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CELL STRUCTURE AND FUNCTION 32: 57–67 (2007)

© 2007 by Japan Society for Cell Biology

Functional Domain Analysis of Human HP1 Isoforms in Drosophila

Masaki Kato1,2,3, Yasuko Kato1,4, Miki Nishida1, Tomohiro Hayakawa6,7, Tokuko Haraguchi6,

Yasushi Hiraoka6, Yoshihiro H. Inoue4,5, and Masamitsu Yamaguchi1,4*

1Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585,

Japan, 2Venture Laboratory, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan, 3Present Address: Department of Biology, Stanford School of Medicine, 279 Campus Dr., Stanford,

CA94305, USA, 4Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku,

Kyoto 606-8585, Japan, 5Drosophila Genetic Resource Center, Kyoto Institute of Technology, Sagaippongi,

Ukyo-ku, Kyoto 616-8354, Japan, 6Kansai Advanced Research Center, National Institute of Information and

Communications Technology, 588-2 Iwaoka-cho, Nishi-ku, Kobe 651-2492, Japan and 7Present address:

Laboratory for Chromatin Dynamics, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minami-

machi, Chuo-ku, Kobe, Hyogo 650-0047, Japan

ABSTRACT. Three subtypes of HP1, a conserved non-histone chromosomal protein enriched in heterochromatin,

have been identified in humans, HP1α, β and γ. In the present study, we utilized a Drosophila system to

characterize human HP1 functions. Over-expression of HP1β in eye imaginal discs caused abnormally patterned

eyes, with reduced numbers of ommatidia, and over-expression of HP1γ in wing imaginal discs caused abnormal

wings, in which L4 veins were gapped. These phenotypes were specific to the HP1 subtypes and appear to reflect

suppressed gene expression. To determine the molecular domains of HP1 required for each specific phenotype,

we constructed a series of chimeric molecules with HP1β and HP1γ. Our data show that the C-terminal chromo

shadow domain (CSD) of HP1γ is necessary for HP1γ-type phenotype, whereas for the HP1β-type phenotype both

the chromo domain and the CSD are required. These results suggest human HP1 subtypes use different domains

to suppress gene expression in Drosophila cells.

Key words: heterochromatin/chromo domain/chromo shadow domain/transgenic fly

Introduction

Heterochromatin protein 1 (HP1) is a major constituent of

heterochromatin and plays a key role in its formation and

maintenance (Eissenberg and Elgin, 2000; Grewal and

Elgin, 2002). HP1 is highly conserved from yeast to human

(Li et al., 2002). HP1 was first identified as a gene product

of an allele of Su(var)2-5 in Drosophila melanogaster

(James and Elgin, 1986; Eissenberg et al., 1990). In

Drosophila, it is localized in pericentric and telomeric

heterochromatin and plays a role in condensation and seg-

regation of chromosomes and telomere capping (Kellum

and Alberts, 1995; Fanti et al., 2003; Perrini et al., 2004).

Recruitment of HP1 to certain sites of the genome involves

interaction with various chromatin components and depends

on methylation of lysine 9 on histone H3 (H3K9) (Petters et

al., 2003; Thiru et al., 2004; Stewart et al., 2005). Both

trimethylated H3K9 and HP1 appear to be important for

establishing and maintaining domains of heterochromatin

(Sims et al., 2003). In human cells, three subtypes of HP1

have been identified and designated as HP1α, HP1β and

HP1γ (Singh et al., 1991; Saunders et al., 1993; Ye and

Worman, 1996). All of these HP1 family proteins consist of

two highly conserved regions, the N-terminal chromo

domain (CD) and the structurally related C-terminal chromo

shadow domain (CSD), connected by a hinge region

(Eissenberg and Elgin, 2000). The CSD motif appears

unique to the HP1 family. In contrast, the CD motif is also

found in Su(var)3-9 and Polycomb family proteins (Jones et

al., 2000). It is reported that HP1 family members interact

*To whom correspondence should be addressed: Masamitsu Yamaguchi,Department of Applied Biology, Kyoto Institute of Technology, Matsu-gasaki, Sakyo-ku, Kyoto 606-8585, Japan.

Tel: +81–75–724–7781, Fax: +81–75–724–7760E-mail: myamaguc@kit.ac.jp

Abbreviations: HP1, heterochromatin protein 1; CSD, chromoshadow domain;CD, chromodomain; H3K9, histone H3 lysine 9; DAPI, 4,6-diamidino-2-phenylindole; CKII, casein kinase II.

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M. Kato et al.

with various other proteins through CSD (Le Douarin et

al., 1996; Murzina et al., 1999; Li et al., 2002; Vassallo and

Tanese, 2002), while the hinge region appears to be impor-

tant for nuclear targeting (Smothers and Henikoff, 2001)

and RNA-binding (Muchardt et al., 2002). In addition, CD

is known to mediate the interaction of HP1 family proteins

with H3K9 (Bannister et al., 2001; Lachner et al., 2001).

HP1α and β are concentrated in pericentric hetero-

chromatin, but HP1γ may exhibit differential localization,

always with some accumulation in pericentromeric hetero-

chromatin (Minc et al., 1999; Minc et al., 2000). HP1β and

γ can also be found in euchromatic regions (Minc et al.,

1999; Minc et al., 2000; Nielsen et al., 2001a), where they

are presumably involved in gene repression (Nielsen et al.,

2001b; Li et al., 2002). Intracellular localization of HP1 sub-

types dynamically changes during the cell cycle (Hayakawa

et al., 2003). HP1β is replaced at the centromere by HP1α

as the cell enters metaphase (Hayakawa et al., 2003). The

N-terminal region of HP1β, including the CD, is respon-

sible for localization to interphase centromeres and the C-

terminal region, including the CSD, is responsible for local-

ization to metaphase centromeres (Hayakawa et al., 2003).

However, it has yet to be determined in detail whether dif-

ferences in sub-cellular localization of HP1 family proteins

relate to their functions in tissues of multi-cellular organisms.

Human HP1α, HP1β and HP1γ have been successfully

expressed in transgenic Drosophila and are associated with

heterochromatin in Drosophila chromosomes (Ma et al.,

2001; Norwood et al., 2004). Ectopic expression of HP1α

and HP1γ significantly enhanced heterochromatic silencing

in Drosophila, but that of HP1β failed (Ma et al., 2001;

Norwood et al., 2004). In addition, expression of HP1α

rescued the lethality of homozygous Su(var)2-5 mutants

lacking HP1 (Norwood et al., 2004). These results indicate

that at least some aspects of heterochromatic structure are

highly conserved throughout the evolution of eukaryotes.

In the present study, we utilized a Drosophila system to

characterize actions of human HP1α, HP1β and HP1γ when

over-expressed in different tissues and developmental

stages. Our results suggest that HP1β and γ each cause tis-

sue-specific phenotypes due to suppressed gene expression.

To determine the molecular domains of HP1 required for

these actions, we constructed a series of chimeric molecules

between HP1β and HP1γ. Our data show that CSD of HP1γ

is necessary for the HP1γ-type phenotype (wing phenotype),

while both CD and CSD are required for the HP1β-type

phenotype (eye phenotype). These results suggest human

HP1 subtypes use different domains to suppress gene expres-

sion in Drosophila cells.

Materials and Methods

Fly stocks

Fly stocks were maintained at 25°C on standard food. The Canton

S fly was used as the wild-type strain. The GMR-GAL4 line (num-

ber 16) was described previously (Takahashi et al., 1999). The

following enhancer-GAL4 lines were kindly provided by Dr N.

Dyson (MGH Cancer Center): decapentaplegic-GAL4 (dpp-GAL4),

sevenless-GAL4 (sev-GAL4), eyeless-GAL4 (ey-Gal4), 32B-GAL4,

71B-GAL4, Hsp70-GAL4 (hs-GAL4), engrailed-GAL4 (en-GAL4),

apterous-GAL4 (ap-GAL4), twist-GAL4, Act88F-GAL4, veinlet-

GAL4 (ve-GAL4), wingless-GAL4 (wg-GAL4), 3735-GAL4, 2689-

GAL4, Sg-GAL4.

Plasmids

To construct the plasmids pUAST-HP1α, pUAST-HP1β, pUAST-

HP1γ and pUAST-dHP1a, cDNA fragments of human HP1α,

HP1β, HP1γ and Drosophila HP1a (dHP1a) were obtained by PCR

using appropriate oligonucleotide primers with EcoRI and XbaI

linkers and HP1α, HP1β, HP1γ and dHP1a cDNAs as templates.

The PCR products were digested with EcoRI and XbaI and

inserted into the pUAST vector. To construct chimera HP1s, the

following regions were used: BG2, amino acids 1 to 116 of HP1β

and amino acids 111 to 173 of HP1γ; BG1, amino acids 1 to 76 of

HP1β amino acids 76 to 173 of HP1γ; GB2, amino acids 1 to 110

of HP1γ and amino acids 117 to 185 of HP1β; and GB1, amino acids

1 to 75 of HP1γ and amino acids 77 to 185 of HP1β (Hayakawa et

al., 2003).

Establishment of transgenic flies

Transgenic lines for pUAST-HP1α, -HP1β, -HP1γ, -dHP1a and

various chimera HA-tagged HP1s were generated by P element-

mediated germ line transformation as described earlier (Spradling,

1986). F1 transformants were selected on the basis of white eye

color rescue (Robertson et al., 1988). Several independent lines for

each construct were established and confirmed to exhibit essen-

tially the same phenotype.

Immunostaining of polytene chromosomes

Salivary glands from third instar larvae were dissected in 0.7%

NaCl, fixed in 45% acetic acid and squashed in 33.3% lactic acid/

50% acetic acid. Squashes were incubated with mouse monoclonal

antibody specific for HP1β and HP1γ (Euromedex, Souffelweyer-

shim, France) at 1:1,000 dilution, or culture supernatant of hybri-

doma cells-producing mouse anti-HA monoclonal antibody (sup-

plied by M. Inagaki, Aichi Cancer Center Research Institute)

(1:100) at 25°C for 2 h. After extensive washing with PBS contain-

ing 0.2% Tween 20 and 1% BSA, samples were incubated at 25°C

for 2 h with anti-mouse IgG conjugated with Alexa488 (Molecular

Probes [Invitrogen], Carlsbad, CA, USA) at 1:400 dilution. Slides

were mounted in Vectashield (Vector Laboratory, Peterborough,

Analysis of Human HP1 Isoforms in Drosophila

59

England) with 4,6-diamidino-2-phenylindole (DAPI) (0.05 µg/ml)

for microscopic observation. The microscopic images were

obtained by Olympus BX-50 microscope equipped with cooled

CCD camera (Hamamatsu Photo., Shizuoka, Japan).

Western immunoblot analysis

To perform Western blots, third instar larvae were homogenized in

SDS sample buffer and heated at 95°C for 10 min. Proteins were

applied to SDS-polyacrylamide gels containing 12% acrylamide.

After SDS-PAGE, proteins were transferred to polyvinylidene dif-

luoride membranes (Bio-Rad, Hercules, CA, USA). The blotted

membranes were incubated with mouse monoclonal anti-HP1β

antibody (Euromedex, Souffelweyershim, France) at 1:2,000

dilution, mouse monoclonal anti-HP1γ antibody (Euromedex,

Souffelweyershim, France) at 1:4,000 dilution or mouse mono-

clonal anti-α tubulin antibody (Sigma, St. Louis, MO, USA) at

1:2,000 dilution. After washing, the membranes were incubated

with HRP-conjugated anti-mouse IgG (GE Healthcare, Bucking-

hamshire, England). Visualization was carried out using ECL (GE

Healthcare, Buckinghamshire, England) and images were analyzed

with a Lumivision Pro HSII image analyzer (Aisin Seiki, Aichi,

Japan).

Over-expression experiments

Transgenic lines carrying various UAS-HP1s or UAS-dHP1a were

crossed with the different GAL4-driver lines described above and

raised at 28°C. For heat shock induction of proteins, third instar

larvae were heat-shocked for 1 h at 37°C, recovered for 2 h at

25°C, then subjected for either immunostaining or Western immu-

noblot analyses.

Results

Functional differences in human HP1 isoforms

In order to examine the effects of human HP1 isoforms on

Drosophila development, we generated transgenic fly lines

carrying UAS-HP1α, UAS-HP1β or UAS-HP1γ and crossed

them with fifteen different GAL4-driver strains. HP1β and

HP1γ generated abnormality in specific tissues (Table I).

Over-expression of HP1β with the ey-GAL4 driver caused

abnormally patterned eyes (100% penetrance) (Fig. 1A),

with an approximately 50% reduction in the number of

ommatidia, and an abnormal head phenotype (Fig. 1B)

(the HP1β-type phenotype). In contrast, no changes were

observed with over-expression of HP1α and γ under control

of the ey-GAL4 driver (Table I).

With 32B-GAL4/UAS-HP1γ and en-GAL4/UAS-HP1γ,

flies exhibited abnormal wings, in which wing vein L4

was gapped (32B-GAL4: male 100% (n=32), female 19.1%

(n=62), en-GAL4: male 40.9% (n=44), female 60% (n=60))

(Fig. 1C, arrows) (the HP1γ-type phenotype). In addition, a

curled wing phenotype was observed with dpp-GAL4/UAS-

HP1γ (data not shown). In contrast, over-expression of

HP1α and β with the 32B-GAL4, the en-Gal4 or the dpp-

Table I. SUMMARY OF EFFECT OF hHP1β, γ EXPRESSION WITH EACH GAL4 DRIVER LINES

GAL4 line Chromosome linkagePhenotype

hHP1α hHP1β hHP1γ dHP1a

GMR X — — — ND

sevenless II — — — ND

eyeless II — small eye — ND

en II — — vein wing

32B III — — vein pupa lethal

71B III — — — ND

wg III — — — ND

ve II — — — ND

3735 X — — — ND

31 III — — — ND

Hsp70 III — — — ND

2689 X — — — ND

dpp II — wing — wing

twist X — — — ND

act88F II — — — ND

— No detectable phenotype, ND: Not determined

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M. Kato et al.

GAL4 did not have any discernible influence on the phe-

notype (Table I). These results suggest that human HP1

isoforms have functional differences in Drosophila, as

observed with cultured mammalian cells (Hayakawa et al.,

2003).

Distribution patterns of HP1β and HP1γ on polytene chromosomes

For some reason, no alteration was observed on ectopic

expression of HP1α with any of the GAL4 drivers used. We

therefore focused on differences between HP1β and HP1γ

by immunostaining salivary gland polytene chromosomes

from transgenic larvae (hs-GAL4/UAS-HP1β or hs-GAL4/

UAS-HP1γ) with HP1β- and HP1γ-specific antibodies

(Figs. 2A and 3A). Neither the anti-HP1β nor the anti-HP1γ

antibodies are cross-reactive to Drosophila HP1 family pro-

teins, since neither of these antibodies detected the endoge-

nous HP1 family proteins on the yw salivary gland polytene

chromosomes in immunostaining (Figs. 2A and 3A, g to I)

and Western immunoblot analyses (Figs. 2B and 3B). HP1β

and HP1γ were both associated primarily with heterochro-

matic chromocenters (Figs. 2A and 3A, arrows), localized

in the telomeric regions of the chromosomes (Figs. 2A and

3A, arrowheads).

While both were found in euchromatic regions, the

Fig. 1. Over-expression of HP1β results in small eyes and an abnormal head phenotype while that of HP1γ results in loss of the L4 wing vein phenotype.

(A) Scanning electron micrographs of adult compound eyes with HP1β over-expressed using the eyeless-GAL4 driver. UAS-HP1β/CyO (upper), eyeless-

GAL4/UAS-HP1β (bottom). (B) abnormal head phenotype. (a) UAS-HP1β/CyO, (b) eyeless-GAL4/UAS-HP1β. (C) HP1γ was over-expressed with the

following GAL4 drivers in wing discs. (a) wild type, (b) en-GAL4/UAS-HP1γ, (c) 32-B GAL4/UAS-HP1γ Arrows indicate loss of L4 wing veins.

A B

Ca b c

b

a

Analysis of Human HP1 Isoforms in Drosophila

61

HP1β-staining was in sharp bands (Fig. 2A, d to f). In

contrast, HP1γ-staining was more diffuse (Fig. 3A, d to f).

To test the possibility that HP1γ does not bind tightly to

chromatin or chromosome, we decreased the amount of

HP1γ expression in salivary gland cells. Without heat shock

induction with the hs-GAL4 driver, only 38% of HP1γ was

expressed in compared with the heat-shocked samples (Fig.

3B). Under these conditions, HP1γ was mainly localized at

Fig. 2. Localization of HP1β in Drosophila polytene chromosomes. (A) Polytene chromosomes of yw (g to i) or transgenic third instar larvae carrying the

HP1β gene (hs-GAL4/UAS-HP1β with no heat shock (hs-, a to c) or after incubation for 1 h at 37°C (hs+, d to f) were processed for indirect

immunofluorescence with anti-HP1β antibodies (a, d and g). DNA was counterstained with DAPI (b, e and h). Merged images are also shown (c, f and i).

Arrows indicate chromocenters and arrowheads indicate the telomeric regions of the chromosomes. (B) Western immunoblot of third instar larvae protein

extracts from Canton S (CS) and hs-GAL4/UAS-HP1β with (hs+) or without (hs-) heat shock for 1 h at 37°C. The blots were probed with anti-HP1β and

anti-α tubulin antibodies. The arrowhead indicates HP1β band.

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M. Kato et al.

chromocenters (Fig. 3A, a to c). With heat shock induction,

HP1γ localization was expanded through the euchromatic

region (Fig. 3A, d to f). In contrast, no difference of HP1β

localization on the chromosomes was observed with and

without heat shock and only the signal intensity changed

(Fig. 2A, a and d), despite the 2.6 fold increase of HP1β

after the heat shock (Fig. 2B). These data suggest that HP1β

and HP1γ localize on chromosomes differently and bind in

different ways.

The CSD of HP1γ is required for the HP1γ-specific phenotype

Although HP1 isoforms share significant similarities in

their amino acid sequences (Fig. 4A), their over-expression

using several GAL4 drivers in Drosophila here resulted in

Fig. 3. Localization of HP1γ in Drosophila polytene chromosomes. (A) Polytene chromosomes of transgenic third instar larvae carrying the HP1γ gene

(hs-GAL4/UAS-HP1γ with no heat shock (hs-, a-c) or after incubation for 1 h at 37°C (hs+, d to f) were processed for indirect immunofluorescence with

anti-HP1γ antibodies (a, d and g). DNA was counterstained with DAPI (b, e and h). Merged images are also shown (c, f and i). Arrows indicate

chromocenters and arrowheads indicate the telomeric regions of the chromosomes. (B) Western immunoblot of third instar larvae protein extracts from

Canton S (CS) and hs-GAL4/UAS-HP1γ with (hs+) or without (hs-) heat shock for 1 h at 37°C. The blots were probed with anti-HP1γ and anti-α tubulin

antibodies. The arrowhead indicates HP1γ band.

Analysis of Human HP1 Isoforms in Drosophila

63

different phenotypes. Thus, we determined the necessary

molecular domains of HP1 required for each specific pheno-

type. To this end, we constructed a series of chimeric mole-

cules between HP1β and HP1γ (Fig. 5A). Over-expression

of BG1 (HP1β CD+HP1γ hinge and CSD) and BG2 (HP1β

CD and hinge+HP1γ CSD) using 32B-GAL4 driver resulted

in the HP1γ-specific wing phenotype (BG1; males 55.8%

(n=43), females 30.3% (n=79), BG2; males 82.4% (n=34),

females 23.5% (n=51)) (Fig. 5B, a and b). In contrast, no

detectable wing phenotype in adult external morphology

was observed on over-expression of GB1 (HP1γ CD and

HP1β hinge+CSD) or GB2 (HP1γ CD+hinge and HP1β

CSD) using 32B-GAL4 driver (Fig. 5B, c and d). These

results suggest that CSD of HP1γ is required for the HP1γ-

specific wing phenotype.

Next we examined the molecular domains of HP1

required for the HP1β-specific eye phenotype induced by

over-expression with eyeless-GAL4. However, expression

of chimeric proteins resulted in no detectable phenotype

(Fig. 5A). These results suggest that the whole of HP1β is

required for the HP1β-specific eye phenotype, although the

possibility remains that the hinge domain is also indispens-

able for it can be excluded.

Fig. 4. (A) Sequence alignment and schematic representation of human HP1s and Drosophila HP1a. Amino acid sequences of human HP1α, β, γ and

dHP1a are aligned. Black boxes and asterisks indicate identical residues. Conserved and semi-conserved substitutions are indicated by gray boxes, double

dots and single dots, respectively. (B) Comparison of CSD sequences between HP1β and γ. HP1β Ser-172 and Ser-175 are indicated by closed and open

triangles, respectively.

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M. Kato et al.

Molecular domains in HP1 necessary for their localization on polytene chromosomes in Drosophila

One possible explanation for the above-described results is

that the specific localizations of HP1 isoforms cause HP1

isoform-specific phenotypes. To address this question, we

examined the localization of HP1 chimeric molecules on

polytene chromosomes. Over-expression was attained using

the hs-GAL4 driver. Immunostaining with anti-HA antibod-

ies demonstrated localization of all chimeric HP1 proteins

in chromocenter and telomeric regions (Fig. 6). However,

the sharp and restricted bands in euchromatic regions spe-

cifically observed with HP1β (HP1β-specific localization)

(Fig. 2A) were not apparent with any of the chimeric pro-

teins (Fig. 6). These results again suggest that the whole

region of HP1β is required for the HP1β-specific localiza-

tion on polytene chromosomes. In addition, we could not

observe any variation in the localization of chimeric pro-

teins on polytene chromosomes (Fig. 6). The localization of

HP1 chimeric proteins therefore does not explain the HP1γ-

specific phenotype.

Fig. 5. Schematic representation of chimera HP1β and HP1γ proteins and summary of effects of chimera HP1 expression with ey-GAL4 and 32B-Gal4

driver lines (A). (B) Chimera HP1 was over-expressed with the following GAL4 drivers in wing discs. (a)32B-GAL4/UAS-BG1, (b)32B-GAL4/UAS-BG2,

(c)32B-GAL4/UAS-GB1, and (d)32B-GAL4/UAS-GB2. Arrows indicate loss of L4 wing veins.

Analysis of Human HP1 Isoforms in Drosophila

65

Discussion

Localization of human HP1 on Drosophila polytene chromosomes

While HP1γ is predominantly linked to heterochromatic

chromocenters in Drosophila polytene chromosomes under

weak expression conditions, it becomes more diffusely

located among euchromatin regions, unlike HP1β (Fig. 3A).

One possible mechanism by which HP1 associates with

chromosomes is through an interaction of its CD with meth-

ylated H3K9, due to a modification generated by the

Su(var)3-9 (Bannister et al., 2001; Lachner et al., 2001;

Jacobs and Khorasanizadeh, 2002). This interaction is con-

sistent with the histone code hypothesis stating that histone

tail modifications serve as specific recognition markers for

chromosomal proteins (Jenuwein and Allis, 2001).

However, other experimental data suggest that HP1 uses

alternative mechanisms for localization in non-centric

regions (Li et al., 2002). On Drosophila polytene chromo-

somes, methylated H3K9 and HP1 do not exhibit complete

co-localization within euchromatin (Cowell et al., 2002; Li

et al., 2002). Furthermore, a mutation in CD that abolishes

interactions with methylated H3K9 does not eliminate

association at telomeres (Fanti et al., 1998; Jacobs and

Khorasanizadeh, 2002). In the present study, none of the

chimera HP1s exhibited HP1β-specific localization on

Drosophila polytene chromosomes, suggesting that both of

CD and CSD are required for site determination.

What are the functional differences between HP1 subtypes?

In our chimera HP1γ experiment, CSD was required for the

HP1γ-specific wing phenotype. A large number of CSD

interaction partners contain a PXVXL motif, including:

TIF1α, TIF1β and TAFII130, which regulate transcription;

IDN3, which may play a role in chromosome segregation;

and the large subunit of CAF1, which contributes to

nucleosome assembly during DNA replication and repair

(Le Douarin et al., 1996; Murzina et al., 1999; Vassallo and

Tanese, 2002). The functions of many of these proteins rely

on their interactions with CSD, linking them to H3K9 meth-

ylation and the establishment/maintenance of gene silencing.

Surprisingly, however, comparison of CSD amino acid

sequences between HP1β and HP1γ showed only limited

variation (Fig. 4B). In Drosophila, casein kinase II (CKII)

phosphorylation of at least two distinct regions of dHP1a

affects targeting to heterochromatin: Ser-15 in the N-terminal

region as well as Ser-169 and Ser-172 at CSD (all HP1β

numbering) (Zhao and Eissenberg, 1999). Ser-172 is within

a consensus CKII site (S/TXXD/E), and it is thought that

Ser-169 becomes a consensus CKII site following phospho-

rylation of Ser-172 (Zhao and Eissenberg, 1999). In HP1β,

there are potentially three such interdependent phosphoryla-

tion sites, at Thr-169, Ser-172 and Ser-175. The last is miss-

ing in HP1γ, so that it could conceivably be responsible for

the difference in affinity for CSD interactions and thus phe-

notypic differences in Drosophila tissues. Further analysis

is necessary to clarify this point.

Fig. 6. Localization of chimera HP1 in Drosophila polytene chromosomes. Polytene chromosomes from salivary glands of transgenic larvae expressing

HA-BG1 (a and b), HA-BG2 (c and d), HA-GB1 (e and f), and HA-GB2 (g and h) are shown. Staining with an anti-HA antibody (a, c, e and g) and merged

images with DAPI-staining (b, d, f and h).

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M. Kato et al.

Acknowledgements.We thank Dr M Moore (Aichi Cancer Center Research

Institute) for comments on the English language in the manuscript. This

study was partially supported by grants from the Ministry of Education,

Culture, Sports, Science and Technology, of the Japanese Government.

References

Bannister, A.J., Zegerman, P., Partridge, J.F., Miska, E.A., Thomas, J.O.,

Allshire, R.C., and Kouzarides, T. 2001. Selective recognition of

methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature,

410: 120–124.

Cowell, I.G., Aucott, R., Mahadevaiah, S.K., Burgoyne, P.S., Huskisson,

N., Bongiorni, S., Prantera, G., Fanti, L., Pimpinelli, S., Wu, R., Gilbert,

D.M., Shi, W., Fundele, R., Morrison, H., Jeppesen, P., and Singh, P.B.

2002. Heterochromatin, HP1 and methylation at lysine 9 of histone H3

in animals. Chromosoma, 111: 22–36.

Eissenberg, J.C. and Elgin, S.C. 2000. The HP1 protein family: getting a

grip on chromatin. Curr. Opin. Genet. Dev., 10: 204–210.

Eissenberg, J.C., James, T.C., Foster-Hartnett, D.M., Hartnett, T., Ngan,

V., and Elgin, S.C. 1990. Mutation in a heterochromatin-specific

chromosomal protein is associated with suppression of position-effect

variegation in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA, 87:

9923–9927.

Fanti, L., Berloco, M., Piacentini, L., and Pimpinelli, S. 2003. Chromo-

somal distribution of heterochromatin protein 1 (HP1) in Drosophila: a

cytological map of euchromatic HP1 binding sites. Genetica, 117: 135–

147.

Fanti, L., Giovinazzo, G., Berloco, M., and Pimpnelli, S. 1998. The

heterochromatin protein 1 prevents telomere fusions in Drosophila.

Mol. Cell, 2: 527–538.

Grewal, S.I. and Elgin, S.C. 2002. Heterochromatin: new possibilities for

the inheritance of structure. Curr. Opin. Genet. Dev., 12: 178–187.

Hayakawa, T., Haraguchi, T., Masumoto, H., and Hiraoka, Y. 2003. Cell

cycle behavior of human HP1 subtypes: distinct molecular domains of

HP1 are required for their centromeric localization during interphase and

metaphase. J. Cell Sci., 116: 3327–3338.

Jacobs, S.A. and Khorasanizadeh, S. 2002. Structure of HP1 chromo-

domain bound to a lysine 9-methylated histone H3 tail. Science, 295:

2080–2083.

James, T.C. and Elgin, S.C. 1986. Identification of a nonhistone

chromosomal protein associated with heterochromatin in Drosophila

melanogaster and its gene. Mol. Cell. Biol., 6: 3862–3872.

Jenuwein, T. and Allis, C.D. 2001. Translating the histone code. Science,

293: 1074–1080.

Jones, D.O., Cowell, I.G., and Singh, P.B. 2000. Mammalian chromo-

domain proteins: their role in genome organization and expression.

Bioessays, 22: 124–137.

Kellum, R. and Alberts, B.M. 1995. Heterochromatin protein 1 is required

for correct chromosome segregation in Drosophila embryos. J. Cell Sci.,

108 (Pt 4): 1419–1431.

Lachner, M., O’Carroll, D., Rea, S., Mechtler, K., and Jenuwein, T. 2001.

Methylation of histone H3 lysine 9 creates a binding site for HP1.

Nature, 410: 116–120.

Le Douarin, B., Nielsen, A.L., Garnier, J.M., Ichinose, H., Jeanmougin, F.,

Losson, R., and Chambon, P. 1996. A possible involvement of TIF1

alpha and TIF1 beta in the epigenetic control of transcription by nuclear

receptors. EMBO J., 15: 6701–6715.

Li, Y., Kirschmann, D.A., and Wallrath, L.L. 2002. Does heterochromatin

protein 1 always follow code? Proc. Natl. Acad. Sci. USA, 99 Suppl 4:

16462–16469.

Ma, J., Hwang, K.K., Worman, H.J., Courvalin, J.C., and Eissenberg, J.C.

2001. Expression and functional analysis of three isoforms of human

heterochromatin-associated protein HP1 in Drosophila. Chromosoma,

109: 536–544.

Minc, E., Allory, Y., Worman, H.J., Courvalin, J.C., and Buendia, B. 1999.

Localization and phosphorylation of HP1 proteins during the cell cycle

in mammalian cells. Chromosoma, 108: 220–234.

Minc, E., Courvalin, J.C., and Buendia, B. 2000. HP1gamma associates

with euchromatin and heterochromatin in mammalian nuclei and chro-

mosomes. Cytogenet. Cell Genet., 90: 279–284.

Muchardt, C., Guilleme, M., Seeler, J.S., Trouche, D., Dejean, A., and

Yaniv, M. 2002. Coordinated methyl and RNA binding is required for

heterochromatin localization of mammalian HP1alpha. EMBO Rep., 3:

975–981.

Murzina, N., Verreault, A., Laue, E., and Stillman, B. 1999. Heterochro-

matin dynamics in mouse cells: interaction between chromatin assembly

factor 1 and HP1 proteins. Mol. Cell, 4: 529–540.

Nielsen, A.L., Oulad-Abdelghani, M., Ortiz, J.A., Remboutsika, E.,

Chambon, P., and Losson, R. 2001a. Heterochromatin formation in

mammalian cells: interaction between histones and HP1 proteins. Mol.

Cell, 7: 729–739.

Nielsen, S.J., Schneider, R., Bauer, U.M., Bannister, A.J., Morrison, A.,

O’Carroll, D., Firestein, R., Cleary, M., Jenuwein, T., Herrera, R.E., and

Kouzarides, T. 2001b. Rb targets histone H3 methylation and HP1 to

promoters. Nature, 412: 561–565.

Norwood, L.E., Grade, S.K., Cryderman, D.E., Hines, K.A., Furiasse, N.,

Toro, R., Li, Y., Dhasarathy, A., Kladde, M.P., Hendrix, M.J.,

Kirschmann, D.A., and Wallrath, L.L. 2004. Conserved properties of

HP1Hsα. Gene, 7: 37–46.

Perrini, B., Piacentini, L., Fanti, L., Altieri, F., Chichiarelli, S., Berloco,

M., Turano, C., Ferraro, A., and Pimpinelli, S. 2004. HP1 controls

telomere capping, telomere elongation, and telomere silencing by two

different mechanisms in Drosophila. Mol. Cell, 15: 467–476.

Petters, A.H., Kubicek, S., Mechtler, K., O’Sullivan, R.J., Derijck, A.A.,

Perez-Burgos, L., Kohlmaier, A., Opravil, S., Tachibana, M., Shinkai,

Y., Martens, J.H., and Jenuwein, T. 2003. Partitioning and plasticity of

repressive histone methylation states in mammalian chromatin. Mol.

Cell, 12: 1577–1589.

Robertson, H.M., Preston G.R., Philips, R.W., Johnson-Schlitz, D.M.,

Benz, W.K., and Engels, W.R. 1988. A stable genomic source of P-

element transposase in Drosophila melanogaster. Genetics, 118: 461–

470.

Saunders, W.S., Chue, C., Goebl, M., Craig, C., Clark, R.F., Powers, J.A.,

Eissenberg, J.C., Elgin, S.C., Rothfield, N.F., and Earnshaw, W.C. 1993.

Molecular cloning of a human homologue of Drosophila heterochromatin

protein HP1 using anti-centromere autoantibodies with anti-chromo

specificity. J. Cell Sci., 104: 573–582.

Sims, R.J., Nishioka, K., and Reinberg, D. 2003. Histone lysine methylation:

a signature for chromatin function. Trends Genet., 19: 629–639.

Singh, P.B., Miller, J.R., Pearce, J., Kothary, R., Burton, R.D., Paro, R.,

James, T.C., and Gaunt, S.J. 1991. A sequence motif found in a

Drosophila heterochromatin protein is conserved in animals and

plants. Nucleic Acids Res., 19: 789–794.

Smothers, J.F. and Henikoff, S. 2001. The hinge and chromo shadow

domain impart distinct targeting of HP1-like proteins. Mol. Cell. Biol.,

21: 2555–2569.

Spradling, A.C. 1986. P-element-mediated transformation. In: Roberts,

D.B. (Ed.), Drosophila: a practical approach. IRL Press, Oxford,

pp.175–197.

Stewart, M.D., Li, J., and Wong, J. 2005. Relationship between histone H3

lysine 9 methylation, transcription repression, and heterochromatin

protein 1 recruitment. Mol. Cell. Biol., 25: 2525–2538.

Takahashi, Y., Hirose, F., Matsukage, A., and Yamaguchi, M. 1999. Iden-

tification of three conserved regions in the DREF transcription factors

Analysis of Human HP1 Isoforms in Drosophila

67

from Drosophila melanogaster and Drosophila virilis. Nucleic Acids

Res., 27: 510–516.

Thiru, A., Nietlispach, D., Mott, H.R., Okuwaki, M., Lyon, D., Nielsen,

P.R., Hirshberg, M., Verreault, A., Murzina, N.V., and Laue, E.D. 2004.

Structural basis of HP1/PXVXL motif peptide interactions and HP1

localisation to heterochromatin. EMBO J., 23: 489–499.

Vassallo, M.F. and Tanese, N. 2002. Isoform-specific interaction of HP1

with human TAFII130. Proc. Natl. Acad. Sci. USA, 99: 5919–5924.

Ye, Q. and Worman, H.J. 1996. Interaction between an integral protein of

the nuclear envelope inner membrane and human chromodomain pro-

teins homologous to Drosophila HP1. J. Biol. Chem., 271: 14653–

14656.

Zhao, T. and Eissenberg, J.C. 1999. Phosphorylation of heterochromatin

protein 1 by casein kinase II is required for efficient heterochromatin

binding in Drosophila. J. Biol. Chem., 274: 15095–15100.

(Received for publication, October 30, 2006 and accepted June 5, 2007)