1 Max Planck Institute for Biophysical Chemistry, Department of Molecular
Biology, 37070 Göttingen, Germany
2 King's College London, Randall Division of Cell and Molecular Biophysics, New
Hunt's House Guy's Campus, London SE1 1UL, UK
* Author for correspondence (e-mail: djovin{at}gwdg.de)
Accepted 21 June 2005
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SUMMARY |
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PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo.
We determined the translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria.
Key words: Polycomb group proteins, FRAP, Inverse FRAP, iFRAP, Transcription, Repression, Homeotic genes
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Introduction |
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The Polycomb group (PcG) and Trithorax group (trxG) of proteins are
chromatin-binding proteins responsible for conserving the transcriptional
state of the HOX genes and thereby cell identity. PcG proteins are responsible
for the persistence of silencing whereas the trxG proteins are required for
transcription in the active domains
(Francis and Kingston, 2001;
Levine et al., 2004
;
Orlando, 2003
). PcG proteins
are targeted to particular regions of the genome called Polycomb response
elements (PREs) (Chan et al.,
1994
; Orlando et al.,
1998
; Strutt et al.,
1997
) where they act in multicomponent complexes to repress
transcription of their target genes. The continued presence of PcG proteins on
the PREs throughout development is required for silencing since deletion of
the PRE (Busturia et al.,
1997
) or individual PcG genes
(Beuchle et al., 2001
) anytime
during development of the organism results in gene derepression.
Interestingly, although PcG complexes maintain the repression pattern for up
to 10 cell generations most of the PcG protein complement dissociates at every
mitosis (Buchenau et al.,
1998
).
There exist experimental data for the association of the PcG proteins with
specific chromatin sequences, including the first observations by
immunofluorescence on polytene chromosomes
(Chiang et al., 1995;
Franke et al., 1992
;
Rastelli et al., 1993
). In
vivo crosslinking and chromatin immunoprecipitation (ChIP analysis) of PcG
proteins have preferentially detected high levels of proteins of the PCC
(Polycomb core complex), and recently, also of Pleiohomeotic (Pho) and
Enhancer of zeste [E(Z)], on PREs and promoters of known homeobox genes
(Breiling et al., 2004
;
Ringrose et al., 2003
;
Strutt and Paro, 1997
;
Wang et al., 2004
). Several
models have been proposed for the mechanism of PcG-mediated repression, such
as (1) heterochromatinization or formation of a closed chromatin conformation
that does not allow access to promoters; (2) inhibition of the assembly of the
preinitiation transcription complex; and (3) interference with transcription
initiation and/or elongation (Min et al.,
2003
; Paro and Hogness,
1991
; Simon and Tamkun,
2002
). Experimental evidence can be found to support each of the
models. For example, PcG complexes reduced accessibility for large RNA
polymerases over large stretches of DNA in the bithorax homeobox gene cluster
(BX-C) (Fitzgerald and Bender,
2001
), thereby inhibiting transcription of reporter genes,
although restriction enzymes retained DNA access. However, the presence of PcG
proteins at the Ubx promoter in wing imaginal discs
(Wang et al., 2004
) lends
support to a direct inhibition of transcription, although perhaps only at the
elongation rather than at the initiation step as has been suggested for the
heat shock protein 26 (hsp26) promoter
(Dellino et al., 2004
).
Two different multiprotein polycomb repression complexes (PRCs) have been
isolated and characterized biochemically. PRC2
(Ng et al., 2000) is composed
of the PcG proteins, Extra sex combs (Esc), Suppressor (12) of zeste [Su(z)12]
and histone-binding Nurf-55 and Enhancer of zeste [E(Z)], the latter of which
methylates histone H3 at lysine 27 both in vivo and in vitro
(Cao et al., 2002
;
Czermin et al., 2002
;
Kuzmichev et al., 2002
;
Müller et al., 2002
;
Yamamoto et al., 2004
), thus
marking nucleosomes for assembly of repression complexes. PRC1, which contains
equimolar quantities of Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs
(Psc) and Sex combs extra (Sce/dRing1), all of which have been shown to be
essential for PcG silencing. Other PcG and non-PcG proteins such as Sex combs
on midleg (Scm), heat-shock protein cognate 4 (Hsc4) and Zeste (Z), and some
transcription factors have been isolated with PRC1 in non-stoichiometric
amounts, implying the presence of more than one type of polycomb repression
complex (Levine et al., 2002
;
Mulholland et al., 2003
;
Saurin et al., 2001
).
Whether the in vitro isolated or assembled complexes represent truly
competent repression machineries is a matter of debate, as will be discussed
later. In vivo data imply that functional complexes are assembled
sequentially, directly and with a particular hierarchy, on the chromatin
itself (Buchenau et al., 1998;
Wang et al., 2004
) and single
PcG gene deficiencies such as E(Z) result in loss of complex
formation on PREs, although all of the proteins involved in the PCC or PRC1
are still present (Rastelli et al.,
1993
; Wang et al.,
2004
). For a complete understanding of the repression mechanism,
we need to know the stability and lifetime of functional repression complexes
in the living organism. Recently, it was reported that Polycomb can be
competed away from genomic sites by methylated histone tail peptides in
permeabilized salivary gland nuclei
(Ringrose et al., 2004
).
However, no data have been available about binding equilibria and dissociation
rate constants of any multiprotein PcG chromatin complex in vivo. In this
study, we addressed this problem by performing photobleaching experiments
(fluorescence recovery after photobleaching, FRAP) on GFP fusion proteins of
Polycomb (Pc) and Polyhomeotic (Ph), two essential members of the PCC in whole
living Drosophila embryos and larval tissues to determine their
diffusion, binding equilibria and residence times. We measured these values in
living organisms at different stages of development to determine whether there
are changes in the stability of the complexes. By taking advantage of the
polytene nature of the salivary gland chromosomes, we assessed the uniformity
of the complexes between individual bands. The actual forward and reverse rate
constants for complex formation were determined. Most of the complexes
exchange within a period of 1 minute and all of the complexes in less than 10
minutes. We discuss the compatibility of these data with present models for
repression and draw inferences about the homogeneity of the repression
complex.
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Materials and methods |
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Fly strains and culture
The following strains were used in this study: w1118;
P{pPc-PcGFP,w+}; w1118; P{UAS,Pc-PhGFP,
w+}; yw; P{en2.4-GAL4}e22c/SM5 (to drive expression
of phGFP in embryos);
P{Gal4;w+}BxMS1096 (to drive expression of
phGFP in wing imaginal discs) where it drives the expression of Gal4
in the whole wing blade (Capdevila and
Guerrero, 1994).
All strains were maintained on standard corn-agar medium at 18°C and
experiments were carried out at 25°C. The PcGFP stock was kindly
provided by R. Paro (Dietzel et al.,
1999) and the en:Gal4 and
BxMS1096:Gal4 (Milan
et al., 1998
) drivers were provided by H. Jäckle.
Mounting of specimens for microscopy and imaging
For live imaging dechorionated embryos were transferred to a chamber with a
coverslip bottom (LabTek) in oxygenated Tyrode's buffer (135 mM NaCl, 10 mM
KCl, 0.4 mM MgCl2, 1 mM CaCl2, 5.6 mM glucose, 10 mM
HEPES, pH 7.2). In order to prevent movement and buffer evaporation, they were
covered with a polycarbonate membrane with 8 µm pores that allowed oxygen
exchange (Nucleopore). Larval imaginal and salivary gland tissues were
dissected in PBS and immediately transferred to similar chambers and covered
with a Whatman 3M filter paper soaked in Tyrode's buffer. Imaging was
performed at 21°C for a maximum of 2 hours after mounting with a 63x
N.A. 1.2 water immersion objective using an inverted Zeiss LSM 510META
microscope. GFP was excited with the 488 nm line of an Ar ion laser and
emission collected between 505 and 545 nm with a pinhole equivalent to 2 Airy
discs.
Photobleaching methods and image processing
FRAP images in somatic cell nuclei were performed with an XY sampling of
0.10 µm/pixel and in polyploid salivary gland nuclei, at 0.14 µm/pixel.
Photobleaching was carried out for 200 ms (FRAP in salivary gland nuclei)
at
200 µW laser power (measured through the objective). Pre-bleach and
post-bleach images were acquired at high scanning speed with minimal laser
intensity (AOTF 2%,
5 µW). At later measurement times (after frame
20), the interval between scans was increased in order to reduce bleaching
during monitoring.
3D-iFRAP
Three dimensional inverse FRAP (3D-iFRAP) experiments in embryos and
imaginal discs were carried out by bleaching the whole nucleus except for a
small region surrounding a fluorescent locus of interest for 4 seconds. A
time series of seven confocal z sections (with 5 second intervals
between stacks for PhGFP and 10 seconds for PcGFP) were recorded for
120
seconds after bleaching. The time stacks were aligned using a 3D tracking
algorithm (View5D information can be found at
http://wwwuser.gwdg.de/
rheintz/View5D/)
developed in this laboratory. After alignment, the spot intensity was
calculated using a weighted region of interest.
Image processing and fitting algorithms
Background fluorescence in all photobleaching experiments was measured in a
user-defined field outside the tissue for each experiment separately or
estimated directly from the acquired image. An average loss of fluorescence
intensity during imaging was corrected for in the evaluation of FRAP and iFRAP
data via normalization to time-dependent average intensity plots from separate
nuclei imaged under identical conditions to the FRAP and iFRAP experiments.
This correction was always less than 10%. The relative increase (FRAP) or
decrease (iFRAP) in fluorescence intensity, corrected for background and
bleaching during recovery, was normalized to the pre-bleach value and these
Inorm,i values were plotted for each time point:
![]() | (1) |
![]() | (2) |
![]() | (3) |
In FRAP experiments on bands of PcG proteins in salivary gland nuclei, the free signal was estimated by averaging the intensity in each frame near the spot in a region as defined by the lowest 30% voxels of the sum intensity projection over all aligned pre-bleach frames (see Fig. 7). This nucleoplasmic signal of the free protein estimated frame by frame was subtracted from each pixel and the total bound protein was determined as the sum of all pixels in the mask region of the 30% brightest pixels in the projection over all aligned pre-bleach frames.
Recovery curves were fitted with a single exponential function after
excluding the first 30 seconds after bleaching, during which diffusion still
influences the data in spite of the correction for free protein:
![]() | (4) |
![]() | (5) |
Simulations showed an approximately linear dependence of the
t0 values to the Bratio of a locus
using several fixed dissociation rate constants in the range previously found
from iFRAP measurements in imaginal discs and a diffusion constant derived
from the experimental data (D=0.5 µm2
second1) as is shown by the lines in
Fig. 8. The recovery times
(t0) for the measured data were corrected for this
dependence according to the approximation (Eqn 6):
![]() | (6) |
The pseudo reassociation rate constant kon* was
calculated according to the method described by Sprague et al.
(Sprague et al., 2004) that
defines a pseudo-first-order rate constant given by
![]() | (7) |
![]() | (8) |
Western blots
Crude extracts were prepared from embryos of different developmental stages
and larval tissues using lysis buffer [20 mM HEPES-KOH pH 7.5, 100 mM KCl, 2
mM EDTA, 0.5% Triton X-100, 0.3 U/ml aprotinin, 10 µg/ml leupeptin, 100
µg/ml soy bean trypsin inhibitor, protease inhibitor cocktail tablets
(Roche Diagnostics), 5 mM DTT, 1 mM MgAc2]. Proteins were separated
on NuPage 4-12% Bis-Tris or NuPage 3-8% Tris-acetate polyacrylamide gels, and
western blots were probed with primary polyclonal anti-Pc (kind gift of Renato
Paro), anti-Ph or anti-GFP and HRP-conjugated secondary antibodies by
chemiluminescence (Amersham Pharmacia Biotech). PABP
(Roy et al., 2004), S6 (Santa
Cruz Biotechnology) and eIF4A
(Hernández et al.,
2004
) antibodies were used as loading controls. The intensities of
the signals on the x-ray films were quantified on a scanning densitometer
(G-710, BioRad). Optical density values were extracted and normalized to the
loading controls indicated in Table
1.
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Results |
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Quantitation of western blots of imaginal discs or salivary glands from wild-type and transgenic flies revealed slightly lower levels of GFP-labeled proteins than the endogenous ones, such that the ratio of total Pc protein in transgenics (including PcGFP) was only 1.6 times that in wild type and the ratio of total Ph (including PhGFP) in transgenics was 1.7 times that of wild type (Fig. 1A,B; Table 1). PhGFP expression in wing imaginal discs was induced by BxMS1096:Gal4 driver that induces expression in the whole wing blade. The wing blade represents 60-70% of the wing disc. Therefore, the amount of PhGFP expressed per nucleus is comparable to the amount of total untagged Ph per nucleus. There was a change in the relative expression of the proximal to the distal Ph genes in the transgenic line as seen in Fig. 1A. The relative expression levels of PcGFP and PhGFP in transgenic salivary glands determined by western blotting using an anti-GFP antibody was 1 to 2.2 (Fig. 1C). In the salivary gland nuclei both PcGFP and PhGFP expressions are induced by the Pc promoter (no Gal4 driver used in this case).
Diffusion constants of free PcGFP and PhGFP in early embryos and salivary gland nuclei
Before cellularization in Drosophila, the PcG proteins are all of
maternal origin and their binding to chromatin is restricted to a few PRE
sites (Orlando et al., 1998).
It is not clear if the repression complexes formed are functional as zygotic
transcription has not yet begun. Thus, there exists a window in development
(early division cycles) in which one can measure the diffusion of the fusion
proteins by classical FRAP techniques. In the preblastoderm embryos the
distribution of PcGFP is rather homogeneous throughout the nucleus
(Dietzel et al., 1999
) and the
nuclear size is large relative to somatic diploid nuclei later in development
(Fig. 2A). At cycle 10, we
observed a few faint aggregates of PcGFP in a uniform fluorescent nucleoplasm.
We measured the diffusion constant in regions without aggregations by
conventional FRAP. The number and intensity of the PcGFP aggregated loci
increases as embryonic development proceeds and as nuclei decrease in size.
Another development stage providing access to the free protein is in the
larval salivary gland nuclei, where the chromatin is condensed in polytene
chromosomes leaving regions of free nucleoplasm. From FRAP experiments in both
early embryos (Fig. 2) and
salivary gland nucleoplasm (not shown), we obtained similar diffusion
constants for PcGFP of 0.74 µm2 second1 and
0.41 µm2 second1, respectively
(Table 2). The amount of PhGFP
induced in early pre-blastoderm embryos was insufficient for obtaining
reproducible FRAP measurements. Thus, the free diffusion constant was derived
exclusively from salivary gland nuclei. The value, 0.22 µm2
second1, is only twice as slow as that of PcGFP
(Table 2) as would be expected
for a protein three times larger than Pc. The values for both proteins are
slower than expected for free, monomeric, diffusing proteins
(Verkman, 2002
), indicating
that the proteins may interact non-specifically in the nucleus with histones
or other chromatin-bound proteins, although no specific binding to PREs occurs
at this stage.
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As shown in the following sections, the recovery times of the PcGFP and
PhGFP complexes were at least an order of magnitude longer than the free
diffusion of the macromolecules. Thus, it was necessary to measure for more
than 50 seconds in order to reach equilibrium between the redistribution of
bleached and unbleached proteins. A preferred method for determining
dissociation rate constants under such conditions is to use inverse FRAP
(iFRAP), whereby the entire nucleus (except for a small region surrounding the
fluorescent complex of interest) is bleached and the depletion of fluorescence
from this region is monitored over time. The rate of disappearance of the
fluorescent locus will be a direct measure of the first order dissociation
rate constant of the protein from the complex
(Dundr et al., 2002). We found
that this type of photobleaching technique fitted our system best due to the
reasons described below. Nuclei and chromatin itself are not stationary in
live Drosophila tissues, as shown in
Fig. 3. Core histone-GFP that
does not dissociate from chromatin in interphase showed similar dynamics,
indicating that the movement we observe in our cells is not due to
dissociation of whole complexes from the chromatin
(Post et al., 2005
) (see Movie
1 in the supplementary material). In addition, photobleaching of the nuclear
lamin fused to RFP in embryonic and 2N larval disc Drosophila nuclei
revealed no rotation of the nuclei over a period of more than 3 minutes (C.
Fritsch, personal communication). In order to overcome the problem of
chromatin mobility, the dissociation and residence times of the PcG
chromatin-bound proteins were analyzed in three dimensions by adapting the
iFRAP procedure to a version denoted 3D-iFRAP that tracks the fluorescent
locus over time (see Materials and methods). The fluorescence decay of the
unbleached locus and the increase in fluorescence in the bleached nucleus were
monitored in the movement corrected data
(Fig. 4) and the average
intensities of the locus of interest were plotted over time to derive the rate
constants (Fig. 5).
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PcG complexes have different residence times on individual bands in salivary gland nuclei
PcGFP and PhGFP bind to distinct (Fig.
6) overlapping loci in salivary gland nuclei. Classical FRAP
experiments (intense photobleaching of the band and monitoring of the
fluorescence recovery over time) (Fig.
6E) were conducted on individual bands to determine the
dissociation rate constants for PcGFP and PhGFP complexes. Individual bands
showed consistent recovery times after multiple bleachings
(Fig. 6F), although not all
bands in the same nucleus exhibited the same recovery times
(Fig. 6B,D). The equilibrium
association constant of the complex is given by the ratio of a pseudo
first-order forward rate constant (which includes the concentration of the
free protein) and a first-order dissociation rate constant. In the case of
PcGFP, a large amount of the labeled protein is free, i.e. leading to a lower
fraction of bound protein than for PhGFP (bound ratio,
Bratio, see Materials and methods,
Fig. 8B). Therefore, the shape
of the FRAP recovery curve will be dominated by the fast diffusion of the free
protein, as is evident from the comparison of the curves in
Fig. 6B,D. We accounted for the
free protein component by segmenting out the bound fraction of proteins from
the bleach box (Fig. 7) (as
described in the Materials and methods, and further explained in the
Discussion). A comparison of the recovery curves for the bound fraction (blue
curve) and the unbound free component (green curve) of PhGFP and PcGFP
(Fig. 7D,F) shows that the
segmentation separated the fast recovery process of the free protein (which
occurs in the first seconds) from the actual binding reaction. By fitting the
recovery curve for the bound fraction, we computed recovery times
(t0, the time required for the fluorescence intensity to
reach 63% of the final height of the recovery curve for the bound
molecules) for both PhGFP and PcGFP from such curves
(Fig. 8A). The distribution of
values was very similar for both proteins with t0 recovery
times ranging from 50 to 350 seconds.
In the case of salivary gland polytene chromosomes, the individual bands
represent complexes binding to one or a few genes. Thus, we can ask if the
range of measured recovery times represents different exchange rates for
different genes (PREs) or the same exchange rate influenced by the density of
binding sites. Simulations revealed that for interpreting these FRAP
experiments the complex interplay between unbleached free protein, the total
amount of bound protein at a locus and the free diffusion constant must be
taken into account. We simulated the expected FRAP behavior for loci with
different amounts of bound protein at a locus (thus different intensities), as
briefly described in the Materials and methods, using a diffusion constant of
0.5 µm2 second1 of the free protein determined
experimentally and by systematically varying the dissociation rate constants
around the experimentally determined values (a complete description of the
simulations will be presented elsewhere).
Fig. 8B demonstrates that with
a single dissociation rate constant we would expect the recovery times to vary
approximately linearly with the ratio of bound to free protein,
Bratio, as defined in the Materials and methods, Eqn 5,
and results in the data shown by the connected points. In
Fig. 8B, the experimentally
measured recovery times are also plotted against the
Bratio for each locus. In the experimental data, no strong
correlation was found between the recovery time and the intensity of the locus
(number of binding sites), indicating that the complexes on different genes
have different stabilities, and implying that they differ in composition.
Including the dependence of the dissociation time on the density of binding
sites, Bratio, and the free diffusion component
(determined experimentally to be 0.5 µm2
second1) as predicted from the simulations, we calculated
dissociation rate constants for each of the analyzed loci using Eqn 6. These
values are independent of the local concentration of sites and the effect of
diffusion. Bound protein ratios were normalized to the highest
Bratio value for each protein separately and are plotted
in Fig. 9A. The dissociation
rate constants are similar for both proteins but about one-fifth the values
found for the complexes in 2N wing disc nuclei. The forward reaction is 2nd
order and the rate is dependent on the concentration of binding sites and on
the local concentration of the free protein. The amount of unbound protein in
the nucleus is sufficient for the binding process to occur undisturbed for
both PhGFP and PcGFP cases. From the fluorescence intensity ratios, we
calculated that
10% of the total PhGFP and
2% of the PcGFP are in a
bound state in a salivary gland nucleus at equilibrium. As the absolute number
of binding sites (Cs) is unknown, the association rate
constant could not be determined independently. Instead, a pseudo-association
rate constant was calculated (which is related to the actual
kon by the equality
kon*=kon·Cs, see
Materials and methods, Eqn 7) (Sprague et
al., 2004
) for each locus and these values for PhGFP are plotted
in Fig. 9B. The
pseudo-association rate constants are an order of magnitude larger than the
dissociation rate constants, confirming that dissociation is rate
limiting.
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![]() |
Discussion |
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Quantitative FRAP and 3D-iFRAP
The application of fluorescence recovery after photobleaching (FRAP) to
fusion proteins of GFP (and its analogs) by confocal microscopy has allowed
the study of the dynamics of the steady-state distribution of nuclear proteins
in living cells (Houtsmuller and
Vermeulen, 2001; Phair and
Misteli, 2001
; White and
Stelzer, 1999
). Although most analyses have been qualitative
(Cheutin et al., 2003
;
Dou et al., 2002
;
Festenstein et al., 2003
;
Houtsmuller et al., 1999
;
McNally et al., 2000
;
Misteli et al., 2000
), a
quantitative analysis can be used to obtain diffusion constants and
dissociation rate constants by the application of combined techniques and
appropriate models (Carrero et al.,
2003
; Dundr et al.,
2004
; Dundr et al.,
2002
; Phair et al.,
2004
; Rabut, 2004). In most cases, however, a reaction dominant
model has been adopted. That is, because diffusion is faster than most
dissociation rates, it is often, albeit incorrectly, neglected in the
analyses. Recently, Sprague et al. presented a full analytical treatment for
uniformly dispersed binding sites and showed simulations for various boundary
conditions and rate constants (Sprague et
al., 2004
). Because our complexes are not uniformly distributed we
have extended this treatment to a model with discrete binding loci and present
here simulations using diffusion and binding rate constants derived from our
experimental data. (A full description of the model will be presented
elsewhere.)
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|
The influence of diffusion and free protein on FRAP data and binding equilibria
The level of Pc is crucial for the maintenance of a competent complex as
can be deduced from the fact that Pc+/
heterozygotes show homeotic transformations
(Lewis, 1978). Western
blotting revealed that the fusion proteins do not reach levels greater than
the wild type in non-transgenic animals
(Table 1). That is, the total
PcGFP protein content in the mutants was 0.53 that of wild type and PhGFP was
comparable with the wild-type level.
The diffusion constants for PcGFP in early embryos before complex formation
(0.74 µm2 second1) and in the nucleoplasm of
salivary gland nuclei (0.41 µm2 second1) are
smaller than one might expect for a protein of 62 kDa, indicating that
the protein may exhibit non-specific binding to chromatin. Breiling et al.
(Breiling et al., 1999
)
demonstrated that Pc has an affinity for nucleosomes without histone tails and
that the C terminus was crucial for this interaction. However, the Pc
chromodomain, essential for complex assembly, has a strong preference for
trimethylated Lys 27 over other methylated sites or unmodified H3 showing a
KD of 5 µM and >1000 µM, respectively, in in
vitro binding studies (Fischle, 2003). As seen in
Fig. 6, the diffusion of the
free protein obscures the recovery kinetics of the binding process measured on
individual bands in salivary glands and the curve must be decomposed to fit
the recovery kinetics (Fig. 7). The fitting assumes an excess of free protein, which is true for both of our
transgenic proteins, despite the lower nucleoplasmic fluorescence in the case
of PhGFP (see below). In an ideal situation with infinitely fast diffusion,
loci with different concentrations of binding sites of identical affinity
would recover within the same time. At the diffusion constants measured
experimentally in our nuclei (
0.5 µm2
second1), we found by simulation that two loci with the same
size but with different concentrations of binding sites will recover with
different times: i.e. higher concentration, longer recovery time
(Fig. 8). We created masks in
the images to separate out pixels that contained predominantly non-bound
protein from that involved in complexes. By first fitting and removing the
diffusion component we were able to fit the resulting recovery curves to a
single exponential, as in a kinetic process in a standard chemical equilibrium
(Fig. 7). The dissociation rate
constants were in the same range as those measured in the 2N cells of embryos
and imaginal discs but the means were shifted towards a value of around
one-third of that for PhGFP to one-quarter of that for PcGFP. These
differences could reflect some unintended bias in the selection of the bleach
loci in either the 2N nuclei or the polytene bands. However, each polytene
band represents thousands of complexes at one or a few PREs, rather than an
average of many different complexes; thus, these data may be more robust. In
either case, even when PREs are in close proximity, such as is the case of a
thousand chromatids closely aligned in the polytene chromosomes, the PcG
proteins are in a chemical equilibrium with unbound protein. The reduced rate
constants may reflect the large local binding sites, whereby a dissociated
protein does not immediately join the `free pool' but has a higher probability
to rebind in the close vicinity. However, the proteins are not `trapped' in
the complex but rather are able to completely exchange in under 6 minutes. The
reproducibility of the recovery times of individual bands subjected to
successive FRAP measurements is shown in
Fig. 6F, indicating that the
differences of two- to threefold in recovery times
(Fig. 8) between different
bands can be considered reproducible and significant.
The t0 values calculated from these recovery curves
are, however, not a direct measure of residence time because of their
dependence on the effect of diffusion transport in combination with ongoing
depletion from the free pool. As could be demonstrated using simulations, if
the dissociation rate constant and the concentration of free protein were the
same for all complexes then one would expect the recovery rate to depend
linearly on the amount of bound protein (lines in
Fig. 8B), which is essentially
a measure of the ability of a spot to deplete the free protein pool during
recovery. As seen in the same figure, the experimental data do not show such a
correlation, implying that there are differences in the composition of the
complexes on different genetic loci and that the dissociation rate constants,
though similar (within a factor of 5, Fig.
9A), reflect the specific mixture of PcG and non-PcG auxiliary
proteins on the polytene bands. Such an interpretation is compatible with the
data of Rastelli et al., who showed varying occupancy of PcG proteins and
Zeste on more than 100 bands by immunohistochemistry on polytene chromosomes
(Rastelli et al., 1993). To
rule out the possibility of a very slow component that would appear as an
immobile fraction in a single exponential fit, we also fitted the data with a
sum of two exponentials, but did not find a consistent second time in this
case and less precision of the first time. Thus, we conclude that both PcGFP
and PhGPF in repression complexes exchange within a few minutes in live
Drosophila cells.
In Fig. 9B, the
pseudo-association rate constants as described in the Materials and methods
are plotted for PhGFP. The values are an order of magnitude larger than the
dissociation rate constants and thus, dissociation is rate-limiting. We have
not attempted to present pseudo-association rate constants for PcGFP for the
following reasons. We assume that the number of binding sites for Pc and Ph
are approximately equal as the proteins bind to overlapping sites on polytene
chromosomes (Rastelli et al.,
1993), isolated complexes of the proteins contain equimolar
quantities of both proteins (Saurin et
al., 2001
) and they are targeted to the same PREs by ChIP analysis
(Breiling et al., 2001
). As
discussed above, the off-rates are similar for the two proteins. However, we
can see a larger pool of unbound PcGFP compared with PhGFP in both embryos and
larval tissue (Figs 1 and
6). As determined from western
blots in salivary glands, PcGFP is not present in amounts higher than PhGFP
(Fig. 1C). Wang et al.
(Wang et al., 2004
) have shown
that there is sequential recruitment of PcG complexes to the PREs, whereby Pc
targets the complex to chromatin by binding to trimethylated H3K27
(Cao et al., 2002
;
Czermin et al., 2002
;
Fischle et al., 2003
;
Wang et al., 2004
). From these
considerations, we postulate that the PcGFP fusion protein, although competent
to target PREs with modified histones and engage in a competent complex,
cannot substitute for all Pc molecules in the complexes (perhaps owing to
steric hindrance of adjacent GFP moieties). Our data suggest that the
unmodified Pc is preferred in the complex by a factor of about 4 or 5; thus,
association rate constants calculated for PcGFP will not properly reflect the
true on rate of the unmodified protein, whereas off rates should not be
adversely affected.
We calculated the ratio of the bound/free fusion proteins from the
segmentation of the salivary gland prebleach FRAP images to be 1:10 and from
the western blots (Fig. 1) the
ratio of the fusion protein to wild-type protein of 1:1. We estimated the
absolute concentration of GFP protein in the salivary gland nuclei to be
2-4 µM by comparison to the intensity of droplets of purified GFP
protein in an immiscible solution in our microscope system. If the total
concentration of binding sites is equivalent to the concentration of the bound
Ph, we can estimate the KD for the protein in vivo in salivary
gland nuclei to be
5 µM.
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most FRAP studies of nuclear proteins have involved components in
transcription complexes or transcriptional activators that exchange in less
than 2 minutes (Phair et al.,
2004). The only repressor protein that has previously been
investigated is heterochromatin protein 1 (HP1), a protein targeted to
heterochromatin in higher eukaryotes
(Cheutin et al., 2003
;
Festenstein et al., 2003
).
Although HP1 is loaded directly onto the chromatin during replication, it was
found by FRAP to bind only transiently to chromatin with a maximum residence
time of
60 seconds. Thus, both HP1 and PcG repression complexes appear to
function by dynamic competition with other chromatin-binding proteins rather
than by formation of a static, higher-order chromatin structure with
immobilized bound repressors. Our FRAP measurements on polytene chromosomes
revealed differences in the dissociation rate constants between individual
bands that imply a flexible repression system of complexes with various
compositions that influence the binding affinity of other members and whose
turnover is in the order of a few minutes.
We conclude that: (1) activation and repression can be dynamically
controlled by simple chemical equilibria; (2) reduction in PcG levels will
facilitate epigenetic change and may explain why non-cycling cells can be
reprogrammed more easily than cycling cells
(Baxter et al., 2004); and (3)
PcG complexes are exchangeable protein assemblies that maintain repression
over many cell cycles by simple chemical equilibria.
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ACKNOWLEDGMENTS |
---|
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