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Originally published In Press as doi:10.1074/jbc.M505636200 on July 8, 2005

J. Biol. Chem., Vol. 280, Issue 37, 32141-32147, September 16, 2005
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DNA-induced Secondary Structure of the Carboxyl-terminal Domain of Histone H1*

Alicia Roque{ddagger}, Ibon Iloro§, Imma Ponte{ddagger}, José Luis R. Arrondo§, and Pedro Suau{ddagger}1

From the {ddagger}Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Barcelona, 08193 Bellaterra, Barcelona, Spain and the §Departamento de Bioquímica, Universidad del País Vasco, Apartado. 644, E-48080, Bilbao, Spain

Received for publication, May 23, 2005 , and in revised form, June 27, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have studied the secondary structure of the carboxyl-terminal domains of linker histone H1 subtypes H10 (C-H10) and H1t (C-H1t), free in solution and bound to DNA, by IR spectroscopy. The carboxyl-terminal domain has little structure in aqueous solution but becomes extensively folded upon interaction with DNA. The secondary structure elements present in the bound carboxyl-terminal domain include the {alpha}-helix, {beta}-structure, turns, and open loops. The structure of the bound domain shows a significant dependence on salt concentration. In low salt (10 mM NaCl), there is a residual amount of random coil, 7% in C-H10 and 12% in C-H1t. In physiological salt concentrations (140 mM NaCl), the carboxyl termini become fully structured. Under these conditions, C-H10 contained 24% {alpha}-helix, 25% {beta}-structure, 17% open loops, and 33% turns. The latter component could include a substantial proportion of the 310 helix. Despite their low sequence identity (~30%), the representation of the different structural motifs in C-H1t was similar to that in C-H10. Examination of the changes in the amide I components in the 20–80 °C temperature interval showed that the secondary structure of the DNA-bound C-H1t is for the most part extremely stable. The H1 carboxyl-terminal domain appears to belong to the so-called disordered proteins, undergoing coupled binding and folding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
H1 linker histones are thought to be primarily responsible for the condensation of the thick chromatin fiber. It is currently accepted that histone H1 could have a regulatory role in transcription through the modulation of chromatin higher order structure. H1 has been described as a general transcriptional repressor because it contributes to chromatin condensation, which limits the access of the transcriptional machinery to DNA. However, H1 may regulate transcription at a more specific level, participating in complexes that either activate or repress specific genes (1–8). Binding to scaffold-associated regions and participation in nucleosome positioning are other mechanisms by which H1 could contribute to transcriptional regulation (9, 10).

H1 has multiple isoforms. In mammals, six somatic subtypes (designated H1a–H1e and H10), a male germ line-specific subtype (H1t), and an oocyte-specific subtype (H1oo) have been identified (11–14). The subtypes differ in timing of expression (15), extent of phosphorylation (16), turnover rate (17, 18), binding affinity (19), and evolutionary stability (20). Differences in DNA condensing capacity have also been demonstrated for some subtypes (21–23).

Linker histones contain three distinct domains: a short amino-terminal domain (20–35 amino acids), a central globular domain (~80 amino acids, consisting of a helix bundle and a {beta}-hairpin), and a long carboxyl-terminal domain (~100 amino acids) (24). The amino- and carboxyl-terminal domains are highly basic. The distribution of charge in the carboxyl-terminal domain is extremely uniform despite the variation in sequence in the different subtypes (25).

The carboxyl-terminal domain is the primary determinant of histone H1 binding to chromatin in vivo (19, 26). Several studies indicate that the ability of linker histones to stabilize chromatin folding resides in the carboxyl-terminal domain of the protein (27–29). The preferential binding of histone H1 to scaffold-associated regions appears to be determined by the carboxyl-terminal domain (30). The carboxyl-terminal domain is also responsible for the activation of apoptotic nuclease (31). Knowledge of the structure of the carboxyl-terminal domain of H1 once bound to DNA is thus important to our understanding of H1 function.

The amino- and carboxyl-terminal domains have little defined structure in solution, although they may contain a considerable amount of turn-like conformations in rapid equilibrium with unfolded states. Molecular modeling (32) and induction of {alpha}-helix in the presence of secondary structure stabilizers (33), such as 2,2,2-trifluoroethanol (TFE)2 and HClO4, suggest that the carboxyl termini would acquire a folded conformation on binding to DNA. We have shown previously that interaction with DNA induces stable helix and turn structures in a 23-residue peptide from the H10 carboxyl terminus (CH-1, residues 99–121, a peptide studied previously (34, 35)). This region, adjacent to the globular domain, is shown to behave as a specific subdomain in the stabilization of folded chromatin structures by histone H1 (27). Inducible helical elements have also been characterized in the amino-terminal domains of H10 and H1e (36, 37).

In the present study we have used infrared spectroscopy to study the secondary structure of the carboxyl-terminal domains of H10 and H1t in aqueous solution and in the complexes with DNA. The carboxyl-terminal domains have little structure in solution but become fully structured upon interaction with DNA in physiological salt (140 mM NaCl). The secondary structure elements present in the complexes include {alpha}-helix, {beta}-structure, turns, and open loops. The study of the entire carboxyl-terminal domain has provided new information, not readily accessible to studies with peptides, on the variety of structural motifs present in the DNA-bound domain, the salt dependence and thermal stability of the folded structure, and the conservation of the protein structure among divergent subtypes. It has also shown that the property of coupled binding and folding, already demonstrated for the previously studied CH-1 peptide (35), is shared by the entire domain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning, Expression, and Purification of the Carboxyl-terminal Domains of Histone H1 Subtypes—The sequences encoding the carboxyl-terminal domains of histones H10 and H1t were cloned and expressed. All gene fragments were amplified from mouse genomic DNA by PCR. The primers were 5'-CCACCATGGATGAGCCTAAAAGGT-3' and 5'-GGAGATCTTTACTTCTTCTTGCTGGCCCTCT-3' for the carboxyl-terminal domain of H10 and 5'-GTACCATGGCGGCTTCAGGGAACGAC-3' and 5'-ACGGATCCTTACTTCCTCCCTGCTGCCTTCCT-3' for the carboxyl-terminal domain of H1t. The amplification products were cloned in the pQE60 vector (Qiagen) using the NcoI and BglII restriction sites to yield the expression vectors pCTH10 and pCTH1t.

The recombinant plasmids were transformed into Escherichia coli M15 (Qiagen). Cells were grown to an A600 of 0.8 and then induced with 1mM isopropyl 1-thio-{beta}-D-galactopyranoside, allowing the expression to proceed for 4 h at 37 °C. Cells were lysed in 10 mM potassium phosphate plus 4 M guanidinium hydrochloride for 15 min at room temperature. The extract was centrifuged at 20,000 x g for 25 min. The supernatants were loaded on a CHT-II cartridge filled with ceramic hydroxyapatite type II (Bio-Rad) equilibrated with lysis buffer. The bound fraction, corresponding to the recombinant proteins, was eluted with 200 mM potassium phosphate, 4 M guanidinium hydrochloride, pH 7.0. Finally, the proteins were desalted by gel filtration through Sephadex G-25 (Amersham Biosciences).

Circular Dichroism Spectroscopy—Samples for CD spectroscopy were 2 x 10–5 M carboxyl-terminal domain in 10 mM phosphate buffer, pH 7.0. Samples in aqueous solution and in 60% TFE (v/v) were prepared. Spectra were obtained on a Jasco J-715 spectrometer in 1 mm of cells at 20 °C. The results were analyzed with Standard Analysis software (JASCO) and expressed as mean residue molar ellipticity [{theta}]. The helical content was estimated from the ellipticity value at 222 nm ({theta}222), according to the empirical equation of Chen et al. (38): % helical content = 100 [{theta}222/–39,500 x ((1–2.57)/n)], where n is the number of peptide bonds.

Infrared Spectroscopy Measurements—The carboxyl-terminal domains of histone H1 were measured at 5 mg/ml in 10 mM HEPES plus 10 or 140 mM NaCl. DNA-protein complexes contained 7.0 mg/ml mouse DNA and the appropriate amount of protein.

Measurements were performed on a Nicolet Magna II 550 spectrometer equipped with a MCT detector, using a demountable liquid cell with calcium fluorine windows and 50-µm spacers for D2O medium and 6-µm spacers for H2O medium measurements. Typically, 1000 scans for each background and sample were collected, and the spectra were obtained with a nominal resolution of 2 cm–1 at 22 °C. Data treatment and band decomposition have been described previously (39). The DNA contribution to the spectra of the complexes with the carboxyl-terminal domain was subtracted using a DNA sample of the same concentration; the DNA spectrum was weighted so as to cancel the symmetric component of the phosphate vibration at 1087 cm–1 in the difference spectra as described in Vila et al. (35). In addition, spectra of complexes of different ratios of protein to DNA recorded (0.4 and 0.7 (w/w)). The resulting protein spectra were independent of the protein/DNA ratio inside the statistical error, indicating that the amide I region was not significantly affected by DNA spectral changes induced by interaction with the protein.

For the analysis of the thermal stability of the DNA-C-H1t complexes, a tungsten-copper thermocouple was placed directly onto the window, and the cell was placed on a thermostatted cell mount. Thermal analyses were performed by heating from 20 to 80 °C at a rate of 1 °C/min. Spectra were recorded using a Rapid Scan software running under OMNIC (Nicolet). For each degree of temperature interval, 305 interferograms were averaged, Fourier-transformed, and ratioed against background, obtaining the spectra with a nominal resolution better than 2 cm–1 (40). The solvent contribution was subtracted as described earlier (41). Thermal analyses were performed only in D2O. The DNA contribution to the spectra at each temperature was subtracted using a DNA sample at the same temperature and concentration. The subtraction was weighted using the phosphate symmetric vibration at 1087 cm–1 as described above. In the amide I' regions, the spectra of native and denatured mixed-sequence calf thymus DNA were similar enough not to affect the protein difference spectrum.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD and IR Spectroscopy Analysis of the Carboxyl-terminal Domain of Histone H1 in Aqueous Solution and in TFE—We have used IR spectroscopy to examine the secondary structure of the carboxyl-terminal domains of the histone H1 subtypes H10 (C-H10) and H1t (C-H1t) (Fig. 1) in aqueous (D2O) and in TFE solutions (Fig. 2). Values corresponding to band position and percentage area are given in TABLE ONE.



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  		FIGURE 1.
Alignment of the carboxyl-terminal domain sequences of mouse histone H1 subtypes H10 and H1t. The conserved positions are indicated by an asterisk.

 



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  		FIGURE 2.
Amide I decomposition of the carboxyl-terminal domain of histone H1. The left column shows the spectra of the C-H10, and the right column shows the spectra of the C-H1t. The spectra were recorded in D2O and in 60% TFE solution at 20 °C. The buffer was 10 mM HEPES plus 10 mM NaCl, pH 7.0. The protein concentration was 5 mg/ml.

 


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  		TABLE ONE
Percentages of secondary structure of the carboxyl-terminal domain of histones H10 and H1t in aqueous and TFE solutions

 
In aqueous solution, the amide I' of both C-H10 and C-H1t was dominated by the random coil at 1643 cm–1 with about 50% of the total amide I' intensity. The other main component at 1661–1662 cm–1, with 35% of the total area in C-H10 and 29% in C-H1t, was assigned to turns. In previous studies with H1 peptides, the component at 1661 cm–1 was related to the nascent helix, turns in rapid equilibrium with the unfolded state that in the presence of secondary structure stabilizers are transformed into helical structure (35, 36, 42). Small amounts of turns at 1670 cm–1 (6–9%) and extended structure (8–9%) were also present.

In 60% TFE, C-H10 and C-H1t were extensively folded. Turns (40–38%), {alpha}-helix (23–22%), and {beta}-structure (21–25%) were identified, together with a component of random coil/open loops (16%) that are not resolved in D2O. The fraction of turns at 1659–1660 cm–1 represented 13% in C-H10 and 21% in C-H1t. In addition to turns, this component could contain a variable proportion of the 310 helix (43–45), in which case, the proportion of helical structure would increase considerably. The values of {alpha}-helix estimated by CD from the ellipticity value at 222 nm (38) were similar to those estimated by IR spectroscopy for both C-H10 and C-H1t (TABLE ONE, Fig. 3).



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  		FIGURE 3.
TFE-dependent conformational transition of the carboxyl-terminal domain of histone H1 measured by CD. Far-UV CD spectra in water and 60% TFE in 10 mM NaCl, both in 10 mMphosphate buffer, pH 7.0, at 20 °C. The numbers 0 and 60 refer to the TFE concentration in percentage by volume.

 
IR Spectroscopy of the Carboxyl-terminal Domain of Histone H1 Bound to DNA—We have examined the secondary structure of the carboxyl-terminal domain of histone H1 bound to DNA. The spectra of the complexes were recorded both in H2O and D2O to facilitate the assignment of the amide I components. Deuteration has a large effect on the position of some components of the amide I, whereas others are shifted slightly or not at all (39). The vibrations of loops and the {alpha}-helix are only slightly affected by isotopic substitution and are thus found at similar positions in H2O and D2O, the {alpha}-helix at about 1652 cm–1 and loops at about 1643 cm–1. In contrast, deuteration has a large effect on the position of the random coil so that in H2O it overlaps with the {alpha}-helix, whereas in D2O it overlaps with loops. Therefore, the {alpha}-helix could be estimated directly in D2O, whereas loops could be estimated directly in H2O. When both {alpha}-helix and loops are present in the protein structure, the percentage of random coil can be obtained either from the difference of the components around 1652 cm–1 in H2O and D2O or from the difference of the components around 1643 cm–1 in D2O and H2O, as indicated by the expression in Equation 1,

(Eq. 1)
where cb is the percentage of the assigned amide I component band in either H2OorD2O and cbRC is the percentage of random coil.

Because the percentages of the different secondary structure elements in H2O and D2O should be comparable, the similarity of the percentages of random coil estimated from the differences of the components either around 1643 cm–1 in D2O and H2O or around 1652 cm–1 in H2O and D2O warrants the proper assignment of the amide I components containing contributions of {alpha}-helix, loops, and random coil. {beta}-structure components appear approximately at the same frequency in D2O and H2O, whereas turn components in D2O are slightly shifted to lower frequencies.

The complexes of the carboxyl-terminal domain with DNA were examined in 10 and 140 mM NaCl. In 10 mM NaCl, the analysis of the complexes of C-H10 with DNA in D2O and H2O yielded 27% turns, 19% {alpha}-helix, 25% open loops, 22% {beta}-structure, and 7% random coil. The amount of random coil was calculated from the difference of the components around 1654 cm–1 in H2O and D2O and from the difference of the components around 1643 cm–1 in D2O and H2O. The values obtained were equal within 1% (Figs. 4 and 5, TABLE TWO). The representation of the different types of secondary structure in C-H1t bound to DNA was similar to that of C-H10, although some small differences were observed, namely, a higher amount of random coil (12%), a lower amount of {alpha}-helix (15%) and open loops (14%), and a higher amount of {beta}-structure (25–27%) and turns (32–34%) (Figs. 4 and 5, TABLE TWO).



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  		FIGURE 4.
Amide I decomposition of the spectra of the C-H10 bound to DNA. The left column shows the spectra measured in D2O, and the right column shows those measured in H2O. The salt concentration, 10 or 140 mM NaCl, is indicated. The DNA contribution to the spectra of the complexes was subtracted as described under "Materials and Methods." r (protein/DNA ratio) = 0.7 (w/w).

 



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  		FIGURE 5.
Amide I decomposition of the spectra of the C-H1t bound to DNA. The left column shows the spectra measured in D2O, and the right column shows those measured in H2O. The salt concentration, 10 or 140 mM NaCl, is indicated. The DNA contribution to the spectra of the complexes was subtracted as described under "Materials and Methods." r (protein/DNA ratio) = 0.7 (w/w).

 


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  		TABLE TWO
Percentages of secondary structure of the carboxyl-terminal domain of histones H10 and H1t bound to DNA

Band position (cm-1), percentage area (%) and assignment of the components obtained after curve fitting of the amide I band of the C-H1o and C-H1t bound to DNA in H2O and D2O and in 10 mM and 140 mM NaCl. The values were rounded off to the nearest integer.

 
When the salt concentration was increased up to 140 mM NaCl, the DNA-bound carboxyl-terminal domains of both H10 and H1t underwent significant structural changes. In the first place, the protein became fully structured, as judged by the total absence of a random coil component. In C-H10, the disappearance of the random coil was correlated with an increase of {alpha}-helix, {beta}-structure, and turns, which reached values of 24, 24–25, and 33–34%, respectively. A decrease in the percentage of open loops from 25% in 10 mM to 18% in 140 mM was also observed. The increase of turns was most apparent for C-H1t, reaching 40–42%; the {alpha}-helix also increased up to 21%; the contributions of the remaining structural motifs did not change significantly (Figs. 4 and 5, TABLE TWO). As in TFE solution, the component at ~1660 cm–1 could contain a variable proportion of the 310 helix.

Melting Experiments—We monitored the changes in the amide I' components of the DNA-bound C-H1t upon heating in the 20–80 °C interval. The DNA contribution to the spectra at each temperature was subtracted using a DNA sample at the same temperature and concentration; the subtraction was adjusted using the phosphate symmetric vibration at 1087 cm–1 as described under "Materials and Methods." Although free DNA melted before complexed DNA, the spectra of native and denatured DNA were similar enough not to affect the amide I' region of the difference spectra. Melting experiments were performed in either 10 or 140 mM NaCl at a protein/DNA ratio (w/w) of 0.4. In 10 mM NaCl, the {alpha}-helix and {beta}-sheet components remained essentially unaltered up to 78 °C. Between 78 °C and 80 °C, they decreased sharply down to a residual 2–3%, which may disappear at higher temperatures. Concomitantly with the decrease of the {alpha}-helix and the {beta}-sheet, the random coil component increased from 25 to 45%. The low frequency {beta}-sheet (~1620 cm–1) melted along a wide temperature interval with a transition midpoint of ~60 °C; an equivalent increase in turns was observed coinciding with the melting of the low frequency {beta}-sheet (Fig. 6, A and C).



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  		FIGURE 6.
Thermal profiles of the amide I band of the secondary structure motifs of the C-H1t bound to DNA. The symbols of the different structural motifs are shown in the inset. A, melting in 10 mM NaCl. B, melting in 140 mM NaCl. The amide I bands of C-H1t at 20 °C (continuous line) and 80 °C (dotted line) are shown superimposed, both in 10 mM (C) and in 140 mM NaCl (D). The DNA contribution to the spectra of the complexes was subtracted as described under "Materials and Methods."

 
In 140 mM NaCl, the structure of the DNA-bound protein was even more stable, the proportions of {alpha}-helix, {beta}-sheet, and random coil remaining essentially constant up to 80 °C. A small decrease of the low frequency {beta}-sheet and a concomitant increase of the turn component was observed (Fig. 6, B and D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have examined the secondary structure of the carboxyl-terminal domain of linker histone subtypes H10 and H1t free in solution and bound to DNA. The use of the entire carboxyl-terminal domain is important in the study of properties that are not localized to any specific subdomain (27) but rather are determined by the whole domain, such as differential affinity (19) or apoptotic nuclease stimulation (31).

The IR spectra of the carboxyl-terminal domain in aqueous solution were dominated by the random coil (1643–1644 cm–1) with 50% of the total amide I intensity. However, even in this most highly unfolded state, an intense band (35% in C-H10 and 29% in C-H1t) at 1662 cm–1 was observed, suggesting the presence of short-lived turn-like conformations in rapid equilibrium with extended forms (35, 36). Small amounts of local structure consisting of turns at 1670 cm–1 (6–10%) and {beta}-structure (8–9%) were also present in the free protein.

Upon interaction with DNA, the carboxyl-terminal domain acquired an extensively folded conformation. The structure of the bound domain showed a significant dependence on salt concentration. In 10 mM NaCl the DNA-bound C-H10 contained {alpha}-helix (19%), {beta}-structure (22%), turns (27%), and open loops (25%), together with a residual 7% random coil. In physiological salt (140 mM), the carboxyl terminus became fully structured, as indicated by the complete absence of random coil. Under these conditions, C-H10 contained 24% {alpha}-helix, 25% {beta}-structure, 18% open loops, and 33–34% turns. The component at ~1660 cm–1 in TFE and in the complexes with DNA could arise totally or in part from the 310 helix. This position has been assigned to the 310 helix in several studies (43, 45). The presence of the 310 helix was demonstrated in a peptide from the H10 carboxyl-terminal domain (residues 99–121) (34, 35). The amount of {alpha}-helix should thus be considered as a lower limit of the amount of helical structure. All of the structural motifs found in the carboxyl-terminal domain have the potential to interact with the major or minor groove DNA phosphates (46). The dependence of the secondary structure of the DNA-bound carboxyl-terminal domain on salt concentration, although moderate, is an indication of conformational versatility, which could have a role in chromatin structure and dynamics.

Despite the large sequence divergence between C-H10 and C-H1t (30% sequence identity) (Fig. 1), the two appear to have common secondary structure components, in both 10 mM and 140 mM NaCl. This suggests that selective constraints may act to preserve the secondary structure of the DNA-bound domain in different subtypes.

The induced secondary structure of the DNA-bound domain appears to be extremely stable. In 10 mM NaCl, the {alpha}-helix and the {beta}-sheet (1630 cm–1) begin to melt at 78 °C; only the low frequency {beta}-sheet (1620 cm–1) melts earlier with a transition midpoint of ~65 °C. In 140 mM, the {alpha}-helix and the {beta}-sheet are even more stable, and no changes are observed in their proportions up to the experimental temperature limit of 80 °C. The low frequency {beta}-sheet melts in a temperature interval similar to that observed at low salt, with a concomitant increase in the proportion of turns. The high thermal stability of the DNA-bound form may result from increased hydrophobicity following extensive compensation of the positive charge of the protein by the DNA phosphates. Considering the high proportion of Lys residues in the carboxyl terminus (~40%), charge neutralization should have a large effect on the mean complex hydrophobicity. Evidence for extensive reciprocal charge compensation in C-H1-DNA complexes is given by the +/– ratio of ~1.0 in saturated complexes (30, 47). In addition, C-H1-DNA complexes form the kind of toroidal complexes predicted by counterion condensation theory when the DNA charge is fully neutralized (>90% charge compensation) (47–49).

Folding could contribute to hydrophobicity in a more specific way. In the C-H1 peptide, helical folding (from Glu-99 to Ala-117) creates a hydrophobic patch because of the marked amphipathic character of the helix with all of the positively charged residues on one face of the helix and all of the hydrophobic residues on the other face (34).

The carboxyl-terminal domain appears to belong to the group of "disordered proteins," also known as natively unfolded or intrinsically unstructured (50–52). In free solution, these proteins lack specific secondary or tertiary structure and are composed of an ensemble of conformations. Many disordered proteins fold into stable secondary or tertiary structures upon binding to their targets, such as other proteins, nucleic acids, or membranes. Intrinsic disorder is characterized by a low sequence complexity, a low content of hydrophobic amino acids, and a high content of polar and charged amino acids. All of these features are present in the carboxyl-terminal domains and also in the amino-terminal domains of H1 (53). In addition, disordered regions are often present in proteins regulated by phosphorylation, which is also the case with H1 (54).

The H1 globular domain is believed to be involved in specific binding site recognition in chromatin. A stably folded carboxyl-terminal domain with its high charge density could interfere with binding site recognition. Given that the folding of the carboxyl terminus is associated with charge neutralization, binding to unspecific sites, such as nucleosomal DNA, that is already involved in interactions with core histones may lead to partial folding and lower affinity of the carboxyl terminus. One of the possible advantages of intrinsic disorder would thus be to kinetically favor the recognition of specific H1 binding sites in chromatin.


   FOOTNOTES
 
* This work was supported by Ministerio de Educación y Ciencia Grants BMC2002-00087 and BMC2002-01438, Generalitat de Catalunya Grant 2001SGR 00199, and University of the Basque Country Grant 00042.310-13552/2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 34-93-5811391; Fax: 34-93-5811264; E-mail: pere.suau{at}uab.es.

2 The abbreviations used are: TFE, 2,2,2-trifluoroethanol; C-H10, carboxyl-terminal domain of histone H10; C-H1t, carboxyl-terminal domain of histone H1t. Back



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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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