© 2019. Published by The Company of Biologists Ltd.

Comparison of three Heterochromatin Protein 1

homologs of Drosophila

Dong Hoon Lee1,2, Hyun Wook Ryu1, Go Woon Kim1, and So Hee Kwon1, 2,*

1College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei

University, Incheon, 21983, Republic of Korea;

2Department of Integrated OMICS for Biomedical Science, Yonsei University,

Seoul 03722, Republic of Korea

To whom correspondence should be addressed:

85 Songdogwahak-ro, Yeonsu-gu,

Incheon, 21983,

Republic of Korea;

Tel: 0082-32-749-4513

Fax: 0082-32-749-4105

Email: soheekwon@yonsei.ac.kr

Dong Hoon Lee and Hyun-Wook Ryu are equally contributing first authors.

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JCS Advance Online Article. Posted on 18 January 2019

Abbreviations: CD, chromodomain; CSD, Chromoshadow domain; CTD, C-

terminal domain; CTE, C-terminal extension region; DBD, DNA-binding domain;

FACT, facilitates chromatin transcription; HP1, heterochromatin protein 1;

H3K9me, histone H3 lysine 9 methylation; KMT, histone methyltransferase; NLS,

nuclear localization sequence; PTM, post-translational modification; Pol II, RNA

polymerase II

ABSTRACT

Heterochromatin protein 1 (HP1) is an epigenetic regulator of chromatin structure

and genome function in eukaryotes. Despite shared features, most eukaryotes

have minimum three HP1 homologs with differential localization patterns and

functions. Most studies focus on Drosophila HP1a, and little is known about the

properties of HP1b and HP1c. To determine the features of the three HP1

homologs, we performed the first comprehensive comparative analysis of

Drosophila HP1 homologs. HP1 differentially homodimerizes and

heterodimerizes in vivo and in vitro. HP1b and HP1c, but not HP1a, are localized

to both the nucleus and cytoplasm. The C-terminal extension region (CTE)

targets HP1c and HP1b to the cytoplasm. Biochemical approaches show that

HP1 binds to various interacting partners with a differential binding affinity. Each

HP1 associates differently with RNA polymerase II; a gene reporter assay

revealed that HP1a and HP1b, but not HP1c, inhibit transcriptional activity,

suggesting that HP1c serves as a positive regulator in transcription. Thus, these

studies provide the basic clues pertaining to the molecular mechanism by which

HP1 might control cellular processes in a homolog-specific manner.

Keywords: HP1a; HP1b; HP1c; H3K9 methylation; transcription; CTE

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INTRODUCTION

The compaction of chromatin into different structural states is a highly dynamic

process subject to different levels of regulation. One of the most important factors

is the post-translational modifications (PTMs) of the histone tail, which modulate

chromatin structure and function. Methylation of histone H3 at lysine 9 (H3K9me)

is specifically recognized by heterochromatin protein 1 (HP1) (James et al., 1989;

James and Elgin, 1986). HP1, encoded by Su(var)2-5, was first discovered in

Drosophila and is a key regulator of heterochromatic silencing and chromatin

condensation (Eissenberg et al., 1990; James et al., 1989; James and Elgin,

1986). HP1 comprises at least three HP1 homologs (Lomberk et al., 2006) in

almost all eukaryotes and is a phylogenetically highly conserved protein.

Molecular studies have shown that HP1 homologs have two prominent structural

motifs, a chromodomain (CD) and a chromoshadow domain (CSD), that are

separated by a variable length hinge region (H), and are important for chromatin

binding and protein–protein interaction respectively (Lee et al., 2013). HP1

proteins specifically bind to di- and trimethylated H3K9 (H3K9me2 and

H3K9me3) via the CD (Grewal and Moazed, 2003; Paro and Hogness, 1991).

The dimeric CSD recruits many proteins in a conserved penta-peptide motif,

PXVXL-dependent or independent manner (Brasher et al., 2000; Lomberk et al.,

2006; Smothers and Henikoff, 2000; Stephens et al., 2005).

The Drosophila genome encodes five HP1 paralogs, HP1a, b, c, d, and e,

compared with the three vertebrate paralogs, HP1α, ß, and γ. HP1a, b, and c,

are ubiquitously expressed in the adult fly, whereas HP1d/rhino and HP1e are

expressed in the ovaries and testes, respectively (Vermaak et al., 2005).

Drosophila HP1 homologs are different from each other and may not be

orthologs to any of the vertebrate paralogs (Vermaak and Malik, 2009). Although

the HP1 paralogs have similarities in their amino acid sequence and structural

architecture, there are some differences among the three main Drosophila HP1

homologs (Kwon and Workman, 2011b). First, the Drosophila HP1 proteins differ

in size; all are small (206–240 amino acids and 28–35 kDa), but HP1b and HP1c

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are larger than HP1a. Second, the CD domain starts at approximately 20 amino

acids from the N-terminus of HP1a, whereas it is located at the very N-terminal of

HP1b and HP1c. Thus, there is no highly acidic N-terminal region in HP1b or

HP1c. Third, the hinge region is shorter in HP1b and HP1c (40 and 18 residues,

respectively) than in HP1a (65 residues). Likewise, the sequence of the hinge is

not well conserved, with HP1a and HP1c sharing only 33% identity (Kwon and

Workman, 2011b; Li et al., 2002). The hinge region linking the two main domains

seems to enable the CD and CSD to move independently of each other in the

native protein (Brasher et al., 2000). Finally, HP1b and HP1c possess a C-

terminal extension region (CTE, 85 and 96 amino acids, respectively), whereas

HP1a has a 3–residue C terminal tail. However, no significant sequence

similarities for these CTEs, either to each other or to reported proteins or

characterized motifs in current databases have been reported. Moreover, little is

known about the role(s) of the short hinge and long CTEs of HP1b and HP1c.

In addition to the differences in structural architecture, HP1 paralogs

display marked differences in localization. The Drosophila polytene chromosome

HP1a localizes to heterochromatin (Fanti et al., 2003), whereas HP1c localizes to

euchromatin (Font-Burgada et al., 2008) and HP1b localizes to both (Zhang et al.,

2010). The observation of a differential structure and localization implies multiple

functions for HP1 proteins in vivo: heterochromatin formation and gene silencing,

telomere capping and silencing, DNA replication and repair, and positive

regulation of gene expression (Kwon and Workman, 2011a). First, HP1a has

been well characterized as a silencer protein involved in heterochromatin

formation and epigenetic gene silencing (Ayyanathan et al., 2003) and is found in

pericentric heterochromatic regions. Histone methyltransferases (KMTs)

methylate histone H3 at lysine 9, creating a docking site for themselves and for

the CD of HP1a (Bannister et al., 2001). This complex forms a higher–order

chromatin structure to repress gene expression. However, in contrast to the key

role of HP1a in heterochromatin formation, different sets of data have revealed

that the ability of HP1a to regulate active transcription (Bannister et al., 2001; De

Lucia et al., 2005; Lin et al., 2008). For instance, depletion of HP1a causes

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downregulation of a subset of active genes (De Lucia et al., 2005). In line with its

euchromatin function, HP1a is a positive regulator of transcription by facilitating

H3K36 demethylation at active and/or heterochromatic regions (Lin et al., 2008)

and of euchromatic gene expression by interacting with RNA transcripts and

heterogeneous nuclear ribonucleoproteins in Drosophila (Piacentini et al., 2009).

Second, HP1b overexpression decompacts pericentromeric heterochromatin and

decreases binding to HP1a and H3K9me2 to it, suggesting that the presence of

HP1b counteracts the function of HP1a in heterochromatin (Zhang et al., 2010).

Third, euchromatic HP1c functions in a complex with two related Zn–finger

containing transcription factors, Without Children (WOC) and Relative of WOC

(ROW) (Font-Burgada et al., 2008) and ubiquitin receptor protein Dsk2 (Kessler

et al., 2015), as a positive regulator of transcription. A recent comprehensive

bioinformatic analysis has revealed significant similarity and uniqueness in genes

altered in HP1-knockdown S2 cells of Drosophila (Lee et al., 2013). HP1

depletion resulted in up-regulated and down-regulated gene profiles, indicating

that HP1 modulates both repression and activation of gene expression.

All HP1 proteins are associated with chromatin regions enriched in

H3K9me2,3 and recruit various factors through the CSD domain resulting in

multiple biological functions. However, the molecular mechanisms that determine

the differential localization of HP1 homologs and their differential functional

properties remain elusive. Compared with wealth of studies accumulated for

Drosophila HP1a, there is little direct biochemical evidence to prove an exact

overall comparison of HP1–homolog specific–properties and functions. To

investigate these issues, we performed a comprehensive comparative analysis of

the Drosophila HP1 homologs using biochemical and genomic approaches. Our

data show that HP1 binds to various interacting partners with differential binding

affinity. More importantly, each HP1 associates with RNA polymerase II (Pol II)

with a different binding affinity and possesses different transcriptional activities.

Moreover, all three HP1 proteins show different subnuclear localization patterns

in S2 cells. Surprisingly, we observed that unlike heterochromatin-specific HP1a,

HP1b and HP1c localize to the cytoplasm as well as the nucleus. Expression of

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truncated and domain-swapped chimeric HP1 proteins in vivo demonstrates that

the targeting of HP1b and HP1c to the cytoplasm appears to be due to CTE.

Collectively, these results are first to systematically analyze the characterization

of HP1 paralogs.

RESULTS

HP1 homodimerizes and heterodimerizes in vivo and in vitro

Previous observations that HP1α, β, and γ can form homodimers as well as

heterodimers in vitro and in vivo (Brasher et al., 2000; Nielsen et al., 2002; Ye et

al., 1997) prompted us to investigate whether these interactions could occur with

Drosophila HP1. To confirm HP1 interaction in vivo, we developed anti-HP1b-

and HP1c-specific antibodies because anti-HP1a mouse monoclonal antibodies

are available from the Developmental Studies Hybridoma Bank. These

antibodies recognize a protein of the expected molecular weight in extracts from

wild-type flies, that is absent in extracts prepared from HP1c−/− strains and from

S2 cells and do not recognize any of the other HP1 isoforms (Fig. S1). When

HP1 protein was immunoprecipitated from S2 cell nuclear extracts with each anti-

HP1 antibody, endogenous HP1a and HP1b proteins were found in the HP1

immunoprecipitates. HP1b bound to both HP1a and HP1c. Interestingly, HP1a

did not coprecipitate with HP1c and vice versa (Fig. 1A,B). The co-IP was

specific because none of the HP1 proteins could be detected in the control. Thus,

HP1a, b, and c differently homodimerize and heterodimerize in vivo.

In vitro binding assays were then performed to test for direct interactions

between all three Drosophila HP1 proteins. Purified baculovirus-expressed Flag-

tagged HP1 proteins were mixed with purified Escherichia coli (E.coli)-expressed

glutathione S-transferase (GST)-HP1 fusion proteins, followed by anti-Flag

immunoprecipitation (Fig. 1C,D). The matrix-associated GST-HP1 proteins were

revealed by western blotting. GST-HP1a bound to Flag-HP1a, b, and c but not

GST alone (Fig. 1E, lanes 2). Similarly, GST-HP1b and GST-HP1c bound to the

three Flag-HP1 proteins (Fig. 1E, lanes 8-10, and lanes 13-15, respectively) but

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not to GST (lanes 7 and 12). These results clearly show that purified Drosophila

HP1a, b, and c bind directly to each other and to themselves.

HP1a, HP1b, and HP1c localize to distinct cellular compartments in S2 cells

In Drosophila embryonic Kc cells (Smothers and Henikoff, 2001) and polytene

chromosomes, it has previously been indicated that the three HP1 proteins

associate with different chromatin states: HP1a with heterochromatin (Fanti et al.,

2003), HP1c with euchromatin (Abel et al., 2009; Font-Burgada et al., 2008), and

HP1b with both (Zhang et al., 2010). However, there is no direct evidence of the

cellular localization of the three HP1 proteins in Drosophila embryonic S2 cells,

except for HP1a. Thus, we analyzed the subcellular localization of the three HP1

proteins in S2 cells using conventional immunofluorescence microscopy. HP1a

was distributed in the heterochromatin region corresponding to condensed

chromatin masses, as revealed by DAPI staining of S2 cells. Intriguingly, HP1b

and HP1c distributions in S2 cell were different, being distributed in the

cytoplasm as well as nucleus (Fig. 2A,B and Fig. S2). This result was further

confirmed using antibodies made by other labs (Fig. S3A).

To further assess the respective localizations of the HP1 homologs, S2

cells were double-labeled with an antibody specific for each HP1 and then

examined by confocal immunofluorescence microscopy. Figure 2A shows that in

S2 cells, there was obvious overlap of HP1a and HP1b in the nucleus, whereas

the majority of HP1c did not colocalize with the heterochromatic foci containing

HP1a (Fig. 2A). HP1b and HP1c strongly colocalized in nuclear euchromatin and

in the cytoplasm. Furthermore, as confirmed by immunoblot analysis, HP1a was

found only in the nuclear fraction, whereas HP1b and HP1c were found in both

the cytoplasm and nucleus (Fig. 2C and Fig. S3B). The different distribution of

the three HP1 proteins was observed and confirmed in S2 cells.

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To delineate the cytoplasmic targeting segments of the HP1 homologs, we

swapped or deleted domains between heterochromatin-specific HP1a and

cytoplasmic-specific HP1b and HP1c (Fig. 3A) and performed

immunofluorescence assays using C-terminally Flag-tagged HP1 chimeric

constructs. As shown in Figure 3B, HP1a primarily localized to the

heterochromatin and nucleus, whereas HP1a-ΔC+HP1b-CTE and HP1a-

ΔC+HP1c-CTE localized to both the nucleus and cytoplasm. In particular, HP1c

CTE targeted HP1a to the cytoplasm more than HP1b CTE. Alternatively, the

addition of cytoplasmic targeting segments to HP1a caused mislocalization to the

euchromatin. To further clarify whether the CTEs of HP1b and HP1c are

responsible for their cytoplasmic distribution, we generated CTE deletion mutants

HP1b (HP1b-ΔC) and HP1c (HP1c-ΔC) as well as CTE swapped mutants HP1b-

CTE (HP1b-ΔC+HP1c-C) and HP1c-CTE (HP1c-ΔC+HP1b-C). Interestingly,

HP1b-ΔC localized to only the nucleus, whereas HP1b-ΔC+HP1c-C re-localized

to the cytoplasm (Fig. 3C). However, HP1c-ΔC and HP1c-ΔC+HP1b-C localized

to both the nucleus and cytoplasm (N > C) (Fig. 3D). In addition, HP1b-CTE

localized to the nucleus and cytoplasm, whereas HP1c-CTE localized exclusively

to the cytoplasm (Fig. 3C-E). The expression levels of the swapped or deleted

domain mutants in S2 cells were confirmed by western blotting (Fig. S4). These

results indicate that the CTE of HP1c is more efficient for cytoplasmic targeting

than the CTE of HP1b. Taken together, these data suggest that the HP1

homologs each have a special distribution and localization.

HP1a, b, and c bind to H3K9me with differential binding affinity

HP1 binds to di- or trimethylated H3K9 via its CD. It has been reported that HP1a

has slightly higher binding affinity for H3K9me3 than for H3K9me2 in vitro

(Jacobs and Khorasanizadeh, 2002). However, little is known about the

methylated H3K9-binding affinity for each Drosophila HP1 homolog. To address

this issue, we tested the binding of HP1 to H3K9me2 and H3K9me3 in histone

peptide pull-down experiments. To clearly dissect the interactions between the

three HP1 homologs and the H3K9me peptide, we introduced a point mutation

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within the CD that disrupted the interaction with H3K9me. HP1a V26M contains

an amino acid substitution within CD that disrupts its interaction with H3K9me

(Jacobs et al., 2001). This residue was chosen by aligning the amino acid

sequences of HP1b and HP1c (Fig. S5) with Drosophila HP1a, wherein the

residue was shown to be important (Brasher et al., 2000; Thiru et al., 2004).

Therefore, the analogous V6M and V10M amino acid substitutions were

generated in HP1b and HP1c, respectively. These point mutations were used as

negative controls. As shown in Figure 4A, the three HP1 proteins possess slightly

differential binding affinity for H3K9me2 and H3K9me3 peptides in vitro. HP1a

showed the strongest binding to methylated H3K9 peptide, whereas HP1c

presented the weakest binding to the peptide. In contrast to the wild-type HP1s,

the HP1a V26M, HP1b V6M, and HP1c V10M mutants failed to interact with

H3K9me2 and H3K9me3 peptides in vitro. Although all of the three HP1

homologs bound to the H3K9me2 and H3K9me3 peptides, HP1a appeared to

possess the strongest affinity for the methylated H3K9 peptides.

Next, we investigated to what extent the degree of methylation affected

HP1 binding to the H3K9 using fluorescence polarization binding assays. We

used differently methylated H3K9 peptides to measure the relative binding

affinities of HP1 CD to these H3K9 peptides. Fluorescence polarization

measurements showed that HP1 CD possesses significant differential binding

affinity for the methylated H3K9 peptides. The measured dissociation constant

(KD) of ~6.6 μM was similar to that reported for the HP1a/H3K9me2 interaction

(Fischle et al., 2003). HP1b CD bound to the H3K9me2 peptide with affinity of 33

μM and to the H3K9me3 peptide with affinity of 3.0 μM (Fig. 4B). Similarly, the

dissociation constant of HP1c CD for H3K9me2 peptide was 24 μM, and that for

the H3K9me3 peptides was 5.6 μM. Binding of HP1b and HP1c to the H3K9me2

peptide was weakened 5.0-fold and 3.6-fold, respectively, compared to HP1a. In

contrast to HP1a (KD = 1.0 μM), HP1b CD and HP1c CD bound to the H3K9me3

peptide with 3.0-fold and 5.6-fold weaker affinities, respectively but still much

stronger binding to the H3K9me3 peptide than H3K9me2 peptide was observed.

Furthermore, the three HP1 CD weakly bound to the H3K9me1 peptide with

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similar binding affinity (KD of 52~74 μM, 1.4~1.5-fold). No significant interactions

between the three HP1 CD and unmodified (unmethylated) control H3 peptide

were observed. To further confirm this, we performed surface plasmon

resonance analysis using the H3K9me2 peptide and revealed that HP1b (KD = 37

μM, 68.5-fold) and (KD = 9.33 μM, 17.3-fold) HP1c had lower binding affinities

than HP1a (KD = 0.54 μM) (Fig. S6).

Because of this difference in affinity, we hypothesized that the three HP1

homologs might compete for binding to methylated H3K9. To test this hypothesis,

we conducted in vitro competitive peptide binding assays mixing differentially

tagged recombinant HP1 proteins and H3K9me2 peptide. We added different

combinations of recombinant HP1 homologs to the same reaction and pulled

down with streptavidin beads. When mixed with HP1a and HP1b, HP1a and

HP1b efficiently bound to the H3K9me2 peptide and both proteins were absent in

the unbound fraction (Fig. 5C, lanes 3 and 4). While HP1a (lanes 5 and 6) or

HP1b (lane 7 and 8) was found only in the bound fraction, HP1c was found in

both the bound and unbound fractions when mixed with HP1c and HP1a or HP1b,

indicating that H3K9me2 peptide had a lower binding affinity for HP1c than HP1b

and HP1a. When HP1a:HP1b:HP1c were all present, all HP1a still bound to the

H3K9me2 peptide but remaining amounts of HP1b and HP1c increased in the

unbound fraction (~5.9-fold and ~2.0-fold (level of lane 10/level of lane 8),

respectively; lanes 9 and 10). Consistent with KD value, HP1a showed the

strongest binding to the H3K9me2 peptide under mixture of three HP1 proteins

(Fig. 4C, lanes 9 and 10). Taken together, these data indicate that the CDs of the

three HP1 homologs possess a differential binding affinity for differently

methylated histone H3K9 peptides.

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Three HP1 homologs associate with interacting partners with differential

binding affinities

A subset of the main histone H3K9 methyltransferases (KMT1s),

Suv39h1/KMT1A, G9a/KMT1C, GLP/KMT1D, and SETDB1/KMT1E, coexist in

the same megacomplex in mammals (Fritsch et al., 2010). Like mammals,

Drosophila possesses KMT1 homologs, and Su(Var)3-9/KMT1 interacts with

HP1a (Schotta et al., 2002). Thus, we wondered whether the three HP1

homologs associate with these KMT1s. To test for direct interaction between the

HP1 homologs and KMT1s, we performed the in vitro binding assays using

recombinant KMT1s and HP1s, followed by immunoprecipitation with the

indicated antibodies. As a positive control, we used HA-dSSRP1 and confirmed

the specificity of the interactions (Fig. 5D). Our result showed that Su(Var)3-

9/KMT1 presented stronger binding to HP1a and HP1b than to HP1c (Fig. 5A).

Similarly, dG9a/KMT1C bound strongly to HP1a and HP1b (Fig. 5B). In contrast,

dSETDB1/KMT1E bound the three HP1 proteins equally (Fig. 5C). None of the

tested KMT1s interacted with GST. Collectively, these data suggest that the

three HP1 homologs possess different binding affinities for KMT1s.

The two main domains of HP1 have a differential preference for interacting

partners

In an attempt to define the HP1 domain responsible for interaction with other

KMT1s, we purified truncated forms of HP1 contained either CD, hinge, or CSD

alone or in combination and examined them in in vitro binding assays. As seen in

Supplemental Figure S7, CSD is sufficient and necessary for the binding of HP1a

to the three KMT1s, whereas neither CD nor hinge interact with KMT1s under the

same conditions. In case of Su(var)3-9/KMT1 and dG9a/KMT1C, the full-length

protein or the CSD+Hinge protein appeared to bind the two KMT1s with a higher

affinity than the CSD domain alone (Fig. S7C and D, lanes 2, 4, and 7,

respectively). In contrast, dSETDB1/KMT1E was not affected by the hinge

regions (Fig. S7D). Altogether, the CSD of HP1 is necessary and sufficient for

the interaction between these proteins.

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Drosophila HP1c and mammalian HP1γ physically interact with

phosphorylated Pol II (Kwon et al., 2010; Vakoc et al., 2005). We sought to

determine whether the three HP1 homologs possess differential binding affinities

for Pol II. To examine this, we performed immunoblot assays and CTD peptide

pull-down assays with a different modified CTD of Pol II and recombinant HP1

proteins. Although three HP1 proteins bound to the phosphorylated Pol II in vivo

and in vitro (Fig. 6A,C,D), HP1c more strongly bound to the active forms of Pol II

(phosphorylated PoI II at serine 2 and serine 5: ~2-fold) compared with HP1a and

HP1b when HP1 was overexpressed (Fig. 6A). HP1b and HP1c but not HP1a

interacted with the phosphorylated Pol II (1.21-fold vs 2.5-fold: serine 5 and 1.75-

fold vs 2.5-fold: serine 2, respectively) and unmodified (unphosphorylated) Pol II

at the endogenous protein levels and HP1c more robustly bound to Pol II

compared to the HP1b (Fig. 6B). This finding suggests that HP1c may be the

most robust binding affinity for active form of PoI II among three HP1s. Next, to

map the domain of HP1 required for interaction with Pol II, we were tested in a

similar manner in Supplemental Figure S7. The CD contained within the N-

terminal region is sufficient and necessary for the binding of HP1b and HP1c to

phosphorylated CTD peptides (Fig. 6D). In contrast, HP1a CD associates with

the unphosphorylated and phosphorylated CTD peptides. This result indicates

that the CD of each HP1 homolog possesses differential affinity for Pol II CTD

peptides.

HP1a and HP1b repress transcription, whereas HP1c activates transcription

HP1a has been considered a silencing protein involved in transcription

repression and heterochromatin formation (James et al., 1989; James and Elgin,

1986). However, little is known about the transcription activity of the other HP1

homologs. Thus, we investigated the effects of three HP1 proteins on

transcription activity using a luciferase reporter assay. HP1a, HP1b, and HP1c

were fused via their N-termini to the DBD of yeast GAL4. Expression vectors

bearing either GAL4-DBD or GAL4-DBD-HP1 were transfected into HEK 293T

cells with a GAL4-responsive luciferase reporter, pGL4.35-GAL4UAS.

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Transfection of the pGL4.35 reporter into HEK 293T cells with or without GAL4-

DBD elicited the same basal level of luciferase (data not shown). Addition of

GAL4-DBD HP1a, HP1b, or HP1c resulted in differential transcription compared

with the basal level elicited in the presence of GAL4-DBD alone. As expected,

HP1a and HP1b repressed the expression of the reporter construct by > 86%

and 50% in HP1a and HP1b, respectively. In contrast, HP1c increased the

transcription level approximately 4.0-fold compared with the control (Fig. 7A).

GAL4-HP1 expression levels in the cells were similar when measured by western

blotting (Fig. 7B). To further confirm this result, we also assessed the interplay of

HP1a or HP1b with HP1c. Eliciting heteromerization between HP1a and HP1b by

transfecting various ratios of HP1a and HP1b expression vectors did not alter the

degree of repression compared with HP1a or HP1b alone. However,

cotransfecting with different molar ratios of HP1a and HP1c expression vectors

relieved repression; no such effect was observed with HP1a and HP1b. This

indicates that homodimers of repressing HP1a or homodimers of activating HP1c

compete with one another. However, cotransfecting with the HP1b and HP1c

expression vectors, transcription was lower than that observed upon

cotransfection with HP1a and HP1c, indicating that the heterodimer of HP1b and

HP1c represses transcription. Therefore, we conclude that HP1a and HP1b are

important for the repression of transcription, whereas HP1c is important for the

activation of transcription. However, this effect of HP1 on transcription depends

on differentially combined heterodimers of the three HP1 homologs.

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DISCUSSION

HP1 homologs share extensive structural identity, whereas they differ in

localization, interacting proteins, gene regulation patterns, and functions.

Compared with the abundance of studies conducted for Drosophila HP1a,

information about the other HP1 homologs is scarce. Moreover, an overall

comparison of the three HP1 homologs has not been reported. Here, we

exhaustively and comparatively characterize three main Drosophila HP1

homologs. In this study, we provide biochemical and molecular data to

complement our previous proteomic (Ryu et al., 2014) and genomic data (Lee et

al., 2013) showing that the three HP1 homologs have differential features and

properties despite their similar structure.

The CD domain is responsible for HP1a binding to H3K9me2, 3 and this

interaction is required for the localization of HP1a to heterochromatin (Bannister

et al., 2001; Lachner et al., 2001). Although all three HP1 homologs contain a CD

and are able to bind methylated histones H3K9, HP1 CD in particular displayed

significant differential binding affinity for H3K9me2 (Fig. 4B). Compared with

HP1a, the affinity of HP1b CD and HP1c CD for H3K9me2 was much weaker.

These data suggest that CDs of the three HP1 homologs possess a differential

binding affinity for differently methylated histone H3K9 proteins, resulting in

different localization patterns. In agreement with these observations, the

localization of HP1 is likely to be paralog-specific: Drosophila HP1a

predominantly localizes to heterochromatin, whereas HP1c localizes to

euchromatin and HP1b localizes to both regions (Kwon et al., 2010; Kwon and

Workman, 2011a; Smothers and Henikoff, 2001). Truncation and domain-

swapping studies show that both the HP1a hinge and CSD contain a nuclear

localization sequence (NLS) and can separately target heterochromatin, whereas

CSD alone targets HP1c to euchromatin (Powers and Eissenberg, 1993;

Smothers and Henikoff, 2001). Comparative sequence analyses of the HP1

homologs reveal a conserved sequence block within the HP1a and HP1b hinge

that contains a conserved sequence (KRK) and an NLS. However, this sequence

block is absent from the HP1c hinge (Smothers and Henikoff, 2001), which is

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only 20 amino acids long. In addition, Smothers et al. have reported that the CD

domain from either HP1a or HP1c has no apparent targeting activity (Smothers

and Henikoff, 2001). Several other possibilities could explain the inability of HP1c

to localize to the heterochromatin. Intriguingly, HP1c and HP1b possess a CTE

(96 and 85 amino acids, respectively), whereas HP1a has no CTE. However,

little is known about the role(s) of the CTE of HP1c and HP1b. In this study, we

have shown that HP1b and HP1c partially localize to the cytoplasm, although

both proteins are primarily found in the nucleus (Fig. 2). As shown in Figure 3,

the CTE regions cause HP1c and HP1b to localize to the cytoplasm, resulting in

their lower affinity to histone. In a good agreement with this result, we have

reported the specific interaction and colocalization of dTCTP and HP1b or HP1c

to the cytoplasm (Ryu et al., 2014). Second, the interaction of HP1c with other

factors such as WOC, ROW (Font-Burgada et al., 2008), Dsk2 (Kessler et al.,

2015), facilitates chromatin transcription (FACT) or Pol II (Kwon et al., 2010) may

occur at higher affinity than its interaction with H3K9me2. Third, unlike

mammalian HP1, HP1c does not form a heterodimer with HP1a in vivo, whereas

the heterodimer with HP1b except was overexpressed (Fig. 1B,E), resulting in

euchromatin and cytoplasmic targeting of HP1b and HP1c. Consistently other

group reported that HP1c interacts with HP1b (Kessler et al., 2015). However, in

vitro pull-down experiments detected the interaction of HP1a and HP1c. This

observation suggests that additional factors prevent the interaction in vivo.

Fourth, an unidentified mechanism as well as PTMs of HP1c and HP1b may

regulate these interactions and their localization. Thus, these data might account

for the distinct targeting of Drosophila HP1 homologs.

Similar to HP1a, the other HP1 homologs can act as transcriptional

repressors or/and activators depending on their chromatin localization. Specific

HP1c-interacting proteins have been identified by biochemical and proteomic

approaches in Drosophila (Abel et al., 2009; Font-Burgada et al., 2008; Kessler

et al., 2015; Kwon et al., 2010; Ryu et al., 2014; Vakoc et al., 2005). HP1c

exclusively forms a complex with WOC/ROW/Dsk2 (Kessler et al., 2015) and

interacts with FACT and Pol II (Kwon et al., 2010), which are markers for active

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transcription. These data suggest that HP1c acts as a positive regulator in active

transcription in euchromatin. Zhang et al. have reported that HP1b plays

important roles in transcriptional activation and development (Zhang et al., 2010).

In full agreement with these previous reports, a reporter gene assay showed that

HP1a and HP1b repress transcription, whereas HP1c activates transcription (Fig.

7A). When HP1b formed a heterodimer with HP1a or HP1c, it repressed

transcription (Fig. 7C). Similarly, the differential effects of HP1c on transcription

have been reported (Kessler et al., 2015). Interestingly, mammalian HP1γ and

Drosophila HP1c physically interact with the active form of Pol II (Kwon et al.,

2010; Vakoc et al., 2005). Although the three HP1 homologs bind to

phosphorylated Pol II in vitro and in vivo, they appeared to have different

affinities for Pol II in our experiments (Fig. 6). Compared with HP1b and HP1c,

HP1a bound more non-specifically to Pol II in a phosphorylation-independent

manner (Fig. 6A,C), suggesting that the recruitment of HP1a to chromatin does

not rely on Pol II or its phosphorylation status. Consistent with reporter assay

result, HP1c more robustly bound to active forms as well as unmodified form of

Pol II compared to the HP1b at the endogenous protein level (Fig. 6B). This

finding suggests that HP1c may be the most robust binding affinity for active form

of PoI II among three HP1s. Thus, these data imply that phosphorylated Pol II

preferentially recruits HP1c proteins at the transcriptionally active chromatin

region and HP1b may be recruited through forming heterodimer with HP1c at the

region. Taken together, these data indicate that HP1b and HP1c may counteract

HP1a function both in heterochromatin formation and in the active transcriptional

regulation of euchromatic genes.

Our observation that the differential binding affinity of each HP1 for Pol II

and KMT1s indicates that the three HP1 homologs might regulate different sets

of genes. Our previous microarray analysis showed that HP1 regulates different

target genes as well as common target genes (Lee et al., 2013). For instance,

the percentage of fourth chromosome genes misregulated in HP1a- or HP1b-

knockdown experiments was greater than that observed in HP1c-knockdown

studies. In line with these results, high-resolution mapping experiments have

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shown that HP1a and chromosome 4-specific protein Painting of fourth (POF)

interdependently bind to the fourth chromosome and regulate the expression of

genes on it (Johansson et al., 2007; Tzeng et al., 2007). Contrary to expectations

from the gene silencing role of HP1, depletion of HP1 caused more

downregulation than upregulation of genes. This data strongly suggests that HP1

enhances the expression of target genes, although it could also act to repress

the expression of a subset of genes in Drosophila. First, high-resolution mapping

shows that besides forming large heterochromatic domains in pericentric regions,

Drosophila HP1a preferably associates with a subset of euchromatic exon-dense

genes located on the chromosome arms and binds along the entire active

transcriptional units of genes (de Wit et al., 2007). Consistent with this, the

euchromatic functions of HP1a are concentrated within the bodies of euchromatic

genes rather than in the promoter regions (Piacentini et al., 2003) and are

primarily independent of HP1c and KMT1 location (Cryderman et al., 2005),

implying different ways of targeting HP1a to these euchromatic genes. Second,

HP1c complex interacts with Dsk2, localizes at promoters of its target genes and

is required for transcription. Microarray profiling reveals that the HP1c complex

regulates a common set of genes that function in nervous system development

and morphogenesis (Font-Burgada et al., 2008). In addition, DNA adenine

methyltransferase identification analysis demonstrated that HP1c presents a

profile similar to other cofactors, such as Bcd, Gaf, and Jra, in contrast to HP1a,

which does not bind to the same loci as these transcription factors (Moorman et

al., 2006). These results further support a role for HP1c in transcriptional

activation rather than in repression. Compared with studies performed on HP1a

and HP1c, little is known about HP1b. It has been reported that HP1b is required

for the transcription of both euchromatic and heterochromatic genes, although

the exact role of HP1b in transcription is not clear (Zhang et al., 2010). Indeed,

HP1b partially colocalizes with the elongating form of Pol II and active histone

markers, supporting the notion that HP1b is involved in active transcription. In

agreement with this study, our previous studies showed that the expression of a

number of genes was downregulated upon HP1b depletion (Lee et al., 2013).

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Interestingly, HP1b overexpression disrupts pericentric heterochromatic structure

and transcription of heterochromatic genes (Zhang et al., 2010), suggesting that

HP1b competes for HP1a binding sites in pericentric heterochromatin when

highly expressed. This hypothesis is consistent with the idea that HP1a mutant

elicits disturbed heterochromatic structure (Spierer et al., 2005). In contrast, the

presence of HP1a rescued partially defective transcription of heterochromatic

genes. Thus, these findings strongly support the idea that HP1 differentially

modulates transcription and gene regulation in an HP1 homolog-specific manner.

MATERIALS AND METHODS

DNA constructs

HP1a, HP1b, HP1c, Su(Var)3-9, dSETDB1, dG9a, or dSSRP1 was cloned into

the Drosophila Schneider 2 (S2) cell expression vector pRmHa3-C-HA2Flag2

(Guelman et al., 2006), bacterial overexpression vector pGEX-4T-1, and

pBacPAK8-N-HisFlag or -2xHA vector (Invitrogen, USA) for overexpression in

insect cells. Truncated or swapped domains of HP1 were cloned into the

pRmHa3-C-HA2Flag2 vector. The constructs pFA-CMV-HP1a, -HP1b, or -HP1c

used for the transcriptional reporter assay were generated by polymerase chain

reaction (PCR) amplification of the full-length cDNA and the cloning of this

fragment in–frame with the DNA–binding domain (DBD) of the yeast GAL4

coding sequence in the pFA-CMV vector. Mutations of HP1a, HP1b, and HP1c

were generated using the Quick Change II XL Site-Directed Mutagenesis Kit

(Stratagene, La Jolla, CA, USA).

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Western blotting Analysis

D. melanogaster S2 cells were grown in Schneider’s insect medium

supplemented with 10% serum to a density of 3–6 × 106 cells/ml. Cells grown

and treated as indicated were collected, lysed, and separated by SDS-PAGE;

western blotting was performed as previously described (Kwon et al., 2007). Rat

anti-HP1b with His-HP1bΔCD (Hinge+CSD) truncated recombinant antigens

corresponding to residues 55-240 and rat anti-HP1c antibodies with peptide

antigens (HP1c antigen, CRHIAMRMKGVPEELRLAASR) representing unique

portions of the protein were generated in-house. The source of the other primary

antibodies is available upon the request.

Co-immunoprecipitation (co-IP) assays

S2 cells were transfected using FuGene 6 (Roche, Basel, Switzerland) following

the manufacturer’s protocol and whole-cell extracts were prepared. For co-IP

experiments, the cellular extract (500 µg) was incubated overnight with the

primary antibody at 4°C with gentle agitation. Protein A/G agarose slurry (25 µl)

was then added, and the samples were incubated for 3 h at 4°C with gentle

agitation. Beads were washed three times with NP-40 lysis buffer [0.5% NP-40,

50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 25 mM NaF, 25 mM glycerol phosphate,

1 mM EDTA, 5 mM EGTA] supplemented with Complete protease inhibitor

cocktail tablet (Roche, Basel, Switzerland) and subsequently resuspended and

boiled in 20 µl of loading buffer for SDS–PAGE and western blotting.

Subcellular fractionation

S2 cells were collected and washed with ice-cold PBS before being lysed with

lysis buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1%

NP-40, 1 mM DTT, 1 mM PMSF, and Complete protease inhibitor cocktail tablet

(Roche, Basel, Switzerland). Nuclei were pelleted by centrifugation at 5,000 rpm

for 5 min at 4°C. The supernatant (cytosolic fraction) was removed by a pipette,

and the pellet (nuclei) was resuspended in buffer containing 20 mM HEPES (pH

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7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM DTT,

1 mM PMSF, and Complete protease inhibitor cocktail tablet (Roche, Basel,

Switzerland). The nuclear extract was vortexed at maximum speed for 15 s,

incubated on ice for 1 h, and then ultracentrifuged at 45,000 rpm for 1.5 h at 4 °C.

The resultant supernatant was the final “nuclear fraction”. The NaCl

concentration of the extract was subsequently adjusted to 300 mM.

Immunofluorescence microscopy

Immunofluorescence microscopy was performed as previously described (Kwon

et al., 2010). Briefly, cells were fixed, permeabilized, blocked, and incubated with

a primary antibody, followed by an Alexa Fluor-conjugated secondary antibody

(Molecular Probes, Eugene, OR, USA). Images were obtained with a Zeiss LSM-

710 Meta confocal microscope and processed using ZEN software (Carl Zeiss

AG, Oberkochen, Germany). The following antibodies were used: mouse anti-

HP1a (1:1000), rabbit anti-HP1b (1:1000), rat anti-HP1b (1:1000), rat anti-HP1c

(1:500), and mouse anti-Flag M2 antibody (1:1000).

Expression and purification of recombinant proteins in Sf21 insect cells

cDNAs of dSSRP1, dG9a, dSETDB1, HP1a, HP1b, HP1c and derivatives were

subcloned into vector pBacPAK8 carrying an N-terminal Flag or HA tag.

Recombinant baculoviruses were generated and manipulated according to the

manufacturer’s recommendations (BacPAK expression system, Clontech, Palo

Alto, CA, USA). Sf21 insect cells were cultured at 27 oC in Sf-900 II SFM

(Invitrogen, USA) supplemented with 10% FBS (Invitrogen, USA), and penicillin–

streptomycin (Invitrogen, USA). Forty-eight hours after infection, cells were

collected and washed with ice-cold PBS before being lysed in 20 ml of ice-cold

lysis buffer [50 mM HEPES (pH 7.9), 500 mM NaCl, 2 mM MgCl2, 0.2% Triton X-

100, 10% (v/v) glycerol, 0.5 mM EDTA and protease inhibitors]. Cell lysates were

clarified by ultracentrifugation at 40,000 rpm for 30 min at 4oC, and were

subsequently incubated with anti-Flag M2, or anti-HA-agarose beads overnight at

4oC. The beads were washed three times with lysis buffer, and bound proteins

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were eluted twice with 1 column volume of elution buffer [0.5 μg/ml triple Flag or

HA peptide in 50 mM HEPES (pH 7.9), 100 mM NaCl, 2 mM MgCl2, 0.02% NP-

40 and 10% (v/v) glycerol].

In vitro binding assay

The indicated recombinant proteins were incubated in buffer A [50 mM HEPES

(pH 7.9), 300 mM NaCl, 2 mM MgCl2, 0.05% Triton X-100, 10% (v/v) glycerol, 0.5

mM EDTA, 1 mM PMSF, and 0.1 mg/ml BSA] overnight at 4oC. Proteins were

pulled down with the indicated beads for 2 h at 4oC. Beads were washed four

times using buffer A and eluted by boiling in SDS–PAGE sample buffer. Eluates

and input (2%) were analyzed by western blot using the indicated antibodies.

Peptide pull-down assay

Peptide pull-down assays were performed as previously described (Kwon et al.,

2010). Biotinylated C-terminal domain (CTD) peptides or histone tail peptides

(0.5 µg) were bound to 0.5 mg of streptavidin-coated M280 Dynabeads

(Invitrogen, USA) in 50 µl of high salt binding buffer B [25 mM Tris-HCl (pH 8.0),

1 M NaCl, 1 mM dithiothreitol (DTT), 5% glycerol, and 0.03% NP-40] at 4oC for 3

h. The beads were washed once with buffer B and twice with low-salt peptide

binding buffer C [25 mM Tris-HCl pH (8.0), 50 mM NaCl, 1 mM DTT, 5% glycerol,

and 0.03% NP-40] and finally resuspended in 50 µl of buffer C. Recombinant

protein (0.5 µg) was mixed with the beads and incubated at 4oC for 1 h on a

Dyna-Mixer (Invitrogen, USA). Beads were washed three times with buffer C and

then analyzed by SDS-PAGE and western blot.

GAL4-luciferase reporter assay

HEK 293T cells were seeded into 6–well plates and allowed to reach ~50%

confluence. The cells were contransfected with pFA-CMV or pFA-CMV-HP1 and

pGL4.35-GAL4UAS using ExFectin (Welgene, Daegu, Korea). Twenty-four hours

after transfection, cell lysates were analyzed with a Luciferase Reporter Assay kit

(Promega, Madison, Wisconsin, USA) according to the manufacturer's

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instructions. In all experiments, the luciferase activity was determined using

equal amounts of protein, and relative luciferase activity was measured using a

multiplate reader (TECAN, Maennedorf, Switzerland). Each experiment was

performed at least three different times, and the mean and standard deviations

were calculated. The expression of control of each GAL4 protein was determined

by western blot analysis.

Fluorescence polarization measurements

Fluorescence polarization assays were performed and analyzed essentially as

previously described (Jacobs et al., 2004). Fluorescence polarization binding

assays were performed in the presence of 100 nM fluorescein-labeled peptide

using the HiLite Histone H3 Methyl-Lys9 binding assay kit (Active Motif,

Carlsbad, CA, USA) following the manufacturer’s instructions. A titration series of

10 µl volumes in 384-well plates were read multiple times on a multiplate reader

(TECAN, Maennedorf, Switzerland). Sample plates were kept on ice until reading

fluorescence at room temperature. Multiple readings and the independent

titration series were averaged after data normalization.

Statistical analysis

The data presented are the means ± SD from three or more independent

experiments performed in duplicate. Statistical significance was determined using

the Student’s t-test and P < 0.05 is considered statistically significant.

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Acknowledgments

We wish to thank Dr. Lori Wallrath for contributing anti-HP1a monoclonal

antibody (C1A9). We appreciate Dr. Axel Imhof (University of Munich, Munich,

Germany) and Dr. Steven Henikoff (Fred Hutchinson Cancer Research Center,

Seattle, WA, USA) for providing anti-HP1c and HP1b antibodies, respectively.

Conflict of interest

The authors declare that they have no conflicts of interest.

Author contributions: H.R.W and D.H.L designed and performed experiments,

as well as analyzed the data. G.W.K performed experiments. S.H.K conceived

the general design of the study, participated in the development of the

approaches, wrote the initial draft of the manuscript, extensively edited the

manuscript and supervised the work.

Funding

This research was supported by Basic Science Research Program through the

National Research Foundation of Korea (NRF) funded by the Ministry of

Education, Science and Technology (2016R1D1A1A02937071).

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Figures

Fig. 1. Drosophila HP1 homodimerizes and heterodimerizes in vivo as well

as in vitro. (A) Schematic representation of the three HP1 proteins. Full-length

HP1 (FL) contains a chromodomain (CD), a hinge domain (H) and a

chromoshadow domain (CSD). (B) Co-IP assay with endogenous proteins. S2

cell extracts were immunoprecipitated with anti-HP1a, -HP1b, or -HP1c

antibodies, followed by immunoblotting with the indicated antibodies. Ten percent

of total cell lysates used in the immunoprecipitation are shown as input. (C and

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D) Ectopic-tagged recombinant proteins were purified from Sf21 cells infected

with a baculovirus encoding Flag-HP1 and from E.coli. (C) The purity of

recombinant proteins was determined by SDS-PAGE followed by Coomassie

Brilliant Blue staining (CBB) and (D) by western blot analysis. (E) In vitro binding

assays using recombinant proteins. Recombinant Flag-HP1 and GST-HP1 were

mixed with 0.1 mg/ml BSA to reduce background binding. The resulting

complexes were immunoprecipitated using anti-Flag M2 agarose beads. Beads

and input (2%) were analyzed by western blot using anti-Flag and anti-GST

antibodies. The data presented are representative images from at least three

independent experiments.

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Fig. 2. Intracellular distribution of HP1a, HP1b, and HP1c proteins in S2

cells. (A) S2 cells grown on coverslips were fixed in paraformaldehyde. DNA was

labeled with 6-diamidino-2-phenylindole (DAPI) and HP1 isoforms with affinity-

purified antibodies directed against HP1a, HP1b, and HP1c as indicated. The

dense, darkly staining regions are heterochromatic. Scale bar = 5 μm. (B)

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Quantification of the percentage of HP1 distribution in S2 cells. Intracellular

distribution of HP1 homologs in S2 cells is determined by ZEN software. The

percentage of HP1 distribution in nucleus and cytoplasm was obtained for

fluorescence intensity in S2 cells. More than 40 cells from 10 random fields per

sample were analyzed. The data are expressed as the means ± SD from at least

three independent experiments. (C) Subcellular distribution of HP1a, HP1b, and

HP1c in S2 cells was assessed by cell fractionation and western Blot analysis.

The data presented are representative images from at least three independent

experiments. Acetylated histone H4 and α-tubulin are markers for the nuclear

and cytoplasmic fractions, respectively. N, nucleus; C; cytoplasm.

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Fig. 3. Intracellular distribution of HP1 chimeric or truncated proteins in S2

cells. (A) Schematic representation of the HP1 chimeric or truncated constructs.

Chimeric or truncated constructs are grouped according to HP1a, HP1b, and

HP1c. (B-D) Immunofluorescence of HP1 chimeric or truncated proteins. S2 cells

were transfected with the indicated chimeric or truncated constructs fused to the

Flag epitope and incubated with CuSO4 (500 μM) for 24 h. Cells were fixed in

paraformaldehyde and stained with monoclonal Flag M2 antibody. Nuclei were

stained using DAPI. Each image was captured by confocal microscopy and 10

images per sample were analyzed. The data presented are representative

images from at least three independent experiments. Scale bar = 5 μm. (E)

Summary table of the intracellular distribution of HP1 chimeric or truncated

proteins in S2 cells.

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Fig. 4. CD of HP1 binds to H3K9me peptide with differential binding affinity.

(A) In vitro histone peptide binding assays using recombinant HP1 proteins and

differently methylated H3K9 and unmethylated H3 peptides. Mock is in the

absence of peptide. HP1 proteins co-precipitated with H3K9 peptides were

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detected by western blot using anti-Flag antibody. (B) Binding of the recombinant

CD of HP1 proteins to differently methylated and unmethylated H3K9 peptides in

fluorescence polarization binding assays. The average bound fraction of H3

peptides from three independent experiments is plotted (binding curves: right

panel). Dissociation constant values (KD in μM) for the interaction of the CD of

the different HP1 proteins with the indicated H3 peptides are listed on the right.

Values represent the means ± S.D from three independent experiments. (C) In

vitro competitive histone peptide binding assays using differentially tagged

recombinant HP1 proteins and H3K9me2 peptide. The molar ratio of HP1a,

HP1b, and/or HP1c is 1:1 or 1:1:1. Recombinant HP1a and either HP1b or HP1c

and/or all three HP1s were incubated with 0.5 μg of H3K9me2 peptide and pulled

down with streptavidin beads. Input (2%) was analyzed by SDS-PAGE followed

by Coomassie Brilliant Blue staining (CBB). B represents the bound fraction and

S indicates the unbound fraction. The binding and unbinding levels of HP1

protein to H3K9me2 peptide were quantified and the levels in the bound fraction

were set at 1. The data presented are representative images from at least three

independent experiments.

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Fig. 5. Three HP1 homologs have different binding affinities for different

interacting partners. In vitro binding assays using recombinant HP1 and HP1

interacting proteins. Recombinant Su(Var)3-9 (A), dSETDB1 (B), dG9a (C), or

dSSRP1 (D) and HP1a, HP1b, or HP1c were mixed with 0.1 mg/ml BSA to

reduce background binding. The resulting complexes were immunoprecipitated

using the indicated beads. Beads and input (2%) were analyzed by western blot

using anti-Flag, anti-HA, anti-His, and anti-GST antibodies. The relative binding

levels of HP1 to partner protein were quantified and the levels in the HP1a were

set at 1. The data presented are representative images from at least three

independent experiments.

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Fig. 6. Three HP1 homologs interact with phosphorylated Pol II. (A)

Coimmunoprecipitation of HP1s with Pol II. S2 nuclear extracts from cell lines

stably expressing Flag-HP1 homologs were immunoprecipitated with an anti-Flag

antibody, followed by immunoblotting with the indicated Pol II antibodies. Ten

percent of total cell lysates used in immunoprecipitation are shown as input (in).

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The relative binding levels of HP1 protein to Pol II were quantified and the levels

in the HP1a were set at 1. (B) Coimmunoprecipitation of endogenous HP1s with

Pol II. S2 nuclear extracts were immunoprecipitated with an anti-HP1 antibody,

followed by immunoblotting with the indicated Pol II antibodies. Ten percent of

total cell lysates used in immunoprecipitation are shown as input (in). The relative

binding levels of HP1 protein to Pol II were quantified and the levels in the HP1b

or unmodified (unphosphorylated) Pol II at each IP sample were set at 1. Pol II

proteins are denoted with asterisks (*). (C) CTD peptide pull-down assays of

recombinant Flag-HP1 and unmodified and phosphorylated CTD peptides of Pol

II. The binding and unbinding levels of HP1 protein to CTD peptides were

quantified and the levels of each HP1 to unphosphorylated CTD peptide were set

at 1. (D) CTD peptide pull-down assays of full-length HP1 (FL), CD+H, and

CSD+H of HP1s. The relative binding levels of HP1 protein to CTD peptides were

quantified and the levels in the HP1 (FL) were set at 1. The data presented are

representative images from at least three independent experiments.

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Fig. 7. Three HP1 homologs play a distinct role in transcription. (A) HEK

293T cells were transfected with GAL4-DBD and GAL4-HP1 expression

constructs and a GAL4-response luciferase reporter. After 24 h, luciferase

activity was measured and normalized using a luciferase reporter system. The

graph shows relative luciferase levels, as measured with a luminometer, of

GAL4-DBD-HP1s compared with the GAL4-DBD control. The results are mean ±

S.D. of at least three independent experiments. *** P < 0.001 vs the GAL4-DBD

control; Student t-test. (B) Expression levels of GAL4-DBD and GAL4-DBD-HP1

were assayed by SDS-PAGE gel and immunoblotting with GAL4-specific

antibodies. Western blot analysis shows expression of the HP1 constructs. α-

tubulin is used a loading control. (C) GAL4-DBD luciferase reporter construct was

cotransfected with the indicated amounts (ng) of GAL4-HP1 expression vectors.

Changes in luciferase activity compared with 100 ng of GAL4-DBD control vector

are shown. The graph shows relative luciferase levels, as measured using a

luminometer, of GAL4-DBD-HP1s compared with those of the GAL4-DBD control.

The results are mean ± S.D. of at least three independent experiments. * P <

0.05 and ** P < 0.01 vs the GAL4-DBD control; Student t-test.

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Figure S1. Specificity of the anti-HP1b and HP1c antibodies, related to

Figure 1.

Western Blot on HP1 isoforms using the rat polyclonal anti-HP1b and -HP1c antibodies

used in this study. (A and B) The purity of the antibody was determined by SDS-PAGE

followed by Coomassie Brilliant Blue staining (CBB). (C) Whole extract of S2 cells and

purified recombinant GST-HP1b and GST-HP1c were subjected to SDS-PAGE and

blotted using an anti-HP1b antibody. (D) Whole extract of S2 cells or HP1c−/− mutant flies

and purified recombinant GST-HP1c were subjected to SDS-PAGE and blotted using an

anti-HP1c antibody and anti α-tubulin antibody.

J. Cell Sci.: doi:10.1242/jcs.222729: Supplementary information

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Figure S2. Distribution analysis of HP1a, HP1b, and HP1c proteins using anti-

HP1a, -HP1b, and -HP1c antibodies made by other lab in S2 cells, related to

Figure 2.

(A) Enlargement immunofluorescence images (lower) represent the yellow dotted box

(upper) in each image. S2 cells grown on coverslips, were fixed in paraformaldehyde.

DNA was labeled with 6-diamidino-2-phenylindole (DAPI) and HP1 isoforms with each

affinity purified antibodies directed against HP1a, HP1b (two different antibodies used),

and HP1c as indicated. The dense, darkly staining regions are heterochromatic. Scale

bar = 5 μm. (B) Quantification of the percentage of HP1 distribution in S2 cells.

Intracellular distribution of HP1 homologs in S2 cells is determined by Zen software. The

percentage of HP1 distribution in nucleus and cytoplasm was obtained for fluorescence

intensity in S2 cells. More than 40 cells from 10 random fields per sample were analyzed

and representative results are shown above. The data presented are representative

images from at least three independent experiments.

J. Cell Sci.: doi:10.1242/jcs.222729: Supplementary information

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Figure S3. Distribution analysis of HP1a, HP1b, and HP1c proteins using anti-

HP1a, -HP1b, and -HP1c antibodies made by other lab in S2 cells, related to Figure

2. (A) S2 cells grown on coverslips, were fixed in paraformaldehyde. DNA was labeled

with 6-diamidino-2-phenylindole (DAPI) and HP1 isoforms with each affinity-purified

antibodies directed against HP1a, HP1b, and HP1c as indicated. The dense, darkly

staining regions are heterochromatic. Scale bar = 10 μm. (B) Subcellular

J. Cell Sci.: doi:10.1242/jcs.222729: Supplementary information

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distribution of HP1a, HP1b, and HP1c in S2 cells was assessed by cell fractionation and

Western blot analysis. The data presented are representative images from at least

three independent experiments. Acetylated histone H4 and α-tubulin are markers for

the nuclear and cytoplasmic fractions, respectively. N, nucleus; C, cytoplasm.

J. Cell Sci.: doi:10.1242/jcs.222729: Supplementary information

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Figure S4. The expression levels of the HP1 constructs, related to Figure 3.

(A) Schematic representation of the HP1 chimeric or truncated constructs. Chimeric or

truncated constructs are grouped according to HP1a, HP1b, and HP1c. (B) The

expression levels of the swapped or deleted domain mutants (1-8) in S2 cells were

assayed by SDS-PAGE gel and immunoblotting with an anti-Flag antibody. Western blot

shows expression of the HP1 constructs. Expressed recombinant proteins are denoted

with asterisks (*). α-Tubulin is used a loading control.

J. Cell Sci.: doi:10.1242/jcs.222729: Supplementary information

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Figure S5. The Alignment of Drosophila HP1s, related to Figure 4. The primary

amino acid sequences of Drosophila HP1a (205), HP1b (241), and HP1c (237) were

aligned using Vector NTI.

J. Cell Sci.: doi:10.1242/jcs.222729: Supplementary information

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Figure S6. The HP1 binds to H3K9me2 peptide with differential binding affinity,

related to Figure 4. SPR responses for binding of HP1 to an H3K9me2 surface. The

increasing concentrations of H3K9me2 peptide was injected to a surface of HP1a (A),

HP1b (B), or HP1c (C) for 200 s. The protein concentrations are rom bottom to top 0, 3,

10, 30, 60, 90 μM. (D) Dissociation constants values (KD in μM) are listed in the table.

J. Cell Sci.: doi:10.1242/jcs.222729: Supplementary information

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Figure S7. The different regions of the three HP1 homologs interact with

various partners, related to Figure 6.

(A) Schematic representation of HP1a or HP1c truncation mutants. (B) Input of HP1s

truncation mutants. (C-F) Indicated deletion derivatives of GST-HP1a or -HP1c were

examined for binding to indicated recombinant protein. Beads and input (2%) were

analyzed by western blot using the indicated antibodies in C-F.

J. Cell Sci.: doi:10.1242/jcs.222729: Supplementary information

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