From the School of Molecular and Microbial Biosciences, G08, University of Sydney, New South Wales 2006, Australia
Received for publication, January 19, 2003
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ABSTRACT |
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The ability of DNA-binding transcription factors
to recruit specific cofactors is central to the mechanism by which they
regulate gene expression. BKLF/KLF3, a member of the Krüppel-like
factor family of zinc finger proteins, is a potent transcriptional
repressor that recruits a CtBP co-repressor. We show here that BKLF
also recruits the four and a half LIM domain protein FHL3. Different but closely linked regions of BKLF mediate contact with CtBP2 and FHL3.
We present evidence that CtBP2 also interacts with FHL3 and demonstrate
that the three proteins co-elute in gel filtration experiments. CtBP
and FHL proteins have been implicated in both nuclear and cytoplasmic
functions, but expression of BKLF promotes the nuclear accumulation of
both FHL3 and CtBP2. FHL proteins have been shown to act predominantly
as co-activators of transcription. However, we find FHL3 can repress
transcription. We suggest that LIM proteins like FHL3 are important in
assembling specific repression or activation complexes, depending on
conditions such as cofactor availability and promoter context.
The Sp/Krüppel-like factor
(KLF)1 family of mammalian
DNA-binding proteins consists of the Sp1-related proteins (Sp1-6) and a subfamily termed Krüppel-like Factors (KLF1-17), consisting of
erythroid Krüppel-like factor (EKLF/KLF1), lung
Krüppel-like factor/KLF2, basic Krüppel-like factor/KLF3,
and others (1-4). The Sp/KLF proteins play diverse roles in regulating
gene expression during development. For example, EKLF/KLF1 is important
for Once bound to DNA the proteins are thought to regulate transcription by
recruiting co-regulatory molecules. The N-terminal domains of different
Sp/KLF family members exhibit little or no homology, and differential
cofactor recruitment by these divergent domains may in part explain why
some members behave as activators, whereas others act as repressors.
The situation is complex as other regions of the proteins, such as the
zinc finger domain also mediate protein-protein interactions, and
individual proteins may recruit both co-activators and co-repressors.
For instance, EKLF/KLF1 has been shown to activate transcription by
recruiting the histone acetylase proteins p300/CBP and P/CAF through
its N terminus and the chromatin remodeling complex E·RC1
through its zinc finger domain (10). The zinc finger domain can also recruit a histone deacetylase complex to silence gene expression (11).
Other KLFs that can repress transcription include BKLF/KLF3, KLF8, and
Ap-2rep/KLF12. These proteins recruit co-repressors of the
C-terminal-binding protein (CtBP) family (12-15). Their interaction
with CtBP is mediated through a short amino acid motif, of the form
Pro-X-Asp-Leu-Ser (PXDLS), that is
present in all three proteins (13). Aside from this motif, there is
very little homology between the N-terminal repression domains of
BKLF/KLF3, KLF8, and Ap-2rep/ KLF12.
The CtBP co-repressors bind numerous other regulatory proteins,
including many conventional DNA-binding proteins, and accessory molecules, such as polycomb and the viral protein E1A (13). The mode of
action of CtBP is yet to be fully elucidated. CtBP proteins can recruit
histone deacetylases (16, 17), however, deacetylase-independent
repression has also been observed. CtBP interacts with polycomb group
proteins (18) and proteins such as Ikaros (19) that are contained in
chromatin remodeling complexes, suggesting that CtBP proteins may also
participate in regulation of gene expression through the non-covalent
modification of chromatin structure.
We have found that BKLF/KLF3 is a potent transcriptional repressor, and
during our analysis of transcriptional repression by BKLF (14), we
noted that abrogation of CtBP recruitment does not entirely abolish
this function. This suggested that BKLF may recruit one or more
additional cofactors to regulate transcription. A two-hybrid screen
against BKLF identified the LIM-only protein FHL3 as another BKLF
partner protein.
FHL3 is a member of the recently recognized four and half LIM domain
(FHL) family, which consists of FHL1-4, ACT, and KyoT1 (20-26). These
proteins are made up of four LIM domains, plus one N-terminal
"half" LIM domain whose amino acid sequence resembles that
of a single GATA type zinc finger. LIM domains are composed of a double
zinc finger motif that co-ordinate two zinc ions and are primarily
thought to mediate protein-protein interactions (27). The term LIM
originates from the isolation of three Caenorhabditis elegans transcription factors, LIN-11,
Isl-1, and Mec-3, proteins in which this domain
was first described (28). FHL1 and 2 are highly expressed in skeletal
muscle and have been observed to be present in the cytoplasm.
Consequently FHL proteins were originally hypothesized to play a role
in cytoskeletal function in muscle cells (24, 25). Recently, however,
FHL family members have also been detected in the nucleus, and it now
appears that in this compartment several FHL family members function as
transcriptional co-regulators. The testis-specific protein ACT serves
as a co-activator of CREM and CREB (21), and its restricted expression
pattern helps explain how testis-specific gene activation by CREM is
mediated. FHL2 has been shown to act as a specific co-activator of the
androgen receptor (AR) (23) and the zinc finger protein Wilms tumor-1 (WT1) (29) and as a co-repressor of the multi-zinc finger protein PLZF
(30). Sub-cellular partitioning also appears to be important for the
regulation of FHL function. FHL2 has been observed in both nuclear and
cytoplasmic fractions, and its translocation to the nucleus in response
to Rho signaling can lead to potent stimulation of AR-mediated gene
expression (31). Additionally, ACT interacts with the kinesin KIF17b,
and this interaction is thought to regulate its nuclear/cytoplasmic
partitioning (32). To date, the FHL proteins described have been found
to behave as conventional transcriptional co-regulators, raising the
possibility that they may be involved in recruiting the basal
transcriptional machinery or chromatin remodeling enzymes, but their
precise mechanism of action has not yet been defined.
Here we show that FHL3 binds BKLF and acts as a co-repressor. We also
demonstrate that FHL3 contacts CtBP2, a separate BKLF co-repressor.
Because sub-cellular distribution of FHL factors may affect their
function, we were interested in examining the cellular location of
FHL3. We present evidence that FHL3, CtBP2, and BKLF proteins exist in
a large complex only in the nucleus and that FHL3 is significantly
enriched in the nucleus only when it is co-expressed with both BKLF and
CtBP2. We suggest that FHL3, and other members of the FHL family,
operate as linking modules that stabilize multiprotein transcriptional
complexes in the nucleus in addition to their reported roles in the cytoplasm.
Yeast Two-hybrid Screening and Assays--
The
Clontech two-hybrid system was used according to
the manufacturer's instructions. A human erythroleukemia (K562) cell cDNA library in the gal4 activation domain (AD) fusion vector, pGAD10, was transfected into the yeast strain HF7c harboring the gal4DBD-BKLF-(1-268) fusion protein expressed from pGBT9.
Candidate interactors were re-tested by co-transfecting gal4DBD-bait
proteins and gal4AD-prey proteins into HF7c. Transformants were
selected on Trp/Leu-deficient plates and patched onto
Trp/Leu/His-deficient media. Growth was scored after 30 h.
Controls containing bait and prey alone were also conducted and were
negative for growth up to more than 72 h.
In Vitro Binding Assays--
Full-length BKLF and
FHL3 were cloned into the vector pcDNA3 (Invitrogen),
and 35S-labeled in vitro translated proteins
were generated using T7 polymerase and the TNT system from
Promega. GST fusion proteins were prepared and binding assays were
carried out as previously described (33).
Northern Blotting--
A 32P-labeled 300-bp fragment
from the coding sequence of FHL3 was used to probe two
commercial filters, a multiple tissue Northern blot containing mRNA
from human adult tissues, and a blot containing mRNA from various
cancer cell lines, both from Clontech.
Hybridization was performed at high stringency, using ExpressHyb
(Clontech). These membranes were stripped and
re-probed using a human Mammalian Cell Transfections and Reporter Assays--
NIH-3T3
cells were cultured and transfected, using the calcium phosphate
method, as described previously, as were chloramphenicol acetyltransferase and growth hormone reporter assays (14). COS cells
were cultured and transfected, using the DEAE-dextran method, as
previously described (15). K562 stable cell lines expressing BKLF were generated as described (34), using the expression vector pEF1 Nuclear and Cytoplasmic Extract Preparation--
Nuclear
extracts were prepared as previously described (35). Cytoplasmic
extracts were prepared during the procedure for preparation of nuclear
extracts as above.
Antibody-Bead Cross-linking and Co-immunoprecipitation--
To
generate antibody-bead conjugates, 100 µl of pre-immune or immune
sera was incubated with 100 µl of protein-A-agarose beads (Roche
Molecular Biochemicals) to a final volume of 1 ml with PBS, for 60 min
at 4 °C. Beads were then washed twice with 10 volumes of 200 mM sodium borate, pH 9.0, and were resuspended in 10 volumes of 200 mM sodium borate, pH 9.0, with
dimethylpimilimidate (Sigma) at a final concentration of 20 mM. Cross-linking was performed for 30 min at room
temperature, and the reaction was stopped by washing once with 10 volumes of 200 mM ethanolamine, pH 8.0, and then incubation
with 200 mM ethanolamine, pH 8.0, for 2 h at room temperature. Beads were then washed twice with 10 volumes of 100 mM glycine, pH 2.8, to remove non-cross-linked antibody,
washed once with 10 volumes PBS, and resuspended in PBS.
For immunoprecipitation of nuclear extracts, 80-100 µl of extract
from transfected COS cells were diluted 1:3 with Nonidet P-40 buffer
(0.5% Nonidet P-40/Igepal, 150 mM NaCl, 50 mM
Tris, pH 8.0, 1 µg/µl leupeptin, 1 µg/µl aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The solution was
pre-cleared with 10 µl each of protein-A and protein-G beads at
4 °C for 30 min. The supernatant was then immunoprecipitated with 10 µl of anti-BKLF or anti-FHL3 beads, or the corresponding pre-immune
beads, with incubation at 4 °C for 1 h. The beads were washed
four times with Nonidet P-40 buffer and were resuspended in SDS-PAGE
loading buffer with 100 mM dithiothreitol. The samples were
treated at 60 °C for 5 min, separated by SDS-PAGE, and transferred
to nitrocellulose overnight at 100 mA. Blots were then probed by
standard Western blotting techniques with the appropriate primary
antibody then bound with the appropriate secondary antibody linked to
horseradish peroxidase and were detected using the PerkinElmer Life
Sciences Chemiluminescence Reagent Plus kit. For
immunoprecipitation of cytoplasmic extracts, a higher salt buffer was
used during the immunoprecipitation step to maintain the salt
concentration equivalent to that used for nuclear extracts. Subsequent
washes were performed with the 150 mM NaCl Nonidet P-40 buffer.
Gel-filtration Chromatography--
Nuclear and cytoplasmic
extracts were applied to a Superose6TM column (Amersham
Biosciences) equilibrated with buffer containing 20 mM
HEPES, pH 7.9, 10% glycerol, 420 mM NaCl, 1.5 mM MgCl2, and 0.2 mM EDTA and run
at 0.25 ml·min Anti-FHL3 Antibody Generation--
A fusion protein between GST
and full-length FHL3 was prepared as described (14). The GST fusion
protein was then eluted from the GSH-agarose beads using 25 mM glutathione (GSH) in elution buffer (100 mM
Tris-HCl (pH 7.5), 120 mM NaCl) for 1 h at 4 °C with rotation. The GSH-agarose beads were pelleted and re-extracted. The eluates were pooled, and the protein concentration was determined. The protein was lyophilized, and four 300-µg doses were used as antigens for rabbit inoculation. Inoculation and sera collection were
performed by the Veterinary Services Division of the Institute of
Medical and Veterinary Sciences (101 Blacks Rd., Gilles Plains, S.A.,
5086, Australia).
BKLF Interacts with the LIM Protein FHL3--
We have previously
shown that BKLF can recruit the co-repressor protein CtBP2 to repress
transcription (14). A short amino acid motif in BKLF,
Pro-Val-Asp-Leu-Thr (PVDLT), is required for the interaction between
BKLF and CtBP2, and when this motif is disrupted, repression by BKLF is
compromised. However, some repression by BKLF is retained even when the
PVDLT motif is mutated (14). This suggested that BKLF might
recruit additional cofactors to repress transcription. We thus
carried out a further two-hybrid screen to identify additional BKLF cofactors.
The bait used in this screen consisted of the gal4DBD fused to amino
acids 1-268 of BKLF (encompassing the repression domain of BKLF (14)).
We screened a human erythroleukemia cell (K562) cDNA library. K562
cells express embryonic and fetal globins and are frequently used as a
model of erythroid cells at the fetal stage of development (36, 37).
This library was used as BKLF is highly expressed in erythroid cells
(35) and is believed to play a role in hematopoiesis (38, 39). We
screened 4 × 105 clones: 132 primary
His+ colonies were isolated, 10 of which were
also positive for the lacZ reporter. Sequencing revealed two
clones that carried an 899-bp cDNA insert that matched the human
gene termed four and a half LIM protein 3 (FHL3) (25). As a
first test for the specificity of the interaction, these isolates were
tested against the gal4DBD alone and negative control baits, including
gal4DBDs fused to p53, the N finger of GATA-1, and lamin C protein. No
interactions were observed (data not shown). We also swapped FHL3 into
the bait plasmid and tested the ability of a gal4DBD-FHL3 bait to interact with a gal4AD-BKLF prey. As shown in Fig.
1A the interaction is again
observed, whereas there is no interaction with the negative control
prey, gal4AD alone.
The clones we isolated lacked half of the C-terminal LIM domain of
FHL3. To isolate the full-length clone, primers were designed to the
5'- and 3'-ends of the coding sequence (GenBankTM accession
number HSU60116), and the K562 cDNA library was used as a
template in a polymerase chain reaction. The resulting product was
cloned, and sequencing confirmed the presence of a full-length cDNA
encoding human FHL3.
To verify this interaction in another system, a GST-FHL3 fusion protein
was produced by expression in Escherichia coli. The protein
was immobilized on GSH-agarose beads (Fig. 1B, lower
panel) and mixed with radiolabeled BKLF generated by in
vitro transcription and translation in the presence of
[35S]methionine. GST-FHL3 strongly retained
[35S]BKLF (Fig. 1B, lane 3,
upper panel), whereas a negative control protein GST alone
retained no BKLF protein (lane 2, upper panel). Thus, BKLF can interact with FHL3 in both a yeast two-hybrid assay, as
either bait or prey, and in GST pull-down assays.
FHL3 mRNA Expression in Cell Lines and Adult
Tissue--
Because FHL3 was first identified as a gene
that was highly expressed in muscle cells, we wished to test whether it
was expressed at appreciable levels in K562 and other selected cells.
We probed a Northern blot containing RNA from various cancer cell
lines, including K562 cells, with a probe generated from a portion of the coding sequence of FHL3 (Fig.
2A). FHL3 message
was detected in the K562 sample, and low level expression was also
found in HeLa (fibroblast), SW480 (colorectal adenocarcinoma), A599
(lung carcinoma), and MOLT-4 (melanoma) RNA. We also probed a Northern blot of mRNA from various adult tissues and observed high level expression in skeletal muscle as well as lower levels in heart (Fig.
2B). Thus, FHL3 mRNA is present not only in
muscle tissue, as previously observed, but in erythroid cells of fetal
type and other cancer cell lines. This result is consistent with other reports that FHL proteins function in several different cell types in
addition to muscle cells.
Mapping the Interacting Domains of BKLF and FHL3--
BKLF
interacts with CtBP2 through a PVDLT motif centered around amino acid
72 (14). To determine whether FHL3 interacts with BKLF through this
CtBP-contact region or through a distinct region, a series of BKLF
deletions was generated. These constructs produce gal4DBD-BKLF fusion
proteins and were tested for their ability to interact with gal4AD-FHL3
in the yeast two-hybrid system (Fig. 1C). This experiment
indicated that FHL3 interacts strongly with BKLF in a region that lies
between amino acids 160 and 224, but that amino acids 75-160 also
contribute to binding. Importantly, this region is distinct from the
domain in BKLF that binds CtBP2 (amino acids 51-75 (14)), and mutation
of the PVDLT motif, which abrogates CtBP2 binding, has no effect on the
interaction of BKLF with FHL3 (Fig. 1C). This raises the
possibility that BKLF may be able to interact with both FHL3 and CtBP2
simultaneously, and the proximity of the FHL3 and CtBP2 binding sites
within BKLF suggested that FHL3 might also make contacts with CtBP2
(see below).
FHL3 Also Interacts with CtBP2--
To determine whether FHL3
could bind CtBP2, we first used the yeast two-hybrid assay. We tested
whether a gal4DBD-FHL3 protein could interact with a gal4AD-CtBP2
protein. A strong interaction was observed between FHL3 and CtBP2, and
no interaction was seen with the gal4AD alone (Fig.
3A). This interaction was also
observed in a GST pull-down assay (Fig. 3B), as GST-FHL3
retained radiolabeled CtBP2 (lane 3), whereas GST alone did
not (lane 2). The retention of radiolabeled CtBP2 by FHL3 in
GST pull-down assays was generally low but was reproducible (see also
Fig. 4C). Thus, FHL3 can also interact with CtBP2 in both a yeast two-hybrid assay and in GST pull-down assays. Interestingly, although the vast majority of CtBP-interacting proteins contain a PXDLS-like amino acid
motif required for binding CtBP (13), FHL3 has no obvious
PXDLS motif (see "Discussion").
Mapping the Interaction between FHL3 and BKLF and CtBP2--
We
used both GST pull-down assays and the yeast two-hybrid assay to
determine which domains in FHL3 interacted with BKLF and which with
CtBP2. A series of GST-FHL3 deletion constructs were generated and used
in GST pull-down assays with radiolabeled BKLF and CtBP2 (Table
I, and Fig. 4, B and
C). The same domains of FHL3 were also used in the yeast
two-hybrid system, as gal4DBD fusions, and were tested against galAD
fusions with both BKLF (amino acids 1-268) and CtBP2. A summary of the
GST pull-down assays and yeast two-hybrid assays is shown in Table I.
The results indicate that LIM domains half, 1, and 2 (H-1-2) are
sufficient for interaction with BKLF, whereas, only LIM domains half
and 1 of FHL3 are sufficient for the interaction with CtBP2. Other domains of FHL3, however, also appear to contact BKLF and CtBP2 (for
instance LIM domains 3-4 contact both BKLF and CtBP2, and LIM domain 4 contacts CtBP2), and in some instances results of the yeast two-hybrid
assay and the GST pull-down assays are not consistent. Overall, these
experiments suggest that folding of the entire FHL3 protein may be
important and that the complete binding surfaces may be more complex
than just one discreet contiguous domain. Our results are similar to
previous attempts to map the interaction between FHL2 and the AR (23)
or PLZF (30). In these cases, deletion studies showed that the LIM
domains H-1-2 and 3-4 of FHL2 both interact with the AR with similar
affinities (22), and no single LIM domain of FHL2 was capable of
interacting with PLZF (29). Taken together these experiments are
consistent with the view that the four and a half LIM domains in these
proteins fold into a complex protein interaction surface that may wrap around and make multiple specific contacts with its partner
proteins.
FHL3 Can Homodimerize--
It has previously been reported that
FHL proteins can dimerize (22). We tested whether this was the case for
FHL3. A strong interaction was observed between the proteins
gal4DBD-FHL3 and gal4AD-FHL3 in the yeast two-hybrid assay. Deletion
studies show that LIM domains H-1-2 of FHL3 are sufficient for
contact with full-length FHL3 but that dimerization can also occur
between LIM domains 3-4 and full-length FHL3 (Table I). Again this
result demonstrates that the series of contact surfaces is likely to be
complex. A full understanding of the contact surfaces will require
structural analysis of the protein complexes.
BKLF, CtBP2, and FHL3 Form a Nuclear-specific Complex in
Vivo--
We next used co-immunoprecipitation assays to assess the
in vivo interactions between BKLF, CtBP2, and FHL3 (Fig.
5). COS cells were co-transfected with
pMT2 expression vectors for BKLF, plus HA-tagged CtBP2 and HA-tagged
FHL3, or were mock transfected with equivalent amounts of empty vector.
Nuclear extracts were prepared 48 h post-transfection and were
immunoprecipitated with either anti-BKLF antibody or pre-immune
anti-sera. Immunoprecipitates were probed by Western blotting with
anti-HA antibody. Both CtBP2 and FHL3 are efficiently
immunoprecipitated by anti-BKLF antibody (Fig. 5A,
lane 6), whereas neither protein is immunoprecipitated with
pre-immune sera (lane 4). Additionally, CtBP2 and FHL3 are not precipitated from mock transfected cells with either anti-BKLF or
pre-immune sera (lanes 3 and 5). This same
membrane was subsequently stripped and re-probed with anti-BKLF
antibody (Fig. 5B). As expected, BKLF is efficiently
immunoprecipitated with anti-BKLF antibody (Fig. 5B,
lane 6) but not with pre-immune sera (lane 4),
and no BKLF protein was precipitated from mock transfected cells
(lanes 3 and 5). These results are consistent
with previous in vitro experiments and confirm that BKLF can
associate with both CtBP2 and FHL3 in vivo.
To further understand the interaction between BKLF, CtBP2, and FHL3, we
used native gel-filtration chromatography to analyze whether these
three proteins co-elute (Fig. 6). In this
experiment we analyzed first nuclear and then equivalent amounts of
cytoplasmic extract. Extracts were separated by gel-filtration
chromatography, and the fractions were precipitated with
trichloroacetic acid, separated by SDS-PAGE, and probed by Western
blotting with anti-HA antibody (to detect HA-CtBP2 and FHL3-HA), then
stripped and re-probed with anti-BKLF antibody. The monomer masses of
BKLF, CtBP2, and FHL3 are 39, 49, and 31 kDa, respectively, but as
described above FHL3 can self-associate, as can CtBP2 (14) and
BKLF.2 Analysis of nuclear
extracts indicated that BKLF, CtBP2, and FHL3 first co-elute in
fraction 17, with BKLF and CtBP2 peaking in this fraction (Fig.
6A). Examination of standards included in the experiment
suggest that fraction 17 contains protein in excess of 232 kDa, so the
most simple explanation is that this complex may consist of dimers of
each protein (expected molecular mass, 239 kDa). All three proteins
also co-elute in fraction 19, between the 67- and 158-kDa standards,
with significant amounts of FHL3 found in this fraction. This fraction
may contain all three proteins eluting independently as homodimers.
Interestingly, CtBP2 and FHL3 also co-elute independently of BKLF in
fraction 14 and 15 between the 440- and 670-kDa standards, suggesting
these two proteins may be found in additional higher molecular weight complexes within the nucleus. The elution profile from cytoplasmic extracts is significantly different (Fig. 6B). In
particular, the majority of BKLF elutes in fraction 19 (i.e.
between 67 and 158 kDa) and FHL3 is predominantly found in fractions 18 and 19. This observation suggests that a complex of around 240 kDa
containing BKLF, CtBP2, and FHL3 may form in the nucleus but not in the
cytoplasm.
To confirm this, co-immunoprecipitations were performed with
cytoplasmic extract from COS cells transfected with BKLF, HA-CtBP2, and
FHL3-HA using anti-BKLF antibodies, or pre-immune anti-sera (Fig.
7). Immunoprecipitates were probed with
anti-HA antibody (Fig. 7A). No FHL3 protein is found to be
associated with BKLF in the cytoplasmic extracts (Fig. 7A,
lane 5), and only very small amounts of CtBP2 (lane
5). Additionally, neither CtBP2 nor FHL3 is immunoprecipitated
with pre-immune sera (lane 3), or from mock transfected
cells (lanes 2 and 4). The blot was stripped and
re-probed with anti-BKLF antibody, showing that BKLF is efficiently
precipitated from cytoplasmic extract (Fig. 7B, lane
5). Thus, the in vivo interaction between BKLF and both
FHL3 and CtBP2 seen in the nucleus (above) is not observed in the
cytoplasm.
Co-expression of BKLF and CtBP2 Leads to the Nuclear Enrichment of
FHL3--
CtBP proteins are found distributed throughout the cell and
have been hypothesized to have roles both in the nucleus and the cytoplasm (13). As shown above the DNA-binding protein BKLF is also
detectable in both the nucleus and the cytoplasm, although it is more
abundant in the nucleus (Fig. 6). Previous work on FHL proteins has
also suggested that they exist in both the nucleus and the cytoplasm
(20-23, 31, 40-42). Moreover, it has been shown that Rho-signaling
can promote nuclear enrichment of FHL2 protein (31). Thus, it appears
that the sub-cellular localization of FHL proteins may be subject to
regulation. In the gel-filtration experiments discussed above, we noted
that the elution profile for FHL3 is markedly different between nuclear
and cytoplasmic fractions and that BKLF, CtBP2, and FHL3 only co-elute
in high molecular weight fractions in nuclear extracts. We thus
examined the levels of BKLF, CtBP2, and FHL3 in the nucleus and
cytoplasm when different combinations of these three proteins are
co-expressed in COS cells.
When FHL3 is expressed on its own, it is found predominantly in the
cytoplasm (Fig. 8A, compare
lanes 1 and 2). When co-expressed with CtBP2,
again, the majority of FHL3 is found in the cytoplasm, as is CtBP2
(compare lanes 3 and 4). When FHL3 is
co-expressed with BKLF, an increased fraction of FHL3 is found in the
nucleus, although it is still primarily cytoplasmic, whereas BKLF is
predominantly nuclear (Fig. 8A, upper and
lower panels, compare lanes 5 and 6).
However, when FHL3 is co-expressed with both BKLF and CtBP2, FHL3 is
significantly enriched in the nucleus, as is CtBP2 (Fig. 8A,
upper and lower panels, compare lane 7 with 1 and 3). Taken together with the
co-immunoprecipitation assays and gel-filtration data above, it appears
that a specific complex of ~240 kDa containing BKLF, CtBP2, and FHL3
may accumulate in the nucleus only when all three proteins are present
and that this complex is not present in the cytoplasm.
To examine whether BKLF promotes nuclear accumulation of endogenous
FHL3 and CtBP2 protein, we generated four stable cell lines expressing
BKLF using K562 hematopoietic cells, which normally express very low
levels of BKLF protein (Fig. 8B, lane 1,
upper panel). K562 cells express FHL3 mRNA
message (see above), and the original FHL3 prey plasmid, and
full-length FHL3 were isolated from a K562 cell cDNA
library. We generated an anti-FHL3 antibody and used this to test the
effect of BKLF expression on nuclear localization of FHL3. The four
K562 cell lines carrying the stable transgene for BKLF show
high level BKLF expression in the nucleus, compared with the parental
line (Fig. 8B, upper panel, compare lane
1 with lanes 2-5). Importantly, these same BKLF stable
cell lines show a marked increase in FHL3 protein in nuclear extracts, compared with the parental cell line (Fig. 8B, lanes
1-5, middle panel). Additionally, nuclear levels of
CtBP2 are increased in the BKLF stable lines (Fig. 8B,
lanes 1-5, lower panel). Thus, in a similar
manner to that observed in transfected COS cells, BKLF can promote the
nuclear accumulation of endogenous FHL3 and CtBP2 in erythroleukemia cells.
FHL3 Can Repress Transcription--
Previous studies have shown
that FHL proteins can function as transcriptional regulators. The FHL
protein ACT serves as co-activator of CREM and CREB (21), and FHL2 as a
co-regulator for the androgen receptor (22, 23), WT1 (29), and PLZF
(30). We thus examined the transcriptional activity of FHL3 (Fig.
9). Because FHL proteins are not known to
bind DNA, we first fused FHL3 to the gal4DBD and tested its ability to
regulate basal transcription of two artificial test promoters carrying
gal4 binding sites. The gal4DBD-FHL3 fusion protein repressed the
thymidine kinase (tk) promoter by greater than 12-fold in a
dose-dependent manner (Fig. 9A, columns 5-7), whereas the gal4DBD alone has no effect on this promoter (Fig. 9A, columns 2-4). Additionally, the FHL3
fusion protein repressed the minimal promoter containing only a TATA
box to about 15-fold (Fig. 9B, columns 5-7),
again in a dose-dependent manner, whereas the gal4DBD only
marginally affected its activity (Fig. 9A, columns
2-4).
We next tested the ability of FHL3 to regulate activated transcription
in the context of a natural promoter. The erythropoietin promoter is
strongly activated by the erythroid-specific transcription factor
GATA-1 (43). The promoter contains multiple CACCC elements and we have
found that BKLF can potently repress GATA-1-mediated activation (Fig.
9C, columns 1-3).2 The level of
GATA-1 activation of the basal activity of this promoter is ~20-fold
(Fig. 9C, column 1) and is assigned a -fold repression of 1. BKLF represses the promoter ~7-fold (Fig.
9C, columns 2-3). We tested the ability of FHL3
to repress GATA-1-mediated activation of this promoter. To direct FHL3
to the promoter, we generated a chimeric fusion protein between FHL3
and the zinc finger DBD of BKLF. As judged by electrophoretic mobility
shift assays, this FHL3-zinc finger fusion protein can efficiently bind CACCC sites in DNA in a similar manner to BKLF (data not shown). As
shown in Fig. 9C (columns 4 and 5),
the FHL3-zinc finger chimera represses GATA-1 activation as efficiently
as BKLF, whereas FHL3 alone (columns 8 and 9) and
the BKLF zinc finger DBD alone (columns 6 and 7)
have no effect on GATA-1 activation.
To further test the activity of FHL3, we examined whether FHL3 (without
a linked DBD) could potentiate BKLF repression (Fig. 9D).
GATA-1 strongly activates the erythroid-specific A Transcriptional regulation is mediated in part by the recruitment
of multiprotein complexes to regulatory regions in DNA. Revealing the
composition and activities of these complexes will aid our
understanding of how DNA-binding transcription factors regulate gene
expression in a tissue- and temporal-specific manner. Here we show that
the Krüppel-like factor BKLF/KLF3 can bind to two co-regulatory
molecules, FHL3 and CtBP2, both of which can repress transcription.
This work expands our understanding of the activities of the FHL class
of proteins: FHL proteins were previously known to behave primarily as
co-activators; we show here that FHL3 can behave as a transcriptional
repressor and forms a physical link between BKLF and its co-repressor CtBP2.
FHL proteins were initially identified as being highly expressed in
muscle cells (24, 25) and were originally proposed to play roles in
cytoskeletal regulation. This proposal was primarily based on the
observation that, like the cytoskeletal-associated LIM proteins zyxin
and paxillin, the FHL proteins were also composed primarily of LIM
domains, and were developmentally regulated during muscle hypertrophy
(24). Recently, however, it has become clear that many FHL proteins
participate in the regulation of gene expression within the nucleus
where they serve as co-regulators of DNA-binding transcription factors.
ACT binds the testis-specific factor CREM (21), and ACT, FHL2, and FHL3
all bind CREB (22). Additionally, FHL2 binds Wilms Tumor-1 (WT-1) (29),
and the androgen receptor (in an agonist-dependent manner)
(23). In these cases, the FHL proteins behaved as co-activators, but
whether they act as conventional co-activators, recruiting components
of the basal transcriptional machinery, or chromatin-modifying proteins
or act in a different manner has not been established. There are also
two reports of FHL proteins functioning to counter gene activation.
KyoT2, a splice variant of FHL1, antagonizes the activity of RBP-J by
displacing co-activators EBNA2 or Notch (20). Furthermore, FHL2 has
been demonstrated to function as a co-repressor of the multi-zinc
finger repressor PLZF (30), and in this same report, FHL3 was also shown to function as a PLZF co-repressor. These data support our observations that FHL3 binds the transcriptional repressor BKLF and its
co-repressor CtBP2 and aid in the direct repression of transcription by
BKLF. The finding that FHL proteins participate both in activation and
repression fits best with the view that FHL proteins are neither
primarily co-activators or co-repressors but, rather, serve as adaptor
molecules that stabilize large transcriptional complexes with differing activities.
We have previously shown that BKLF recruits the protein CtBP2 as a
co-repressor and that this interaction is dependent on the integrity of
a PXDLS-CtBP recognition element in the repression domain of
BKLF (14). CtBP proteins have been shown to function as co-repressors
for a large number of regulatory molecules that contain
PXDLS motifs in their repression domains (13). Having observed that CtBP2 binds FHL3, we searched the FHL3 sequence for
PXDLS type sequences that might mediate the interaction.
None were detected. This suggests that FHL3 binds a distinct region of
CtBP proteins rather than occupying the putative PXDLS
peptide pocket of CtBP (13, 45). Deletion analysis of FHL3 is
consistent with this view, because no single small peptide in FHL3
contacts CtBP2. Instead, it appears that several different LIM domains of FHL3 are required for contact, and it is likely that different LIM
domains of FHL3 may make several contacts with different surfaces of
CtBP2. BKLF binds CtBP2 using a PVDLT motif (amino acid residues 70-75) (14) and binds FHL3 through a domain that may extend from
around amino acids 80-260. FHL3 also uses several different LIM
domains to contact BKLF. The mode of contact utilized by FHL3 is
reminiscent of that seen with other members of the FHL family that also
seem to utilize multiple different LIM domains to contact their partner
proteins (22, 23, 41, 42, 46). Detailed structural studies will be
necessary to fully elucidate the configuration by which FHL proteins
contact other proteins.
We have found that FHL3 behaves as a potent transcriptional repressor
of both basal and activated transcription when tethered to a promoter
region either by the gal4DBD or by the zinc finger DBD of BKLF.
Furthermore, FHL3 expression (without a linked DBD) can potentiate BKLF
repression of GATA-1 activation. Taken together with the in
vitro and in vivo binding data between BKLF and FHL3 presented above, this argues strongly that FHL3 functions as a BKLF
co-repressor. This is further supported by recent observations that
FHL3 (and FHL2) can also act as a co-repressor for the multi-zinc finger transcription factor PLZF (30). Because FHL3 binds CtBP, the
transcriptional repression observed may be due, at least in part, to
its ability to recruit one or more endogenous CtBP proteins to the
promoter. In a previous study, FHL3 behaved as transcriptional activator (23). Differences in the context of the promoter are most
likely to explain these different findings. We have chosen promoters
that BKLF and CtBP2 can potently repress in cellular assays, and these
may be particularly sensitive to FHL3. Differences in cell lines, and
hence other accessory proteins, may also be important. Furthermore, in
the context of recruitment by DNA-binding factors, unique interactions
with different FHL3 target proteins may alter the conformation of FHL3,
allowing it to interact with either co-activators or co-repressors
through the presentation of distinct protein interaction domains.
FHL proteins are not exclusively nuclear and have been shown to bind
both nuclear and cytoplasmic partner proteins (20-23, 41, 42, 46-48).
Furthermore, sub-cellular partitioning of FHL proteins has been
implicated in the regulation of their function. For example,
Rho-signaling promotes the nuclear localization of FHL2, and subsequent
co-activation of the AR (31), and the kinesin KIF17b binds ACT and is
thought to counter co-activation of CREM by ACT by shuttling it from
the nucleus to the cytoplasm (32). We have found that the sub-cellular
localization of FHL3 is influenced by the availability of its
cofactors. When FHL3 is expressed in COS cells on its own, or with
either BKLF or CtBP2, it is primarily cytoplasmic. However,
co-expression with both BKLF and CtBP2 markedly increases the amount of
FHL3 (and CtBP2) found in the nucleus. Additionally, stable transgene
expression of BKLF in erythroleukemia cells promotes the nuclear
accumulation of endogenous FHL3 and CtBP2. Indeed, gel-filtration
chromatography shows that a large complex of ~240 kDa containing
BKLF, CtBP2, and FHL3 is found in the nucleus and not in the cytoplasm.
The full composition of this complex is not certain. However, given the
molecular weights of the three proteins, BKLF (at 39 kDa), FHL3 (at 31 kDa), and CtBP2 (at 49 kDa), and the knowledge that each can
self-associate, the simplest explanation is that the complex contains a
dimer of each component (totaling 239 kDa). The presence of a
nuclear-specific complex containing BKLF, CtBP2, and FHL3 is supported
by co-immunoprecipitation data, showing that BKLF in the nucleus binds
both CtBP2 and FHL3. Cytoplasmic BKLF behaves very differently in gel
filtration and, as judged by co-immunoprecipitation studies, does not
bind appreciable amounts of FHL3 or CtBP2. Post-translational
modification of BKLF may be important in directing it to the nucleus
and allowing it to associate with different partner proteins.
Alternatively, BKLF, carrying a putative nuclear localization signal
(49), may form a complex with CtBP2 and FHL3 in the cytoplasm, and this
complex may then be shuttled to the nucleus. We have previously found that BKLF can behave as both a transcriptional activator
(35)2 and as a repressor (14). These intriguing results may
be accounted for by the presence of different post-translational states
and/or the availability of partners such as FHL3 and/or CtBP2.
Members of the Sp/KLF family, numbering greater than 20, share related
DNA-binding domains composed of three Krüppel-like zinc fingers,
and all bind to similar sites in DNA (1-4). Many of these proteins are
co-expressed in various cell types, and a current challenge is to
understand whether this co-expression facilitates redundant regulation
of similar genes or regulation of specific genes. So far there is
strong evidence that some KLF members, such as EKLF/KLF1, have specific
target genes, like
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin gene expression (5, 6), and LKLF/KLF2 plays critical
roles in lung development, T cell maturation, and in endothelial cells (7-9). Sp/KLF proteins contain a characteristic DNA binding domain (DBD) at or near their C terminus that consists of three
Krüppel-type Cys2His2 zinc fingers that
bind GC and CACCC boxes in regulatory elements of genes. Different
members of the family exhibit similar DNA-binding specificity, but in
general the Sp1-like subgroup has a higher affinity for GC boxes and
the KLF subgroup proteins bind more strongly to CACCC sequences.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin probe supplied by Clontech.
.neo.BKLF.
1 at 4 °C. The eluant was monitored at
280 nm. 1-ml fractions were collected, and constituent proteins were
precipitated with 0.5 ml of 20% trichloroacetic acid (Sigma). After
washing with cold ethanol, samples were resuspended in SDS-PAGE loading
buffer, separated by SDS-PAGE, and treated as described above for
immunoprecipitation experiments. Protein standards were applied to the
column separately (blue dextran, 2000 kDa; thyroglobulin, 670 kDa;
ferritin, 440kDa; catalase, 232 kDa; aldolase, 158 kDa; albumin, 67 kDa; chymotrypsinogen A, 25 kDa; ribonuclease A, 14 kDa; Amersham
Biosciences).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BKLF/KLF3 binds the LIM protein FHL3 in
vivo (in yeast) and in vitro.
A, yeast two-hybrid assay shows that FHL3 binds BKLF. Yeast
carrying the plasmids shown were grown on media lacking leucine and
tryptophan ( L
T) (left panel) or histidine, leucine, and
tryptophan (
H
L
T) (right panel). Growth of yeast
carrying the gal4DBD-FHL3 and gal4AD-BKLF fusions on -H-L-T
medium show that the bait and prey proteins interact.
B, GST pull-down assay (upper panel) shows BKLF
and FHL3 interact in vitro. 35S-Labeled BKLF was
retained by GST-FHL3 protein (lane 3), but not by GST alone
(lane 2). The input (lane 1) contains 20% of the
radiolabeled BKLF used in the assay. Equivalent amounts of GST
proteins, shown in the lower panel by Coomassie Blue
staining, were used in each case. Lane 1 in the lower
panel contains molecular weight markers (MW). C,
mapping of the domain in BKLF that binds FHL3. Yeast were
co-transformed with the gal4AD-FHL3 plasmid and the gal4DBD fusions
with the BKLF deletion constructs shown. The black box
represents the PVDLT motif in BKLF, and the 1-268mut construct carries
a mutation in this motif that abolishes CtBP binding. Interactions, as
measured by growth of yeast carrying the plasmids on SD-H-L-T medium,
are shown in the right column.
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Fig. 2.
FHL3 mRNA is expressed in K562
(erythroleukemia) cells and is highly expressed in adult heart
tissue. A, FHL3 message is expressed in K562
cells and other cancer cell lines. A Northern blot carrying mRNA
from various cancer cell lines (Clontech) was
probed with a radiolabeled probe from the coding sequence of
FHL3. B, FHL3 message is expressed in
adult skeletal muscle. A Northern blot carrying mRNA from various
adult human tissues (Clontech) was probed with a
radiolabeled probe from the coding sequence of FHL3. For
A and B, both membranes were stripped and
re-probed with a human -actin probe (Clontech)
(lower panels) to test for equal loading of mRNA on the
membrane.
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Fig. 3.
FHL3 binds the co-repressor CtBP2 in
vivo (in yeast) and in vitro.
A, Yeast two-hybrid assay shows that FHL3 binds
CtBP2. Yeast carrying the plasmids shown were grown on medium lacking
leucine and tryptophan (left panel) or histidine, leucine,
and tryptophan (right panel). Growth of yeast carrying the
gal4DBD-FHL3 and gal4AD-CtBP2 fusions on SD-H-L-T medium show that the
bait and prey proteins interact. B, GST pull-down assay
shows FHL3 and CtBP2 interact in vitro.
35S-Labeled CtBP2 was retained by GST-FHL3 protein
(lane 3) but not by GST alone (lane 2). The input
(lane 1) contains 20% of the radiolabeled CtBP2 used in the
assay. GST proteins used were the same as shown in Fig. 1B,
lower panel.
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Fig. 4.
Mapping of the domains in FHL3 that bind BKLF
and CtBP2. A, GST fusion proteins were prepared with
various combinations of LIM domains from FHL3 and tested for binding to
35S-labeled BKLF (B) and 35S-labeled
CtBP2 (C) using GST pull-down assays. Results are summarized
in Table I. Lane 1 in A contains molecular weight
markers, and the Input lanes (lane 1) in
B and C contain 20% of the radiolabeled BKLF and
CtBP2, respectively, used in the assay. Equivalent amounts of
GST-fusion proteins were used in each assay.
Mapping of the LIM domains in FHL3 that bind BKLF and CtBP2 and that
homodimerize with full-length FHL3, using GST pull-down assays and
yeast two-hybrid assays
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Fig. 5.
BKLF binds both FHL3 and CtBP2 in
vivo. COS cells were co-transfected with expression
vectors for BKLF, and HA-tagged CtBP2 and FHL3, or were mock
transfected. Nuclear extracts were immunoprecipitated with either
anti-BKLF antibody (lanes 5 and 6) or pre-immune
Ig (lanes 3 and 4), and the Western blot was
probed with anti-HA antibody (upper panel), stripped and
re-probed with anti-BKLF antibody (lower panel). Five
percent of the extract used for the immunoprecipitations is shown in
lanes 1 and 2. The asterisk shows the
immunoglobulin heavy and light chains.
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Fig. 6.
BKLF, FHL3, and CtBP2 are part of a
nuclear-specific complex of ~240 kDa. COS cells were
co-transfected with expression vectors for BKLF and HA-tagged CtBP2 and
FHL3. Nuclear (A) and cytoplasmic (B) extracts
were prepared and separated by native gel-filtration chromatography.
Proteins in each fraction were precipitated, and probed by Western
blotting, first with anti-HA antibody (upper panel in
A and B), then stripped and re-probed with
anti-BKLF antibody (lower panel in A and
B). Nuclear BKLF, FHL3, and CtBP2 first co-elute in fraction
17, at greater than 232 kDa, whereas the elution profile for
cytoplasmic BKLF, FHL3, and CtBP2 is distinctly different, with the
three proteins predominantly co-eluting in fraction 19, between 67 and
158 kDa.
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Fig. 7.
BKLF does not bind FHL3 in the
cytoplasm. COS cells were co-transfected with expression vectors
for BKLF, and HA-tagged CtBP2 and FHL3, or were mock transfected.
Cytoplasmic extracts were immunoprecipitated with either anti-BKLF
antibody (lanes 4 and 5) or pre-immune Ig
(lanes 2 and 3), and the Western blot was probed
with anti-HA antibody (upper panel), stripped, and re-probed
with anti-BKLF antibody (lower panel). Lane 1 contains 5% of the extract used for the immunoprecipitation. The
asterisk shows the immunoglobulin heavy chain.
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Fig. 8.
Co-expression of BKLF, FHL3, and CtBP2
promotes nuclear localization of FHL3 and CtBP2. A, COS
cells were transfected with expression vectors as shown, and nuclear
and cytoplasmic extracts were prepared and probed by Western blotting
as shown. FHL3 is predominantly cytoplasmic (upper panel)
when expressed on its own (lanes 1 and 2) or
co-expressed with either CtBP2 (lanes 3 and 4) or
BKLF (lanes 5 and 6). When FHL3 is co-expressed
with both BKLF and CtBP2, FHL3 is significantly enriched in the
nucleus, as is CtBP2 (lanes 7 and 8, upper
panel) and BKLF (lanes 7 and 8, lower
panel), albeit to a lesser extent. B, stable expression
of BKLF in K562 cells promotes nuclear accumulation of endogenous FHL3
and CtBP2. Nuclear extracts were prepared from four K562 cell lines
carrying stable BKLF transgenes and parental K562 cells and were probed
by Western blotting as shown. BKLF stable lines (lanes 2-5,
upper panel) express high levels of BKLF protein in the
nucleus, whereas K562 cells express little BKLF (lane 1,
upper panel). FHL3 expression in the nucleus is
significantly increased in the nucleus in the BKLF stable cell lines
(lanes 1-5, middle panel), as is CtBP2
(lanes 1-5, lower panel). Lane 6 contains nuclear extract form COS cells transfected with expression
vectors for BKLF, FHL3-HA, and HA-mCtBP2 (note that the small
discrepancy in migration between HA-mCtBP2 and endogenous CtBP2 is due
to addition of the HA tag).
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Fig. 9.
FHL3 can repress both basal and activated
transcription when tethered to a promoter with a heterologous DNA
binding domain, and can potentiate BKLF repression. A,
FHL3 represses the thymidine kinase (tk) promoter. NIH-3T3 cells were
transfected with 5 µg of the reporter plasmid
p(gal4)4tkCAT (column 1), along with 0.5, 2, and
4 µg of expression vectors for the gal4DBD (columns 2-4)
or a fusion between the gal4DBD and FHL3 (columns 5-7). The
gal4DBD-FHL3 fusion efficiently repressed the basal activity of this
promoter, whereas the gal4DBD alone has no effect. B, FHL3
represses a basal promoter carrying the -globin TATA box. NIH-3T3
cells were transfected with 5 µg of the reporter plasmid
p(gal4)5GH (column 1) along with 0.5, 2, and 4 µg of expression vectors for the gal4DBD (columns 2-4) or
a fusion between the gal4DBD and FHL3 (columns 5-7). The
gal4DBD-FHL3 fusion efficiently repressed the basal activity of this
promoter, whereas the gal4DBD alone has minimal effect on the promoter.
C, FHL3 represses the erythropoietin promoter, when
activated by GATA-1. NIH-3T3 cells were transfected with 1 µg of the
reporter plasmid p(EpoR)GH and 1 µg of the GATA-1 expression vector
pRcCMV.GF1 (columns 1-9) together with 0.5 and 1 µg of
expression vectors for BKLF (columns 2 and 3),
the FHL3-BKLF DBD chimera (columns 4 and 5), the
DBD of BKLF (columns 6 and 7) or FHL3
(columns 8 and 9). D, FHL3 potentiates
repression by BKLF. COS cells were transfected with 200 ng of the
reporter plasmid pA
GH and 2 µg of the GATA-1 expression vector
pXM.GF1 (columns 1-5) together with 100 ng of the
expression vector pcDNA3.BKLF (columns 2-5) and the 500 ng and 1 and 2 µg of the FHL3 expression vector pMT2.FHL3-HA
(columns 3-5).
-globin promoter,
a known GATA-1 target gene (44). Again, GATA-1 activation of this
promoter, at ~20-fold, is set to a -fold repression of 1 (Fig.
9D, column 1). We have found that BKLF can
potently repress this natural promoter, through binding to multiple
CACCC sites in the promoter.2 As shown in Fig.
9D (column 2), low levels of BKLF expression vector (100 ng) repress GATA-1 activity ~1.5-fold. Note that a low
level of BKLF expression was chosen for minimal repression of the
promoter, allowing further super-repression by FHL3 to be analyzed.
Importantly, when FHL3 is co-expressed with BKLF (Fig. 9D,
columns 3-5), BKLF repression is enhanced up to 4-fold. Thus FHL3 can repress both basal and activated transcription when targeted to a promoter through heterologous DNA binding domains, and
the native FHL3 protein can potentiate BKLF repression. This fits well
with recent data, showing that FHL3 (and FHL2) can also act as
co-repressors for the multi-zinc finger repressor PLZF (30).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin, that are not activated by other members
of the family. The observation that different family members share
little homology outside their DNA-binding domains and hence interact
with distinct cofactors may be of central importance. Potentially, each
KLF may interact with a unique set of cofactors that allows it to
regulate a specific subset of genes. On this note, it has been shown
that FHL3 does not bind the related KLF Sp1 (22). Thus, the complex we
observe between BKLF, CtBP2, and FHL3 may be unique to BKLF, allowing it to repress a specific set of target genes. Future work correlating target genes with cofactor interactions, for example using chromatin immunoprecipitation with antibodies to a specific KLF and its cofactors, may afford insight into this complex problem.
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ACKNOWLEDGEMENTS |
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We are grateful to members of the laboratory, and to Melissa Holmes, for careful reading of the manuscript and helpful suggestions.
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FOOTNOTES |
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* This work was supported by a grant from the Australian National Health and Medical Research Council (to M. C.).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. Tel.: 61-2-9351-2233;
Fax: 61-2-9351-4726; E-mail: M.Crossley@mmb.usyd.edu.au.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M300587200
2 J. Turner, H. Nicholas, D. Bishop, J. M. Matthews, and M. Crossley, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: KLF, Sp/Krüppel-like factor; EKLF, erythroid Krüppel-like factor; DBD, DNA binding domain; CtBP, C-terminal-binding protein; FHL, four and half LIM domain; AR, androgen receptor; WT1, Wilms tumor-1; AD, activation domain; PBS, phosphate-buffered saline; GST, glutathione S-transferase; GSH, glutathione; tk, thymidine kinase; CREB, cAMP-response element-binding protein; CREM, cAMP-responsive element modulator.
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