| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 281, Issue 24, 16279-16288, June 16, 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom
Received for publication, January 23, 2006 , and in revised form, April 10, 2006.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
In addition to undergoing spontaneous thermal movements, nucleosomes can be repositioned by ATP-dependent chromatin remodeling enzymes. These enzymes consist of a catalytic subunit with a region of homology to the yeast Snf2 protein and a variable number of accessory subunits. Snf2 family proteins fall into distinct subfamilies. For example the ISWI subfamily is named after its founding member, the Drosophila ISWI protein (5). The ISWI protein was subsequently found to be a component of several distinct protein complexes that have the ability to alter chromatin structure in an ATP-dependent reaction (6). Related ISWI complexes have since been identified in a broad spectrum of eukaryotes from yeast to humans (7). These complexes have been found to function in a range of processes ranging from the regulation of transcription and DNA replication to the maintenance of chromatin structure (7).
Characterization of ISWI-driven chromatin remodeling reactions has revealed that one outcome is the repositioning of nucleosomes along DNA (8–10). Although the redistribution of nucleosomes may represent the major means by which these complexes alter chromatin structure, this is not necessarily the case for other subfamilies of Snf2 proteins which have been shown to cause other transitions in chromatin structure (11–15). In addition, there are differences in the way that different ISWI containing complexes redistribute nucleosomes. For example, while the Drosophila ISWI-containing complex NURF and its isolated catalytic subunit redistributes nucleosomes to positions closely related to those observed in thermal nucleosome redistribution reactions (4, 16), the ACF, CHRAC, and Isw2 complexes have been reported to preferentially move nucleosomes to positions closer to the center of short DNA fragments (9, 17, 18).
Yeast Chd1p represents another, less well characterized subfamily of remodelers, which shows genetic interactions with ISWI factors (19, 20). This subfamily is also represented in multicellular eukaryotes and in Drosophila and yeast appears to be mainly monomeric (21, 22).
The differences in the directionality with which nucleosomes are redistributed are likely to significantly influence the functions for which these complexes are used. To understand what these differences, we have systematically analyzed the positions to which nucleosomes are redistributed in different contexts using yeast Isw1a, Isw1b, Isw2, and Chd1 remodeling enzymes. We find that the enzymes that relocate nucleosomes to more central locations preferentially engage with nucleosomes bearing linker DNA. This may explain why these enzymes are unable to move nucleosomes to positions close to DNA ends where linker DNA would be lost. In addition to this inability to move nucleosomes close to DNA ends we find that the inherent nucleosome positioning properties of the DNA play an important role in determining where nucleosomes are moved to.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Enzymes—Isw1a and Isw1b were purified from yeast strains (YTT1168 and YTT1167) in which the Ioc3p or Ioc2p factors, respectively, were expressed as fusions with the TAP2 double affinity tag (23). Chd1 and Isw2 were purified from Chd1-TAP or Isw2-TAP strains, which were purchased from Euroscarf. Remodelers were purified by IgG and calmodulin affinity chromatography from 20 liters of yeast (24). Supplemental Fig. 1 illustrates the purity of these complexes.
Nucleosomes—Histone octamers were assembled from individual Xenopus laevis histones expressed in bacteria (25). Octamers for high resolution repositioning assays contained a S47C mutation in H4 and a C110A change in H3. These octamers were subsequently reacted with the thiol-reactive EDTA derivative (EDTA-2-aminoethyl)2-pyridyl disulfide (26). Octamers were reconstituted into nucleosomes using PCR prepared DNA derived from the MMTV nucleosome A (4) or 601.3 (27) positioning sequences. Reconstitutions were performed at 1 µM concentration and pH 7.5 by stepwise dialysis from 2 M NaCl or KCl to 0.85, 0.65, 0.5, and finally 0 M.
Mononucleosome Repositioning Assays—Nucleosome repositioning was carried out in 10-µl reactions containing 20 mM Tris-Cl, pH 8, 50 mM KCl, 1 mM MgCl2, 1 mM MgATP, 1 pmol of nucleosome (32P-end-labeled at a single 5'-DNA end) and various concentrations of enzyme as described in the legends to Figs. 1, 2, 3, 4, 5. After 20-min incubation at 30 °C reactions were stopped by transfer to ice and addition of competitor DNA (0.1 µg/µl final) and additional salt (200 mM KCl final). One-tenth of the reaction was electrophoresed through 0.2x Tris borate-EDTA, 5% acrylamide gels for 3.5 h at 300 V at 4 °C with running buffer recirculation; the remainder was subjected to site-directed mapping to determine the exact nucleosome position (28). This involved the addition of 1 µl of 40 µM ammonium ferrous sulfate and 5 µl each of 19.2 mM ascorbic acid and 0.2% hydrogen peroxide to each reaction followed by a 1-h incubation on ice. DNA was extracted with phenol:chloroform: isoamyl alcohol (25:24:1), recovered by ethanol precipitation, and resuspended in 5 µl of formamide loading dye (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue). Samples were run on 8% acrylamide sequencing gels containing 6 M urea, fixed with 10% methanol, 10% acetic acid, dried, and exposed to image plates. Markers for sequencing gels were obtained by limited cleavage at G residues of the appropriate DNA template.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
It is known that when a histone octamer is positioned close to the end of a DNA fragment the mobility through a gel is greater than when the octamer is close to the center of the same piece of DNA (3). Therefore these observations suggest that Isw1a and Chd1 relocate nucleosomes closer to the center of the DNA ("end to center" type sliding) and that Isw1b causes nucleosomes to move to locations that are near to, or at, the ends of the DNA ("center to end" type sliding). To study this in more detail, site-directed nucleosome mapping was used to determine the positions nucleosomes were relocated to at high resolution. Briefly, this involves the tethering of cysteaminyl EDTA to a recombinant histone octamer via a thiol group introduced at H4 cysteine 47 (29). The chelation of Fe2+ ions by this reagent provides a means of generating localized hydroxyl radicals that are capable of cleaving DNA but only within a range of less than 1.5 nm. The site at which the reagent is attached on histone H4 comes closest to DNA 2 bp from either side of the nucleosome dyad, meaning that the sites of cleavage can be used to determine nucleosome positions at base pair resolution.
In Fig. 2A the positions to which Isw1a and Isw1b reposition nucleosomes on an DNA fragment designed to position nucleosomes initially at an off center location with 54 bp of DNA on one side and 18 bp on the other. The untreated nucleosomes display a characteristic pattern of strong and weak DNA cleavage separated by 7 bp, indicating that nucleosomes assembled onto this DNA fragment were initially positioned predominantly at +70 relative to the MMTV transcription start site (Fig. 2A, lanes 1, 6, and 10). Following incubation with Isw1b, the cleavage at the +70 location is reduced, and new cleavage sites indicating predominant new locations at +22 and +27 are detected. These position nucleosomes close to the end of the fragment, consistent with the increased mobility following electrophoresis (Fig. 1B). Nucleosomes are also redistributed to these positions following thermal equilibration (Fig. 2A, lanes 10 and 11). However, the positions following redistribution by Isw1b exhibit a subtle bias for the locations closest to the end of the fragment. The movement of nucleosomes to positions closely related to those used during thermal redistribution reactions is consistent with previous studies of NURF (16) and recombinant Drosophila ISWI protein (4). In contrast, Isw1a relocates this nucleosome to a series of more centrally located positions at +39, +42, +47, and +58 (Fig. 2A, lanes 5–8). This cluster of locations is too close together to be resolved by native gel electrophoresis (Fig. 1A). Isw1a moves nucleosomes to a similar distribution of locations when the nucleosome is initially located at the end of the DNA fragment (Fig. 2B, lanes 1–3). The enzymes Chd1 and Isw2 also move nucleosomes to the same distribution of locations (Fig. 2B, lanes 4–9).
The finding that the Isw1 protein when associated with different accessory subunits in the Isw1a and Isw1b complexes moves nucleosomes with different directionalities illustrates that it is not the catalytic subunit alone that is responsible for this. While the locations to which nucleosomes are moved by Isw1b are clearly related to the most favorable locations available on these fragments, the mechanism for selecting the sites used by Isw1a, Chd1, and Isw2 is less clear. Nethertheless, the observation that these enzymes use the same subset of locations suggests that sequence or structural properties of the DNA fragment may contribute to the selection of sites to which nucleosomes are repositioned.
Isw1a Moves Nucleosomes to a Subset of Locations No Closer than 15 bp from a DNA End—To investigate what underlies the selection of these more central positions, nucleosome repositioning by Isw1a was investigated on a series of nucleosomes with successively shorter DNA extensions. Nucleosomes were redistributed predominantly to the +39, +42, and +47 locations on the template with a 54-bp extension (Fig. 2B, lanes 1–3). As the length of the DNA extension is reduced to 44 and 38 bp, there is a progressive decrease in the proportion of nucleosomes accumulating at the +39 and +42 locations, while nucleosomes are still relocated to the +47 position efficiently (Fig. 3, lanes 4–9). It is also notable that the proportion of nucleosomes accumulating at the +58 location increases as the length of the linker DNA is reduced. For the fragments with 34- and 31-bp extension nucleosomes that have relocated 12 bp to the +58 location are the major new species observed following remodeling. It is possible that this 12-bp movement represents a minimal distance that the mechanical action of the Isw1a ATPase motor can move a nucleosome. However, it is also possible that this is the first location that is sufficiently stable to be detected.
|
The nearest to a DNA end that we have observed Isw1a relocate nucleosomes is 15 bp (Fig. 3, lanes 7–9). However, the range over which DNA ends influence positioning varies depending on the context. For example, the +47 location is disfavored despite being 31 bp from a DNA end in the presence of a 34 or 54 bp downstream linker (supplemental Fig. 2, lanes 6–13).
Isw1a Repositions Nucleosomes to Thermodynamically Favorable Locations—In addition to the exclusion of nucleosomes from regions close to the DNA ends, the data in Figs. 2 and 3 show that Isw1a, Isw2, and Chd1 move nucleosomes to discrete locations that are not located at the geometric center of the DNA fragment. We next sought to investigate what underlies the selection of these more central locations. We first characterized the positions to which nucleosomes relocate during thermal incubation in more detail. Nucleosomes were assembled onto series of DNA fragments designed to form nucleosomes with progressively shorter DNA extensions. On nucleosomes with 48 and 44 bp extensions we observed nucleosomes being redistributed to the positions at +22 and +27 as reported previously (4) (Fig. 4, A and B, lanes 2–6). To determine the location of less favorable positions, thermal redistribution was carried out on shorter fragments where these most favorable locations were no longer present. Reduction of the DNA extensions to 38 and 36 bp revealed new locations at +31, +39, +42, and +47 (Fig. 4, A and B, lanes 8–13). It is notable that of these less strongly preferred positions, the +39, +42, and +47 locations were also observed during Isw1a-driven redistribution. This supports the hypothesis that the positioning properties of the DNA contribute to the sites selected by Isw1a. In fact, the positions observed following redistribution by Isw1a appear to result from rearrangement between favorable locations with the exception that locations too close to a DNA ends are excluded.
A prediction of this hypothesis would be that if DNA fragments were designed on which the most highly preferred +22 and +27 locations were sufficiently far from DNA ends, then they would be occupied following redistribution by Isw1a. Fig. 4C shows that this is indeed true. Nucleosomes initially located predominantly at the +39 position with a 44-bp DNA extension are redistributed to the highly favorable positions at +27 and +22 (Fig. 4C, lanes 13–16). Similarly it would be anticipated that the bias against an unfavoured location would be reduced by increasing the linker DNA that flanks it. This is illustrated for the +70 location. Although nucleosomes are deposited during assembly at this location, it is not favored following redistribution on a DNA fragment with an asymmetric extension. However, extension of linker DNA on the downstream side of this location such that it is placed more centrally progressively increases occupancy at this site (supplemental Fig. 2).
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
To illustrate how enzymes such as Isw2 select more central locations, we generated a plot illustrating the location and relative preference for the different locations along MMTV DNA that we have detected (Fig. 6A). Remodeling enzymes might cause redistribution between these sites according to a number of different schemes. For example, an enzyme able to rapidly associate and dissociate from nucleosomes along an idealised DNA with no preferential nucleosome positioning properties would redistribute nucleosomes along the DNA fragment uniformly as shown in Fig. 6B, trace 1. However, certain DNA motor proteins have been observed to undergo a prolonged dissociation and reassociation reaction upon reaching a DNA end (31). Such behavior would result in an idealised distribution similar to that shown in Fig. 6B, trace 2. Superposing this with the nucleosome positional preference shown in Fig. 6A results in a distribution of nucleosomes with a bias toward DNA ends (Fig. 6C). This is very similar to the pattern of redistribution observed by Isw1b (Fig. 2A) and previously observed for NURF and ISWI (4, 16). Note that while the enzyme itself exhibits no preference for either end of the DNA, the structural properties of the DNA fragment result in the preferential accumulation of nucleosomes at the end with more favorable nucleosome positioning sequences.
In contrast, the inability to move nucleosomes closer than about 15 bp from DNA ends gives rise to an idealized distribution illustrated in Fig. 6B, trace 3. However, the 15-bp exclusion limit we found for Isw1a is a lower limit, and DNA ends were observed to reduce occupancy at otherwise favorable locations over distances of at least 34 bp (data not shown). Such behavior is represented by the idealised distribution in Fig. 6B, trace 4. If this trace is scaled to fit a DNA fragment with a 54-bp extension on one side, it can be superposed with the nucleosome positional preferences shown in Fig. 6A to provide a means of modeling the outcome of Isw1a redistribution on this fragment (Fig. 6D). The modeled outcome fits well with that obtained experimentally (Fig. 2B, lanes 1–3). If a similar process is performed on a DNA fragment with a shorter extension, then the idealized trace has to be adjusted to meet the new dimensions of the DNA fragment (supplemental Fig. 3C), but again, the outcome of superposition is in good agreement with the experimental data. In fact this holds true for all fragments we have studied (supplemental Fig. 3). This correlation strengthens support for the hypothesis that redistribution between thermally favorable locations that do not encroach upon DNA ends underlies the repositioning of nucleosomes by Isw1a. However, it is important to point out that this model remains qualitative because we have not quantitatively measured either the free energy of nucleosome positioning (32) or the energetic penalty associated with Isw1a moving nucleosomes close to DNA ends. Nonetheless, it provides a basis for understanding the outcome of remodeling carried out by enzymes such as Isw1a.
While most of the data we have presented has been obtained using Isw1a, we believe that the principles involved are likely to be applicable to other enzymes that act with this apparent directionality. For example, the Isw2 remodeling complex has previously been found to redistribute nucleosomes to positions no closer than 13 bp from DNA ends (30). This is very similar to the limit of 15 bp we have observed for Isw1a (Fig. 3). In the case of Isw2 it has been proposed that preferential binding to nucleosomes containing linker DNA may underlie the exclusion of nucleosomes from locations close to DNA ends (18). The rationale for this is that if a remodeling enzyme requires contact with linker DNA on one side of a nucleosome to move it in that direction, the DNA available will be reduced as the nucleosome approaches the end of the fragment making it difficult to move any further (30) (Fig. 6E). Our observation that the Isw1a and Chd1 activities preferentially bind to nucleosomes bearing linker DNA but the Isw1b complex does not (Fig. 5) provides further support for this model and suggest it may be generally applicable. In fact published observations suggest that it applies for ACF and Mi-2 (16, 33). Our observation that positioning sequences also contribute to the process explains why nucleosomes are not always moved to the geometric center of relatively short mononucleosomal DNA fragments.
Interestingly, Bartholomew and co-workers (30) previously observed that while extending linker DNA to 20 bp most significantly improves binding of Isw2 to nucleosomes, additional linker DNA extending to over 60 bp has more subtle effects. The strong requirement for a short length of linker DNA together with an extended region over which more subtle effects are observed is consistent with the broad range over which we observe that DNA ends can influence nucleosome positioning by Isw1a.
We also report here the yeast Chd1 protein can slide nucleosomes and does so with a directionality to move nucleosomes away from DNA ends in a manner that is very similar to the Isw2 and Isw1a complexes. This similarity in action may be related to the fact that these complexes perform partially redundant functions in vivo (19).
Although our observations are made using short DNA fragments on which DNA ends are encountered at a far higher frequency than would be expected in a physiological setting, we believe that they reflect important mechanistic differences in the way these complexes function. The behavior of remodeling enzymes as they encounter a DNA end may be relevant to nucleosome remodeling in proximity to other barriers which in vivo are most likely to be adjacent nucleosomes or bound transcription factors (Fig. 6E). Supporting this, many of the enzymes that move nucleosomes to more central locations have the ability to space arrays of nucleosomes (19, 21, 23). Although some enzymes that have the ability to move nucleosomes to positions adjacent to DNA ends have also been reported to be able to space nucleosomes, they appear to be less efficient in this assay (23). If the variable range over which we observe DNA ends influence nucleosome positioning also applies in the context of a nucleosome spacing reaction, we anticipate that these enzymes would be capable of establishing nucleosome spacings over the range 15 to at least 34 bp.
In biological contexts, movement of nucleosomes away from barriers may act to organize chromatin, while enzymes that move nucleosomes close to barriers may be more disruptive. An example where this may hold true is provided by the Saccharomyces cerevisiae MET16 promoter (34). Here, Isw1a is involved in establishing nucleosome positioning that is refractory to transcription initiation. When expression is induced, the repositioning of a nucleosome by Isw1b appears to play an important role in regulating the amount of RNA polymerase II that is able to enter productive elongation. Further studies will be required to establish whether remodeling enzymes that remove nucleosomes from barriers generally act to organize chromatin.
|
|
FOOTNOTES |
|---|
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.
1 To whom correspondence should be addressed. Tel.: 44-1382-385796; Fax: 44-1382-388072; E-mail: t.a.owenhughes{at}dundee.ac.uk.
2 The abbreviations used are: TAP, tandem affinity purification; MMTV, murine mammary tumor virus; ATP
S, adenosine 5'-O-(thiotriphosphate).
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Biswas, R. Dutta-Biswas, and D. J. Stillman Chd1 and yFACT Act in Opposition in Regulating Transcription Mol. Cell. Biol., September 15, 2007; 27(18): 6279 - 6287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ferreira, J. Somers, R. Webster, A. Flaus, and T. Owen-Hughes Histone Tails and the H3 {alpha}N Helix Regulate Nucleosome Mobility and Stability Mol. Cell. Biol., June 1, 2007; 27(11): 4037 - 4048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. K. Gangaraju and B. Bartholomew Dependency of ISW1a Chromatin Remodeling on Extranucleosomal DNA Mol. Cell. Biol., April 15, 2007; 27(8): 3217 - 3225. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
