Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP163, 67404 Illkirch-Cedex, France
* Author for correspondence (e-mail: chambon{at}igbmc.u-strasbg.fr)
Accepted 12 June 2002
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Summary |
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Key words: Nuclear compartmentalization, Transcriptional silencing, Endodermal differentiation, Transcriptional intermediary factor 1 ß, Heterochromatin protein 1
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Introduction |
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Pericentromeric heterochromatin represents a specialized nuclear
compartment known to play a role in gene silencing
(Henikoff, 2000). When placed
next to or within pericentromeric heterochromatin, either by chromosomal
rearrangement or trans-recruitment, euchromatic genes usually undergo a
stochastic, but clonally heritable, inactivation, leading to a variegated
pattern of expression (Wallrath,
1998
). Dominant genetic modifiers of this phenomenon, known as
position effect variegation (PEV), were isolated in Drosophila as
mutations that can alter the proportion of cells in which inactivation occurs.
One of these modifier genes, Su(var)2-5, encodes the
heterochromatin-associated protein HP1
(Eissenberg et al., 1990
).
Su(var)2-5 suppresses PEV when deleted or enhances PEV when
duplicated (Eissenberg et al.,
1990
; Eissenberg et al.,
1992
), indicating that HP1 is an essential component of
heterochromatin required in a precise stoichiometry to properly set and/or
maintain the inactivated state of genes subject to PEV. Three distinct HP1
proteins, HP1
, ß and
, have been described in mammals
(Eissenberg and Elgin, 2000
).
Supporting the notion that these HP1s also play a role in gene silencing, they
have been reported (i) to be associated although not exclusively
with pericentromeric heterochromatin
(Nielsen et al., 1999
;
Minc et al., 2000
); (ii) to
silence transcription in a deacetylase-dependent manner when directly tethered
to DNA (Nielsen et al., 1999
);
(iii) to cause dose-responsive silencing of centromeric transgenes
(Festenstein et al., 1999
);
(iv) to colocalize in primary B-cells with inactive genes
(Brown et al., 1997
); and (v)
to exhibit conserved heterochromatin targeting and silencing when ectopically
expressed in Drosophila (Ma et
al., 2001
). Recent studies have demonstrated that mouse HP1
proteins self-associate and associate with nucleosomal core histones in
interphase nuclei (Nielsen et al.,
2001
). A selective binding to the tail domain of H3 methylated at
lysine 9 (Bannister et al.,
2001
; Lachner et al.,
2001
) and a direct interaction with several non-histone proteins,
including components of the replication machinery, proteins of the nuclear
envelope and various transcriptional cofactors have also been described
(reviewed in Eissenberg and Elgin,
2000
).
Initially identified in a two-hybrid screen for proteins interacting with
mouse HP1 (Le Douarin et al.,
1996
), transcriptional intermediary
factor 1 (TIF1) ß (also known as KAP-1 or KRIP-1) was reported
to function as a bona fide corepressor for the large family of
Krüppel-associated box (KRAB)-domain-containing zinc finger proteins
(Friedman et al., 1996
;
Kim et al., 1996
;
Moosmann et al., 1996
).
TIF1ß is a member of an emerging family of transcriptional regulators
that includes TIF1
and TIF1
in mammals
(Le Douarin et al., 1995a
;
Venturini et al., 1999
) and
Bonus in Drosophila (Beckstead et
al., 2001
). All members of this family are composed of a
N-terminal RBCC (RING finger, B boxes, coiled coil) motif and a C-terminal
bromodomain preceded by a PHD finger. They have all been reported to repress
transcription when tethered to DNA through fusion to a heterologous
DNA-binding domain. Coimmunoprecipitation experiments showed that TIF1ß
is associated with HP1
, ß and
in interphase nuclei of
various mammalian cell lines (Nielsen et
al., 1999
; Ryan et al.,
1999
). In vitro, TIF1ß interacts directly with and
phosphorylates the HP1 proteins (Nielsen
et al., 1999
). This interaction is mediated by a conserved PxVxL
motif and is required for the repression activity of TIF1ß in transiently
transfected cells (Nielsen et al.,
1999
; Ryan et al.,
1999
). Recently, TIF1ß has also been reported to be an
intrinsic component of the histone deacetylase N-CoR1 complex
(Underhill et al., 2000
) and
to interact both physically and functionally with the Mi-2
subunit of
the nucleosome remodeling and deacetylation (NuRD) complex
(Schultz et al., 2001
). Taken
together, these biochemical data strongly suggest that TIF1ß may exert
its corepressor function via the organization and/or maintenance of
higher-order, heterochromatin-like structures.
Recent genetic studies of TIF1ß in mice have provided evidence that
TIF1ß is a developmental regulatory protein that exerts cellular
function(s) essential for early embryogenesis
(Cammas et al., 2000). To
further investigate the functions of TIF1ß in mammalian cells, we have
now examined its subnuclear distribution in undifferentiated and
differentiated F9 embryonal carcinoma (EC) cells, which represent a well
established model system of early embryonic development and cellular
differentiation (Hogan, 1977
;
Hogan, 1983
). The F9 EC cells
closely resemble the pluripotent inner cell mass (ICM) cells of the early
mouse embryo and can be induced to differentiate into primitive endoderm-like
cells when grown as monolayer in the presence of retinoic acid (RA)
(Strickland et al., 1978). Our data show that, during RA-induced primitive
endoderm differentiation, TIF1ß is relocated into regions of centromeric
heterochromatin. Dissection of the molecular mechanism underlying this
differentiation-induced relocation reveals that TIF1ß becomes centromere
associated through HP1 interaction. We discuss the implications of these data
for mechanistic models of TIF1ß function in mammalian cells.
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Materials and Methods |
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Antibodies
Monoclonal antibodies (mAbs) used include: mouse anti-TIF1ß mAb, 1Tb3,
raised against recombinant E. coli-expressed mouse
TIF1ß(123-834) (Nielsen et al.,
1999); mouse anti-HP1
mAbs, 2HP1-1H5 for
immunocytochemistry and 2HP-2G9 for western blot analysis
(Nielsen et al., 1999
); mouse
anti-RPB1 mAb, 1PB-7C2, raised against the heptad repeat CTD-containing
peptide of the RPB1 largest subunit of the human RNA polymerase II
(Nguyen et al., 1996
); rat
anti-BrdU mAb (Becton Dickinson); rat anti-Endo A, TROMA 1 [kindly provided by
R. Kemler (Department of Molecular Embryology, Max-Planck Institute, Freiburg,
Germany)]; rat anti-laminin B1 (Chemicon, Harrow, UK); mouse anti-FLAG mAb, M2
(Sigma-Aldrich, France); mouse anti-ER
mAb, F3, against the F region of
human ER
(Le Douarin et al.,
1995b
); and mouse anti-VP16 mAb, 2GV-4
(Le Douarin et al., 1995b
).
The rabbit polyclonal antibodies (pAbs) used include anti-TIF1ß pAb,
PF64, raised against TIF1ß (amino acids 141-155) and anti-fibronectin (a
gift of R. Hynes, MIT, Massachusetts).
F9 cell culture and establishment of stably transfected cell
lines
Wild-type and mutant F9 cells were grown as monolayers on gelatinized
surfaces in Dulbecco's modified Eagle medium (DMEM, Gibco, Invitrogen, France)
supplemented with 10% fetal calf serum as previously described
(Boylan and Gudas, 1991). To
induce differentiation, cells were plated at a density of 102 to
103 cells/cm2 and treated with 1.0 µM all-trans
retinoic acid (Sigma) or with vehicle (control untreated) for the indicated
times, with a change of media every two days. Isoleucine deprivation was
performed as described previously (Hosler
et al., 1989
; Faria et al.,
1998
). Cells plated 24 hours earlier were incubated with
isoleucine-free DMEM supplemented with 10% dialyzed fetal calf serum and grown
for 48 or 72 hours. To establish stably transfected cell lines,
5x106 exponentially growing F9 EC cells (106 per
10 cm plate) were transfected by electroporation with 5 µg of expression
plasmids (pCX-FLAG-TIF1ßWT or FLAG-TIF1ßV488A/L490A) along with 0.1
µg of a plasmid conferring resistance to hygromycin (pPGK-hygro; a gift of
D. Metzger, IGBMC, Strasbourg, France). Cells were selected in the presence of
250 µg/ml hygromycin (Euromedex) added to the growth medium 24 hours after
transfection over a period of 2 weeks with regular medium changes. Several
drug-resistant colonies were subsequently picked in 24-well plates and
expanded for western blot and immunofluorescence analysis.
ES cell culture and induction of neuronal differentiation
Mouse ES cells, strain D3, were used in this study. The routine growth and
in vitro differentiation of these cells were performed as previously described
(Boeuf et al., 2001). In brief,
neuronal differentiation was induced by culturing the cells as embryoid bodies
for 4 days in the presence of 1.0 µM all-trans RA. After RA treatment, the
embryoid bodies were dispersed by trypsinization and transferred to
gelatinized tissue culture plates (or glass coverslips for
immunocytochemistry) to allow cell attachment and neuronal outgrowth.
Immunofluorescence and confocal microscopy
Cells grown on gelatinized glass coverslips were washed once with
phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde in PBS (pH
7.5) for 10 minutes at room temperature. Samples were then permeabilized twice
with 0.1% Triton X-100 in PBS for 5 minutes, washed in PBS and incubated for
16 hours with primary antibodies appropriately diluted in PBS at room
temperature. Following two consecutive 5 minute washes in PBS 0.1% Triton
X-100, cells were incubated for 1 hour with fluorochrome-conjugated secondary
antibodies. Slides were washed twice (5 minutes/wash) in PBS 0.1% Triton
X-100, stained for DNA with Hoechst 33258 at 5 µg/ml and mounted in PBS 5%
propyl gallate 80% glycerol. Image acquisition was performed using a Leica
TCS-4D confocal scanning microscope (Leica Microsystems, Heidelberg,
Germany).
Cell cycle analysis
The cell cycle profile of a cell population was determined by measuring
cell DNA contents in different phases by flow cytometry. Cells were
trypsinized, washed twice with cold PBS and fixed in 70% ethanol for 30
minutes at 4°C. Following two washes in PBS, cells were treated with 1
µg/ml RNase A (Macherey-Nagel, France) for 30 minutes at 37°C, pelleted
by centrifugation and resuspended in PBS at a concentration of 106
cells/ml. Propidium iodine was added at a final concentration of 15 µg/ml
for 30 minutes at room temperature, and the samples were analyzed on a FACScan
(Beckton Dickinson, Mountain View, CA) using the CellQuest software.
In situ replication assay
5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co) incorporation was
performed by incubating untreated and RA-treated cells in the presence of 40
µM BrdU for 10 minutes. Cells were then washed with PBS, fixed with 2%
paraformaldehyde in PBS for 10 minutes at room temperature and permeabilized
twice with 0.1% Triton X-100 in PBS for 5 minutes. DNA was denatured with 4 M
HCl for 10 minutes at room temperature. Neutralization was achieved by three
washes with PBS, and the slides were further processed as described above.
Note that the HCl treatment did not interfere with the detection and
localization of TIF1ß.
Immunoprecipitation and western blot analysis
Isolation of whole cell extracts from F9 cells and transfected COS-1 cells,
immunoprecipitation and western blot detection were performed as previously
described (Chiba et al., 1997;
Nielsen et al., 1999
).
Yeast two-hybrid interaction assays
Yeast cells grwon in YEPD or selective medium were transformed by the
lithium acetate procedure (Gietz et al.,
1995). Yeast PL3 (Mat
ura3-
1 his3-
200
leu2-
1 trp1::3ERE-URA3) transformants were grown exponentially for
about five generations in selective medium supplemented with uracil. Yeast
extracts were prepared and assayed for OMPdecase activity as described
previously (Le Douarin et al.,
1995b
).
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Results |
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To determine how rapidly RA induced TIF1ß heterochromatin association,
individual F9 cell nuclei (>1000) were examined for TIF1ß staining and
Hoechst counterstaining at several time points after RA addition. TIF1ß
heterochromatic foci were detected in 1% of the nuclei 12 hours of treatment,
the earliest time examined in this experiment
(Fig. 1D). The percentage of
TIF1ß-foci-containing nuclei increased progressively to a maximum of
45-50% after 96 hours and remained unchanged at all subsequent time
points (Fig. 1D), providing
evidence for a stable change in TIF1ß distribution. Interestingly, a
similar proportion of cells exhibiting heterochromatic TIF1ß (44%) was
obtained by maintaining the differentiated cultures for up to six days in a
RA-free medium after an RA treatment of two days (data not shown). This result
indicates that the continued presence of the ligand is not required for
maintenance of the heterochromatin association of TIF1ß in the
differentiated cells.
TIF1ß heterochromatin association occurs in response to
differentiation
Because differentiating F9 cells divide more slowly than F9 EC cells
(Strickland et al., 1980), the
RA-induced heterochromatin association of TIF1ß could be the result of a
decline in growth rate. To test this possibility, the subnuclear distribution
of TIF1ß was investigated in F9 cells starved of the animo acid
isoleucine. Isoleucine starvation has previously been reported to cause
partial growth arrest without inducing differentiation
(Dean et al., 1986
;
Hosler et al., 1989
). Cell
cycle progression of isoleucine-starved F9 cells was determined by flow
cytometry, using untreated and RA-treated F9 cells as controls
(Table 1). As expected, RA
treatment caused a gradual decline in growth rate, indicated by an increase in
the percentage of the cell population in the G1 phase from
20 to 60% at
72 hours. A similar decline in growth rate was observed in F9 cells grown for
48 hours in medium without isoleucine
(Table 1). However, none of
these isoleucine-starved cells exhibited a change in the nuclear distribution
of TIF1ß when compared to exponentially growing F9 cells
(Table 1). Thus, the
heterochromatin association of TIF1ß observed in RA-treated F9 cells does
not result from the slower growth rate associated with differentiation.
|
Because F9 EC cells are transformed, whereas their differentiated
derivatives are not, it was also important to be sure that the RA-induced
heterochromatin association of TIF1ß in F9 endodermal cells is not due to
the loss of their transformed state. To test this possibility, we have
investigated the subnuclear distribution of TIF1ß in embryonic stem (ES)
cells, which are pluripotent stem cell lines derived directly from early mouse
embryos without the use of immortalizing or transforming agents. These cells
can be propagated as undifferentiated stem cell cultures
(Fig. 2A) and can be induced to
differentiate into neuronal cells in the presence of RA
(Bain et al., 1995)
(Fig. 2D). TIF1ß was found
to be uniformly distributed throughout the nucleoplasm in undifferentiated ES
cells (Fig. 2B,C) and
concentrated into the large blocks of centromeric heterochromatin in their
neuronal derivatives (Fig.
2E,F). It therefore seems clear that the change in TIF1ß
distribution following F9 cell differentiation reflects a bona fide
differentiation response resulting from the change in cell type rather than
loss of the transformed state.
|
To further support this conclusion, we also examined TIF1ß
localization in compound RXR-/-/RAR
-/-
mutant F9 cells, which are refractory to RA-induced differentiation
(Chiba et al., 1997
). In these
mutant cells, no significant change in the staining pattern of TIF1ß was
observed in the presence of RA (data not shown). Thus, the selective
heterochromatin association of TIF1ß in the nuclei of RA-treated F9 cells
requires that the F9 cells respond to RA.
TIF1ß heterochromatin association correlates with changes in
both proliferation and differentiation-specific gene expression
Because only half of the RA-treated F9 cells displayed a change in
TIF1ß distribution (Fig.
1C), the cell population was further characterized at the single
cell level with respect to both proliferation and differentiation. As
determined by bromodeoxyuridine (BrdU) pulse-labeling and double-label
immunofluorescence, S-phase cells could be detected in both subpopulations of
differentiated cells characterized by a diffuse or a focal TIF1ß staining
pattern (Fig. 3A,B). However,
the proportion of BrdU-positive cells was significantly lower in cells
exhibiting a focal TIF1ß staining as compared with those in which
TIF1ß distribution was diffuse (24% versus 43%), indicating that cells
containing heterochromatin-associated TIF1ß have a decreased
proliferative activity. To monitor their differentiation state, cells were
double-labeled with antibodies to TIF1ß and a panel of specific
endodermal markers. After a 6 day treatment with 1 µM RA, similar
proportions of fibronectin-positive cells exceeding 80% were observed in the
two TIF1ß cell subpopulations (Fig.
3C,D). By contrast, the proportion of cells positive for the
cytokeratin filament EndoA was much higher in cells containing heterochromatic
TIF1ß as compared with those in which TIF1ß was localized in
euchromatic areas (80% versus 10%) (Fig.
3E,F). By contrast, most of the cells with unfocused TIF1ß
staining were found to produce large amounts of laminin B1, whereas the cells
containing heterochromatic TIF1ß showed barely detectable amounts of this
marker (Fig. 3G,H). Taken
together, these results confirm the notion that RA-induced F9 cell cultures
are heterogeneous with respect to both proliferation and differentiation
(Moore et al., 1986;
Kurki et al., 1989
) and
furthermore indicate that two types of differentiated cells can also be
characterized with respect to TIF1ß localization in euchromatin versus
heterochromatin.
|
Targeting TIF1ß to centromeric heterochromatin requires HP1
interaction
TIF1ß interacts with HP1, ß and
through a 25
amino-acid segment containing the conserved PxVxL motif
(Nielsen et al., 1999
;
Smothers and Henikoff, 2000
;
Lechner et al., 2000
) (see
also Fig. 4A). To investigate
whether an interaction with the HP1s is required for the
differentiation-induced heterochromatin association of TIF1ß, we
generated a stable F9 EC-derived cell line expressing a FLAG epitope-tagged
TIF1ß mutant, f:TIF1ßVL/AA, in which the conserved hydrophobic
residues V488 and L490 in the PxVxL motif were replaced by Alanine residues
(Fig. 4A).
|
The f:TIF1ßVL/AA mutant was initially characterized for HP1
interaction using the yeast two-hybrid system. In contrast to FLAG-tagged
TIF1ß wild-type (f:TIF1ßWT), f:TIF1ßVL/AA did not interact with
HP1, HP1ß and HP1
, and binding to the KRAB domain of KOX1
was not affected (Fig. 4B). The
effect of the double mutation VL/AA on TIF1ß-HP1 interaction in mammalian
cells was also examined. Expression vectors encoding HP1
and either
f:TIF1ßWT or f:TIF1ßVL/AA were transiently cotransfected in COS-1
cells. The localization of the FLAG-tagged TIF1ß proteins in nuclei was
confirmed by immunocytofluorescence (data not shown). When these proteins were
immunoprecipitated from the respective whole cell extracts with an immobilized
monoclonal antibody directed against the FLAG epitope, HP1
was found in
the f:TIF1ßWT immunoprecipitate (Fig.
4C, lane 4), but not in the f:TIF1ßVL/AA immunoprecipitate
(Fig. 4C, lane 6), indicating
that, as observed in yeast, f:TIF1ßVL/AA does not stably associate with
HP1
in mammalian cells.
The subnuclear distribution of f:TIF1ßVL/AA was then compared to that
of the wild-type protein, f:TIF1ßWT, using F9 EC-derived cell lines that
stably express similar amounts of each FLAG-tagged protein (see Materials and
Methods). On the basis of morphological criteria, both f:TIF1ßWT- and
f:TIF1ßVL/AA-expressing cells differentiated into primitive endoderm-like
cells in response to RA treatment to the same extent as the parental F9 EC
cells (data not shown). As revealed by confocal immunofluorescence microscopy
using the anti-FLAG antibody, the f:TIF1ßWT protein exhibited in nearly
50% of the RA-treated cells the focal staining pattern characteristic of
centromeric localization (Fig.
5H), similar to that observed for the endogenous TIF1ß
protein (Fig. 5J). By contrast,
in both induced and uninduced f:TIF1ßVL/AA-expressing cells, the
f:TIF1ßVL/AA mutant protein displayed a granular distribution pattern
throughout the nucleoplasm, with little or no colocalization to the
heterochromatic regions (Fig.
5N,T). It should be noted that, in these
f:TIF1ßVL/AA-expressing cells, neither the average number of HP1
heterochromatic foci (Fig.
5R,X) nor the distribution and dynamic behavior of endogenous
TIF1ß in response to RA treatment
(Fig. 5, compare P with V) was
altered. Taken together, these results indicate that the PxVxL motif of
TIF1ß is essential for centromeric targeting in RA-induced F9 cells and
support the hypothesis that during differentiation TIF1ß is targeted to
centromeric heterochromatin through HP1 interaction.
|
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Discussion |
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As measured by the number of nuclei in which TIF1ß is localized at
centromeric heterochromatin in the differentiated F9 cells, we found that
induction of differentiation by a single application of RA was as effective as
continuous treatment in stimulating TIF1ß redistribution. This result is
in full agreement with the hypothesis that this redistribution corresponds to
an event related to the differentiation program in which RA triggers the first
step of the differentiation process. However, our finding that RA caused a
significant change in TIF1ß distribution (>10%) only at late times
(48 hours) strongly suggests that relocation of TIF1ß does not
represent a primary response to RA. It is noteworthy that a similar time
course was obtained by using cells synchronized in different phases of the
cell cycle (F.C. and R.L., unpublished), indicating that the ability of RA to
induce TIF1ß relocation may not be cell-cycle dependent. Although cell
cycle regulation may occur during the process of differentiation, our present
data suggest other levels of regulation. Indeed, we found that RA triggers a
nuclear redistribution of TIF1ß only in a subpopulation of cells
exhibiting specific changes in both proliferation and differentiation. Most
cells with heterochromatic TIF1ß were found to contain a well developed
cytokeratin intermediate filament system, unlike the cells with euchromatic
TIF1ß. Moreover, the cells containing heterochromatic TIF1ß were
characterized by a decreased expression of laminin and a low proliferation
rate. On the basis of these criteria, these cells strongly resemble the DifB
cells, which have previously been described in the differentiating F9 cell
cultures treated with RA (Moore et al.,
1986
; Kurki et al., 1986). Interestingly, these DifB cells were
also shown to differ from the other cells, referred to as DifA cells, by their
inability to undergo further differentiation in response to cAMP analogs
(Moore et al., 1986
). Thus,
cells with either heterochromatic or euchromatic TIF1ß although
exhibiting both a primitive endoderm-like morphology may be the
product of two alternative differentiation pathways, which suggests that
changes in TIF1ß distribution are related to specific differentiated cell
types. Supporting this hypothesis, an heterochromatin association of
TIF1ß was observed in the outer layer of visceral endoderm generated in
RA-treated F9 embryoid bodies but not in parietal endodermal cells derived
from monolayer F9 cell cultures upon treatment with RA and dibutyryl-cAMP
(F.C. and R.L., unpublished).
The finding that the PxVxL motif of TIF1ß is essential for both HP1
interaction and centromeric localization provides evidence that a major
mechanism by which TIF1ß is targeted to centromeric heterochromatin is
through HP1 interaction. We have shown that TIF1ß colocalizes with the
heterochromatic HP1 protein in F9 differentiated cells but not in F9
stem cells. Thus, the centromeric targeting of TIF1ß following F9 cell
differentiation may be a consequence of its direct interaction with the
HP1
isoform. Note, however, that in addition to HP1
, F9 cells
express HP1ß and HP1
(see the references in the Introduction), to
which TIF1ß also associates (Nielsen
et al., 1999
). HP1ß, like HP1
, localizes predominantly
into the regions of centromeric heterochromatin
(Wreggett et al., 1994
;
Nielsen et al., 1999
;
Minc et al., 1999
), whereas
HP1
is present in both euchromatin and heterochromatin
(Nielsen et al., 1999
;
Nielsen et al., 2001
;
Minc et al., 2000
). These
data, together with the recent finding that HP1
, HP1ß and
HP1
are capable of mediating self-associatino in vivo
(Nielsen et al., 2001
),
suggest another model of TIF1ß centromeric targeting, which is consistent
with our data demonstrating that one important criterion for targeting is the
HP1 binding capability. In this alternative model, TIF1ß, as a complex
with euchromatic HP1
, is targeted to regions of centromeric
heterochromatin through an association of HP1
with the other
heterochromatic HP1 proteins.
Centromeric localization has recently been reported for a number of
trans-acting factors (Francastel et al.,
2000). For instance, Ikaros, a sequence-specific transcriptional
factor required for proper lymphoid development, has been shown to localize to
areas of centromeric heterochromatin in B lymphocytes
(Brown et al., 1997
;
Brown et al., 1999
). The
CCAAT/enhancer-binding proteins
and ß (C/EBP
/ß)
associate with centromeric heterochromatin during hormone-induced
differentiation of pre-adipocytes (Tang
and Lane, 1999
). A dynamic redistribution from heterochromatin to
euchromatin has recently been described for the subunit NF-E2p18 of the
transcriptional activator NF-E2 during MEL cell differentiation
(Francastel et al., 2001
).
Other proteins involved in gene silencing have also been shown to localize
with centromeric heterochromatin (for review, see
Francastel et al., 2000
).
These include DNA methyltransferases [Dnmts
(Bachman et al., 2001
)],
methyl-DNA-binding domain proteins [MBDs
(Hendrich and Bird, 1998
)],
histone methyltransferases [SUV39H (Aagaard
et al., 1999
)], histone deacetylases [HDAC1
(Kim et al., 1999
;
Francastel et al., 2001
)], and
chromatin remodeling factors [Mi2 and ATRX
(Kim et al., 1999
;
McDowell et al., 1999
)]. In
the case of Ikaros, centromeric targeting has been demonstrated to take place
concomitantly with that of several genes that become silenced during
lymphocyte differentiation (Brown et al.,
1997
; Brown et al.,
1999
). Upon MEL cell differentiation, a concomitant relocation
away from subnuclear compartments enriched in heterochromatin has also been
observed for the human ß-globin locus and NF-E2p18
(Francastel et al., 2001
), and
this relocation results in transcriptional activation
(Schübeler et al., 2000
).
By analogy, it is tempting to speculate that TIF1ß, when targeted to
centromeric heterochromatin, may mediate cell-type-specific gene silencing by
recruiting target genes to this transcriptionally inert compartment.
Consistent with this hypothesis, two KRAB-containing zinc finger proteins,
KRAZ1 and KRAZ2, have recently been demonstrated to repress transcription and
to be targeted to foci of centromeric heterochromatin through TIF1ß
interaction in transiently transfected NIH3T3 cells
(Matsuda et al., 2001
).
Alternatively, the centromeric heterochromatin compartments in which
TIF1ß accumulates during cell differentiation may represent storage sites
that regulate the nucleoplasmic concentration of TIF1ß. From our
immunocytofluorescence data, it is clear that TIF1ß is not limited to
regions of centromeric heterochromatin in differentiated F9 cells, as it also
binds to numerous diffuse sites dispersed throughout the nucleoplasm. Thus, it
is conceivable that the differentiation-induced accumulation of TIF1ß
into regions of centromeric heterochromatin may control TIF1ß levels
within the nucleus and to create compartments, in which TIF1ß may exert
differential function(s). In this respect, it is noteworthy that TIF1ß
has recently been shown to associate with and to act as a transcriptional
coactivator for C/EBPß (Rooney and
Calame, 2001
), indicating that TIF1ß can mediate both
activation and repression of transcription. Whether TIF1ß has both
functions in the same nucleus, depending on its localization with respect to
centromeric heterochromatin, is presently unknown and will require further
biochemical and genetic studies to be determined.
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Acknowledgments |
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References |
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