Chromatin Fiber Folding: Requirement for the Histone H4 N-terminal Tail

doi:10.1016/S0022-2836(03)00025-1

J. Mol. Biol. (2003) 327, 85–96

Chromatin Fiber Folding: Requirement for the Histone H4 N-terminal Tail Benedetta Dorigo†, Thomas Schalch†, Kerstin Bystricky† and Timothy J. Richmond* ETH Zu¨rich, Institute for Molecular Biology and Biophysics ETH-Ho¨nggerberg CH-8093 Zu¨rich Switzerland

We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higherorder structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53 S when maximally folded in 1 mM or 100 mM MgCl2. The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14 –19. q 2003 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: chromatin fiber; higher-order structure; nucleosome array; histone tails; sedimentation

Introduction Regulation of eukaryotic genomes in a chromatin context is a fundamental issue in biology. Highly conserved histone proteins function as building blocks to package eukaryotic DNA into repeating nucleosome units that are folded into a higherorder structure. While the exact arrangement of nucleosomes in this chromatin fiber is unknown, experimental data as well as modeling support a helical packing of nucleosomes.1 – 4 In addition to their structural role, histones are integral and dynamic components of the machinery responsible for regulating gene transcription.5 The nucleosome comprises approximately 200 bp of DNA, an octameric core of two copies each of histones H2A, H2B, H3 and H4, and the linker histone H1. Each of the core histone proteins contains a histone-fold domain and histone-fold extensions responsible for histone –histone and histone – DNA interactions, and an N-terminal “tail” domain. The highly basic histone tail sequences account for 28% of the total amino acid † These authors contributed equally to this work. Present address: K. Bystricky, Departement de Biologie Mole´culaire, Universite de Gene`ve, Science II rm 352, 30 quai Erneste Ansermet, CH-1211 Gene`ve, Switzerland. Abbreviation used: wt, wild-type. E-mail address of the corresponding author: [email protected]

content of the core histones.6 The crystal structure of the nucleosome core particle shows that the N-terminal tails are predominantly external to the particle and do not have a well-defined structural motif, suggesting that they are highly flexible.6,7 The tails contain the sites for a series of posttranslational modifications, which have been implicated in chromatin assembly, replication, transcription, and silencing.8 – 12 The molecular interaction partners of the histone tails in different states of modification are being elucidated and have been shown to play key roles in the regulation of all nuclear processes, leading to an “epigenetic code” or “histone code” hypothesis.10,13 – 16 Although the complete sequences of histones are generally highly conserved throughout evolution, implying essential structural and functional roles, the N-terminal tails of histones H3 and H4 are individually dispensable for growth in the yeast Saccharomyces cerevisiae and have redundant functions in chromatin assembly.17 – 19 Point mutations or deletions of the H3 and H4 tails influence gene expression, replication, nuclear division and silencing.20 – 24 H3 and H4 tails have been shown to interact with heterochromatin components such as SIR3, SIR4 and TUP1 in yeast, and HP1 in mammals.25 – 27 The terminal regions of the H2A and H2B histone tails show some sequence variation between species, but otherwise are also highly conserved.17 As with H3 and H4, the H2A and H2B N-terminal tails have been shown to have essential but redundant functions in yeast.28

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

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Histone Tails and Nucleosome Array Compaction

Table 1. Recombinant histone octamers Octamer name wt

gH2A

gH2B

gH3

gH4

Histones included

Amino acids

wtH2A wtH2B wtH3 wtH4 gH2A wtH2B wtH3 wtH4 wtH2A gH2B wtH3 wtH4 wtH2A wtH2B gH3 wtH4 wtH2A wtH2B wtH3 gH4

1–129 1–122 1–135 1–102 13 –118 1–122 1–135 1–102 1–129 22–122 1–135 1–102 1–129 1–122 27–135 1–102 1–129 1–122 1–135 20–102

Octamer name gH2AgH2B

gH3gH4

gH2BgH3gH4

gH2AgH3gH4

gH2AgH2BgH4

Histones included

Amino acids

gH2A gH2B wtH3 wtH4 wtH2A wtH2B gH3 gH4 wtH2A gH2B gH3 gH4 gH2A wtH2B gH3 gH4 gH2A gH2B wtH3 gH4

13–118 22– 122 1– 135 1– 102 1– 129 1– 122 27– 135 20– 102 1– 129 22– 122 27– 135 20– 102 13–118 1– 122 27– 135 20– 102 13–118 22– 122 1– 135 20– 102

Octamer name gH2AgH2BgH3

gH2AgH2BgH3gH4

H4N11

H4N14

Histones included

Amino acids

gH2A gH2B gH3 wtH4 gH2A gH2B gH3 gH4 wtH2A wtH2B wtH3 H4N11 wtH2A wtH2B wtH3 H4N14

13–118 22–122 27–135 1–102 13–118 22–122 27–135 20–102 1–129 1–122 1–135 11 –102 1–129 1–122 1–135 14–102

Histone amino acid sequences correspond to a Xenopus laevis histone gene cluster.70 The globular domains of the histone proteins (gH2A, gH2B, gH3, gH4) were previously defined.71 gH2A has both an N-terminal and a C-terminal deletion.

The mechanism by which post-translational modifications of the histone tails affect regulation of nuclear processes is still unclear. It may proceed via direct or indirect alteration of the histone – DNA and histone– histone interactions within the chromatin fiber or the nucleosome itself.29,30 Alternatively, the modified histone tails may specifically bind nuclear factors such as ATPdependent chromatin remodeling complexes to change the state of chromatin.31 A major barrier to understanding the mechanistic basis of posttranslational modifications is the present lack of knowledge of the actions of the unmodified tail domains. The consequences of histone tail modifications on chromatin fiber stability are likely to be of key importance in the mechanisms of gene activation and repression, and chromosome condensation. However, the roles of the histone tails in higherorder structure assembly have not yet been rigorously defined in any system. Studies conducted so far have shown that the histone tail regions are of critical importance to nucleosome array folding and that the H3-H4 tetramer tails play a more important role than the H2A – H2B dimer tails.32 Here, we evaluate for the first time the detailed contribution of individual histone tails to the formation of chromatin higher-order structure using an improved nucleosome array assembly system.

proteins were obtained by standard procedures.33 Hybrid octamers were assembled onto 12 tandem, 177 bp repeats of the 601 sequence (12_177_601) by stepwise salt dialysis. The 601 sequence used is the strongest nucleosome positioning sequence characterized so far34 and yields a single nucleosome position as determined by base-pair resolution mapping with site-directed hydroxyl radicals35 (data not shown). The positioning was influenced neither by the array formation nor by the various versions of the histone octamer used (Table 1). The DNA repeat length was chosen from a list of preferentially quantized linker DNA lengths determined from a statistical analysis of measurements of nucleosome repeat lengths.36 Histone octamer to DNA template stoichiometry was evaluated by Sca I endonuclease digestion in experiments analogous to those designed for a 5 S rDNA repeat array cleaved with Eco RI.37,38 Only purified arrays that after digestion yielded low amounts of free DNA (0 –3%) and no particles larger than a mononucleosome were used for sedimentation experiments (Figure 1(a)). Limited micrococcal nuclease digestion of the chromatin constructs shows a ladder pattern of DNA fragments, characteristic of isolated bulk chromatin, ranging from the intact 12mer to the mononucleosome39 (Figure 1(b)). By these criteria, the nucleosome arrays used in this study are seen to have a stoichiometry of 12:1 for histone octamer to 12_177_601 DNA.

Results Assembly of saturated nucleosome arrays using full length and truncated recombinant core histone proteins

Mg21 concentrations below 2 mM and above 95 mM yield compact and homogeneous nucleosome arrays

Histone octamers which contain full length and truncated recombinant Xenopus laevis histone

The degree of compaction of a nucleosome array can be controlled by adjusting divalent cation

Histone Tails and Nucleosome Array Compaction

87

Figure 1. Purification and uniformity of in vitro assembled nucleosome arrays. (a) Native PAGE of 12_177_601 nucleosome arrays after assembly (crude assembly) and purification by preparative native PAGE (purified array), and following Sca I digestion (Sca I digest). After purification, the array is homogeneous. Digestion with the Sca I endonuclease under conditions in which the array is not compacted cleaves the Sca I site in the linker DNA quantitatively, leaving no uncut fragments, free DNA, or significantly miss-positioned mononucleosomes. Cycles of compaction and unfolding do not affect these results (T.S. & T.J.R., unpublished results). (b) Limited cleavage of 12_177_601 nucleosome arrays with micrococcal nuclease. Arrays lacking individual histone tails yield the same periodic DNA fragment ladder on nuclease cleavage as the wt array. Twelve regularly spaced bands including those from the monomer and the intact array are visible for all constructs. Lanes containing DNA from arrays, digested for 60 – 90 seconds, are labeled corresponding to Table 1. Lanes D1 and D2 are digests of free DNA for 20 and 60 seconds, respectively. The DNA marker lanes contain lengths of from 100 bp to 900 bp by 100 bp steps and 1031, 1200, 1500, 2000 and 3000 bp (MBI Fermentas).

concentration and can be measured by analytical ultracentrifugal sedimentation. Sedimentation velocity experiments were used to determine the sedimentation coefficients of a series of 12_177_601 based arrays (Table 1) under different MgCl2 concentrations. Distributions of sedimentation coefficients (s20,w) were obtained from analysis of the analytical ultracentrifugation data using the van Holde & Weischet method.40 The 5 S rDNA nucleosome arrays were previously shown to precipitate when the MgCl2 concentration was raised above 2 –5 mM, but were again soluble at concentrations over 80 mM MgCl2.41 Since compaction is observed when going from 0 mM to 1 mM MgCl2 and when going from 140 mM to 95 mM MgCl2, the folding dynamics of nucleosome arrays at MgCl2 concentrations both below 1 mM and above 95 mM were investigated in order to bracket the intracellular Mg2þ concentration estimated to be approximately 40 mM.42 The sedimentation coefficient distributions of nucleosome arrays shift towards higher values as the MgCl2 concentration approaches the solubility boundary at either lower or higher divalent cation concentrations (Figure 2). Importantly, the sedimentation coefficient distributions of wild-type (wt) and tail-less (gH2AgH2BgH3gH4) nucleosome arrays are highly homogeneous in either regime of salt concentration. Typically, 80% to 90% of the 12_177_601 array boundary is used to obtain mean sedimentation coefficients, even under conditions that yield a sedimentation coefficient of 53 S indicating a highly compact structure. Above 95 mM MgCl2, nucleosome arrays exhibit a decreasing sedimentation coefficient with increas-

ing MgCl2 concentration (Figure 2(b)). Maximal compaction is indicated by an s20,w value near 53 S occurring at 100 mM MgCl2. Sedimentation coefficients in this high salt range are in most cases comparable to those found at sub-millimolar MgCl2 concentrations. With our system, recombinant tail-less arrays (all globular histones) display compaction behavior without aggregation between 0 mM and 140 mM MgCl2. Over the range of 10– 100 mM MgCl2, they reach s20,w values approaching wt (47 – 51 S; data not shown). At sub-millimolar levels of MgCl2, these arrays exhibit a folding transition, but they do not reach the same extent of compaction as wt (Figure 2(c) and Figure 3(a)). Similarly in the high MgCl2 regime, the tail-less array displays s20,w values corresponding to a less compact form (Figure 2(d) and Figure 3(b)). A larger difference between limiting s20,w values can be obtained in low as compared to high salt, since the histone proteins begin to dissociate from the DNA above 140 mM MgCl2. Since the histone tails account for 28% of the total amino acid content of the core histones and 7.7% of the total mass of the wt array, mean s20,w values for arrays containing globular histones were adjusted for the mass difference relative to wt. Mass-adjusted s20,w values for the tail-less arrays are 2 –4 S higher, depending on the salt concentration, than the unadjusted values (Figure 3). For the sedimentation velocity analyses, it is important to adjust for the mass difference between the different constructs. The resulting mass-adjusted sedimentation coefficients as reported here give a reliable measure of the changes in compaction and frictional coefficient between samples.

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Histone Tails and Nucleosome Array Compaction

Figure 2. Compaction of nucleosome arrays at low and high Mg2þ concentration. Sedimentation coefficient distributions of saturated nucleosome arrays were determined by the method of van Holde & Weischet. The percentage of the sample boundary (% boundary) measured by analytical ultracentrifugation is plotted versus the sedimentation coefficient (s20,w). (a) Wild-type at low salt: zero (B), 0.1 mM (S), 0.5 mM (O), 0.9 mM (V) and 1.0 mM (A) MgCl2. (b) Wild-type (wt) at high salt: zero (B), 140 (V), 125 (O) and 100 ( ) mM MgCl2. (c) Tail-less at low salt: 0.2 mM (B), 0.4 mM (V), 0.5 mM (O), 0.6 mM (A), 0.8 mM (S) and 1.0 mM (K) MgCl2. (d) Tail-less (gH2AgH2BgH3gH4) at high salt: zero (B), 125 (V), 110 mM (O) and 100 ( ) mM MgCl2.

†

†

To prove that the sedimentation coefficients observed were not caused by aggregation, the molecular mass of the wt complex was measured by sedimentation equilibrium experiments under the conditions that induce high sedimentation coefficients. Samples were prepared in 100 mM MgCl2, 0.6 mM MgCl2 or 0.1 mM EDTA (the latter was measured at two different array concentrations). A first set of data was collected at 2400 rpm after equilibrium had been reached and then a second measurement was made at 2700 rpm. This protocol was repeated. Fitting each dataset individually to a single model using Ultrascan V 5.043 yields fits with molecular masses between 2.5 MDa and 2.8 MDa. Fitting all data sets together yielded a global model with molecular mass of 2.65(^ 0.003) MDa (theoretical value ¼ 2.64 MDa). These results show that the faster sedimenting species are not products of aggregation but of compaction.

Sedimentation velocity shows the H4 N terminus has a unique structural role in chromatin fiber folding First, constructs containing a single globular histone were analyzed in the low and high Mg2þ concentration ranges. In the absence of Mg2þ, arrays yield s20,w values of 26 –34 S. With increasing divalent cation concentration, the values increase essentially linearly, reaching a maximum of 53 S for wt, 54 S for gH3 and 44 S for gH4 at 1 mM MgCl2 (Figure 4(a)). Precipitation begins above 1.2 mM MgCl2 for wt and gH3 and above 2 mM MgCl2 for gH4. At 100 mM MgCl2 sedimentation coefficients remain between 53 S and 56 S for wt and gH3, while for gH4 they are shifted to 49 S (Figure 4(b)). gH2A and gH2B behave similarly to the gH3, attaining similar or slightly higher s20,w values than wt (Figure 4(a)). In summary, for octamers containing a single globular histone,

Histone Tails and Nucleosome Array Compaction

89

Figure 3. Adjustment of mean s20,w value for the mass difference from wt demonstrated for the tail-less array. The measured mean s20,w values and standard deviations of the sedimentation coefficient distributions for (a) the low and (b) high MgCl2 range as measured in Figure 2 are shown for wt (V) and tail-less arrays (A). The s20,w values of the tail-less arrays adjusted for their mass difference from the wt (B) can be compared directly to the wt values.

only the array construct gH4 is impaired in attaining a compact structure comparable to wt. At high ionic strength, array compaction is less sensitive to the lack of histone tails. Next, constructs containing multiple globular histones were studied. The folding of recombinant arrays specifically lacking both dimer tails (gH2AgH2B) or both tetramer tails (gH3gH4) was examined. While gH2A and gH2B have sedimentation coefficients that are even higher than wt, gH2AgH2B shows the same folding properties as wt and reaches s20,w values of 53 S at 1 mM MgCl2 before the onset of precipitation (Figure 4(c)). In the high MgCl2 range there are almost no differences in sedimentation coefficients between gH2A, gH2B and gH2AgH2B (Figure 4(d)). Thus, the combined dimer tails are dispensable for full array compaction. Since gH4 and wt display different folding properties, but gH3 compacts readily, it was important to see the effect of the combined removal of the tetramer tails. gH3gH4 reveals almost identical compaction to gH4 in both low and high MgCl2 (Figure 4(e) and (f)). This result indicates that the H4 N terminus is critical for full fiber compaction whereas the H3 N terminus is not. In summary, all constructs analyzed show an overall increase in s20,w as the MgCl2 concentration increases under low salt or decreases under high salt conditions. With the exception of gH4, arrays lacking a single histone tail (gH3, gH2A, gH2B) or the combined dimer tails (gH2AgH2B) reach s20,w values between 53 and 56 S in both MgCl2 concentration ranges used, indicating a similar degree of compaction. Arrays lacking the H4 tail (gH4, gH3gH4) stand out as they attain sedimentation coefficients of only 44 S at 1 mM MgCl2 and 49 S at 100 mM MgCl2. gH4 corresponds to a deletion of the first 19 amino acid residues of histone H4. In order to examine whether or not a smaller region of the H4 N terminus could account for the observed behavior, arrays were prepared containing deletions of the first N-terminal 10 (H4N11) and 13 (H4N14) amino acid residues of H4 (Figure

5(a)). In both cases, folding of the arrays in the low and high Mg2þ concentration ranges leads to the same degree of compaction at 1 mM and 100 mM MgCl2 as wt (Figure 5(b) and (c)). These results show that deletion of two-thirds of the H4 N-terminal tail does not influence array compaction while a region within amino acid residues 14– 19 plays a critical role in obtaining a compact chromatin fiber structure. To test if one histone tail alone can confer wt properties to the system, arrays that contain only one individual histone tail domain were analyzed. While gH2AgH2B exhibits wt folding properties as shown, the additional removal of the H3 tail leads to a decrease in sedimentation coefficient of 4 S at 1 mM and 100 mM MgCl2 (Figure 6). Therefore, an array which contains only the H4 tail (gH2AgH2BgH3) is still substantially more compacted than gH4 in the low salt range (by 4 –5 S at 1 mM MgCl2), while in the high salt range s20,w values are similar to those seen for gH4 and tailless arrays. If the H4 tail is involved in ionic interactions, its contribution at low salt would be greater than at high salt, which could explain the observed behavior. However, this result also shows that the H4 tail alone is not sufficient to exhibit wt folding properties. Although the H2A, H2B and H3 tails alone are dispensable when the other tails are present, they apparently provide non-specific ionic interactions that stabilize the chromatin fiber. Most probably, they play the role of polyamines neutralizing phosphate groups. Indeed, arrays that contain any one of H2A, H2B or H3 intact (gH2BgH3gH4, gH2AgH3gH4 and gH2AgH2BgH4) reach higher s20,w values than the tail-less array (gH2AgH2BgH3gH4) under both low and high salt conditions (Figure 6). Oligomerization assay indicates a special role for the H4 N terminus Chromatin isolated from cells as well as arrays assembled with purified histones have been shown to oligomerize and then to precipitate with

90

Histone Tails and Nucleosome Array Compaction

Figure 4. Influence of individual histone tails on nucleosome array folding. (a) and (b) Effect of single histone tail deletions. The mean s20,w value of the sedimentation coefficient distributions as measured in Figure 2 are shown for wt (V), gH2A (A), gH2B (K), gH3 (£) and gH4 (W) in the low (a) and high (b) MgCl2 range. (c) and (d) Deletion of histone dimer tails. Analogously, the mean s20,w are shown for wt (V), gH2A (A), gH2B (K) and gH2AgH2B (W) at low (c) and high (d) MgCl2 concentrations. (e) and (f) Deletion of histone tetramer tails. The mean s20,w are shown for wt (V), gH3 (A) and gH4 (K) and gH3gH4 (W) at low (e) and high (f) MgCl2 concentrations. Values for arrays containing globular histones have been adjusted to wt mass.

increasing salt concentration.41,44 Divalent rather than monovalent cations are required to achieve oligomerization of 12mer nucleosome arrays.45 Monitoring the Mg2þ dependence of precipitation provides a guide to the relative propensity of different arrays to oligomerize. Using our 12_177_601 DNA and various versions of recombinant histone octamer, nucleosome arrays were incubated in solutions containing increasing amounts of MgCl2, and the precipitate formed after 15 minutes was removed from suspension by centrifugation. The absorbance at 260 nm of the remaining soluble fraction was measured as an indicator of oligomerization. Precipitation is

induced by Mg2þ, as no reduction of absorbance occurs in its absence. The precipitate formed can be quantitatively resolubilized in solutions devoid of divalent cations, as described for the 5 S rDNA array.45 The oligomerization assay was applied to 601 array constructs that lack an individual histone tail. Each of these constructs was characterized by the Mg2þ concentration at which 50% of the initial material remains soluble (M50). M50 was determined to be 1.3 mM MgCl2 for the wt array. The M50 values of gH3, gH2A and gH2B at 1.5 mM MgCl2 are similar to wt, whereas M50 is not reached until 3.4 mM for gH4. The substantially

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Figure 5. Folding of nucleosome arrays containing truncated H4 N-terminal tails. (a) Amino acid sequence of the N terminus of H4 and of the truncated constructs H4N11 and H4N14. (b) and (c) The mean s20,w of the sedimentation coefficient distributions as measured in Figure 2 are shown for wt (V), H4N11 (A), H4N14 (K) and gH4 (W) in the low (b) and high (c) MgCl2 range. Arrays containing globular histones have been adjusted to wt mass.

higher divalent cation concentration required to precipitate gH4 indicates that this construct is more resistant to oligomerization than wt and arrays lacking either the H3, H2A or H2B tails. In contrast to gH4, arrays assembled with H4N14 octamers, which lack only the N-terminal 13 amino acid residues of histone H4, oligomerize with an M50 value of 1.8 mM. Therefore, H4 amino acid residues 14 – 19 contain structural features critical for oligomerization and precipitation.

Discussion The mechanism of nucleosome array folding into compact higher-order structures, the structure of

the chromatin fiber itself, and the role of interconversion between these structures during gene regulation is still a matter of debate. In this study of chromatin fiber formation, we have utilized a DNA sequence which results in a strong, single histone octamer position per repeat, and recombinant histone proteins which are post-translationally unmodified.34,46 Previously established in vitro systems used for this purpose have incorporated a DNA template shown to result in multiple nucleosome positions, and chicken erythrocyte histones which have tail sequences that contain heterogeneous post-translational modifications.35,37,47,48 Our well-defined arrays share folding properties similar with those produced previously, but importantly, the nucleosome arrays used here are more

Figure 6. Folding of nucleosome arrays containing a single histone tail. The mean s20,w value of the sedimentation coefficient distributions as measured in Figure 2 are shown for gH2BgH3gH4 (A), gH2AgH3gH4 (K), gH2AgH2BgH4 (£) and gH2AgH2BgH3 (V) in the low (a) and high (b) MgCl2 range. gH2BgH3gH4 contains both the N and C-terminal tails of H2A. Values for arrays containing globular histones have been adjusted to wt mass.

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homogeneous, and as a consequence highly compacted material with well defined s20,w values of 53 S and higher are obtained reproducibly. In addition, assembly of nucleosome arrays using recombinant core histone proteins facilitates the investigation of the contributions made by specific histone regions to chromatin fiber folding. In previous work, nucleosome arrays constructed from a 208 bp DNA repeat originally derived from the Lytechinus variegatus 5 S rRNA gene, and histone octamer purified from chicken erythrocytes were used.37,41,49 Hansen and colleagues could show three distinct forms of this array with corresponding sedimentation coefficients of 29 S, 42 S and 55 S50,51 (the value of 55 S for this 12_208_5S array drops to 51 S after adjusting to the mass of 12_177_601). The 29 S and 55 S values are the limiting values obtained under conditions that do not result in aggregation, and possibly correspond to an open “beads on a string” form and a highly folded, higher-order structure representing the compact chromatin fiber as observed by electron microscopy of isolated chromatin.52 We do not see a folding intermediate during compaction. The data presented here support a process of gradual compaction or a fast equilibrium between the open and closed state influenced by the divalent cation concentration. We attribute this result mainly to the uniformity of the arrays used here. As determined from sedimentation velocity analyses of 12_177_601 arrays using analytical ultracentrifugation, none of the N-terminal tail deletions (and C-terminal in the case of H2A) completely inhibited compaction of nucleosome arrays compared to those containing full-length histone proteins. Even tail-less nucleosome arrays are able to compact to near wild-type level at intermediate and high MgCl2 concentration. This observation is consistent with the suggestion that compaction is facilitated by the neutralization of the DNA electrostatic charge and augmented by the histone tails at low MgCl2 concentration.53 Intranucleosomal divalent-ion-coordination interactions are likely occurring under both salt conditions,54 – 56 driving the compaction of the arrays. Hansen and colleagues observed that the histone tails are important for chromatin fiber compaction, since proteolytic removal of the tail domains abolished array folding and self-association.45 “Hybrid” typsinized nucleosome arrays containing only H3/H4 tail domains sedimented between 31 S and 42 S in 4 mM MgCl2, while the hybrid arrays containing only H2A/H2B tail domains sedimented between 29 S and 41 S in 8 mM MgCl2 prior to oligomerization. These results suggested that the H3/H4 tail domains are the dominant mediators of the 29 S to 40 S folding transition postulated by the authors.51 The data presented here allow us to extend these observations to significantly higher resolution. As the tetramer tails were shown to have more influence on chromatin fiber condensation than

Histone Tails and Nucleosome Array Compaction

the dimer tails, it was of interest to evaluate the individual roles of the H3 and H4 tail domains. The folding of the wt array and the array lacking the H3 tail shows nearly identical dependence on Mg2þ concentration under high salt conditions. In the low MgCl2 range, gH3 sediments even more rapidly than wt nucleosome arrays. Therefore, the histone H3 N-terminal tail alone is dispensable for, if not inhibitory to, full fiber compaction. Numerous post-translational modifications of this domain linked to interactions with a multitude of heteromeric protein complexes have been described recently.27,57 – 59 In particular, the H3 tail has greater accessibility to an acetyltransferase in oligonucleosomes as compared with core particles.60 Taken together, these results suggest that the H3 tail is a repository for regulatory information rather than a structural element directly involved in the organization of the chromatin fiber. In support of long-standing models attributing a structural role to the histone N termini, our results for gH4 demonstrate for the first time that the H4 N terminus is critically required for full fiber folding. Compaction to the level of the wt array requires a region C-terminal of amino acid residue 13, as full compaction is possible with H4N14. Similar to the tail-less array, the loss of compaction is much greater in the low MgCl2 range, which indicates that the H4 tail is interacting with its counterpart through electrostatic interactions, possibly involving magnesium ions.7,54 Examination of the crystal structure of the nucleosome core particle containing X. laevis histones has shown that basic residues of the histone H4 N-terminal tail (amino acid residues 16 –25) contact an acidic patch of the H2A/H2B dimer from an adjacent nucleosome.6,7 Our results support the idea that this region of H4 may be integral to the formation of nucleosome-nucleosome interactions in chromatin higher-order structure. Deletion of the amino acid stretch 14– 19 could destabilize this putative interaction, and thus impair compaction. A predominantly electrostatic interaction would also account for the fact that the effect for gH4 is much stronger at low salt. At high salt, the reduced metal binding affinity is compensated for by high Mg2þ concentrations. Furthermore, the fact that deletion of amino acid residues 14 – 19 of the H4 tail has an effect on chromatin condensation suggests that small numbers of specific post-translational modifications may also have similar effects and could be critical for epigenetic regulation.30,61 Correlative studies imply that reduction of the positive charge due to the acetylation of lysine amino acid residues in this region may contribute to a less stable, open structure in vivo. For example, acetylation of K5, K8 as well as K16 of histone H4 in yeast is synonymous with transcriptionally active or open chromatin domains.62 Likewise, newly synthesized histone H4 assembled into chromatin in vivo shows a distinct acetylation pattern in this region.63 In the sense of charge reduction, deletion of the positively charged

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amino acid residues in our study may mimic the effect of acetylation. The overly compacted structures observed in the absence of dimer tails seen with gH2A and gH2B may reflect one or more subtle properties of the fiber formed. First, these experiments do not distinguish between contributions to s20,w arising from alterations of the structure, such as minor changes in radius or nucleosome tilt, or changes in the dynamics of the particle due to the loss of a stabilizing or destabilizing interaction. The deletion of the H2A C-terminal tail might be expected to cause such alterations, as according to the solenoid model of the chromatin fiber and the nucleosome core particle structure,7 this region could provide an interface between the DNA of two nucleosomes (T.J.R., unpublished results). In this case, the divalent cations might overcompensate for the loss of stabilizing interactions. Secondly, removal of a N-terminal tail that protrudes from the compact structure might be expected to increase the sedimentation velocity as a consequence of a decreased frictional coefficient. The increase of s20,w for gH2B may be due to this influence. The experiments conducted with more than one tail missing show that tail effects are not simply additive. This can be seen best in the case of the gH2AgH2B, but is also true for gH3gH4. It is therefore likely that one tail compensates for the loss of another in single tail deletion experiments. The presence of only one tail in the cases of H3 and H2B results in a remarkably high degree of compaction. The most obvious feature common to all histone tails that might be responsible for this is that they are all highly basic and could possibly substitute for each other non-specifically as polycations. The contribution of the core histone tails to the folding and dynamics of the chromatin fiber is possibly multifold. First, they may play a structural role in the compaction and ultimate form of the higher-order structure. Second, they are the targets of signaling cascades that are closest to the DNA, mediated by their post-translational modifications. Third, they may provide additional shielding of the DNA beyond the basic structure of the chromatin fiber. Fourth, the tails may link separate fibers together and form contacts with additional structures in the nucleus. The data presented here indicate that the H4 tail is the most important for chromatin fiber condensation in the absence of further factors, whereas the tails of H2A, H2B and H3 most likely contribute to the other processes mentioned. Further studies using defined arrays and recombinant histone proteins will help to clarify the mechanisms involving the histone tails.

Experimental Procedures Materials The DNA template contains 12 tandem 177 bp repeats of the high affinity 601 sequence.34 DNA arrays were

cloned and purified as described.33,64 After excision with Eco RV, the arrays were separated from the remaining plasmid fragments by polyethylene glycol (PEG) precipitation. Full length and truncated histones, as listed in Table 1, are recombinant X. laevis histones, cloned by PCR, expressed in bacteria and purified as described.65 Equimolar amounts of the four histones were co-folded to form an octamer. Intact octamers were purified from aggregates and free H2A-H2B dimers over a Pharmacia Superdexe 200 gel filtration column.33 Nucleosome array assembly The 12_177_601 DNA template was assembled with different versions of recombinant histone octamers. Octamers and DNA were combined in equimolar amounts (5 mM) in TEN2 buffer solution (10 mM Tris – HCl (pH 7.5), 0.1 mM EDTA, 2 M NaCl). Chromatin arrays were assembled by step-wise salt dialysis in TEN1.4, TEN1.2, TEN0.8 and TEN0.6 (the number represents the molar concentration of NaCl) over 12 hours at 48 C followed by a final step in 10 mM Tris – HCl (pH 7.5) for six hours. In order to prevent overloading of the DNA with octamer, 147 base-pair fragments of the weaker binding MMTV A DNA were added to the reconstitution mix as an “octamer buffer”. The small DNA fragments and mononucleosomes were separated from the nucleosome arrays by precipitation of the array with approximately 4 mM MgCl2 (exact amount empirically determined for each type of octamer). Preparative gel electrophoresis using a 1% (w/v) agarose, 2% (w/v) acrylamide composite gel system was used as an alternate purification for arrays that did not precipitate. The stoichiometry of soluble material of 12:1 for histone octamer to 12mer DNA was confirmed by digesting the assembled arrays with Sca I in 50 mM NaCl, 10 mM Tris – HCl (pH 7.5), 0.5 mM MgCl2 for 12 hours at 22 8C. Each 601 repeat is separated by a Sca I restriction site. The ratio of free DNA to nucleosomal DNA was analyzed on a 1% agarose, 2% acrylamide gel. A single octamer per DNA repeat yields a pattern absent in bands corresponding to free DNA and to particles larger than mononucleosomes. Micrococcal nuclease cleavage Chromatin arrays were digested using ten units of micrococcal nuclease (Sigma) per 125 pmol of reconstituted DNA for one minute on ice. The reactions were stopped by addition of an equal volume of 0.4 M NaCl, 0.2% (w/v) SDS, 20 mM EDTA followed by chloroform extraction and ethanol precipitation. The resulting DNA fragments were electrophoresed on a gel composed of 1% acrylamide/bisacrylamide (40:1) and 1.2% agarose at 4 8C and 10 V/cm using 0.25 £ TAE (10 mM Tris – acetate (pH 7.5), 0.25 mM EDTA). Precipitation assay Assembled chromatin arrays were mixed with equal amounts of buffer solutions (10 mM Tris – HCl (pH 7.5)) containing twice the final sample concentration of MgCl2, incubated for 15 minutes and centrifuged at 20,000g for 15 minutes at 22 8C. The absorbance at 260 nm of the supernatant was measured. MgCl2 concentration was controlled by measurement of the conductivity of the stock MgCl2 solution. Control samples lacking divalent cations also included 0.1 mM EDTA.

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Analytical ultracentrifugation Sedimentation velocity experiments were performed using a Beckman XL-I analytical ultracentrifuge equipped with scanner optics. The initial sample absorbance at 259 nm was between 0.6 and 0.8. Samples (400 ml for sedimentation velocity) were equilibrated in their respective buffer solutions in the analytical ultracentrifuge chamber under vacuum for 30 minutes at 20 8C prior to sedimentation at 12,000 rpm in 12 mm double sector cells and an eight-hole rotor. Scans were collected at 259 nm in continuous scan mode using a 0.003 cm radial step size. Boundaries were analyzed by the method of van Holde & Weischet using the program UltraScan v5.0.40,66 Sedimentation coefficients were corrected to s20,w using a partial specific volume of 0.622 ml/g for saturated chromatin arrays and density and viscosity specific to the buffer solution. The partial specific volume is based on 0.499 ml/g for Na-DNA at 0 mM NaCl67 and 0.75 ml/g for the histone octamer.68 The mass of each DNA strand was calculated by the following formula: Mol. Wt ¼ (251 £ nA) þ (245 £ nT) þ (267 £ nG) þ (230 £ nC) þ (61 £ (n 2 1)) þ (54 £ n) þ (23 £ (n 2 1)) (the last three terms account for the phosphate, three bound water molecules and one bound sodium ion per base-pair). The mass of the wt histone octamer is 107,906 Da. Processed data were plotted as boundary fraction versus s20,w to yield the integral distribution of sedimentation coefficients. For calculation of mean sedimentation coefficients, only the dominant homogeneous fraction of the boundary was taken into account. Care was taken to avoid precipitation of the sample during an experiment, since in this case the absorbance will decrease and the analysis of the sedimentation coefficient distribution becomes less reliable.51 All sample points include two to six independent measurements. Sedimentation equilibrium data were collected at 2400 and 2700 rpm at 20 8C in step scan mode with 0.001 cm radial increments at 259 nm and 280 nm (column height 3 mm, 25 replicates) using the same experimental set-up and sample preparation as described above. Equilibrium was reached after 72 hours. Two separate assembly reactions were measured, and the global analysis component of Ultrascan 5.0 was used to determine molecular masses assuming one non-interacting species. Based on the Svedberg equation: s¼M

ð1 2 nrÞ Nf

the measured mean s20,w values of the sedimentation coefficient distributions were adjusted to the mass of the wt array by the following formula: s20,w (adjusted) ¼ s20,w (measured)(MW (wt)/MW (mutant)) (n, v-bar, the partial specific volume per gram of dissolved macromolecule; r, solution density; N, Avogadro’s number; f, frictional coefficient).69 The adjusted values are directly comparable to the measured values for the wt array as demonstrated in Figure 3.

Acknowledgements We thank S. Duda and Y. Hunziker for technical assistance and D. Sargent for comments on the manuscript. Partial financial support from the Swiss National Science Foundation through the

NCCR Structural Biology and a grant to T.J.R. are gratefully acknowledged. K.B. was supported by a post-doctoral fellowship from the Human Frontier Science Program.

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Edited by J. O. Thomas (Received 18 September 2002; received in revised form 11 December 2002; accepted 20 December 2002)