1 Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA
2 Department of Cell and Structural Biology, University of Illinois,
Urbana-Champaign, Urbana, IL 61801, USA
* Author for correspondence (e-mail: lori-wallrath{at}uiowa.edu)
Accepted 13 January 2003
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SUMMARY |
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Key words: Gene silencing, Drosophila, Heterchromatin protein 1 (HP1)
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INTRODUCTION |
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One protein that exhibits a localization bias towards heterochromatic
regions is Heterochromatin protein 1 (HP1)
(Eissenberg and Elgin, 2000).
On Drosophila polytene chromosomes, HP1 localizes to centric regions,
telomeric regions and in a banded pattern along the small fourth chromosome.
In addition to these heterochromatic locations, HP1 is observed at
approximately 200 sites throughout the euchromatic arms
(James et al., 1989
). HP1
contains two domains, the N-terminal chromo domain and the C-terminal chromo
shadow domain, conserved from yeast to humans
(Eissenberg and Elgin, 2000
).
Structural studies show that these two domains form hydrophobic pockets that
are the sites of protein-protein interactions
(Ball et al., 1997
;
Brasher et al., 2000
;
Cowieson et al., 2000
).
Mutations within these domains affect chromosome segregation and gene
silencing (Fanti et al.,
1998b
; Wang et al.,
2000
).
One mechanism by which HP1 associates with chromosomes is through an
interaction of the chromo domain with methylated lysine nine of histone H3 (H3
K9 methylation), a modification generated by the histone methyltansferase
SU(VAR)3-9 (Bannister et al.,
2001; Jacobs and
Khorasanizadeh, 2002
; Lachner
et al., 2001
; Nielsen et al.,
2002
). This interaction is consistent with the histone code
hypothesis that states histone tail modifications serve as specific
recognition markers for chromosomal proteins
(Jenuwein and Allis, 2001
).
Experimental data support an interaction of HP1 with methylated K9 of H3 for
centric localization. For example, Drosophila homozygous
Su(var)3-9 mutant flies show reduced levels of HP1 in centric regions
(Schotta et al., 2002
). By
contrast, other experimental data suggest that HP1 uses alternative mechanisms
for localization at non-centric locations
(Li et al., 2002
). On
Drosophila polytene chromosomes, methylated K9 of H3 and HP1 do not
exhibit complete co-localization within euchromatin
(Cowell et al., 2002
;
Li et al., 2002
). Furthermore,
a mutation in the chromo domain that abolishes an interaction with methylated
K9 of H3 does not eliminate association at telomeres
(Fanti et al., 1998b
;
Jacobs and Khorasanizadeh,
2002
). Direct interactions of HP1 with unmodified histones and
non-histone chromosomal proteins have been proposed as alternate mechanisms of
HP1 association (Nielsen et al.,
2001
; Zhao et al.,
2000
).
To understand the relationship between HP1 and gene expression better we have developed a system to tether HP1 upstream of a reporter transgene inserted at sites within euchromatic domains of the Drosophila melanogaster genome. We determined that tethered HP1 is sufficient to nucleate the formation of silent chromatin at some, but not all sites tested within euchromatin. Silencing of the downstream reporter gene correlated with formation of ectopic fibers that frequently connected the tethered site to other sites, some of which contain HP1. This finding suggests that HP1 can partner with itself to bring distant chromosome sites into close proximity. Such a mechanism might be used for chromatin packaging, regulating gene expression or nuclear organization. Silencing of the reporter gene was independent of SU(VAR)3-9 dosage, suggesting that HP1 functions downstream of SU(VAR)3-9 in the silencing pathway.
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MATERIALS AND METHODS |
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Stocks with transgenes containing 256, 32 and four copies of lac
repeats cloned 500 bp upstream of a mini-white reporter gene were
generated by standard transformation procedures
(Rubin and Spradling, 1982);
these stocks will be referred to as reporter stocks. Expression of the
mini-white gene, required for eye pigmentation, was used in the
silencing assays. Additional reporter stocks with 15-256 copies of
lac repeats were generated by P-element mobilization
(Robertson et al., 1988
) using
reporter stock 157.1, containing a transgene with 256 copies of lac
repeats, as a starting stock. During the process of mobilization, copies of
lac repeats were lost, generating stocks with smaller numbers of
repeats.
In order to score silencing of mini-white in reporter stocks, the
mini-white transformation marker in the expressor stocks needed to be
removed. Imprecise P-element excision
(Harrison and Perrimon, 1993)
was performed on the expressor stocks to recover flies in which the
white+ transformation marker was deleted but the
lacI fusion gene remained intact. The resulting
white- expressor stocks were crossed to reporter stocks,
heat shocked daily, and silencing of the mini-white reporter was
scored by visual inspection of the adult eye.
Rescue of lethality
To determine whether the lacI-HP1 fusion retained functions of
HP1, we tested lacI-HP1 for the ability to rescue the lethal
phenotype of HP1 mutants. Mutations in the gene encoding HP1 are designated as
alleles of Su(var)2-5 (Eissenberg
et al., 1990; Fanti et al.,
1998b
). Females with the genotype lacI-HP1;
Su(var)2-504/CyO, GFP were crossed to males of the genotype
Su(var)2-502/CyO, GFP. CyO is a balancer chromosome
carrying the Cy mutation that generates a curly wing; the GFP
transgene allows for scoring of the larvae containing this marker under a
fluorescent dissecting microscope (FlyBase). Crosses were heat shocked at
37°C for 45 minutes daily. Rescue of lethality was indicated by the
presence of non-GFP, straight winged adult progeny, representing the genotype
lacI-HP1; Su(var)2-504/Su(var)2-502.
Approximately 100 rescued adult progeny (the expected ratio for complete
rescue) were obtained from five independent crosses.
Immunostaining of polytene chromosomes
To examine the chromosomal localization of the lacI-HP1 fusion
protein with respect to the location of endogenous HP1 and H3 K9 methylation,
third instar larvae were heat-shocked at 37°C for 45 minutes and allowed
to recover at room temperature for 2 hours. Salivary glands were dissected,
fixed and squashed as described (Platero
et al., 1995). To detect HP1, the monoclonal antibody C1A9 (gift
of Dr S. C. R. Elgin) was used. To detect the lacI fusion proteins, a
rabbit polyclonal antibody to lacI (Stratagene) was used. To detect
methylated histone H3, a rabbit polyclonal antibody to H3 K9 dimethyl (gift of
Dr C. David Allis) and a rabbit polyclonal antibody to H3 K9 tri-methyl (gift
from P. Singh) were used. To detect SU(VAR)3-9EGFP, a polyclonal antibody to
GFP (Molecular Probes) was used. FITC-conjugated (Sigma) and Cy5-conjugated
(Jackson ImmunoResearch Laboratories) secondary antibodies were used for
detection. Images were photographed using a DMLB fluorescence microscope
(Leica) and a Spot RT slider digital camera (Spot Diagnostic Instruments).
Determination of P-element insertion sites
Inverse PCR was used to determine the P-element insertion sites in reporter
stocks according to published methods
(Cryderman et al., 1998). A
BLAST search was performed with the sequences obtained to determine the
location of the P-element within the Drosophila genome.
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RESULTS |
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Given that the lacI-HP1 fusion protein was functional, we tested for effects on gene silencing. Flies expressing the fusion protein were crossed to flies carrying a reporter transgene with 256 to four copies of lac repeats located 500 bp upstream of a mini-white reporter gene. For 25 out of 26 reporter stocks, tethered HP1 caused silencing of mini-white, as indicated by a reduction in the eye pigmentation observed in the adult progeny (Fig. 2; Table 1). In the single case where silencing was not observed, stock J3.2, the eye phenotype was red in the presence or absence of tethered HP1 (Fig. 2, middle row). Taken together, the results indicate that tethered HP1 is sufficient to nucleate the formation of silent chromatin at the majority of sites tested.
|
|
In contrast to the lines where the nearest gene was greater than 10 kb
away, 13 lines (12 that showed silencing) had the P-element inserted at
distances less than 10 kb from a gene. We then examined the relationship
between the silencing phenotype of mini-white, the distance to the
transcription start site of the nearest gene, and the expression pattern of
the nearest gene. For 11 out of the stocks in which silencing was observed,
the nearest gene was not expressed or expressed only at low levels, during the
time of development when white was expressed (FlyBase and
http://genome.med.yale.edu/
Lifecycle). Two exceptions were stock S9.2 in which the P-element was inserted
48 bp downstream of the agros gene and stock J3.2 in which the
P-element was inserted 62 bp upstream of the Atp gene
(Feng et al., 1997
;
Freeman et al., 1992
). Both of
these genes are expressed in the developing eye at a similar time in
development as the white gene
(Sawamoto et al., 1998
;
Yasuhara et al., 2000
).
Interestingly, only stock S9.2 showed silencing, while stock J3.2 has a red
eye phenotype in the absence or presence of lacI-HP1
(Table 1). The failure of HP1
to nucleate silent chromatin in stock J3.2 suggests that chromatin
modifications associated at the promoter region of an active gene might block
lacI-HP1 association. Alternatively, they might allow association of
lacI-HP1 but prevent the formation or stabilization of a silencing
complex. To investigate these possibilities, polytene chromosome staining
experiments were performed following expression of lacI-HP1 in stock
J3.2. The results indicated that lacI-HP1 was bound at the location
of the Atp
gene (Fig.
2). Thus, association of HP1 to a site might be insufficient to
nucleate the formation or stabilization of silent chromatin in the promoter
region of an active gene.
In contrast to stock J3.2, silencing might be permitted in stock S9.2 because the P-element was inserted within the coding region of a gene. Chromatin modifications that antagonize silencing might be associated with active promoters and not coding regions. Alternatively, a P-element insertion within a gene might disrupt transcription of that gene, resulting in the absence of modifications associated with gene activity.
Tethered HP1 causes ectopic associations
In addition to silencing, a second effect of tethered HP1 was observed when
examining polytene chromosomes that were fixed, squashed, and stained with
antibodies to lacI-HP1. There was an obvious appearance of ectopic
fibers that frequently connected the tethered site to other nearby sites that
contain HP1 (Fig. 3A,B). This
phenomenon was observed in the four stocks (157.1, 157.4.112, P2.5 and 179.1)
that were examined. Both intra- and inter-chromosomal associations were
observed within a given stock, although intra-chromosomal associations were
more frequent. In some cases, chromosomal contacts occurred between the
tethered site and a site that did not show visible staining for HP1
(Fig. 3C), suggesting other
proteins might participate in ectopic associations. The fibers are likely to
be composed of protein because they disappear with acid treatment and do not
stain with dyes used to visualize DNA. For a given stock, 50% of the
nuclei showed HP1-dependent ectopic associations. This frequency became nearly
100% upon increasing SU(VAR)3-9 dosage through introduction of a heat shock
driven Su(var)3-9-EGFP transgene. Under conditions of daily heat
shock treatments, presumably tethered HP1 recruits SU(VAR)3-9-EGFP and
methylation of nearby histone H3 occurs
(Fig. 4C). Importantly, ectopic
associations were not observed when lacI-HP1 was not expressed or
when the control GFP-lacI was expressed (data not shown).
Interestingly, the associations did not appear in chromosome preparations of
J3.2, the stock that did not show silencing with tethered HP1, implying that
these associations correlated with the process of silencing.
|
|
Several possible explanations could account for the lack of enhanced H3 K9
methyl staining at the tethered site. One explanation is the fusion of the
lacI BD to HP1 allows for a bypass of the histone methylation
requirement. This suggests HP1 functions downstream of SU(VAR)3-9 in
silencing. A second explanation is that the fusion of the lacI BD to
HP1 disrupts the ability of HP1 to interact with SU(VAR)3-9, an interaction
shown by two-hybrid analysis and coimmunoprecipitation experiments
(Schotta et al., 2002). We
tested this possibility by adding an hsp70 driven
Su(var)3-9-EGFP transgene into the system. Under conditions of daily
heat shock treatments SU(VAR)3-9-EGFP was recruited to the tethered site,
implying an interaction with lacI-HP1 can occur in vivo
(Fig. 4C). A third explanation
for the lack H3 K9 methylation is that the methyl mark is masked under the
fixation conditions. This seems unlikely as colocalization of the H3 K9
methylation and HP1 was observed within the chromocenter and at discrete
euchromatic sites (Cowieson et al.,
2000
; Li et al.,
2002
). After considering these possibilities, the cytological data
are most consistent with the idea that SU(VAR)3-9 activity is not required for
silencing by tethered HP1.
The lack of SU(VAR)3-9 recruitment predicts that silencing due to tethered
HP1 should be independent of Su(var)3-9 gene dosage. We genetically
tested this by examining the ability of tethered HP1 to silence in a
Su(var)3-9 mutant background. In general, gene silencing by
heterochromatin is sensitive to both the dosage of HP1 and SU(VAR)3-9
(Wallrath, 1998). As examples,
stocks containing a white+ gene juxtaposed to centric
heterochromatin or inserted within fourth chromosome heterochromatin are
dominantly suppressed by Su(var)3-906, a null mutation
(Fig. 4D)
(Schotta et al., 2002
). Three
reporter stocks (157.1, 157.4.112 and 6.4P5) expressing lacI-HP1 that
were either heterozygous or homozygous for Su(var)3-906
showed no suppression of silencing (Fig.
4D; data not shown). Furthermore, a reduction in the intensity of
HP1 staining on polytene chromosomes at the site of the lacI repeats
was not observed (data not shown). Taking the cytological and genetic data
together, HP1 appears to function downstream of SU(VAR)3-9 in the silencing
pathway in Drosophila.
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DISCUSSION |
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Overall, our results using tethered Drosophila HP1 are consistent
with those using tethered mammalian HP1 proteins. Both human and mouse HP1
proteins have been tethered to a small number of binding sites immediately
upstream of a reporter gene on transient transfected plasmids, resulting in
gene repression (Lehming et al.,
1998; Seeler et al.,
1998
; van der Vlag et al.,
2000
). Our studies reported here extend these findings
demonstrating that Drosophila HP1 is sufficient to nucleate the
formation of silent chromatin on a native chromatin template.
When we compare silencing with lacI-HP1 to a study using a
Drosophila Gal4-HP1 tethering system
(Seum et al., 2001), some
interesting differences emerge. In the Gal4-HP1 studies, tethered HP1 silenced
a white reporter gene at only one of six genomic locations tested.
The authors concluded that HP1 was necessary, but not sufficient, to form
silent chromatin. In their study, the one site that supported silencing was
surrounded by middle repetitious sequences thought to promote the formation of
silent chromatin. One explanation for the different results obtained with the
two HP1 fusion proteins is that the Gal4-HP1 fusion protein possessed limited
capabilities, silencing only in a genomic context already favoring the
formation of silent chromatin. In support of this hypothesis, the Gal4-HP1
fusion did not rescue the lethality of an HP1 mutant as did lacI-HP1
in our study. A second explanation for the different results obtained with the
two different HP1 fusion proteins is that the five locations unable to support
silencing by Gal4-HP1 were within active regions of the genome, similar to the
reporter gene insertion of stock J3.2 described here. However, it is difficult
to know whether this is the case as the genomic locations of the sites that
did not support silencing by Gal4-HP1 were not reported.
Gene silencing by heterochromatin frequently is observed by a phenomena
known as position effect variegation (PEV)
(Weiler and Wakimoto, 1995).
PEV is the cell-by-cell variation in expression of a gene that is brought into
juxtaposition with heterochromatin through a chromosomal rearrangement or
transposition event. In the study reported here, the reporter stocks selected
for analysis were those that did not have the P-element inserted near
heterochromatin, or ones in which the insert was within repetitive DNA
sequences typically found within heterochromatin. In all cases, silencing by
tethered lacI-HP1 was observed as a uniform, non-variegated,
reduction in eye pigmentation. By contrast, the single example of silencing by
tethered Gal4-HP1 was in a stock in which the reporter gene was inserted near
repetitive elements. Taking this data together, it is tempting to speculate
that the PEV phenotype might be related to the presence of repetitive DNA
sequences. Such sequences might play a role in the `on/off' decision of a
promoter by `locking in' a particular conformation state.
Previously, we have shown that gene silencing due to an association of HP1
correlates with a `closed' chromatin structure and regular nucleosome arrays
(Sun et al., 2001;
Wallrath and Elgin, 1995
). One
possible mechanism to explain the formation of a closed chromatin
configuration is through HP1-HP1 interactions on adjacent or nearby
nucleosomes. Such interactions might render DNA sequences inaccessible to
transcription factors and/or prevent nucleosome sliding induced by chromatin
remodeling machines (Vignali et al.,
2000
). HP1-HP1 interactions at more distant chromosome locations
might facilitate chromatin folding, leading to a closed chromatin structure.
In addition, HP1-HP1 interactions might facilitate looping of regulatory
elements (both enhancers and silencers) to promoter regions. This mechanism
could explain how HP1 has different effects on distinct genes depending on
their chromatin context. Finally, HP1-HP1 interactions might indirectly
regulate gene expression by directing the localization and arrangement of
chromosomes within the nucleus. This idea is supported by the discovery that
HP1 interacts with the lamin B receptor at the nuclear membrane
(Ye et al., 1997
). Nuclear
organization is thought to play a pivotal role in gene regulation
(Hediger and Gasser,
2002
).
The results from the lacI-HP1 tethering studies suggest that in
the majority of cases silent chromatin `spreads' at least 500 bp downstream of
the lac repeats, hindering the promoter activity of the reporter
gene. In preliminary studies, we have extended these studies showing that
tethered HP1 can silence strong heat shock promoters up to distances of 5 kb
from the lac repeats (J.R.D. and L.L.W., unpublished). Based on a
current model for heterochromatin spreading
(Bannister et al., 2001),
tethered HP1 would be predicted to recruit SU(VAR)3-9, which would in turn
methylate adjacent histone tails, serving as additional substrates for HP1
association. Both our cytological and genetic data do not support this model
for spreading at distances of 500 bp to 5 kb from the tethered site in a
wild-type genetic background. Only upon increased Su(var)3-9 gene
dosage do we observe recruitment of the SU(VAR)3-9-EGFP and subsequent
enhancement of H3 K9 methylation. These results imply that under normal
circumstances, SU(VAR)3-9 is a limiting component for silent chromatin
formation. Therefore, we favor alternative models for spreading that include
the recruitment of histone deacetylases and/or additional proteins that
propagate the silent state along the chromosome. Our tethering system, in
conjunction with the powerful technique of chromatin immunoprecipitaton
(Orlando et al., 1997
), will
allow us to identify the histone modifications and protein components of
silent chromatin extending from the lac repeats and determine the
mechanism of silent chromatin spreading in greater molecular detail.
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ACKNOWLEDGMENTS |
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