From the Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Received for publication, August 28, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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Emerin belongs to the "LEM domain" family of
nuclear proteins, which contain a characteristic ~40-residue LEM
motif. The LEM domain mediates direct binding to barrier to
autointegration factor (BAF), a conserved 10-kDa chromatin protein
essential for embryogenesis in Caenorhabditis elegans. In
mammalian cells, BAF recruits emerin to chromatin during nuclear
assembly. BAF also mediates chromatin decondensation during nuclear
assembly. The LEM domain and central region of emerin are essential for
binding to BAF and lamin A, respectively. However, two other conserved
regions of emerin lacked ascribed functions, suggesting that emerin
could have additional partners. We discovered that these
"unascribed" domains of emerin mediate direct binding to a
transcriptional repressor, germ cell-less (GCL). GCL
co-immunoprecipitates with emerin from HeLa cells. We determined the
binding affinities of emerin for GCL, BAF, and lamin A and analyzed
their oligomeric interactions. We showed that emerin forms stable
complexes with either lamin A plus GCL or lamin A plus BAF.
Importantly, BAF competed with GCL for binding to emerin in
vitro, predicting that emerin can form at least two distinct types of complexes in vivo. Loss of emerin causes
Emery-Dreifuss muscular dystrophy, a tissue-specific inherited disease
that affects skeletal muscles, major tendons, and the cardiac
conduction system. Although GCL alone cannot explain the disease
mechanism, our results strongly support gene expression models for
Emery-Dreifuss muscular dystrophy by showing that emerin binds directly
to a transcriptional repressor, GCL, and by suggesting that
emerin-repressor complexes might be regulated by BAF.
Biochemical roles for emerin in gene expression are discussed.
Emery-Dreifuss muscular dystrophy
(EDMD)1 is characterized by
progressive muscle weakening, contractures of major tendons, and
defects in cardiac conduction that can be life-threatening (1). EDMD is
inherited through mutations in two different genes: LMNA and
STA. LMNA encodes A-type lamins, which are
developmentally regulated nuclear filament proteins, and STA
encodes an integral nuclear membrane protein named emerin (2, 3).
Emerin mutations cause the X-linked recessive form of EDMD (4, 5).
Emerin is expressed in most cell types examined thus far, except
non-myocytes of the heart (6). Because emerin is expressed in most
cells, but its loss affects only a few specific tissues, emerin was
proposed to have roles in tissue-specific gene expression (7).
Emerin belongs to the LEM domain family of nuclear proteins, which
contain a characteristic ~40-residue LEM motif (8, 9). Other family
members include LAP2, MAN1, otefin, Lem-3, and SANE (5, 10-12) Most
LEM proteins are localized at the nuclear inner membrane. The LEM
domains of LAP2 We now report that these "unascribed" domains of emerin mediate
direct binding to germ cell-less (GCL) a transcriptional repressor that
is conserved from Caenorhabditis elegans to humans. Nili et al. (20) showed previously that GCL binds LAP2 Protein Purification--
Human emerin (residues 1-222) was
cloned into pET15 plasmid (Novagen, Inc., Madison, WI), and alanine
substitution mutagenesis was performed as described previously (15).
Wild type emerin and all mutant proteins were expressed in
Escherichia coli strain BL21(DE3):pLysS as described
previously (15). Induced bacteria were collected by centrifugation,
resuspended in PBS, and sonicated (30 s, five times each). The
resulting homogenate was centrifuged (20 min, 40,000 × g, 4 °C). The pellet was washed twice with PBS; resuspended in 8 M urea, 500 mM NaCl, and 20 mM HEPES (pH 7.4); and recentrifuged for 20 min at
40,000 × g. The supernatant was recovered, treated
with 10 volumes of 500 mM NaCl, 20 mM HEPES (pH
7.4), and recentrifuged (40,000 × g for 20 min). This
supernatant was loaded on a Superdex 75 preparative gel filtration
column (Amersham Biosciences), and 1.5-ml fractions were collected.
Emerin elutes at a position consistent with monomers (data not shown). Fractions containing emerin protein were snap-frozen in liquid nitrogen. 35S-labeled proteins were synthesized in
vitro using the TnT® Quick Coupled Transcription/Translation
System according to the manufacturer's instructions (Promega, Madison,
WI). The cDNA encoding human GCL was a kind gift from Amos Simon
(20).
Binding Assays--
Purified recombinant human emerin (residues
1-222) was covalently attached to Affi-Gel-15 beads (Bio-Rad
Laboratories, Inc., Hercules, CA) according to the manufacturer's
instructions. Beads were washed three times in binding buffer (20 mM HEPES, pH 7.4, 110 mM potassium phosphate, 2 mM magnesium acetate, and 0.5 mM EGTA) and
incubated with either 35S-GCL or 35S-BAF for
2 h at 22-24 °C. Beads were washed five times with binding buffer, eluted with SDS sample buffer, and subjected to SDS-PAGE, and
bound proteins were detected by autoradiography.
Co-immunoprecipitations were performed as described previously (15)
using 3.5 mg nuclear extract/experiment. HeLa cell nuclear extracts
were generated by standard cell fractionation techniques, as described
previously (23). Antibodies against human GCL were produced in rabbits
(#4438) immunized against a GCL peptide corresponding to residues
408-420, linked to keyhole limpet hemocyanin. Microtiter well binding
assays were done essentially as described previously (24), with the
following changes. To measure affinities accurately, the amount of
emerin per microtiter well was titrated 10-20-fold: we added lamin A,
GCL, or BAF at several concentrations ranging from 0.5 nM
to 10 µM to microtiter wells containing either 1, 5, 10, 20, or 50 pmol of emerin. These affinity curves were confirmed by
computer modeling (data not shown). When testing for GCL binding to our
collection of emerin mutants, subsaturating amounts of 35S-GCL (50 nM) were used, with 15-25 pmol of
emerin typically present per well. Wells were not allowed to dry at any
time during these assays. In all cases, bound 35S-labeled
proteins were extracted with 5% SDS and counted in a scintillation counter.
To detect three-way complexes, 150 nM lamin A plus 250 nM BAF, 160 nM lamin A plus 54 nM
GCL, or 5 µM BAF plus 60 nM GCL was mixed and
immediately added to 10 pmol of wild type emerin. BAF inhibition of GCL
binding to emerin was quantitated by first mixing 60 nM
35S-GCL with increasing concentrations of purified
recombinant BAF (50 nM to 10 µM), prepared as
described previously (18), and immediately adding the mix to 1.5 pmol
of emerin immobilized on microtiter wells. Wells were washed five times
with binding buffer, eluted with SDS, and separated on SDS-PAGE, and
proteins were detected by autoradiography.
Reverse Transcription-PCR--
Multitissue panels I and II
(Clontech) were used as templates to probe with
oligonucleotide primers specific for a LAP2 Quantitation of BAF--
HeLa cells (109) were
resuspended in sample buffer, and 106 cell equivalents were
subjected to SDS-PAGE along with 1, 2.5, 5, and 10 ng of recombinant
purified human BAF. After transfer to nitrocellulose, blots were probed
with an antibody against human BAF diluted 1:1000 (18). We calculated
the concentration of BAF near the inner nuclear membrane by modeling
the nucleus as a sphere 5 µm in diameter and defining the interaction
zone (IZ) to extend 200 nm from the inner membrane. We
calculated the volume of the IZ by subtracting the volumes of two
spheres, where R1 is the radius to the inner
membrane (2.5 µm), and R2 (2.3 µm) equals
R1 minus the interaction distance (200 nm). The
volume of the IZ of the nuclear envelope was VIZ = V1 Because the GCL-binding region in LAP2
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and emerin mediate their direct binding to barrier
to autointegration factor (BAF) (13-15). BAF is a 10-kDa, highly
conserved chromatin protein essential for the viability of dividing
cells (16). In mammalian cells, BAF recruits emerin to chromatin during
nuclear assembly (17), and this recruitment is somehow essential for
localizing emerin at the reforming nuclear envelope. BAF also has
important roles in higher-order chromatin structure during nuclear
assembly (18). The LEM domain of emerin is essential for binding to
BAF, and the central region of emerin is essential to bind lamin A
(15). These results, coupled with distinct binding regions for BAF and lamins on LAP2
(14, 19), suggest that BAF links chromatin directly
to membrane-anchored LEM proteins and indirectly to lamins. However,
two conserved regions of emerin lacked any ascribed function, suggesting that emerin has additional unknown partners (15).
. GCL
also binds directly to the DP3 subunit of E2F-DP heterodimers
and thereby represses E2F-DP-dependent gene transcription
(20, 21). Independently, E2F-DP-dependent genes are
repressed by retinoblastoma protein, which binds to E2F and recruits
histone-modifying complexes (22). Interestingly, LAP2
can inhibit a
reporter gene regulated by E2F-DP, suggesting that LAP2
itself is a
repressor; furthermore, co-expression of both LAP2
and GCL repressed
transcription as effectively as retinoblastoma protein (20). Because
GCL binds to a region in LAP2
that is conserved with emerin, we
proposed and tested the hypothesis that GCL also binds emerin. We
determined the binding affinities of emerin for GCL, BAF, and lamin A
and studied their oligomeric interactions biochemically. Our findings support molecular models in which emerin and its partners form at least
two types of complexes in vitro. Our results suggest that
emerin has distinct roles in vivo, depending on whether it is complexed with BAF or a transcription factor.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-specific exon or GCL,
according to the manufacturer's instructions. The 5' LAP2
primer
was 5'-CTCAAGCTGGAATAACTGAGACTGAATGG-3', and the 3' primer was
5'-GGGTCAACATGAAGAAAATTAGAGAAGG-3'. The 5' GCL primer was
5'-CCGGGATCCGATGAATATTATTGAACTGGAGATTCC-3', and the 3' primer was
5'-CCGCTCGAGGTTTTCTGGATCTTCTGGGTGGC-3'. Control primers for glyceraldehyde-3-phosphate dehydrogenase were provided by the manufacturer.
V2 = 14.1 fl (10
15 liters), where V1 = 4/3
R13 and V2 = 4/3
R23.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was conserved in emerin,
we tested the hypothesis that GCL can also bind emerin. We synthesized
full-length 35S-labeled human GCL and BAF and incubated
each protein separately with recombinant human emerin conjugated to
Affi-Gel beads. Beads conjugated with BSA served as negative controls.
The emerin beads specifically bound BAF and also bound efficiently to
GCL (Fig. 1A). The equilibrium
affinity of the emerin-GCL interaction was measured using a microtiter
well binding assay. Recombinant soluble purified emerin (residues
1-222) was immobilized in microtiter wells, and different
concentrations of soluble 35S-GCL were added to each well.
In this assay, the affinity of GCL for emerin was 30 nM
(range, 20-60 nM; n = 30; Fig.
1B), and the stoichiometry of interaction was ~0.8-1 mol
GCL/mol emerin (data not shown). These results demonstrated that emerin
can bind a transcriptional repressor, GCL, with high affinity in
vitro. GCL was previously shown to be enriched at the nuclear
envelope in Drosophila and mammalian cells (20, 21, 25). To
determine whether GCL and emerin interacted in vivo, we did
co-immunoprecipitation experiments. HeLa nuclear extract was incubated
with either protein A beads alone or protein A beads plus anti-emerin
antibody. These beads were then washed and resolved by SDS-PAGE,
transferred to nitrocellulose, and probed with antibodies against GCL.
GCL co-immunoprecipitated with emerin; comparatively little GCL was
pulled down by the protein A beads alone (Fig. 1C).
Co-immunoprecipitation of GCL with emerin was evidently efficient
because GCL was present at almost undetectable levels in the starting
lysate (Fig. 1C, load). Thus, emerin and GCL
appear to interact both in vitro and in vivo.
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Fig. 1.
GCL binding to wild type and mutant emerin
proteins. A, affinity binding assays. Affi-Gel
beads coupled to recombinant human emerin (residues 1-222) or BSA were
incubated with either 35S-GCL or 35S-BAF (see
"Experimental Procedures"). B, affinity of GCL for
emerin, determined by adding increasing concentrations of GCL to
constant amounts of emerin immobilized in microtiter wells (see
"Experimental Procedures"). Double reciprocal plots and computer
modeling were used to accurately determine the affinity constant.
C, GCL co-immunoprecipitates with emerin from HeLa
nuclear extract. Extracts were incubated with either protein A beads
alone or protein A beads plus antibodies against human emerin. Pelleted
beads were washed, extracted, resolved by SDS-PAGE, and immunoblotted
using antibodies against human GCL (top panel) or emerin
(bottom panel). load, 70 µg of HeLa nuclear
extract. D, aligned amino acid sequences of human
LAP2 (top row, starting at residue 112) and
full-length emerin (bottom row), showing mutations used to
map the putative GCL-binding domain in emerin. In most cases,
underlined residues were replaced by alanine (A) as
indicated, in clusters. Mutations in residues conserved between emerin
and LAP2
are indicated in black (15). New mutations in
emerin-specific residues are shown in gray. Mutant clusters
are numbered according to their most N-terminal altered residue, as
detailed in Table I. Arrow indicates the last residue (222)
of recombinant emerin protein. E and F,
quantitative microtiter well binding assays using immobilized wild type
(WT) emerin (residues 1-222) and emerin mutants (numbered
as described in D). Emerin proteins immobilized in
microtiter wells were incubated with 35S-GCL, washed, and
counted. Top panels show emerin proteins extracted from
parallel wells and immunoblotted to verify similar amounts of
emerin/microtiter well.
We then mapped the GCL-binding region in emerin. The microtiter well
binding assay was used to test 35S-GCL binding to a
collection of emerin mutants (15), each containing a cluster of alanine
substitutions in residues highly conserved between human LAP2 and
emerin (Fig. 1D, mutations are noted in black). We immobilized either BSA, recombinant wild type
emerin (residues 1-222), or emerin mutant proteins in microtiter wells and added 35S-GCL to the solution in these wells. After
washing, the bound 35S-GCL was removed using 5% SDS and
counted (Fig. 1E, graph). To control for the
amount of emerin present in each well, immobilized emerin proteins were
eluted from parallel wells using 10% SDS, separated by SDS-PAGE,
transferred to nitrocellulose, and probed with an anti-emerin antibody
(Fig. 1D, top). At the concentration of
35S-GCL used for these studies (50 nM), emerin
was in 3-5-fold excess. Mutants 24, 112, 164, and 179 bound 68-84%
as well as wild type emerin (Fig. 1E). Mutants 34, 196, 207, and 214 had weak binding (35-45% of wild type). The weakest binding
among "conserved mutations" was seen for mutants 70 and 76 (Fig.
1E). The background binding of 35S-GCL to BSA
ranged from 16-23% of the wild type emerin signal, suggesting that
mutants 34-76 and 196-214 were severely reduced in binding to GCL. To
refine the putative GCL-binding domain of emerin, we generated 11 new
emerin mutants by alanine substitutions, specifically targeting
clusters of residues that are dissimilar between LAP2
and emerin
(Table I; Fig. 1D,
mutations are shown in gray). We reasoned that
emerin-specific residues might contribute uniquely to its affinity for
GCL and that these new mutations might better define the GCL-binding
domain. Indeed, several emerin-specific mutations disrupted binding
quite effectively. When tested in the well binding assay, mutants 122, 133, 151, 161, 198, and 206 each bound 35S-GCL to at least
65% wild type levels (Fig. 1F, graph). Control blots verified that similar amounts of mutant emerin protein were immobilized in each well (Fig. 1F, top). Mutant
145 was compromised (~50% wild type activity) but still bound
detectably to 35S-GCL. Importantly, mutants 45A, 45E, 175, and 192 had very low or background levels of binding to
35S-GCL (Fig. 1F). Collectively, these results
identified two regions in emerin essential for binding to GCL:
(a) residues 34-83, which overlap the LEM domain, and
(b) residues 175-196 and 207-217 near the transmembrane
domain of emerin. These results also implicated serines at positions
175-176 and 192-196 of human emerin as potentially critical for
binding GCL.
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Most disease-causing mutations in the emerin gene cause cells to be
null for emerin protein. However, there are special mutations in emerin
(point mutants S54F, Q133H, P183H, P183T, and deletion 95-99) that
cause EDMD, wherein the mutant proteins are stable and properly
localized at the nuclear envelope (15). The
95-99 mutation disrupts
emerin binding to lamin A in vitro but has no effect on BAF
binding. Mutants S54F and P183H bind normally to both lamin A and BAF
in vitro (15). To determine whether any disease-causing
mutations disrupted emerin binding to GCL, we used 35S-GCL
to probe mutants S54F,
95-99, and P183H immobilized on microtiter
wells. Similar amounts of emerin protein were present in each well, as
determined by Western blotting of parallel microtiter wells (Fig.
2A,
-emr).
GCL bound to emerin mutants S54F and P183H at levels similar to wild
type (75-85% of wild type). In contrast, GCL binding to
95-99 was
reduced to levels barely above the BSA negative control. The
95-99
disease mutation therefore disrupts emerin binding to two partners:
(a) transcriptional regulator GCL, and (b) lamin
A (15). We considered the possibility that the
95-99 mutation
causes emerin to misfold, nonspecifically disrupting its binding to
both partners. However, any such misfolding must be spatially limited
because the
95-99 mutant is soluble and can still bind BAF (15) and
therefore has a properly folded LEM domain. Alternatively, GCL and
lamin A might both require this region of emerin and might
theoretically compete for binding to emerin. Our GCL binding results
for all emerin mutants are mapped schematically in Fig. 2B,
relative to previous results for BAF and lamin A (15). Residues
required to bind GCL mapped primarily to the two "unascribed"
regions of emerin that flank the lamin-binding region, with some
overlap of both the BAF- and lamin A-binding domains. Thus, this
transcription factor represents an important new type of binding
partner for emerin.
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The identification of a transcription factor as a binding partner for emerin raised important questions about the relationship between GCL and emerin's other partners. To test the hypothesis that GCL, BAF, or lamin A might compete with each other for binding to emerin, we first needed to determine the equilibrium binding affinities of BAF and lamin A for emerin. BAF dimers bind emerin with a Kd of 200 nM (range, 100-550 nM; n = 23; Fig. 2C) and stoichiometry of ~0.75-1 mol BAF dimer/mol emerin in microtiter well binding assays (Fig. 2C; data not shown). Lamin A bound emerin with higher affinity (40 nM; range, 30-80 nM; n = 19; Fig. 2D) and a stoichiometry of ~0.85-1.1 mol lamin A dimer/mol emerin (Fig. 2D; data not shown). Thus, all three partners had reasonable affinities for emerin (Kd of 200, 40, and 30 nM for BAF, lamin A, and GCL, respectively).
We then did competition assays to determine whether BAF competed with
GCL for binding to emerin. Microtiter wells with a constant amount of
immobilized emerin were incubated with buffer containing a constant
amount of 35S-GCL plus increasing concentrations of BAF
(Fig. 3A). Under these conditions, BAF blocked the binding of GCL to emerin, with 50% inhibition of binding at 4.1 µM BAF
(Ki, Fig. 3A). This competition depended
specifically on the BAF-emerin interaction because BAF failed to
compete for GCL binding to emerin LEM domain mutant m24 (Figs.
1D and 3B) (15). We concluded that BAF binding to
the LEM domain of emerin displaces GCL by reducing its affinity because
GCL also depends on residues at the C terminus of the LEM domain.
Because the affinity of BAF for emerin (200 nM) is about
7-fold weaker than that of GCL (30 nM), it was reasonable that higher levels of BAF (4.1 µM) were required to
inhibit GCL binding. To determine whether these concentrations were
physiologically relevant, we did Western blots comparing HeLa cell
lysates against known amounts of purified recombinant BAF and
determined that HeLa cells contain ~7 nM endogenous BAF
(Fig. 3C). However, based on the relative fluorescence
intensities for BAF in cultured Xenopus cells, determined by
indirect immunofluorescence (18), we estimate that ~30% of cellular
BAF is concentrated near the nuclear inner membrane. We therefore
modeled the nucleus as a sphere (diameter, 5 µm) and defined the
interaction zone as extending 200 nm from the inner membrane (Fig.
3C, IZ). Based on these estimates, the concentration of BAF dimers near the nuclear envelope is ~9
µM (see "Experimental Procedures"). However, local
(molecular scale) concentrations of BAF could be even higher because
BAF dimers oligomerize in the presence of DNA in vitro (16).
In contrast, immunoblotting results for GCL suggested that the
concentration of GCL near the nuclear envelope is lower, ~1
µM (data not shown). Thus, the above-determined
affinities of emerin for BAF and GCL are likely to be physiologically
relevant and collectively predict that BAF has the potential to inhibit
GCL binding to emerin at the nuclear envelope in cells.
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Does lamin A influence GCL binding to emerin? To answer this question, we did binding competition assays in microtiter wells, using appropriate concentrations of each protein. Emerin was immobilized in wells and incubated with buffer containing premixed 54 nM 35S-GCL plus 160 nM 35S-lamin A (GCL does not bind detectably to lamin A; data not shown). This concentration of lamin A ensured that over 90% of emerin molecules would be occupied by lamin A, and thus any GCL signal could be validly attributed to the formation of three-way complexes. Interestingly, lamin A and GCL did not compete for binding to emerin but instead bound efficiently and simultaneously to emerin in vitro (Fig. 3D). We concluded that emerin is capable of forming stable complexes with lamin A and GCL at the nuclear envelope, if BAF is absent or negatively regulated. This finding supported "scaffolding" models in which lamin A anchors emerin at the inner nuclear membrane yet does not interfere with emerin binding to the transcriptional repressor.
Can BAF itself form stable complexes with emerin and lamin A? To answer this question, we used a relatively low concentration of lamin A (150 nM dimers) because lamin A binds weakly but directly to BAF with a Kd of 1 µM (Fig. 3E). In the presence of 150 nM lamin A, only one in five BAF molecules would be prebound to lamin A (see Fig. 3E). Under these conditions, we found that BAF (at 250 nM) and lamin A (at 150 nM) bound simultaneously and quantitatively to emerin (Fig. 3F). This work strongly supported our previous model that emerin can bind independently, through distinct domains, to both BAF and lamin A (15).
Because GCL can interact with two different LEM proteins (emerin and
LAP2), the tissues susceptible to EDMD disease may be determined, at
least in part, by the natural presence or absence of LEM domain
proteins, such as LAP2
, whose functions overlap with emerin. To
determine whether LAP2
or GCL was present in tissues affected by
EDMD, we performed quantitative reverse transcription-PCR analysis of
mRNA from 16 human tissues using LAP2
- or GCL-specific primers.
LAP2
expression is relatively low but detectable in heart and
skeletal muscle, which are strongly affected by the loss of emerin
(Fig. 3G). The low expression of LAP2
mRNA in these
tissues was consistent with previously determined low levels of LAP2
protein (26). Human GCL mRNA was abundant in only one tissue
examined (testis). GCL was undetectable in eight tissues (liver,
spleen, thymus, prostate, ovary, small intestine, colon, and peripheral
blood leukocytes), and low expression of GCL was detected in heart,
brain, placenta, lung, skeletal muscle, kidney, and pancreas,
consistent with expression patterns seen in adult mice (27). Thus, GCL
is expressed in a tissue-specific manner and is present (albeit at low
levels) in two tissues (heart and skeletal muscle) affected in EDMD
patients. Obviously, the expression pattern for GCL does not explain
the tissue specificity of EDMD disease. However, as discussed below,
GCL is the first of many transcription factors that may bind emerin
(see "Discussion"). Thus, models based on GCL-emerin interactions
may be generally applicable to the disease mechanism.
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DISCUSSION |
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Our biochemical results suggest that emerin and lamin A are stable partners at the nuclear envelope and can form stable oligomeric complexes with either BAF or GCL, but not both. Interestingly, the stability of BAF-emerin-lamin A complexes might be increased by the low but direct affinity of BAF for lamin A. Based on our estimated physiological concentrations of BAF and GCL, we propose that BAF-mediated chromatin attachment to emerin will dominate and exclude GCL in the absence of modifications or regulators that change their measured affinities. Because GCL localizes to the nuclear envelope (20, 21), we propose that its binding is regulated by posttranslational modifications of BAF, GCL, emerin, or all three, as discussed below. Our findings have important implications for emerin function and for the disease mechanism of Emery-Dreifuss muscular dystrophy.
The structures of GCL and emerin (outside the LEM domain) are not yet known. However, based on our analysis of 24 mutations distributed throughout the nucleoplasmic domain of emerin, we suggest that GCL might contact two surfaces on emerin, which we broadly designate repressor binding domain (RBD)-1 (residues 34-83) and RBD-2 (residues 175-217; Fig. 3H). Alternatively, emerin might fold to form a single repressor binding domain surface (not depicted). As modeled in Fig. 3, the binding of GCL to emerin may recruit E2F-DP heterodimer-chromatin complexes to the nuclear envelope and thereby repress transcription (Fig. 3H, model I). Alternatively, the recruitment of GCL to emerin may cause GCL-E2F-DP3 complexes to dissociate, allowing activation of E2F-DP-dependent genes (Fig. 3H, model II). In either case, GCL or GCL-containing complexes can be competed by BAF (Fig. 3H, model III). Interestingly, BAF was recently shown to bind directly to tissue-specific homeodomain transcription factors and block their activity (28).
GCL appears to be concentrated at the nuclear envelope in
Drosophila (25) and mammalian cells (20). Our biochemical
analysis of recombinant proteins suggests that emerin can form stable
complexes with lamin A and GCL, but only in the absence of BAF. Because many nuclear envelope proteins are differentially phosphorylated during
interphase, including lamin A, LAP2, and emerin (29, 30), we propose
that GCL binding to emerin or displacement of BAF from BAF-emerin-lamin
complexes is regulated by signal transduction in vivo. This
predicted interplay between GCL and BAF for binding to emerin will be
interesting to explore in living cells, where binding affinities may be
further influenced by chromatin. For example, the affinity of BAF for
LEM proteins appears to increase in the presence of DNA (14).
Additional studies of BAF and its regulation are clearly important to
understand how BAF affects chromatin attachment to the nuclear envelope.
The discovery that emerin binds the transcriptional repressor GCL
strongly supports gene expression models for the EDMD disease mechanism. Further supporting this model, the disease-causing mutation
95-99 potently disrupted emerin binding to both GCL and lamin A. Nevertheless, the loss or disruption of GCL binding to emerin is
insufficient to explain disease, for two reasons. First, LAP2
is
theoretically available as backup to bind GCL in skeletal muscle and
heart, although we do not know whether GCL-LAP2
-lamin B complexes
are functionally equivalent to GCL-emerin-lamin A complexes. Secondly,
and more importantly, we and our collaborators recently identified two other
transcription factors that bind emerin.2,3 We therefore
suggest that EDMD disease results from the combined loss of emerin
binding to multiple transcription factors, some of which may be more
"important" than others in maintaining the function of a specific
tissue. Our analysis of GCL-emerin interactions, presented here,
suggests an important testable paradigm for the binding of many
different transcription factors to emerin-lamin complexes.
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ACKNOWLEDGEMENTS |
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We are grateful to Doug Robinson for biochemical advice. We thank the National Cell Culture Center for providing the HeLa cell pellets.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1 GM64535 (to K. L. W.) and T32 HL07227 (to J. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cell Biology,
The Johns Hopkins University School of Medicine, 725 N. Wolfe St.,
Baltimore, MD 21205. Tel.: 410-955-1801; Fax: 410-955-4129; E-mail:
klwilson@jhmi.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M208811200
2 T. Haraguchi, J. M. Holaska, M. Yamane, T. Koujin, N. Hashiguchi, K. L. Wilson, and Y. Hiraoka. Emerin binding to Btf, a death-promoting transcriptional repressor, is disrupted by a missense mutation that causes Emery-Dreifuss muscular dystrophy, submitted for publication.
3 F. L. Wilkinson, J. M. Holaska, A. Sharma, S. B. Manilal, I. Holt, S. Stamm, K. L. Wilson, and G. E. Morrris. Emerin interacts with the splicing-associated factor YT521-B: implications for Emery-Dreifuss muscular dystrophy, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: EDMD, Emery-Dreifuss muscular dystrophy; BAF, barrier to autointegration factor; GCL, germ cell-less; IZ, interaction zone; BSA, bovine serum albumin.
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