Histone H522Chromatin Interactions In Situ AreStrongly Modulated by H5 C-terminal Phosphorylation

Nora N. Kostova,1,2 Ljuba Srebreva,1 Dimiter V. Markov,1 Bettina Sarg,3 Herbert H. Lindner,3*

Ingemar Rundquist2,4*

� AbstractWe used linker histone-depleted normal human fibroblast nuclei as templates to studyhow phosphorylation affects histone H5 binding to chromatin in situ. Permeabilizedcells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 waspurified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phos-phorylated protein contained a mixture of multiply phosphorylated forms. Controlexperiments, using mass spectrometry, revealed that up to five SPXK motifs in the Cterminus were phosphorylated, but also that about 10% of the protein contained onephosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei withnonphosphorylated or phosphorylated H5 was performed at physiological ionicstrength. The bound H5 was then extracted using NaCl concentrations in the range of0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining andimage cytofluorometry. Our results show that H5 phosphorylation substantiallyreduced its affinity for chromatin in situ, which support previous observations indicat-ing that C-terminal phosphorylation may be essential for the biological functions oflinker histones. ' 2012 International Society for Advancement of Cytometry

� Key termschromatin; linker histones; affinity; phosphorylation

THE histone H1 family is the most divergent subgroup of the highly conserved chro-

mosomal histone proteins (1,2). H1 histones are bound to the outer surface of

nucleosomes near the entry/exit point of the linker DNA and are also known as linker

histones (2–4). They have been implicated to participate in determining the higher-

order folding states of chromatin and thus in control of gene activity (5).

In higher eukaryotes, H1 is a heterogeneous family and at least nine subtypes

have been found in mammals (6–10). Although it seems that each subtype may

have a distinct function, the specific role played by linker histones is still enigmatic.

Moreover, individual subtypes or some groups of subtypes are not essential for via-

bility in some systems studied (11–13), but on the other hand it seems now clear

that linker histones in general are essential for proper development of higher organ-

isms (14).

In general, H1 histones consist of a conserved globular domain and variable N-

and C-tails responsible for the H1 family heterogeneity (6,15). The tails, highly

enriched in positively charged lysine and arginine residues, stabilize chromatin fold-

ing by shielding the negative charges on the DNA backbone (16,17) and also by

adopting specific secondary structure upon interaction with DNA (18,19). They are

targets for several postsynthetic modifications, particularly phosphorylation. H1

phosphorylation increases during cell cycle progression and has been observed in a

number of different organisms and cell types (20–22). It occurs at specific serine and

threonine residues located in the tail domains (18,23). This modification is believed

1Institute of Molecular Biology, BulgarianAcademy of Sciences, BG-1113 Sofia,Bulgaria2Division of Cell Biology, Department ofClinical and Experimental Medicine,Link€oping University, SE-58185 Link€oping,Sweden3Division of Clinical Biochemistry,Biocenter, Innsbruck Medical University,Innrain 80-82, A-6020 Innsbruck, Austria4Integrative Regenerative Medicine(IGEN) Centre, Link€oping University,SE-58185 Link€oping, Sweden

Received 6 July 2012; Revision Received21 September 2012; Accepted 22September 2012

Grant sponsor: The Bulgarian NationalScience Fund; Grant number: K-906/1999;Grant sponsor: The Swedish ResearchCouncil; Grant number: 349-2001-6688;Grant sponsor: European ScienceFoundation EUROCORES ProgrammeEuroDYNA (Austrian ScienceFoundation); Grant number: I23-B03;Grant sponsor: The EC SixthFramework Programme; Grant number:ERAS-CT-2003-980409;

Original Article

Cytometry Part A � 83A: 273�279, 2013

to alter linker histone interaction with DNA and thus to mod-

ulate chromatin structure (24,25). High level of H1 phospho-

rylation was observed during mitosis and suggested that it

may play an active role in mitotic chromosome condensation

(26). In some cell systems, however, H1 phosphorylation was

uncoupled from mitosis and highly condensed chromatin was

enriched in unphosphorylated H1 (25).

A specific subtype of linker histones, histone H5, has

been found to accumulate in nucleated avian erythrocytes

(27,28). H5 is a counterpart of mammalian histone H18 andboth of them are considered to be differentiation-specific H1

subvariants. In immature cells, H5 is phosphorylated and then

it becomes dephosphorylated during erythrocyte maturation

(29). H5 shows strong preference for higher-order chromatin

structures (30–32) and is more tightly bound to DNA or chro-

matin compared with other H1 subvariants (33–36), most

probably due to the higher Arg/Lys ratio in its tails.

In principle, H1 phosphorylation should neutralize the

positive charges and weaken the binding to DNA, resulting in

more open, decondensed, chromatin structure (24,25,37–42).

However, differences between N- and C-tail domains in the

binding of phosphorylated H1 histones to DNA have been

described. For instance, Hill et al. (24) found that the phos-

phorylation of isolated N-terminal domains of sea urchin

sperm-specific linker histones abolished their binding to

DNA. In contrast, phosphorylation in the C-terminus had lit-

tle effect on its overall affinity for DNA. Talasz et al. (43)

reported that linker histones with different levels of phospho-

rylation, isolated from cells in different phases of the cell cycle

(22), did not show any differences in their binding to mono-

nucleosomes in vitro.

Most of the previous in vitro studies have been carried

out on isolated chromatin fragments, mononucleosomes, or

naked DNA. However, it seems clear that linker histone affin-

ity may be substrate-dependent, which indicates that in vitro

binding studies should preferably be performed on chromatin

templates that are as intact as possible. Linker histones are

bound to chromatin mainly through ionic interactions and

they can be selectively extracted using salt concentrations in

the range of 0.3 to 0.7 M NaCl. This property of linker his-

tones was used to develop a method to investigate H1-chro-

matin interactions in situ using the DNA-binding fluoro-

chrome DAPI as an indirect probe (44–46). Furthermore, our

method was extended to study the affinity of a particular H1

subfraction reconstituted into nuclei after depletion of the en-

dogenous linker histones (47). We have now applied this

method to study how phosphorylation affects histone H5 af-

finity for chromatin in situ in normal human H1-depleted

fibroblasts.

MATERIALS AND METHODS

Materials

DAPI, digitonin, and Trizma (Tris base) were purchased

from Sigma. Recombinant human cdk5/p35 active kinase (lot

no. 22420AU, specific activity 1,830 U/mg) was purchased

from Upstate (Lake Placid, NY). All other chemicals were pur-

chased from Fluka (Buchs, Switzerland) if not otherwise indi-

cated.

Preparation of H5 Histone

Chicken blood was obtained from a poultry slaughter

house under the regulations of the Bulgarian Veterinary Medi-

cal Activity Law. Linker histones were extracted with 5%

HClO4 and histone H5 was purified by gel exclusion chroma-

tography on a Bio Gel P100 column as detailed by Srebreva

and Zlatanova (48).

In Vitro Phosphorylation of H5 Histones

Four milligrams of purified histone H5 was phosphoryl-

ated in vitro by cdk5/p35 kinase, according to the recommen-

dations of the manufacturer, with the following modifications:

final H5 concentration, 2 mg/ml; total amount of kinase, 4 lgin 2 ml reaction solution; no BSA was added. The phosphoryl-

ation was performed at 308C. The extent of phosphorylation

was monitored by capillary electrophoresis after 2, 4, and 6

hours. After 7 hours, the reaction was stopped by precipitation

of H5 proteins with TCA (final concentration 20%). The mix-

ture was left on ice for 1 h, centrifuged, washed, and lyophi-

lized as detailed previously (49). The same relative conditions

(enzyme/substrate ratio 1:1,000) were used to phosphorylate a

small batch of highly pure recombinant H5 (kindly provided

by Professor Jean Thomas, University of Cambridge), which

was used in control experiments to determine the N- and C-

terminal phosphorylation pattern by mass spectrometry. Such

control experiments were also performed using a tenfold

increase in the enzyme/substrate ratio.

Capillary Electrophoresis

High performance capillary electrophoresis (HPCE) was

performed on a Beckman system P/ACE 2100. Data collection

and postrun data analyses were carried out using P/ACE and

System Gold software (Beckman Instruments). The capillary

cartridge used was fitted with 75 lm internal diameter fused

silica of 67 cm total length (60 cm to the detector). In all

experiments an untreated capillary was used. Protein samples

were injected by pressure and detection was performed by

measuring UV absorption at 200 nm. Separation of H5

was performed as described (50–53). The linker histones were

*Correspondence to: Ingemar Rundquist, Division of Cell Biology, Dep.of Clinical and Experimental Medicine, Faculty of Health Sciences,Link€oping University, SE-581 85 Link€oping, Sweden. or HerbertLindner, Division of Clinical Biochemistry, Biocenter, InnsbruckMedical University, Innrain 80-82, A-6020 Innsbruck, Austria

Emails: ingemar.rundquist@liu.se (or) herbert.lindner@i-med.ac.at

Published online 18 October 2012 in Wiley Online Library(wileyonlinelibrary.com)

DOI: 10.1002/cyto.a.22221

© 2012 International Society for Advancement of Cytometry

ORIGINAL ARTICLE

274 Histone H5��Chromatin Interactions In Situ

analyzed in 0.1 M sodium phosphate buffer (pH 5 2.0) con-

taining 0.02% HPMC. All runs were carried out at a constant

voltage (12 kV) and at a capillary temperature of 258C.

Enzymatic Cleavage and Mass Spectrometry

In vitro phosphorylated H5 was digested with a-chymo-

trypsin [EC 3.4.21.1] (Sigma type I-S, 1/150 w/w) in 100 mM

sodium acetate buffer (pH 5 5.0) for 40 min at room temper-

ature. The peptides obtained were separated using a Nucleosil

300-5 C18 column (150 mm 3 4 mm I.D.; 5 lm particle pore

size; end-capped; Macherey-Nagel, Duren, Germany). Samples

of �50 lg were injected onto the column. Chromatography

was performed within 50 min at a constant flow of 0.5 ml/min

with a two-step acetonitrile gradient starting at solvent A—

solvent B (87:13) (solvent A: water containing 0.1% TFA; sol-

vent B: 85% acetonitrile and 0.1% TFA). The concentration of

solvent B was increased linearly from 13% to 20% during 25

min and from 20% to 50% during 28 min. Fractions obtained

in this way were collected and, after adding 20 ll 2-mercapto-

ethanol (0.2 M), lyophilized and stored at2208C.Determination of the molecular masses of the N- and C-

terminal fragments of H5 obtained by RP-HPLC was carried

out by electrospray ion-mass-spectrometry (ESI-MS) tech-

nique using a Finnigan LCQ ion trap instrument (San Jose,

CA). Samples (5–10 lg) were dissolved in 50% aqueous meth-

anol containing 0.1% formic acid, and injected into ion

source.

Determination of the phosphorylation sites of the N-and

C-terminal fragments of H5 was carried out by further diges-

tion with trypsin [3.4.21.4] (Roche, sequencing grade, 1/50 w/

w) in 5 mM NH4HCO3 (pH 5 8.5) for 1 h at 378C followed

by LC-ESI-MS as described previously (54).

Cell Culture

Human diploid foreskin fibroblasts (AG 1523, passages

14–19) were cultured in Earle’s Minimal Essential Medium

supplemented with 10% fetal bovine serum, penicillin (50 IU/

ml), streptomycin (50 lg/ml), and L-glutamine (2 mM). The

cells were kept at 378C in a 5% CO2 atmosphere and serially

passaged at a 1:2 split ratio every 4th day. Before the experi-

ments, the cells were plated on coverslips in 12-well plates and

used when reaching confluence at a cell density of about 2 3

105 cells/coverslip.

Cell Preparation

The fibroblasts were rinsed in KRG buffer (120 mM

NaCl; 4.9 mM KCl; 1.2 mM MgSO4 3 7H2O; 1.7 mM

KH2PO4; 8.3 mM Na2HPO4 3 2H2O; 10 mM glucose) and

permeabilized with 40 lg/ml digitonin in Tris buffered saline

(TBS; 10 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 0.5

mM MgCl2 for 10 min. The cells were then extracted with 0.7

M NaCl in TBS for 5 min to remove all native linker histones.

The salt extraction buffer contained also 1 M sucrose to pre-

vent cellular disruption. Thereafter, the cells were washed in

TBS and reconstituted by incubation in either phosphorylated

or nonphosphorylated H5 histones (20 lg/ml, dissolved in

TBS) for 1 h. The reconstitution solution was supplemented

with the protease inhibitors AEBSF (69 lg/ml; Calbiochem,

San Diego, CA), pepstatin (2 lg/ml; Boehringer Mannheim,

Mannheim, Germany), and leupeptin (5 lg/ml; Boehringer

Mannheim). The reconstituted cells were extracted for 5 min

with different concentrations of NaCl ranging from 0.15 to 0.7

M and then fixed in 4% paraformaldehyde for 2 days. All pre-

parations were performed on ice.

DAPI Staining and Image Cytofluorometry

The fixed cells were stained with 50 nM DAPI and the flu-

orescence intensity (FI) was measured by image cytofluorome-

try as detailed previously (47). The number of G1 cells per

frame was about 100, and their mean integrated fluorescence

was used for the calculations of H5 affinity. Four frames on

each coverslip were analyzed, and each experiment included

data from duplicate glasses. Thus, about 800 G1 cells were

measured for each data point within a series of measurements.

The analysis of salt extraction curves was performed as

described previously (44,45) using a least-squares curve fitting

to a linker histone binding equation (33). The salt extraction

curves were normalized using an average of the three highest

FIs after extraction as 100%. At this point, all linker histones

were considered to be extracted from chromatin. The average

of the three lowest FIs then represented the level at which all

reconstituted linker histones were considered to be bound to

chromatin. The NaCl concentration required to induce a 50%

increase in FI from this level was then calculated from the

fitted equation and used as a measure of average apparent lin-

ker histone affinity for chromatin in situ. The results are,

unless otherwise indicated, expressed as mean � S.D. The sta-

tistical significance of differences between results was analyzed

using unpaired Student’s t-test.

RESULTS AND DISCUSSION

The electropherogram obtained from purified H5 his-

tones showed N-terminally nonacetylated and acetylated H5

peaks (Fig. 1A) as expected (55). After H5 in vitro phospho-

rylation by cdk5/p35, a number of slower migrating peaks rep-

resenting phosphorylated forms of both nonacetylated and

acetylated forms of H5 appeared (Fig. 1B), indicating that

almost all H5 molecules contained one or more phosphate

groups.

Histone H5 was suggested to become phosphorylated in

vivo at four major sites in a cell cycle-dependent manner and

two of those sites were found to be located in Ser-Pro-X-Basic

motifs in the highly basic C-terminal domain (CTD) (29,56).

The H5 phosphorylation in the present experiments was car-

ried out using cdk5/p35, which is predicted to phosphorylate

five Ser-residues in histone H5 located in SPXK motifs in the

C terminus. To check the enzyme specificity, a small batch of

recombinant H5 was phosphorylated in vitro using the same

phosphorylation conditions. After chymotrypsin cleavage and

subsequent analysis of the fragments using tandem mass spec-

trometry four phosphorylation sites in the C terminus were

detected (Fig. 2A). A large majority of these molecules were

mono- or diphosphorylated. We also found that the N-termi-

nal fragment was a mixture of nonphosphorylated and mono-

ORIGINAL ARTICLE

Cytometry Part A � 83A: 273�279, 2013 275

phosphorylated molecules (Fig. 2B), indicating that the in

vitro phosphorylated H5 contained about 10% H5 where the

N terminus was monophosphorylated in combination with

the C-terminal phosphorylation pattern. To further check the

specificity of the enzyme, we performed in vitro phosphoryla-

tion of recombinant H5 using a tenfold higher enzyme to sub-

strate ratio. After chymotrypsin cleavage and mass spectro-

metric analyses, we found that the C-terminal phosphoryla-

tion pattern was clearly shifted to di-, tri-, and

tetraphosphorylated forms and that also pentaphosphorylated

molecules were present (Fig. 2C). The N- and C-terminal frag-

ments were further isolated by RP-HPLC and digested using

trypsin. The predicted SPXK phosphorylation sites in the

CTD were then verified by MS analysis (data not shown).

Concomitantly, the N-terminal phosphorylation pattern

showed only a small increase in the number of monopho-

sphorylated molecules (Fig. 2D), indicating a substantially

lower specificity for the enzyme to phosphorylate this site in

the N terminus. This may be explained by the absence of

SPXK motifs in the N terminus of H5, and the present phos-

phorylation site in the N terminus was also proved to be Ser7,

which is the only nonmotif site in H5 where serine is followed

by a proline. In conclusion, the phosphorylation conditions

used in our reconstitution experiments resulted in a balanced

mixture of phosphorylated H5 molecules (Fig. 1B) in accord-

ance with a typical average phosphorylation pattern present in

exponentially growing cells containing multiple H1 subtypes

(57,58).

Figure 2. Mass spectrometric analysis of the phosphorylation pattern obtained after phosphorylation of recombinant H5 in vitro by cdk5/

p35 and subsequent cleavage with chymotrypsin. A: C-terminal fragment (enzyme/substrate 5 1:1,000); B: N-terminal fragment (enzyme/

substrate 5 1:1,000); C: C-terminal fragment (enzyme/substrate5 1:100); D: N-terminal fragment (enzyme/substrate5 1:100).

Figure 1. HPCE separation of purified H5 histones from chicken ery-

throcytes with a 0.1 M sodium phosphate buffer (pH5 2) containing

0.02% HPMC. A: nonphoshorylated H5 (peak 1, nonacetylated H5

and peak 2, acetylated H5); B: phosphorylated H5. Running condi-

tions for all samples were as follows: injection time, 5 s; voltage

12 kV; detection at 200 nm; untreated capillary (60 cm3 75 lm).

ORIGINAL ARTICLE

276 Histone H5��Chromatin Interactions In Situ

Permeabilized fibroblasts were treated with 0.7 M NaCl

to extract endogenous linker histones. Thereafter, they were

reconstituted by incubation with either nonphosphorylated or

in vitro phosphorylated H5 histones at physiological ionic

strength. The salt extraction of linker histones leads to an

increase in FI which is proportional to the sum of DAPI bind-

ing sites that become available when H1 is detached from

chromatin (44–46). We have recently applied this method to

study linker histone–chromatin interactions in normal human

fibroblast nuclei after depletion of the endogenous linker his-

tones and reconstitution with H1 subfractions (47). The pre-

sence of exogenous protein in nuclei after reconstitution was

verified by Alexa-labeled H1. The exogenous linker histones

showed a slightly reduced affinity for chromatin compared

with the native linker histones (47), indicating that the recon-

stituted proteins did not bind exactly in the same manner as

in the native state. However, this system allows linker histone–

chromatin interactions to be studied in situ using a chromatin

template that structurally is as close as possible to the intact

cell nucleus.

Histone H5 reconstituted nuclei stained with DAPI were

similar in appearance to native fibroblast nuclei. Background

cytoplasmic fluorescence was negligible in accordance with

previous results (47). The reconstituted cells were then

extracted with NaCl concentrations in the range of 0.15 to 0.7

M. When the relative FIs were plotted against the NaCl con-

centration (Fig. 3), they showed a close fit to the linker histone

binding equation described by Kumar and Walker (33). The

salt concentration needed to induce a half-maximal increase

in FI in nuclei reconstituted with nonphosphorylated H5

(Fig. 3A) was 0.52 � 0.01 M (n 5 5), which was slightly, but

significantly (P \ 0.01), lower than the corresponding value

from native H5 in chicken erythrocytes (0.55 � 0.02 M)

reported previously (35). This reduction in affinity for the ex-

ogenously applied H5 is in accordance with our previous

results with other H1 subfractions used for reconstitution

(47). In contrast, the corresponding salt concentration needed

to induce a 50% increase in FI in nuclei reconstituted with

phosphorylated H5 (Fig. 3B) was substantially lower, 0.41 �0.02 M (n 5 4, P\ 0.0001). Examples of fluorescence images

and their corresponding fluorescence intensity data are

presented in Figure 4.

Thus, we observed that phosphorylated H5 molecules had

a considerably reduced affinity for chromatin in situ compared

with nonphosphorylated ones. This finding is consistent with

the proposal that phosphorylation of H1 tails loosens H1-DNA

interactions, leading to relaxed chromatin structure (25). How-

ever, a number of different phosphorylation sites have been

described and they may have differential effects on the DNA-

binding properties of linker histones. Data in this respect are

controversial. The discrepancies could be due to differences in

linker histone subtypes, the distribution of phosphorylation

sites within them, the number of phosphate groups incorpo-

rated, and the template systems used (43,59). Phosphorylation

has thus been reported to reduce DNA-binding of histone H5

(60). However, these authors used a cAMP-dependent kinase

isolated from calf thymus to introduce phosphate groups in

H5. This enzyme probably phosphorylated H5 at multiple sites,

mainly in the globular domain, as shown previously using a

cAMP-dependent kinase isolated from pig brain (61). In

experiments with sea urchin sperm-specific linker histones, Hill

et al. (24) found that phosphorylation of six sites in the N-ter-

minal domain almost abolished its binding to DNA. Moreover,

SPKK motifs in sea urchin sperm H1 are localized in N-termi-

nal domain (62) and phosphorylation of peptides, containing

SPKK sequences was shown to weaken their binding to DNA

(37). In contrast, phosphorylation of three residues in the iso-

lated CTD from sea urchin sperm H1 had a negligible effect on

binding of this domain to DNA (24), probably because of the

extended lysine rich C-terminal in this species. Interestingly,

the same pattern of phosphorylation in the whole H1 molecule

(nine sites) did not significantly change its binding to DNA,

whereas it showed a clearly reduced affinity for chromatin in

solution (24). This is probably explained by the heterogeneous

pattern of phosphorylation sites in the extended CTD of sea ur-

chin sperm H1, the most distal part containing all phosphoryl-

ation sites while the major remaining part of the highly charged

CTD, lacking phosphorylation sites, determines the binding to

DNA of the whole CTD as well as the entire molecule.

Moreover, Talasz et al. (43) found that linker histones isolated

from cells in G1, S, or mitosis, containing one to five phosphate

groups (22), did not show any differences in their binding to

mononucleosomes in vitro. These authors thus observed

very high binding affinity to a mononucleosome, but on the

other hand a low chromatin aggregation capability, in the

case of highly phosphorylated H1 histones. However, since

mononucleosomes lack linker DNA, the affinity of H1 is prob-

ably not significantly affected by H1 phosphorylation in such

preparations.

Figure 3. Linker histone dissociation curves derived from the rela-

tive fluorescence intensity (FI) as a function of NaCl concentration.

A: after reconstitution with nonphosphorylated H5 (n 5 5); B: after

reconstitution with phosphorylated H5 (n 5 4). Fluorescence in-

tensity values were obtained after staining with 50 nM DAPI. The

vertical bar through each data point indicates its standard error of

the mean.

ORIGINAL ARTICLE

Cytometry Part A � 83A: 273�279, 2013 277

A remaining paradox concerns the relation between H1

affinity for chromatin, H1 phosphorylation, and chromatin

condensation (25). The various H1 subtypes were shown to

have different inherent affinities for chromatin and different

chromatin condensing capacities, and it was concluded that

the CTD was the main determinant of these properties (63).

Interestingly, partial phosphorylation of the H18 CTD did not

cause neither a substantial DNA condensation nor a large

reduction in affinity for naked DNA, whereas the fully phos-

phorylated CTD showed increased DNA condensation and

reduced affinity for DNA (64). However, our findings, using a

chromatin template, clearly show that phosphorylation of

SPXK-motifs in the CTD is a strong modulator of H5 binding

to chromatin. Along this line, recent data challenged the com-

monly accepted idea that the binding, and function, of the lin-

ker histone CTD is mainly regulated by charge neutralization.

For example, C-terminal phosphorylation at two sites directly

modulated the affinity of histone H1.1 for chromatin in vivo

without influencing the charge distribution or the overall net

charge of the tail domain (65). Moreover, histone H1 binds

dynamically to chromatin (66,67) and phosphorylation of the

tails facilitated its mobility (41,42,68).

In conclusion, the present results verify that linker his-

tone affinity for chromatin in situ can be measured using

DAPI as a fluorescent probe. Linker histone-depleted normal

fibroblast nuclei represent relatively intact chromatin tem-

plates well suited for reconstitution experiments. Phosphoryl-

ation by cdk5/p35 resulted in a substantial reduction of H5 af-

finity for chromatin in this template. In line with in vivo

results using green fluorescent protein-tagged H1.1 (65), our

results indicate that phosphorylation of SPXK-motifs in the

CTD is a strong modulator of linker histone binding proper-

ties, which may be responsible for the dynamic regulation of

chromatin structure. Our data thus supply further evidence

for the importance of the CTD in the determination of linker

histone biological functions.

ACKNOWLEDGMENTS

The authors thank A. Devich, Innsbruck Medical Univer-

sity, and A. Lonn, Linkoping University, for excellent technical

assistance. They also thank Professor Jean Thomas, University

of Cambridge, for providing recombinant H5 protein.

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