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C-
0 oqulum
Does heterochromatin protein ~ always follow code?
Yuhong Li*, Dawn A. Kirschmannt, and Lori L. Wallrath*t
Departments of *Biochemistry and "Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242
Heterochromatin protein 1 (HP1) is a conserved chromosomal
protein that participates in chromatin packaging and gene silenc-
ing. A loss of HP1 leads to lethality- in Drosophila and correlates
with metastasis in human breast cancer cells. On Drosophila
polytene chromosomes HP1 is localized to centric regions, telo-
meric regions, in a banded pattern along the fourth chromosome,
and at many sites scattered throughout the euchromatic arms.
Recently, one mechanism of HP1 chromosome association was
revealed; the amino-terminal chrome domain of HP1 interacts with
methylated Iysine nine of histone H3, consistent with the histone
code hypothesis. Compelling data support this mechanism of HP1
association at centric regions. Is this the only mechanism by which
HP1 associates with chromosomes? Interest is now shifting toward
the role of HP1 within euchromatic domains. Accumulating evi-
dence in Drosophila and mammals suggests that HP1 associates
with chromosomes through interactions with nonhistone chromo-
somal proteins at locations other than centric heterochromatin.
Does HP1 play a similar role in chromatin packaging and gene
regulation at these sites as it does in centric heterochromatin? Does
HP1 associate with the same proteins at these sites as it does in
centric heterochromatin? A first step toward answering these
questions is the identification of sequences associated with HP1
within euchromatic domains. Such sequences are likely to include
HP1 "target genes" whose discovery will aid in our understanding
of HP1 lethality in Drosophila and metastasis of breast cancer cells.
n eukaryotes, there are two major types of chromatin: hetero-
~ chromatin and euchromatin (1~. Heterochromatin corre-
sponds to the relatively gene-poor, late-replicating, repetitious
sequences found near centric and telomeric locations. In con-
trast, euchromatin replicates relatively early in the cell cycle and
contains single copy sequences, including the majority of genes.
Both euchromatin and heterochromatin are packaged into nu-
cleosomes, the fundamental packaging unit consisting of a
histone octamer. Euchromatin and heterochromatin can be
distinguished by specific histone tail modifications. In general,
the histone tails in heterochromatin are relatively hypoacety-
lated; however, acetylation of lysine twelve of histone H4 is a
distinguishing mark for heterochromatin (2-4~. In contrast,
histone H3 and H4 tails found in euchromatin are generally
acetylated (4~. Histone H3 acetylation is often linked to H3
phosphorylation and is likely to represent a two-component code
for high levels of gene expression (5, 6~.
In addition to distinct differences in histone modification,
euchromatin and heterochromatin show differences in nonhis-
tone chromosomal protein constituents. One of the best-studied
examples is heterochromatin protein 1 (HP1) first discovered in
Drosophila and named for its predominant localization to centric
heterochromatin (7) (Fig. Lid. Consistent with this localization,
the gene encoding HP1, Su~varJ2-5, was isolated as a dominant
suppressor of position effect variegation (PEV) (8, 9~. PEV is the
mosaic pattern of expression exhibited by genes placed near
centric heterochromatin by chromosomal rearrangements or
transposition events (10~. Overexpression of HP1 leads to en-
hanced silencing of variegating genes. Conversely, a decreased
level of HP1 leads to reduced silencing of variegating genes. A
16462-16469 1 PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4
complete loss of HP1, as in homozygous Su~var>2-5 null mu-
tants, results in lethality. Larvae survive until the late third instar
stage because of maternally supplied HP1 (11, 12~. The cause of
lethality is unknown. Given the centric localization of HP1, and
the interaction between the Schizosaccharomyces pombe HP1-
like protein Swi6 and a cohesion protein, chromosome segre-
gation might be affected (13, 14~. Thus, HP1 levels are critical for
regulating the extent of heterochromatization within centric
regions that is required for proper chromosome segregation.
In addition to centric regions, HP1 is observed at other regions
of the genome known to be heterochromatin The small fourth
chromosome of Drosophila melanogaster, interspersed with het-
erochromatic domains, shows a banded pattern of HP1 local-
ization (7, 15~. Consistent with HP1 having a packaging function
at these locations, transgenes inserted along the fourth chromo-
some exhibit PEV that is suppressed by Su~varJ2-5 mutations
(15, 16~. HP1 localization is also observed et Drosophila telo-
meres that terminate in repetitive arrays of retrotransposons
(17~. Telomeric association, however, appears to be independent
of primary DNA sequence as broken chromosomes lack-
ing terminal retrotransposons retain HP1 association (12~.
Telomere-telomere fusions occur in larval neuralblasts of
Su~var)2-5 mutants, suggesting HP1 plays a role in telomere
capping (12~.
In contrast to these chromosomal domains rich in repetitive
DNA sequences, HP1 is present at approximately 200 sites
within the euchromatic arms of polytene chromosomes that are
relatively poor in repetitious DNA sequences. Do these sites
represent small domains of repressive chromatin? Are there
genes at these sites that are regulated by HP1? These questions
are currently under investigation.
Here we describe current studies on the role of HP1 in gene
regulation at both euchromatic and heterochromatin domains.
We summarize the results from reports that have identified HP1
partner proteins and discuss implications for these findings. Last,
we hypothesize about multiple mechanisms of HP1 chromosome
association and their impact on gene expression.
HP1 Follows Code
HP1 is a highly conserved protein with family members found in
a variety of eukaryotic organisms ranging from S. pombe to
humans (18-214. In Drosophila, two additional HP1-like pro-
teins, HPlb and HPlc, sharing amino acid sequence similarity
and domain structure, have been identified (22) (Fig. 2~.
Whereas HPlb shows localization to both euchromatin and
heterochromatin just as HP1, HPlc is found only in euchromatin
(22~. Mice and humans each have three HP1-like proteins that
possess similarities in amino acid sequence, domain structure,
This paper results from the Arthur M. Sackier Colloquium of the National Academy of
Sciences, "Self-Perpetuating Structurai States in Biology, Disease, and Genetics," held
March 22-24, 2002, at the National Academy of Sciences in Washington, DC.
Abbreviations: CAF1, chromatin assembly factor 1; CD, chrome domain; CSD, chrome
shaclow domain; HP1, heterochromatin protein 1; ORC, origin recognition complex; HOAP,
HP1/ORC-associated protein; PEV, position effect variegation; Rb, retinobiastoma.
tTo whom reprint requests should be acicdressecl. E-maii: iori-wailrath@?ulowa.edu.
www.pnas.org/cgi/cloi/10.1073/pnas.162371699
OCR for page 87
Fig. 1. (A) Pattern of HP1 distribution on Drosophila polytene chromosomes.
D. melanogaster larval polytene chromosomes were stained with mouse
monoclonal C1A9 antibodies against HP1 (gin of Sarah C. R. Elgin) and a
secondary antibody conjugated with rhodamine. The chromocenter (C), the
fourth chromosome (indicated by 4), telomeres (T), and euchromatic sites
associated with HP1. (B) The pattern of HP1 and methylated Iysine nine of
histone H3 on Drosophila polytene chromosomes. D. melanogaster larval
polytene chromosomes were stained with mouse monoclonal C1A9 antibody
against HP1 and a rabbit polyclonal antibody that recognizes methylated
Iysine nine of histone H3 (gin of C. David Allis, University of Virginia, Char-
lottesville). A Cy5-conjugated rabbit secondary antibody and a FITC-
conjugated mouse secondary antibody were used for detection. The chromo-
center (C) and the fourth chromosome (indicated by 4) show strong
colocalization (yellow). Example locations enriched in HP1 are denoted by
green arrows; example locations enriched in methylated Iysine nine of histone
H3 are indicated by red arrows. (C) Same as in B. showing a closer view of the
chromocenter region. (D) Same as in B. showing a closer view of a telomeric
region.
and centric chromosomal localization properties as Drosophila
HP1 (Fig. 2~. Although there are minor differences in chromo-
somal localization and protein interaction partners for HP1-like
proteins within a given species (23), it is not clear whether these
proteins have specific or redundant functions. In flies, mice, and
humans, the HP1-like proteins are small in size, ranging from 173
to 240 aa (Fig. 24. Overall the percent identity of HP1-like
proteins to Drosophila HP1 is approximately 50% for mamma-
lian HP1-like proteins. The majority of conserved amino acids
are concentrated in two domains. The structure of HP1-like
proteins can be simplified as two conserved domains separated
by a less conserved hinge region (Fig. 2~. The conserved
amino-terminal region of HP1-like proteins is termed the
chromo domain (CD) (24~. This domain is present in 20 proteins
in Flybase (www.ebi.ac.uk/proteome/DROME/interpro/
stat.html), many of which play roles in gene regulation. The
conserved carboxyl-terminal region, termed the chromo shadow
Li et a/.
domain (CSD), is related to the CD in primary amino acid
sequence (25~. Both the CD and the CSD have been the subject
of extensive structural analysis (26-30~. Each domain forms a
hydrophobic pocket. The CSD dimerizes (18, 27, 28, 31) as well
as interacts with a wide variety of nuclear proteins (see below).
Cross-species functional studies in which the mouse HP1-like
protein M31 was expressed in S. pombe indicate that species-
specific functions of HP1 reside within the CSD (32~. The CD is
required for chromosome association (33~.
The mechanism~s) by which HP1 establishes the complex
localization pattern on chromosomes remained a mystery for
over a decade since its discovery. For many chromosomal
proteins, localization is achieved through direct interaction with
DNA sequences. Attempts to identify specific interactions be-
tween HP1 and DNA sequences, particularly repetitive DNA
sequences found within heterochromatin, were not particularly
revealing (344. For some chromosomal proteins, localization is
achieved through interactions with DNA binding proteins.
Therefore, a search for HP1 partner proteins might reveal the
"missin~ link" between HP1 and the chromosome. A phage
display assay was performed to identify peptides that interact
with the CD and CSD (31~. This assay revealed peptide se-
quences that showed a specific interaction with the CSD. Com-
parison of the peptide sequences allowed a consensus pentapep-
tide to be generated (31~. Supporting these results, the consensus
pentapeptide was found in several proteins shown to interact
with HP1 by other types of assays (35-37~. To date, the local-
ization pattern of candidate interacting proteins cannot explain
the entire localization pattern observed for HP1. In contrast to
the results obtained for the CSD, no peptides were identified
from the phage display assay that specifically interacted with the
CD. These results were surprising because a point mutation
within the CD of Drosophila HP1 eliminates the majority of
chromosome association, suppresses PEV, and is homozygous
lethal (9~.
The mystery surrounding interactions of the CD was solved by
studies of the murine Suv39hl protein, a homologue of the
Drosophila SU(VAR)3-9 protein (38, 39~. A comparative
genomic approach in combination with biochemical studies
revealed that the SET ta conserved motif in Drosophila
Su~rar'3-9, Enhancer of Zeste, and trithora~c] domain of Suv39hl
contains methyltransferase activity specific for lysine nine of
histone H3. This methylation mark on the histone H3 tail serves
as a specific recognition code for the CD of HP1. This discovery
supports the histone code hypothesis that proposes histone tail
modifications serve as specific recognition motifs for chromatin
proteins (40~. The HP1 CD, but not the CD of several other
proteins, binds methylated lysine nine of histone H3 (41~.
Therefore, the substrate specificity is likely caused by minor
differences in the amino acid sequences of CDs from different
proteins. The connection between Suv39hl and HP1 is consis-
tent with Drosophila research showing that the genes encoding
HP1 and SU(VAR)3-9 genetically interact with the heterochro-
matin silencing system (8) and that the proteins physically
interact (42~. The relationship between HP1 and Suv39hl has
been maintained by the S. pombe homologues, Swi6 and Clr4,
respectively (43, 44), suggesting evolutionary conservation in this
mechanism of chromosome association and heterochromatic
. .
gene S1 encing.
In summary, HP1 serves as a bridging protein, connecting
histones, through interactions with the CD, to nonhistone chro-
mosomal proteins, through interactions with the CSD (Fig. 3A).
In this case, Suv39hl sets the histone code for HP1 association.
Based on these findings, mechanisms for heterochromatin
spreading have been proposed to involve recruitment of Suv39hl
by HP1 and propagation of the methylation mark along the
chromosome (39~. Details of such spreading mechanisms remain
to be elucidated.
~_
~ ~ _
~ ~ -
PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16463
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Drosophila:
HP1 _
18 76 141 203
HP1 b _ _
3 52 94 153
HP1c
8 5~ 79 134
Human:
HP1 HI _
14 71 116 176
HP1 Us, __
15 72 112 172
p1Hs'
14 71 106 166
Mouse:
mHP1a _ _ _
14 71 116 176
_ 1 __
15 72 112 172
M32 __
14 71 106 166
S. pombe:
75 136 199 252
JO Identity
Total AA Total CD Hinge CSD
206 1 00
240 51
237 44
191 49
185
191 49
185 54
328
100
58
100 100
42 58
53 33 36
~0
54 67
52 59
60
67
52 59
25 48
27 54
24
26 56
52
27 54
56
26
24 52
13 17
Fig. 2. Diagram of HP1 proteins in Drosophila, mouse, human, and S. pombe. Total length of each protein is indicated and drawn to relative scale. Percent
identity when compared with Drosophila HP1 over the full length (total), or the CD, CSD, or the hinge region was calculated according to ref. 85.
Are There Multiple Mechanisms for HP1 Association?
HP1 localizes to distinctly different environments throughout
the genome. Has the discovery of the interaction with the
methylated lysine nine of histone H3 cracked the code, or are
there alternative mechanisms of HP1 chromosomal association?
The importance of this question is evident when reviewing data
on the Su~varJ2-502 allele of the gene encoding HP1. This allele
contains the amino acid substitution of a highly conserved valine
to a methionine in the CD. Structural analysis indicates that this
residue plays a critical role in the formation of the hydrophobic
pocket of the CD (30~. In most genetic silencing assays this allele
behaves as an HP1 null; however, an important distinction
between this allele and null alleles was revealed by a cytological
analysis of HP1 staining on chromosomes from HP1 mutants.
Whereas null alleles show no HP1 chromosome association, the
Su~varJ2-502 allele shows diminished HP1 localization to centric
regions, but retains association at euchromatic and telomeric
sites (12~. These data suggest an alternative mechanism of HP1
association might be operating at concentric locations.
Further evidence for alternate mechanisms of association
comes from cytological experiments on polytene chromosomes
in wild-type flies. The pattern of staining observed by antibodies
to HP1 and methylated lysine nine of histone H3 is not com-
pletely coincident. Both antibodies show colocalization to the
chromocenter and along the fourth chromosome, but not
throughout the euchromatic arms and at telomeric regions (Fig.
1 B-D) (45~. One technical explanation for incomplete colocal-
ization is that the epitopes recognized by either antibody are
masked by fixation at specific genomic locations. However, if this
is not the case, sites within the euchromatic arms that stain with
only the HP1 antibody could be generated by HP1 associations
through mechanisms independent of SU(VAR)3-9. Interactions
of HP1 with unmodified histone tails, the histone-fold domain,
and histone H1 might account for the staining pattern observed
(34, 46~. Such possibilities are diagrammed in Fig. 3B. Alterna-
tively, interactions with nonhistone chromosomal proteins might
16464 1 www.pnas.org/cgi/doi/10.1073/pnas.162371699
serve as an additional mechanism of association (Fig. 3C).
Interactions between HP1 and transcriptional corepressors that
associate with DNA binding proteins (see below) (47, 48)
support this hypothesis. The double staining also revealed sites
within the euchromatic arms that are detected only by the methyl
lysine nine histone H3 antibody. These sites could correspond to
different degrees of methylation because the antibody recog-
nizes only dimethylated lysine (Upstate Biotechnology, Lake
Placid, NY); HP1 is thought to recognize both methylated states
with relatively equal affinity (30~. Another explanation for lack
of complete colocalization of HP1 and the methyl lysine nine
histone H3 antibody is that additional histone modifications
might be present that do not permit HP1 association (Fig. 3D).
Clearly the code for chromosomal protein association might
have multiple components.
HP1 Interacts with a Myriad of Proteins
Does the identification of proteins that associate with HP1
provide clues about the mechanisms of silencing? Genetic
analysis of PEV in Drosophila provided a collection of mutations
that encode candidate HP1 interaction partners: for example,
SU(VAR)3-9, the histone methylase discussed above interacts
with HP1 by two-hybrid analysis (42) (Table 1~. A second
example is SU(VAR)3-7, a zinc finger protein that associates
with satellite DNA sequences (49~. HP1 and SU(VAR)3-7
colocalize in the Drosophila embryo and on polytene chromo-
somes (50, 51) and interactions between the two proteins have
been demonstrated by yeast two-hybrid analysis and coimmu-
noprecipitation from embryonic extracts (35, 50~. More specif-
ically, the CSD of HP1 interacts with multiple regions of
SU(VAR)3-7, but it is not yet clear how these two proteins
collaborate to form and/or spread heterochromatin.
In addition to a gene silencing function, HP1 is thought to play
a role in nuclear organization. This hypothesis is based on the
discovery that HP1 interacts with lamin B receptor, either
directly (52, 53) or indirectly through interactions with histones
~i et a/.
OCR for page 89
A) Interaction with methylated histones
B) Interactions with nucleosomes
~ ,~
C) Interaction with DNA binding proteins
_.
D) Exclusion by histone modifications
__ ~
ark 43
Me Ac Me Ac
1 1
Fig. 3. Models for HP1 association and nonassociation with chromosomes.
(A) Interaction between the HP1 CD with the methylated Iysine nine of histone
H3. HP1 serves as a bridge for partner proteins. (B) HP1 interacts with histones
in a nonmethylated-dependent fashion. (C) HP1 associates with chromosomes
through interactions of the CSD and DNA binding proteins, such as zinc-finger
proteins (ZNF). (D) HP1 does not associate with methylated histones that have
additional modifications such as acetylation or phosphorylation.
(54~. In addition, experimental data support an interaction
between HP1 and B-type lamin and Lap2f, lamin-associated
protein, located within the nuclear envelope. In vitro, these
interactions foster nuclear envelope assembly, suggesting HP1
plays a role in organizing nuclear architecture (55~. Given that
heterochromatin localizes to the nuclear periphery in many
eukaryotic cell types, HP1 might tether heterochromatin to the
Li et a/.
nuclear envelope, leaving active regions of the genome free to
coalesce into transcription factories within the interior of the
nucleus (56~.
The localization of HP1 to many sites throughout the Dro-
sophila euchromatic arms brings to question the role of HP1 in
the regulation of gene expression. Supporting a role for HP1 in
transcriptional regulation, HP1 has been shown to interact with
numerous proteins involved in modulating chromatin structure
and gene expression (Table 1~. In mammals, association of HP1,
the retinoblastoma (Rb) protein and SUV39H1 with the cyclin
E promoter correlates with gene silencing (57~. Furthermore,
HP1 has been implicated in gene repression mediated by
Kruppel-associated box (KRAB) zinc finger proteins (47, 48~.
Taken together, these findings suggest that HP1 is recruited to
specific genes by protein-protein interactions, resulting in gene
silencing by an unknown mechanism.
In addition to transcriptional regulators, HP1 interacts with
proteins involved in DNA replication and repair. Chromatin
assembly factor 1 (CAF1) is a three-subunit complex that
assembles histones H3 and H4 onto newly replicated DNA in
both euchromatic and heterochromatic regions of the genome.
In mammals, the large subunit, pl50, contains a domain that
interacts with the CSD of HP1 (37~. Deletion of this domain does
not alter CAF1-mediated chromatin assembly after replication
in vitro or targeting of HP1 to heterochromatin in vivo during
DNA replication. However, deletion of this domain reduces the
amount of CAF1 present in heterochromatin outside of S phase.
Although the significance of CAF1-HP1 interaction is not clear,
the data suggest that CAF1 might stabilize heterochromatin
structure during times of chromosome decondensation and
transcription.
HP1 associates with origin recognition complex (ORC) pro-
teins (58, 59~. This presents an intriguing parallel to the situation
in Saccharomyces cerevisiae where ORC proteins associate with
silent information regulator (SIR) proteins to generate silent
chromatin (60, 61~. A high molecular weight complex isolated
from Drosophila embryos was recently shown to contain HP1/
ORC-associated protein (HOAP) in addition to HP1 and ORCs.
HOAP has sequence similarity to high mobility group proteins
and binds to satellite sequences in vitro (62~. Mutations in the
genes encoding ORC proteins and HOAP are suppressors of
PEV, suggesting a role in heterochromatin formation (624.
What regulates HP1 association with protein partners? Post-
translational modifications are likely to govern interactions
between HP1 and partner proteins and/or itself. In Drosophila,
HP1 is multiply phosphorylated giving rise to at least eight
differently charged isoforms (63~. Drosophila embryonic extracts
possess HP1-containing complexes that differ in HP1 phosphor-
ylation status (58, 62~. For example, hypophosphorylated HP1 is
found in a complex containing ORC and HOAP (58, 62~. In
Drosophila, casein kinase II (CKII) is credited for the phosphor-
ylation of serine residues at the amino and carboxyl termini of
HP1. Mutation of these serine residues to alanine reduces the
amount of HP1 localized to centric heterochromatin and reduces
gene silencing, suggesting phosphorylation plays a role in chro-
mosome association and/or complex stability (63, 64~.
In mammals, HP1 phosphorylation changes through the cell
cycle. HPlHSa and HPlHS~ exhibit increased levels of phosphor-
ylation during mitosis (23~. HPlHS~ is a substrate for Pim-1
kinase that phosphorylates a serine cluster in the center of the
protein (65~. Phosphorylation of HPlHS~ is thought to disrupt
protein-protein interactions that are necessary to maintain most
of the centric localization. In G2, phosphorylated HPlHS~ shifts
from a centric location to being dispersed throughout the
nucleus. Clearly, the role of phosphorylation needs further
investigation to understand the biological significance of this
dynamic process.
PNAS I December ~o, 2002 I vol. 99 | supp~. 4 | 16465
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Table 1. HP1 interacting partners and candidate partners
Protein Organism HP1 variant Methodology HP1 domain Ref(s).
Transcription regulation/
chromatin modifying proteins
H1 Drosophila HP1 rPD nd 46
HP1-BP74 H1-like Mouse mHP1a Y2H, FW, rPD Hinge region 46, 86
H3 Mouse mHP1cz, M31, M32 FW, rPD, exlP CD 46, 54
H3 Drosophila HP1 rPD nd 46
Methylated K9 of H3 S. pombe Swi6 rPD, ChlP CD 38, 43
Methylated K9 of H3 Drosophila HP1 IF, FAITC, NMR CD 26, 41
Methylated K9 of H3 Mouse mHP1a, M31, M32 rPD CD 39
Methylated K9 of H3 Human HP1HS~, HP1Hs0, HPlHsY rPD, SPRA CD 38
H4 Mouse M31 rPD nd 54
H4 Drosophila HP1 /n vitro cross-linking CSD 34
MacroH2A1.2* Mouse M31 IF nd 87
SUVAR3-9 Drosophila HP1 IF, Y2H, exlP CSD 42
Suv39h1 Mouse M31 IF, exlP, SED nd 88, 89
SUV39H1 Human HP1Hsf IF, exlP, SED nd 88, 89
Suvar3-7 Drosophila HP1 IF, Y2H, exlP CSD 35, 50
KAP-1/TIF1,8 Human HP1Hsa, HPlHs~ IF, rPD, exlP, SPRA, GFC CSD 28, 46~8
KAP-1/TIF1,B Mouse mHP1`x, M31, M32 IF, rPD, Y2H, exlP, GFC CSD 28, 37, 47, 86, 90
TRF1 /PIN2 Mouse M31 IF nd 91
TAF''130 Human HP1Hs~, HP1HsY Y2H, transPD, exPD CSD 36
TIF1a Mouse mHP1a~, M31, M32 Y2H, rPD CSD 86, 90
mSNF2,B Mouse mHP1 cr Y2H CSD 86
Rb Human HP1, HP1Hs' Y2H, exPD, exlP, ChlP nd 57, 82
Rb Maize HP1> rPD, Y2H nd 82
Dnmt3a Mouse cells mHP1c~ IF nd 92
Dnmt3b Mouse cells mHP1Oe IF nd 92
ATRX/HP1-BP38 Mouse mHP1c~, M31 Y2H, IF CSD 86, 93
Pim-1 Human HP1Hs~ Y2H, exlP, rPD CSD 65
CKII Drosophila HP1 In vitro phosphorylation nd 63
dAF10 Drosophila HP1 transPD CSD 94
DNA replication and repair
CAF-1 p150 Mouse mHP1a, M31 IF, Y2H, rPD, GFC, NMR CSD 28, 37
CAF-1 p150 Human HP1Hs~ rPD CSD 48
Ku70 Human HP1Hs~ Y2H, rPD, exlP CSD 95
BRCA-1 * Human HP1 Hsa IF nd 96
ORC1 Drosophila HP1 translP CD, CSD 58
ORC2 Drosophila HP1 IF, exPD, exlP, translP CD, CSD 58
ORC3 Drosophila HP1 translP CD, CSD 58
ORC4 Drosophila HP1 translP CD, CSD 58
ORC5 Drosophila HP1 exlP, translP CD, CSD 58
ORC6 Drosophila HP1 exlP, translP CD, CSD 58
Xorc1 Xenopus XHP1~, XHP17 Y2H nd 58
HOAP Drosophi/a HP1 IF, exlP nd 62
Nuclear architecture
Lamin B receptor Human HP1Hs~, HP1Hs0, HP1Hs~ Y2H, rPD, exPD, transPD, exlP CSD 48, 52-54
HP1-BP84 Mouse mHP1ar, M31 Y2H CSD 86
Lamin B Mouse M31 BA CD 55
LAP2,B Mouse M31 BA CD 55
Nuclear envelope Mouse mHP1a~, M31, M32 IF, BA CD 55
Other chromosome-associated
proteins
Psc3 S. pombe Swi6 IF, Y2H, exPD, ChlP CD+Glu-rich 13
DDP1 Drosophila HP1 IF nd 97
Arp4/dArp6 Drosophila HP1 IF nd 98, 99
INCENP Human HP1Hs~, HP1HsY Y2H, transPD Hinge region 100
Ki-67 Human mHP1a, M31, M32 Y2H, exPD, IF CSD 101
SP100B Human HP1Hs~, HP1Hs0, HPlHsY IF, Y2H, rPD, transPD CSD 48, 102, 103
EST AA153281 Mouse mHP1a, M31 Y2H, rPD CSD 37
EST AA003533 Mouse mHP1cY, M31 Y2H, rPD CSD 37
BA, o~nu~ng assay; ChlP, chromatin immunoprecipitation; exlP, co-immunoprecipitation using extract; exPD, pull-down assay using extracts; FAITC, fluorescence
anisotropy, isothermal titration calorimetry; FW, farWestern analysis; GFC, gel filtration chromatography; IF, immunofluorescence colocalization; nd, not determined;
rlP, coprecipitation using recombinant proteins; rPD, pull-down assay using recombinant proteins; translP, immunoprecipitation with in vitro-translated protein;
transPD, pull-down assay using in vitro translated protein; SED, sedimentation assay; SPRA, surface plasmon resonance analysis; Y2H, yeast two-hybrid assay.
*Denotes cell cycle-dependent association.
16466 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 162371699
Li et al.
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HP1 Regulates Gene Expression
Effects on Gene Expression Near Centric Heterochromatin. The gene
encoding HP1, Su~var)2-5, was isolated in a screen for suppres-
sors and enhancers of PEV of the white+ gene brought into
juxtaposition with heterochromatic through a chromosomal
rearrangement (8~. To determine the effects of HP1 on chro-
matin packaging, stocks containing the well-characterized
Drosophila hsp26 gene inserted within centric heterochromatic
were used for chromatin structure analysis (16~. These trans-
genes exhibit less accessibility to nucleases and are packaged into
a more regular nucleosome array than euchromatic insertions. In
an HP1 mutant background the transgenes become more acces-
sible to restriction enzyme digestion, indicating a more "open"
chromatin configuration (664. To determine the transcriptional
mechanism impaired by packaging with HP1, high-resolution
chromatin structure analysis was performed (67~. The results
indicated that general transcription factors such as TFIID and
RNA polymerase II are not associated with heterochromatic
transgenes exhibiting silencing. Thus, association of HP1 corre-
lates with a "closed" chromatin configuration that limits the
accessibility of regulatory sites to trans-acting factors. This is
similar to the mechanism of X-chromosome inactivation in
mammals where transcription factors are also absent from genes
on the inactive X (68), but contrasts the mechanism of silencing
at the mating type loci in S. cerevisiae and Polycomb-mediated
silencing in Drosophila in which general transcription factors and
RNA polymerase II are found in association with silenced
promoters (69-71~. Differences between these systems and HP1
silencing could reflect fundamentally different properties of the
silencing systems or developmentally different stages of silent
chromatin maturation.
In contrast to the silencing effects HP1 has on euchromatic
genes, HP1 is required for the expression of genes that naturally
reside within heterochromatic Over the years genetic and
molecular analyses have revealed genes that reside within centric
heterochromatic (72, 73~. Two well-characterized genes are light,
an essential gene encoding a protein involved in the vesicle
transport pathway (74, 75), and rolled, an essential mitogen-
activated protein kinase (76~. Heterochromatic genes are not
specific for Drosophila; they also have been discovered in
Arabidopsis (77) and are likely to be found in other organisms as
genomic sequence analysis becomes more complete. In Drosoph-
ila, heterochromatic genes appear to be unrelated to each other
in function, however, they do share some common properties.
Structurally, many heterochromatic genes have introns contain-
ing middle repetitive DNA sequences (74) (D. E. Cryderman
and L.L.W., unpublished data). Heterochromatic genes are
inefficiently expressed and sometimes exhibit PEV when trans-
located to euchromatin (78, 79~. In addition, heterochromatic
genes require heterochromatic proteins such as HP1 for
expression (11~.
How does HP1 establish a chromatin configuration that
hinders the expression of euchromatic genes while fostering the
expression of heterochromatic genes? This question will be
better addressed as the promoter regions of heterochromatic
genes are analyzed. Assuming a role for HP1 in chromatin
compaction, HP1 might bring distant regulatory elements in
association with the promoter region of heterochromatic genes.
Alternatively, HP1 may be required to set up a favorable
chromatin configuration within the promoter proximal region
and/or be involved in the recruitment of general transcription
factors as suggested by a recent report showing an interaction
between HP1 and the general transcription factor TF~130 (36~.
Effects of Tethering HP1. What are the effects of HP1 on gene
expression at locations other than centric heterochromatin? One
approach taken to address this question has been to generate
Li et a/.
HP1 fusion proteins containing heterologous DNA binding
domains. In mammalian cell transient transfection assays, effects
of HP1 fusion proteins on the expression of reporter genes
possessing the appropriate DNA binding sequences are assayed.
In these experiments both murine and human HP1 family
members repress transcription when tethered to a small number
of sites located in close proximity to the promoter (36~. Tran-
scriptional repression no longer occurs as the binding sites are
moved to distances more than 2 kb from the promoter (80~. Does
this indicate that HP1-mediated repression has only short-range
capabilities, perhaps operating on a gene-by-gene basis? If this
is the case, HP1 located at euchromatic sites (Fig. 1) might play
a role in the regulation of individual euchromatic genes, rather
than entire domains.
The effects of tethering HP1 in a chromosomal context, as in
transgenic Drosophila, demonstrate the complexities of gene
silencing. A Gal4-HP1 fusion protein tethered upstream of a
reporter gene caused silencing at only one of six genomic
locations tested (51, 81~. Interestingly, the site that supported
silencing was flanked by middle repetitive DNA sequences,
reminiscent of heterochromatic domains. In this case, silencing
could spread in bans to a homologue lacking the tethering sites.
These results suggest that not all chromosomal contexts will
support the formation of silent chromatin by HP1. The inability
to silence at certain locations might depend on the chromatin of
the neighboring region, including the types of histone modifi-
cations, as well as gene density and transcriptional status of the
region.
Identification of HP1 Target Genes. As a second approach to
determine the effects of HP1 on gene expression at locations
other than centric heterochromatin, investigators have identified
potential target genes by their response to HP1 dosage. Repre-
sentational difference analysis identified two genes that are
up-regulated in homozygous HP1 mutant larvae (59~. Interest-
ingly, one of the genes misregulated in the HP1 homozygous
mutant maps to cytological region 31, a chromosome division
that stains intensely with antibodies to HP1 (7~. Two additional
randomly selected genes within region 31 show up-regulation in
HP1 homozygous mutants (59~. For all four candidate HP1
target genes, mutations in additional modifiers of PEV, includ-
ing Su~var)3-9, cause increases in gene expression. These results
suggest that HP1 might function to silence genes located within
euchromatic domains in a mechanism similar to that operating
. . .
In centric regions.
A microarray approach has also been used to identify candi-
date genes regulated by HP1. Several hundred genes mapping
within the euchromatic arms are up-regulated in an HP1 mutant
background (D. E. Cryderman and L.L.W., unpublished data).
In addition, several hundred genes were down-regulated, a
pattern similar to that of heterochromatic genes. For all of the
candidate target genes identified in Drosophila to date, it remains
to be determined whether misregulation is caused by a direct
interaction with HP1. It will be of interest to determine whether
interactions between HP1 and specific genes are conserved
through evolution.
In mammals, it is also likely that HP1 plays a role in the
regulation of genes at concentric locations. HP1 has been
identified as a partner protein for many promoter-associated
factors involved in control of gene repression. These include the
transcription intermediate factor TIF1,B that interacts with zinc
finger proteins containing Kruppel-associated box (KRAB)
domains known to be involved in transcriptional repression. The
role of HP1 in KRAB-mediated repression is not clear, but might
involve recruitment of histone deacetylases (47, 48~.
The first example of a direct association of HP1 with a
promoter region came from studies on the cyclin E gene (574.
Binding of the Rb protein upstream of the cyclin E promoter
PNAS | December 10, 2002 | vol. 99 | suppl. 4 | 16467
OCR for page 92
causes gene silencing, partly through the recruitment of histone
deacetylases. In addition, chromatin cross-linking and immuno-
precipitation experiments place Rb at the promoter with HP1
and methylated lysine nine of histone H3 (57~. Consistent with
the finding, an "Rb binding motif" is present within the amino
acid sequences of HP1 from a variety of species (82~. It is unclear
whether HP1 is recruited to the cyclin E promoter by Rb,
methylated lysine nine of histone H3, SUV39H1, or any com-
bination of these interacting molecules. In support of such
interactions, HP1 possesses the ability to simultaneously interact
with the methylated histone H3 tail and Rb (57~. One hypothesis
is that Rb recruits histone deacetylases first, because the histone
H3 methyltansferase cannot use an acetylated lysine as a sub-
strate for methylation (43), then SUV39H1 methylates the
histone tail which serves as the substrate for HP1 binding.
Interestingly, Drosophila SU(VAR)3-9 was recently purified in
a complex with histone deacetylase HDAC1, suggesting that the
two proteins might cooperate to methylate previously acetylated
histone tails (83~.
The identification of HP1 target genes has implications for
understanding breast cancer metastasis in humans. HPlHS~, but
not HPlHSf or HPlHS7, is down-regulated in highly invasive/
metastatic breast cancer cells compared with poorly invasive/
nonmetastatic breast cancer cells (84~. Introduction of a tagged
HPlHS~ into the highly invasive/metastatic cells, which normally
have low levels of HPlHS~, lead to a less in vitro invasive
phenotype. These results imply that modulation of the levels of
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HPlHS~ alters molecular properties of cells needed for invasion.
Consistent with the cell culture studies, HPlHS~ is down-
regulated in tissues from distant metastatic sites in breast cancer
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Since the discovery of HP1 over 12 years ago by the laboratory
of Sarah C. R. Elgin (Washington University, St. Louis), HP1 has
grown in popularity. In part, this has been caused by the fact that
HP1 has unexpectedly been identified as an interacting partner
for a wide variety of proteins with diverse nuclear functions.
Dissecting the function of HP1 in association with its partner
proteins lies ahead. These experiments will shed light on the
connections between chromatin structure, gene expression,
DNA replication and repair, and nuclear organization.
We thank Sarah C. R. Elgin for the Drosophila HP1 antibody and
C. David Allis for the gift of histone H3 methyl K9 antibodies and the
histone code hypothesis. We thank Pamela Geyer and members of
the Wallrath lab for suggestions regarding this manuscript. Work in the
laboratory of L.L.W. is supported by American Cancer Society Grant
GMC-100527 and National Institutes of Health Grant GM61513. Re-
search into the role of HP1 in breast cancer metastasis was supported by
a Carver Collaborative Pilot grant to L.L.W. and D.A.K. from the Roy
J. and Lucille A. Carver College of Medicine at the University of Iowa.
48
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PNAS 1 December 10, 2002 1 vol. 99 1 suppl. 4 1 16469
Representative terms from entire chapter:
lysine nine
