI may disrupt contact with H4 and possibly also with the DNA.
In contrast to the essential nature of conserved framework residues, mutation of the central amino acids of LI did not affect targeting. These mutationally flexible amino acids coincide with a hypervariable region of LI found in a sequence alignment of Cids from across the Drosophila lineage (22). These amino acids are changing rapidly, perhaps in response to the changing DNA satellites at centromeres (12, 21). However, the hypervariable amino acids within LI did not appear to be crucial for targeting to D. melanogaster centromeres in our assay, because LI from either CidMel or CidSim targeted D. melanogaster centromeres. Since the adaptive evolution between these species has occurred within LI of the HFD (21), it is likely that D. simulans LI mediates discrimination between D. melanogaster and D. simulans centromeric DNA (21). Discrimination might result from subtle differences in binding affinity during assembly or from a step subsequent to targeting, such as nucleosome stability. It should be noted that even a slight advantage in centromere function that would not be detected experimentally will be expected to drive to fixation within a population (12).
Cid LI could be a discriminating factor at any step in nucleosome assembly (Fig. 6). First, LI may dictate interaction of (Cid.H4)2 tetramers with a specialized chromatin assembly factor that targets centromeres (Fig. 6, step I). Currently there are no assembly factors that are known to act exclusively at centromeres (31, 46). Furthermore, overexpressed Cid (Fig. 2) and CENP-A (42) are incorporated into euchromatin, suggesting that (CenH3.H4)2 tetramers can be assembled outside of centromeres by general chromatin assembly factors. The best candidate for a specific assembly factor is Schizosaccharomyces pombe Mis6p, which is required for targeting of SpCENP-A (38). However, it is unclear if Mis6p directly recruits SpCENP-A, and homologs of Mis6p are not involved in targeting CENP-A or Cse4 (23, 26). The inability of CidMel LI to target CenH3 heterologs to D. melanogaster centromeres suggests that LI does not act as a simple recognition site for a centromere-specific Drosophila assembly factor. In addition, such a protein-protein interaction should evolve to an optimal conformation, leading to the conservation of LI rather than the adaptive evolution observed in multiple Drosophila lineages (21, 22).
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LI may also specify targeting by preferential assembly of (Cid.H4)2 tetramers on certain DNAs (Fig. 6, step III) (21, 33). Different DNA sequences may compete for binding of (Cid.H4)2 tetramers during chromatin assembly. Those DNA sequences that are capable of adopting a favorable conformation for interaction could then be strongly preferred during the assembly step. In an equilibrium assembly process, complexes with the lowest free energy will prevail and be available for binding of H2A.H2B dimers and subsequent chromatin maturation. A recent energetic study suggests that the histone-DNA contacts at the central one or two turns of nucleosomal DNA provide a nucleation point for wrapping DNA around the rest of the octamer (4). If assembly indeed starts at the dyad, a noncanonical LI could present a critical energetic barrier 25 bp in either direction from the central contact point. The location of LI at superhelical positions +2.5 and -2.5 of the nucleosome (20) ensures that a DNA molecule will encounter LI at an interval of 50 bp during assembly. Different DNA sequences can have a substantial free-energy difference for canonical nucleosomes (40). In the case of CenH3 nucleosomes, the free-energy differences for different DNAs may be even larger due to constraints imposed by the increased length of LI. This could result in substantial preference for one DNA over another.
Despite differences at the primary sequence level, centromeric satellites contain patterns consistent with a bias in nucleosome assembly: nucleosomal unit repeat lengths are a common occurrence (12), and there is evidence for positioning of CENP-A nucleosomes on -satellite DNA (41). Although some sequences appear to be preferred at centromeres (30, 44), optimal conformations for assembly of (CenH3.H4)2 tetramers need not be specified by just one primary sequence (17, 44). Indeed, a periodic secondary structural feature may be required for optimal binding to LI. It is not surprising that such DNA features have escaped detection: predicting positioning strength based on DNA sequence is notoriously difficult even for canonical nucleosomes (40). Neocentromeres may represent DNA sequences with free energy of nucleosome formation higher than those of bona fide centromeres but lower than those of other sequences in the genome. Although neocentromeres do not contain -satellite DNA, they do show a sequence bias (18, 19), consistent with DNA preference in targeting of (CENP-A.H4)2 tetramers.
The present study underlines the primary importance of the Cid HFD and LI for centromeric targeting. We have suggested that the N-terminal tails of Cid may also bind DNA, specifically in the minor groove (22). Such an interaction may result in a unique higher-order structure of centromeric core chromatin (22). Minor groove binding of the Cid N-terminal tail is likely to occur at a step subsequent to wrapping of the DNA around the octamer (Fig. 6) and may contribute to the stability of Cid nucleosomes.
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ACKNOWLEDGMENTS |
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We were supported by the Cancer Research Fund of the Damon Runyon Cancer Research Foundation, DRG-1554 (D.V.), and by the Howard Hughes Medical Institute (H.S.H. and S.H.).
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FOOTNOTES |
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Present address: LifeSpan BioSciences, Inc., Seattle, WA 98121.
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