Translocation of Histone H1 Subtypes Between

Chromatin and Cytoplasm During Mitosis in Normal

Human Fibroblasts

Anna Green,1 Anita L€onn,1 Kajsa Holmgren Peterson,2 Karin Ollinger,3 Ingemar Rundquist1*

� AbstractHistone H1 is an important constituent of chromatin, which undergoes major struc-tural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes,and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylatedH1 was found located in nuclei during prophase and in both cytoplasm and condensedchromosomes during metaphase, anaphase, and telophase as detected by immunocyto-chemistry. Moreover, we detected remarkable differences in the distribution of the his-tone H1 subtypes H1.2, H1.3, and H1.5 during mitosis. H1.2 was found in chromatinduring prophase and almost solely in the cytoplasm of metaphase and early anaphasecells. In late anaphase, it appeared in both chromatin and cytoplasm and again in chro-matin during telophase. H1.5 distribution pattern resembled that of H1.2, but H1.5was partitioned between chromatin and cytoplasm during metaphase and early ana-phase. H1.3 was detected in chromatin in all cell cycle phases. We propose therefore,that H1 subtype translocation during mitosis is controlled by phosphorylation, in com-bination with H1 subtype inherent affinity. We conclude that H1 subtypes, ortheir phosphorylated forms, may leave chromatin in a regulated way to give access forchromatin condensing factors or transcriptional regulators during mitosis. ' 2010

International Society for Advancement of Cytometry

� Key termshistone H1; chromatin; cell cycle; mitosis

IN the cell cycle, DNA and protein content are duplicated, and the cell divides in two

in a strict sequential order of events. During prophase, the replicated chromosomes

condense, and the nuclear membrane breaks down in prometaphase. At metaphase,

the chromosomes are aligned at the equator of the mitotic spindle, and the sister

chromatids are segregated to the two poles of the spindle during anaphase. Finally,

the separation is completed during telophase by cytoplasmic division. Impaired cell

cycle control or cell cycle progression may result in cell death or malignant transfor-

mation.

DNA is compacted into chromatin by superhelical wrapping of 146 bp of DNA,

1.65 turns around a histone octamer, consisting of two copies of each core histone,

H2A, H2B, H3, and H4 (1). Histone H1 is a fundamental part of chromatin, located

at or near the entry/exit sites of DNA on the nucleosome and also binds to linker

DNA (2,3). The binding of H1 proteins to chromatin is highly dynamic (4,5). His-

tone H1 stabilizes the nucleosome and is important in compaction of chromatin into

higher order structures (6,7). Moreover, it has been implicated in transcriptional reg-

ulation (8).

There are several subtypes of linker histones; H1.1–H1.5, H1t, H18, os-H1, and

H1.X. They have a highly conserved globular domain, and more variable N- and C-

terminal domains. The function of having multiple subtypes has not yet been clari-

fied, [for review see (9)]. Although there seems to be a noteworthy redundancy

1Division of Cell Biology, Department ofClinical and Experimental Medicine,Link€oping University, SE-58185 Link€oping,Sweden2Division of Medical Microbiology,Department of Clinical and ExperimentalMedicine, Link€oping University, SE-58185Link€oping, Sweden3Division of Experimental Pathology,Department of Clinical and ExperimentalMedicine, Link€oping University, SE-58185Link€oping, Sweden

Received 25 September 2009; RevisionReceived 9 November 2009; Accepted 8December 2009

*Correspondence to: Ingemar Rundquist,Division of Cell Biology, Department ofClinical and Experimental Medicine,Faculty of Health Sciences, Link€opingUniversity, SE-58185 Link€oping, Sweden

Email: ingemar.rundquist@liu.se

Published online 26 January 2010 in WileyInterScience (www.interscience.wiley.com)

DOI: 10.1002/cyto.a.20851

© 2010 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 77A: 478�484, 2010

among the histone H1 subtypes (10), evolutionary data indi-

cate that they posses different functional roles (11). They differ

in affinity for chromatin (12,13), ability to affect gene regula-

tion (14), and are found to localize to different parts of the

nuclei (12). Thus, GFP-labeled H1.1, H1.2, and H1.3 were

mainly found in euchromatin, whereas H1.4 and H1.5 were

more common in heterochromatin (12). Some of the H1 sub-

types have been assigned specific roles such as H1.2, which

participates in apoptosis signaling after induction of DNA

double strand breaks (15).

In addition to the microheterogeneity of H1 histones,

they can also be posttranslationally modified in various ways

(16). Phosphorylation is believed to be the most vital modifi-

cation. H1 histones are phosphorylated at multiple sites in the

N- and C-terminal tails, in a cell cycle dependent manner.

During G1 phase, H1 phosphorylation is low, while addition

of phosphate groups increases during the S- and G2-phases to

reach maximum at the M-phase (17). This is executed by one

or more kinases, for example, CDK2 (18). Some phosphoryla-

tion sites have been mapped, namely H1 phosphorylation at

SP(K/A)K sites in interphase chromatin of CEM cells and

H1.5 phosphorylation at threonine residues during mitosis

(19). The significance of H1 phosphorylation is not yet com-

pletely established and contradictory results have been pub-

lished. Increased H1 phosphorylation has been implicated in

condensation of chromatin into mitotic chromosomes (20),

whereas other studies have related increased phosphorylation

levels to decondensation of chromatin (18,21). H1 phospho-

rylation neutralizes positive charges, which is believed to lead

to increase weakening of the H1–DNA interaction, and thereby

increases the access for various DNA binding proteins like tran-

scription and condensing factors. Phosphorylation may also dis-

rupt the interaction between histone H1 and HP1a, which is

located to heterochromatin, leading to relaxation of chromatin

higher order structure (22). Histone H1 phosphorylation is

thought to play a role in gene regulation (23).

Histone H1 is vital to chromatin structure in mammals

(6). During the cell cycle, chromatin undergoes major struc-

tural changes, and the participation and location of H1 in this

process need further investigation. In regard to the differential

affinities of H1 subtypes for chromatin, the presence of phos-

phorylated H1 histones in the cytoplasm during mitosis

(24,25), and previous results from our group demonstrating

that the affinity of histone H1 for chromatin is dramatically

reduced in mitotic cells (26); our aim was to determine the

intracellular localization of individual H1 subtypes during

mitosis.

MATERIALS AND METHODS

Cell Culture

Normal human foreskin fibroblasts, AG-1523 (NIA Aging

Cell Culture Respository, Institute for Medical Research, Cam-

den, NJ) were cultured in Earle’s minimal essential medium

(EMEM; Gibco BRL, Grand Island, NY) supplemented with

10% fetal bovine serum (FBS; Gibco), penicillin (50 IU/mL)-

streptomycin (50 lg/mL; Gibco), and L-glutamine (2 mM;

Gibco) in a humidified incubator at 378C in an atmosphere of

95% air and 5% CO2. For continuous cell culture, the cells

were grown in 25 cm2 flasks, detached with trypsin (0.25%,

Gibco) when considered confluent (4–7 days), and split at 1:2

ratio. For immunocytochemistry experiments, confluent cul-

tures were split and seeded at 7200 cells/cm2 in 12-well plate

(Costar Corning, NY) containing 16-mm coverslips and

grown for 2 days. Fibroblasts in passages 12–22 were used.

Immunocytochemistry

All chemicals were obtained from Sigma, unless otherwise

indicated. The medium was aspirated, and the cells on cover-

slips were washed twice in phosphate-buffered saline (PBS),

fixed in 4% paraformaldehyde in PBS for 20 min at 48C, andthen in 100% methanol for 20 min at 2208C. After washingin PBS (2 3 5 min), the specimens were placed in incubation

buffer (0.1% saponin and 5% fetal bovine serum in PBS) for

20 min at room temperature. Then, the cells were incubated

with primary antibodies diluted in incubation buffer in a

humidified chamber overnight at 48C. The primary antibodies

(all from Abcam, Cambridge, UK unless otherwise indicated)

were: rabbit polyclonal to histone H1.2 (0.4 lg/mL, ab4086);

histone H1.3 (4 lg/mL, ab24174); histone H1.5 (1 lg/mL,

ab24175); phospho-histone H1 (2 lg/mL, Upstate, Waltham,

MA); and mouse monoclonal to histone H1 (clone AE-4,

ICN, Aurora, Ohio) diluted 1:10. The following day, the cover-

slips were washed in incubation buffer (2 3 5 min) and incu-

bated with secondary antibodies in a humidified chamber in

the dark at room temperature for 1 h. The secondary antibo-

dies were: goat-antirabbit Alexa594 and goat-antimouse

Alexa488 (Molecular Probes, Invitrogen, Eugene, OR) diluted

1:400. Finally, the cells were washed in PBS for 5 min, in

MilliQ-water for 5 min, and mounted on slides with printed

rings (Erie Scientific, Portsmouth, NH) using Vectashield con-

taining 4,6-diamidino-2-phenylindole (DAPI: Vector Labora-

tories, Burlingame, CA). For confocal microscopy, the cover-

slips were fixed to the slides using varnish. Normal rabbit se-

rum (4 lg/mL, X0902, Dako Cytomation, Glostrup,

Denmark) was used as a control for the rabbit polyclonal anti-

bodies and for preparations without primary antibodies.

These preparations showed only weak unspecific staining. Pre-

parations without primary antibodies but with secondary

antibody displayed very weak, unspecific staining.

For each antibody, at least 10 slides were prepared and

examined by fluorescence microscopy. Overview fluorescence

micrographs were collected. Representative images were then

collected for each mitotic phase (on average 4–5 cells/phase)

using confocal microscopy.

Confocal Microscopy

Confocal images were generated using a Bio-Rad Radi-

ance 2100 MP confocal system (Carl Zeiss, Jena Germany)

based on a Nikon Eclipse TE2000U microscope (Nikon,

Tokyo, Japan) and equipped with a PlanApo DicH 603 oil

immersion objective (NA 1.40). Alexa488 was detected using

the 488 nm line from the Argon ion laser and the band-pass

emission filter HG515/30. For detection of Alexa594, the 543

ORIGINAL ARTICLE

Cytometry Part A � 77A: 478�484, 2010 479

nm line of the HeNe laser was used in combination with a

HQ600/50 emission filter. The 780 line of a Mai Tai multipho-

ton laser (Spectra-Physics, Mountain View, CA) was used for

activating the DAPI signal, and emission filter HQ450/80 and

blocking filter E625SP were used. Images consisting of 512 3

512 pixels were generated with a zoom factor of four, giving a

final pixel size of 100 nm. Series of sections were generated

with a distance between sections of 1 lm.

RESULTS

Intracellular Localization of Histone H1 and

Phosphorylated H1

We detected the presence of H1 in chromatin during the

cell cycle stages in normal human fibroblasts using a monoclo-

nal antibody toward histone H1. No cytoplasmic pool of H1

was detected during cell division (Fig. 1).

In contrast, staining for phosphorylated H1 revealed

weak nuclear staining in interphase cells and both chromo-

somal and cytoplasmic staining in dividing cells (Fig. 2). Fig-

ure 2A shows a weakly stained interphase cell and a prophase

cell exhibiting strong nuclear fluorescence. In prometaphase/

metaphase, anaphase, and telophase, both chromosomal and

cytoplasmic staining were prominent (Figs. 2B–2D), indicat-

ing that phosphorylated H1 histones appear both bound to

and released from chromatin.

Histone H1.2 Becomes Translocated from Chromatin

to Cytoplasm During Mitosis

Histone H1.2 was found in chromatin during interphase,

as analyzed by staining with a polyclonal antibody (cell marked

with an arrow in Fig. 3B). This staining pattern remained dur-

ing prophase (Fig. 3A). At prometaphase/metaphase, we

detected H1.2 almost exclusively in the cytoplasm and no colo-

calization with DAPI stained DNA could be found (Fig. 3B).

Such staining pattern largely remained during early anaphase

(Fig. 3C). However, in late anaphase, small amounts of H1.2

could be detected in the condensed chromatin (data not

shown). In telophase, H1.2 was largely found in the still con-

densed chromosomes with minor amounts appearing in the

cytoplasm (Fig. 3D). Occasionally, we observed histone H1.2 in

the vicinity of DNA in a structured manner during early telo-

phase (Fig. 3E) suggesting that H1.2 is relocated to chromatin

during a narrow time window in late anaphase/early telophase.

Histone H1.5 is Partitioned Between Chromatin and

Cytoplasm During Mitosis

Histone H1.5 was detected in chromatin during interphase

(not shown) and prophase (Fig. 4A). In prometaphase/meta-

phase and early anaphase, histone H1.5 was found both in the

condensed chromosomes and the cytoplasm (Figs. 4B and 4C).

In telophase, H1.5 was reappearing in chromatin (Fig. 4D).

Figure 1. Confocal images of histone H1 (green) and DNA stained

by DAPI (blue) in mitotic human fibroblasts. One confocal-generated

cross section is shown. (A) prophase, (B) prometaphase/metaphase,

(C) anaphase, and (D) telophase. [Color figure can be viewed in the

online issue, which is available at www.interscience.wiley.com.]

Figure 2. Confocal images of phosphorylated histone H1 (red)

and DNA stained by DAPI (blue) in mitotic human fibroblasts. One

confocal-generated cross section is shown. (A) prophase, (B) pro-

metaphase/metaphase, (C) anaphase, and (D) telophase. [Color

figure can be viewed in the online issue, which is available at

www.interscience.wiley.com.]

ORIGINAL ARTICLE

480 Histone H1 Subtype Location in Mitosis

Histone H1.3 Remains Bound to Chromatin

During Mitosis

Histone H1.3 was detected in chromatin and chromo-

somes during all cell cycle phases (Fig. 5). Small quantities of

H1.3 could be observed in the cytoplasm during late ana-

phase/early telophase (Fig. 5D).

DISCUSSION

During cell division, substantial changes in chromatin

structure occur, mainly by condensation during early mitosis

and decondensation during the late stages. The participation

of different histone H1 subtypes in these processes is not

fully elucidated (6). It has been shown that embryonic his-

tone H1 is vital for correct chromosome segregation in

Xenopus egg extracts (27). Using subtype-specific antibodies,

we found remarkable differences in the histone H1 subtype

distributions between chromatin and cytoplasm during mi-

tosis. The most prominent finding was a total redistribution

of histone H1.2 from the chromosomes to the cytoplasm af-

ter prophase. Histone H1.5 showed a similar distribution

pattern, but a pool of histone H1.5 remained located in the

mitotic chromosomes. In contrast, we detected histone H1.3

almost solely in the mitotic chromosomes throughout cell

division.

Figure 3. Confocal images of histone H1.2 (red) and DNA stained by DAPI (blue) in mitotic human fibroblasts. (A) prophase (one section),

(B) prometaphase/metaphase (4 consecutive sections with 1 lm distance; another cell in interphase is marked by an arrow), (C) anaphase

(6 consecutive sections with 2 lm distance), (D) telophase (one section), and (E) early telophase (5 consecutive sections with 1 lm dis-

tance). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE

Cytometry Part A � 77A: 478�484, 2010 481

There can be at least two explanations for release of his-

tone H1 from chromatin during mitosis; either a decreased

H1 affinity for chromatin or an active exchange of H1 for

other chromatin binding factors. Phosphorylation of H1,

especially in its C terminal, is supposed to decrease its affinity

for chromatin and increase its mobility in vivo (28) and, inter-

estingly, recent experiments in vitro have shown that linker

histones with fully phosphorylated C-terminal domains

showed reduced affinity for DNA in combination with

increased aggregation capacity of DNA fragments (29). In

interphase cells, H1.2, H1.3, H1.4, and H1.5 are phosphoryl-

ated on serine residues (19,30). During mitosis, H1.5 becomes

additionally phosphorylated only on threonine residues and

the same is probably true for H1.2, H1.3, and H1.4 (19,30). As

we detected phosphorylated histone H1 in the cytoplasm of

metaphase cells, in line with previous observations (24,25,31),

we suggest that such mitosis specific phosphorylation, on one

or multiple sites, decreases H1 affinity and promote the release

of H1.2 and part of H1.5 to the cytoplasm. However, recent

results indicate that the pentaphosphorylated form of H1.5 is

located solely in the chromosomes, whereas lower phosphoryl-

ated forms also occur in the cytoplasm (30). It is possible that

this specific form of H1.5, containing a nonmotive phospho-

threonine in the N-terminus, shows a different binding beha-

vior compared with other forms.

It is also possible that H1 subtypes have differential inher-

ent affinities for chromatin. Using GFP-labeled proteins,

Th’ng et al. (12) found histone H1.2 to have lower affinity for

chromatin than histone H1.5, whereas histone H1.3 had inter-

mediate binding affinity. In another study, where unlabeled

H1 subtypes were used in chromatin competition experi-

ments, H1.2 and H1.5 had lower affinity for chromatin than

H1.3 (13). Most likely, differential H1 affinity for chromatin is

a result of a combination of phosphorylation and inherent af-

finity, leading to various distributions of H1 subtypes between

the chromosomes and the cytoplasm during the cell cycle.

The function of the release of H1.2 and H1.5, and/or

their phosphorylated forms to the cytoplasm during mitosis

may be to facilitate chromatin compaction or transcriptional

repression, by giving access to chromatin condensing factors

Figure 4. Confocal images of histone H1.5 (red) and DNA stained by DAPI (blue) in mitotic human fibroblasts. (A) prophase (one section),

(B) prometaphase/metaphase (4 consecutive sections with 2 lm distance), (C) anaphase (5 consecutive sections with 2 lm distance), and

(D) telophase (one section). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE

482 Histone H1 Subtype Location in Mitosis

or transcriptional regulators. Transcription factors and chro-

matin proteins have been shown to have access to mitotic

chromosomes (32). Histone H1.2 has previously been shown

to have the highest turnover rate compared with the other H1

subtypes. Furthermore, H1.2 expression is not restricted to S-

phase, like the other somatic subtypes (33). Clear is, however,

that relocation of H1.2 cannot be essential for mitotic com-

paction as H1.2 knockout mice develop normally (10). In case

there is such a role, other H1 subtypes may have overlapping

functions.

In contrast to our findings, H1.2-GFP was solely located

on chromatin during the cell cycle in transfected HeLa cells

(34) and NIH 3T3 cells (32). Possibly, there are differences

between the binding and phosphorylation of H1.2-GFP and

the native H1.2 but the most likely explanation for this differ-

ence is that the residence time for H1.2 bound to chromatin is

too short to become captured by formaldehyde crosslinking

during fixation in accordance with recent results (35). A frac-

tion of H1.2 may, therefore, be transiently bound to chroma-

tin with a local concentration enough for detection of its GFP

fluorescence, whereas the dispersed H1.2 in the cytoplasm

escapes detection. In contrast, formaldehyde fixation prevents

the rebinding of fixed proteins to chromatin and thus

enhances the picture of cytoplasmic location of the most

dynamic linker histone fractions. Furthermore, overexpression

may lead to increased binding of H1.2-GFP to chromatin. Pos-

sibly, there are also differences among cell types and between

normal and malignant cells. Alterations in chromatin binding

characteristics between the native protein and its GFP-labeled

counterpart have recently been recognized in HMGN proteins

(36). In addition, the rate of H1.2-GFP recovery after photo-

bleaching was higher at metaphase compared with prophase,

anaphase or telophase, indicating that the rate of dynamic

exchange of H1.2-GFP increased during metaphase (32), in

line with our present results.

Immunocytochemical investigations are marred by

questions of antibody specificity and epitope availability.

When using the monoclonal anti-histone H1 antibody

(clone AE-4), we could not detect any histone H1 in the

cytoplasm during mitosis. The reason for this may be that

this antibody does not detect all subtypes, all phosphoryl-

ated variants, or cytoplasmic H1 due to conformational

changes in the H1 structure. In agreement with our results,

this antibody was previously shown unable to detect cyto-

plasmic (possibly phosphorylated) H1 (24). There is a possi-

bility that, for the same reasons, cytoplasmic H1.3 cannot

be detected by the H1.3 antibody. However, because some

cytoplasmic H1.3 was detected in late anaphase/early telo-

phase cells, we conclude the antibody to be able to detect

cytoplasmic protein. Although the antibody is specific for

H1.3, some cross-reactivity has been noted using this anti-

body according to the manufacturer. The probability that

H1.2 is undetected in the highly condensed mitotic chromo-

somes due to their compaction is low, since H1, phospho-

H1, H1.5, and H1.3 antibodies had access to their epitopes,

and there were high levels of cytoplasmic H1.2 and H1.5.

None of the subtype-specific antibodies is supposed to cross

react with other subtypes as shown by the supplier by the

use of recombinant subtypes in Western blots. Moreover, the

specificity of the H1.2 antibody has been shown by blocking

with H1.2 peptide (37).

In conclusion, our data provide further evidence for the

functional difference of H1 subtypes. An interesting finding is

that certainH1 subtypes are kept at their chromatin-binding sites

during mitosis, whereas others are dispersed in the cytoplasm

and later redistributed to chromatin, allowing the exchange of

some H1 subtypes between chromatin regions in the daughter

cells. Probably, the dissociation of H1.2 and H1.5 from chroma-

tin is induced by phosphorylation, because phosphorylated H1

appeared in the cytoplasm during mitosis. In addition, differen-

tial subtype affinity for chromatin may contribute to the ability

of the protein to relocate to the cytoplasm. It is possible that

some H1, or phosphorylated H1, located in the cytoplasm is

necessary for mitotic progression regardless of their identity.

Furthermore, the release of histone H1 from certain chromatin

regionsmay allow the exchange of various chromatin remodeling

factors or transcription factors between specific chromatin bind-

ing sites during mitosis effecting the epigenetic reprograming of

daughter cells as discussed in a recent review (38). However, fur-

ther investigations are needed to explore the biological function

connected to the observed translocation of histone H1 subtypes

during themitotic process.

Figure 5. Confocal images of histone H1.3 (red) and DNA stained

by DAPI (blue) in mitotic human fibroblasts. One confocal-generated

cross section is shown. (A) prophase, (B) prometaphase/metaphase,

(C) anaphase, and (D) telophase. [Color figure can be viewed in the

online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE

Cytometry Part A � 77A: 478�484, 2010 483

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ORIGINAL ARTICLE

484 Histone H1 Subtype Location in Mitosis