From the Section on Molecular Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, September 12, 2002, and in revised form, November 26, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
General transcription factor 3 (GTF3) binds
specifically to the bicoid-like motif of the troponin
Islow upstream enhancer. This motif is part of a sequence
that restricts enhancer activity to slow muscle fibers. GTF3 contains
multiple helix-loop-helix domains and an amino-terminal leucine zipper
motif. Here we show that helix-loop-helix domain 4 is necessary and
sufficient for binding the bicoid-like motif. Moreover, the
affinity of this interaction is enhanced upon removal of amino-terminal
sequences including domains 1 and 2, suggesting that an unmasking of
the DNA binding surface may be a precondition for GTF3 to bind DNA in vivo. We have also investigated the interactions of six
GTF3 splice variants of the mouse, three of which were identified in this study, with the troponin enhancer. The The establishment and maintenance of mature fast- and
slow-twitch muscle fibers require the expression of distinct sets of genes for contractile proteins, metabolic enzymes, and ion channels. These expression patterns are mainly controlled at the level of transcription. Fiber type specificity of muscle genes can be
recapitulated in transgenic reporter mouse models or in vivo
transfection assays by using respective transcription control regions
(see, for example, Refs. 1-8). Over the past years, signaling proteins
such as calcineurin and Ras, and transcription factors
GTF31/MusTRD1, MEF-3, MEF-2,
NFAT, and PGC-1 The troponin Islow gene (TnIs) is activated during terminal
myogenic differentiation in all skeletal muscles regardless of their
future fiber type. Its expression is then confined to prospective slow
fibers during fetal development (11, 21). The enhancer that confers
slow fiber specificity to TnIs expression is located ~800 bp upstream
of the gene and was termed SURE (for slow
upstream regulatory element; Refs.
3 and 22). Using a transgenic approach, we showed that the downstream
half of the 128-bp SURE, including binding sites for myogenic
regulatory factors (i.e. MyoD and myogenin) and MEF-2, is
necessary for general muscle-specific activity, but not sufficient to
restrict transcription to specific fiber types (11). Rather, a 36-bp
upstream region of the SURE is required in addition to downstream
sequences to re-establish slow fiber-specific reporter expression.
Within this sequence, a bicoid-like motif (BLM; CGGATTAAC)
was found in a yeast one-hybrid screen to interact with the general
transcription factor GTF3. In a similar approach, the corresponding
sequence of the human TnIs upstream enhancer (equivalent to
SURE) was used to isolate a cDNA encoding MusTRD1 (12). GTF3,
MusTRD1, GTF2ird1, WBSCR11, CREAM, and the mouse ortholog BEN are
synonyms for proteins encoded by the same gene (12, 23-26). GTF3 is
ubiquitous in rodent tissues (11, 27), whereas MusTRD1 has been
suggested to be muscle-specific in humans (Ref. 12; but see Ref. 24).
As we showed previously, the highest expression of GTF3 in rodent
muscle occurs during fetal development, after which it is down
regulated to very low levels in mature muscle fibers. In transfected
rat muscle, GTF3 significantly reduces the transcriptional activity
from the SURE. This is consistent with the idea that a repressive
mechanism establishes slow fiber-specific TnIs expression during
myogenic development. We therefore proposed that GTF3 is involved in
the confinement of TnIs expression to slow-twitch fibers (11).
A characteristic feature of GTF3, and its paralog TFII-I, is the
presence of reiterated helix-loop-helix (HLH) domains, so-called I-repeats (R1-R5/R6; for review, see Ref. 28). Most of these repeats
are believed to function as protein-protein interaction surfaces
because they lack a basic domain. In TFII-I, a basic motif precedes R2,
and its deletion abrogates binding to V GTF3 and TFII-I were mapped along with at least 21 other genes as part
of a 1.5-Mb microdeletion in persons with Williams syndrome (WS) (32,
33). Given the potential importance of GTF3 and TFII-I for the
pathology of WS, and the suggested role of GTF3 in regulating slow
fiber-specific gene expression, a better understanding of the
biochemical properties of GTF3 and its functional relation to TFII-I is
necessary. Therefore, the goal of this study was to characterize the
interactions between GTF3 and the TnI SURE. We mapped the DNA binding
domain of GTF3 to HLH domain 4 that lacks a consensus basic region.
Interestingly, affinity of GTF3 for the BLM was dramatically augmented
upon removal of NH2-terminal sequences. We also show that
rodent skeletal muscles express at least five different GTF3 isoforms
that exhibit distinct DNA binding properties in EMSAs; three variants
( Cell Culture
C2C8 myogenic cells were propagated in low glucose Dulbecco's
modified Eagle's medium (Invitrogen) supplemented with 20% fetal bovine serum (FBS; Invitrogen) and 2 mM
L-glutamine. Cells were maintained at 37 °C in an 8%
CO2 environment. Cell density was kept between 20 and 80%
confluence to prevent terminal myogenic differentiation. HEK293 cells
were propagated in minimal essential medium (Invitrogen) supplemented
with 10% FBS and maintained in a 37 °C, 10% CO2
environment. Primary myotube cultures were prepared from rat embryonic
day 19 hindlimbs and grown for 10 days in 10% FBS/Dulbecco's modified
Eagle's medium at 37 °C and 8% CO2 (34). Cytosine
Plasmids
Plasmids Expressing Truncated Human GTF3
Proteins--
Generation of full-length human (h) GTF3 and hGTF3 hGTF3 hGTF3 hGTF3 hGTF3 hGTF3 hGTF3.4--
Repeat domain 4 was amplified from hGTF3 Cloning of Mouse Alternative Splice Variants--
Total RNA from
mouse tissues and rat primary myotubes (days in vitro (DIV)
10) was extracted using RNA Wiz reagent (Ambion). 2 µg from each
preparation were reverse transcribed with Superscript IITM
(Invitrogen) using oligo(dT) primer. Oligonucleotides to amplify 3'-sequences from GTF3 Mouse GTF3 Deletion Constructs--
Full-length and truncated
mouse GTF3 expression plasmid were based on I.M.A.G.E. clone
555547 (see above). The generation of carboxyl-terminal deletion
mutant mGTF3
A full-length TFII-I cDNA ( Quantitative Analysis of mGTF3 Relative abundance of mouse GTF3 Electrophoretic Mobility Shift Assays
Full-length and partial GTF3 proteins used for EMSAs were
generated in vitro from cDNAs subcloned into pCMV-Sport2
(see above). Proteins were synthesized from 0.5 µg of plasmid DNA
using 14 µl of TNT® SP6 reticulocyte coupled
transcription-translation system (Promega). Relative efficiency of
translation was monitored in parallel reactions by the addition of
[35S]methionine. Radiolabeled proteins were fractionated
on 4-20% gradient SDS-polyacrylamide gels (SDS-PAGE), and gels dried
and exposed to autoradiographic film for visualization of proteins. The
relative levels of translated protein were determined by quantification using the phosphorimager and normalization for the number of
methionines in each GTF3 construct.
Double-stranded complementary oligonucleotide used in EMSAs was
SURE Antibody Production
A cDNA fragment encoding the amino-terminal 130 amino acids
of mouse GTF3 was amplified from I.M.A.G.E. clone 555547 using the
following primers: GTF3-N.Cod (5'-CAC TAG GAA TTC GGA TCC GCC TTG CTG
GGG AAG CAC TGT TGA C-3') and GTF3-N.NCod (5'-TGG TAC GAA TTC ATC TTC
TGC AGC AGG TAC ACA TCC-3'). The PCR product was digested with
BamHI and EcoRI and inserted between the
corresponding sites of pGEX-2T (Amersham Biosciences). Glutathione
S-transferase fusion protein was expressed in
Escherichia coli BL21 cells and extracted from lysates on
glutathione-Sepharose (Amersham Biosciences). The immunogen was further
purified by preparative SDS-PAGE followed by electroelution of the
specific 40-kDa band. This preparation was used to immunize rabbits.
Immunoglobulins were purified from whole antiserum on Protein
A-Sepharose columns (Pierce). Antibody specificity was confirmed by
Western blotting of whole extracts from HEK293 and C2C8 cells
transfected with expression constructs for human and mouse GTF3 as well
as mouse TFII-I (see above). No cross-reactivity of anti-GTF3
antibodies toward TFII-I was observed.
Western Blots and Immunoprecipitations
HEK293 cells grown in 35-mm dishes were transfected at 50%
confluence using FuGENE 6 transfection reagent (Roche). Crude nuclear extracts were made 48-60 h post-transfection from three or four dishes
using the NE-PER reagent kit (Pierce). Affinity-tagged GTF3 proteins
were immunoprecipitated with 1.5 µg of anti-Pk antibody (sv5-pk,
Serotec) and 25 µl of Protein A-Sepharose (Santa Cruz Biotechnology)
from 100 µg of nuclear proteins in 500 µl of binding buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 1%
Triton® X-100). Nuclear extracts and immune complexes were
separated by 10% SDS-PAGE, Western-blotted onto nitrocellulose
membranes, and probed with anti-GTF3 polyclonal antibody (1:2000) or
anti-TFII-I monoclonal antibody (clone 42, 1:1000; BD Transduction
Laboratories). A duplicate set of Western blots was probed with anti-Pk
antibody (1:5000).
Transient Transfections and Immunofluorescence
C2C8 myoblasts were transfected at 20% confluence with plasmids
expressing affinity-tagged GTF3 proteins. The next day, cells were
fixed with 4% paraformaldehyde/phosphate-buffered saline, permeabilized with 0.25% Triton® X-100, and blocked in
10% normal goat serum (Sigma). Cells were incubated overnight at
4 °C with the anti-Pk antibody (1:1000). Alexa 488 goat-anti-mouse
secondary antibody (Molecular Probes) was used at 1:500. Nuclei were
stained with Hoechst 33258.
For immunofluorescence cytochemistry of endogenous GTF3 and TFII-I
expression, C2C8 cells were prepared as above and incubated overnight
at 4 °C with polyclonal anti-GTF3 antibody (1:100) and monoclonal
anti-TFII-I antibody (1:25). Secondary antibodies were goat-anti-rabbit
Alexa 488 (1:500; Molecular Probes) and goat-anti-mouse Cy3TM (1:100; Jackson Immunoresearch), respectively.
Location of DNA Binding Domain in Human GTF3--
We have
previously demonstrated that GTF3 interacts specifically with the BLM
of the TnIs upstream enhancer (11). We concluded that the DNA binding
domain of GTF3 must be located downstream of R2 because many GTF3
clones obtained from our yeast one-hybrid screen lacked sequences
upstream of R3, and EMSA experiments confirmed that the
carboxyl-terminal half of GTF3 (including R3-R5) bound to the BLM, but
not the amino-terminal half (including R1 and R2). To map the location
of its DNA binding domain, we have generated a series of truncated
human GTF3 expression plasmids (Fig. 1). The ability to interact with the BLM was tested in EMSAs using in
vitro translated proteins and an oligonucleotide probe that encompasses the sequence between
As shown in Fig. 2A,
full-length GTF3 produced a relatively weak specific shift (lanes
2 and 13) similar to that observed previously (11).
GTF3 proteins lacking the NH2 terminus plus the first two
(hGTF3
The weak binding of hGTF3
We then asked whether the apparent augmentation of protein-DNA
interaction upon removal of amino-terminal sequences represents a true
increase in the affinity of GTF3 for the BLM, or an artifact caused by
a concomitant increase in translation efficiency for the smaller GTF3
proteins. We expressed the affinities of the respective full-length and
truncated proteins for the BLM as the fraction of bound probe relative
to the total probe count (free plus bound probe) per arbitrary protein
unit and defined full-length GTF3 as 1. As shown in Fig. 2B,
affinities were 6-fold higher for hGTF3 Cloning of Mouse GTF3
All reactions produced at least four discrete fragments that were
confirmed by Southern blotting using an internal probe to represent
specific GTF3 sequences (data not shown). Sizes of predominant fragments were 1100 and 1019 bp, and the weaker bands were 791 and 710 bp, respectively. RT-PCR reactions from perinatal hindlimbs and testis
were used for shotgun subcloning. At least two clones were sequenced
for each of the four size categories. We identified cDNA fragments
representing GTF3
The relative intensity of GTF3
The overall abundance of DNA Binding Properties of Mouse GTF3 Splice Variants--
Next, we
asked how these differently spliced sequences affect the interaction
between GTF3 and the BLM. Cloned fragments of GTF3 splice variants were
excised from the PCR vector and used to replace the corresponding
sequence of GTF3
The common feature distinguishing strongly binding GTF3 Homomerization of GTF3 Polypeptides--
The amino-terminal LZ
domain in TFII-I is required for homomeric interactions (30). Given the
conservation of this domain in GTF3 and TFII-I, we asked whether the LZ
in GTF3 functions as a dimerization motif as well. To this end, we
tested the ability of affinity-tagged GTF3 baits to pull down untagged
GTF3 proteins in co-immunoprecipitation assays. We co-transfected
HEK293 cells with constructs expressing carboxyl-terminally truncated
GTF3 (mGTF3
We next sought to confirm this interaction in myoblasts using
immunofluorescence cytochemistry. We speculated that the bait protein
mGTF3 Analysis of GTF3 Interactions with TFII-I--
The conservation of
the LZ domain in GTF3 and TFII-I raises the question whether these two
proteins can associate to form heteromeric complexes. Again, we
utilized a co-immunoprecipitation assay to test whether the bait
protein mGTF3
Based on experiments that utilized heterologous expression of both
proteins in COS-7 cells, it was recently proposed that the presence of
GTF3 excludes TFII-I from the nucleus (39). The lack of a detectable
interaction between GTF3 and TFII-I in co-immunoprecipitation
experiments would be in general agreement with this notion. To test
whether nuclear exclusion of TFII-I occurs in myogenic cells, we
performed double immunofluorescence cytochemistry for endogenous GTF3
and TFII-I in C2C8 myocytes. Fig. 7 shows
that both proteins predominantly locate to the nucleus. Nuclear
residency of TFII-I (b) appeared to be more pronounced than
that of GTF3 (a). However, it is unclear whether cytoplasmic staining seen with the GTF3 antibody represents background because heterologous epitope-tagged GTF3 detected with the Pk antibody is
confined to the nuclear compartment (see Fig. 5C). The
co-localization of GTF3 and TFII-I is demonstrated by overlaying both
images (c). Similar results were obtained with terminally
differentiated multinuclear C2C8 myotubes, and with non-myogenic cell
lines such as HEK293 and 3T3 (data not shown). In conclusion, our data
indicate that GTF3 and TFII-I represent separate entities that, in the
cells and conditions tested, do not exhibit a strong affinity for each other, and that both factors possess distinct DNA binding properties (see above). However, this statement does not preclude the possibility that GTF3 and TFII-I indirectly interact with each other to
synergistically regulate gene transcription.
Because of the potential importance of GTF3 and TFII-I for the
pathology of WS, and the suggested role of GTF3 in regulating slow
fiber-specific gene expression, a better understanding of the
biochemical properties of GTF3 and its functional relation to TFII-I is
necessary. This study focused on three aspects of GTF3 biochemistry:
(i) location of the DNA binding domain, (ii) expression of GTF3
isoforms in muscle and analysis of their DNA binding properties, and
(iii) assessment of potential homo- and heteromeric interactions
between GTF3 proteins and TFII-I.
GTF3 Binds to the BLM via HLH Domain 4--
We have mapped the
region in GTF3 that interacts with the TnIs BLM to HLH domain 4. R4 is
necessary and sufficient for DNA binding because mutant GTF3 proteins
that lack this sequence fail to bind the BLM, and a protein fragment
that merely encompasses the R4 domain is sufficient to mediate this
interaction. This result is consistent with data from our previous work
demonstrating that GTF3 binds to the TnIs enhancer through an area
located in its carboxyl-terminal half (11). Interestingly, R4 is not
preceded by a sequence that would conform to the consensus for a basic domain as delineated by Atchley et al.
(K/R88%-K/R94%-(X)4-E93%-K/R95%-X-R91%-X; Ref. 40). Thus, it seems unlikely that the DNA binding domain represents a classical bHLH motif. Because the BLM does not resemble an
E-box element (CANNTG) either, we speculate that the mode of GTF3
binding to the BLM does not conform to classical bHLH/E-box interactions. Site-directed mutagenesis of R4 and resolution of the
three-dimensional structure of the protein-DNA complex will be required
to understand the structural basis of this interaction. Unlike GTF3,
TFII-I binds to target sequences via a bHLH motif located in R2 (30).
The corresponding repeat in GTF3 lacks a basic domain and is not
required for interaction with the BLM (see above). Dot matrix analyses
revealed that R2 in TFII-I is most homologous to R3 in GTF3 and not R4
(data not shown), arguing against the idea that DNA binding domains in
both proteins were swapped during evolution. It therefore appears that
GTF3 and TFII-I bind distinct DNA elements via different domains,
despite their close structural relationship. In support of this notion,
we were unable to produce a gel shift with GTF3 proteins and the
initiator element of the adenovirus major late promoter, a well
characterized target sequence for TFII-I (41), or to use the adenovirus
major late promoter oligonucleotide to compete for complex formation between hGTF3
Mapping of the DNA binding domain in GTF3 also revealed a pronounced
increase (~10-fold) in avidity of protein-DNA interaction upon
removal of amino-terminal sequences, suggesting that this region
somehow impedes the ability of R4 to bind DNA. In agreement with this
observation, none of the six independent GTF3 clones we isolated
previously from the yeast one-hybrid screen contained the entire open
reading frame but rather lacked 300 bp or more from the 5' coding
region (11). In addition, Bayersaihan et al. (27) isolated a
5'-truncated GTF3 clone that interacts with the early enhancer of the
Hoxc8 gene from a yeast one-hybrid screen of a mouse embryo
library. In vitro translated full-length TFII-I did not
shift the adenovirus major late promoter oligonucleotide in our
conditions (data not shown). Other groups have used highly purified
proteins from transfected eukaryotic cells or E. coli to
obtain complexes between TFII-I and its target sequences (see, for
example, Ref. 29). Thus, it is possible that both GTF3 and TFII-I
per se exhibit modest affinities for their target sequences, and that concentrations of in vitro translated proteins in
the reticulocyte lysate are too low to drive efficient protein-DNA complex formation. It is therefore conceivable that DNA binding of GTF3
is conditional and requires a conformational modification of the
protein (e.g. post-translational modification or binding of
other proteins), or the disruption of GTF3 protein complexes associated
via the leucine zipper (see below). NH2- and COOH-terminal domains with autoinhibitory properties have been identified in other
transcription factors such as Ets-1, Smad2/Smad4, and Nkx2.5 (42-44).
They regulate transcription factor function through inhibition of DNA
binding (as in Ets-1) or transactivation (as in Smad 2/Smad4), or both
(as in Nkx2.5). A more detailed analysis of the interaction between
amino-terminal sequences and the DNA binding domain R4 in GTF3 will be
necessary to test these different scenarios.
Muscle and Non-muscle Cells Express Multiple GTF3 Splice
Isoforms--
The existence of multiple GTF3 isoforms in rodent tissue
had been reported previously (31), but their expression in different tissues and during development was not investigated. Because our interest in GTF3 stems from its proposed role in fiber type-specific gene expression, we explored by RT-PCR whether isoforms other than
GTF3
No human splice isoforms have been identified to date that contain
sequences corresponding to mouse exons 23 and 26-28. By aligning the
region of the mouse GTF3 gene that harbors the alternatively spliced
exon 23 with the corresponding region of the human locus, we were
unable to detect sequences that resemble mouse exon 23 or flanking
intronic segments. Similarly, no evidence was found for a duplication
of human GTF3 exons 23-25 (encoding R5) that would suggest the
existence of human splice variants that contain a sixth HLH domain. We
therefore speculate that these exons were fairly recently added to the
mouse gene or lost from the human locus.
GTF3 Splice Isoforms Exhibit Distinct DNA Binding
Properties--
In EMSAs, full-length mouse GTF3 splice variants bound
poorly if at all to the BLM. This result is in agreement with the low affinity of human full-length GTF3 for the BLM in vitro. A
surprising variability in their affinity for the BLM was revealed after
ablation of amino-terminal sequences. GTF3
The presence of multiple exon configurations in the vicinity of the DNA
binding domain could conceivably generate GTF3 proteins with different
DNA sequence preferences, and splice variants that interact poorly with
the BLM may bind other DNA targets more efficiently. In this context,
it will be interesting to compare the relative binding affinities of
mouse GTF3 splice isoforms between the TnI SURE and different
bona fide GTF3 target sequences, such as the early enhancer
of the Hoxc8 gene (27). Alternatively, as discussed for the
role of amino-terminal GTF3 sequences, access of weakly binding
isoforms to DNA may be regulated and requires prior association of
other factors or posttranslational modification.
The Leucine Zipper Is Important for GTF3 Homomerization--
We
have demonstrated both in vitro using co-immunoprecipitation
and in vivo using immunofluorescence cytochemistry that the amino-terminal LZ domain functions as a homomerization motif. This
result is in agreement with the demonstration that the LZ in TFII-I is
required for dimer formation as well (30). Interestingly, we observed
no stable interaction between GTF3 and TFII-I, suggesting that either
their respective LZ domains are structurally too diverse or that
sequences outside the LZ interfere with heteromerization. It is also
possible that TFII-I splice variants other than the one we used in our
experiments (TFII-I
In conclusion, GTF3 emerges as a transcription factor with interesting
properties including the location and sequence features of the DNA
binding domain, as well as the potential autoinhibition by
NH2-terminal and possibly COOH-terminal domains. Future
studies will have to focus on the regulation of DNA binding and
transcription effector functions of GTF3 in skeletal muscle and other
tissues to better understand its role in fiber type-specific gene
expression and its possible involvement in the pathology of Williams syndrome.
-isoform lacking exon
23, and exons 26-28 that encode domain 6, interacted most avidly with
the bicoid-like motif; the
- and
- isoforms that include these exons fail to bind in gel retardation assays. We also
show that GTF3 polypeptides associate with each other via the leucine
zipper. We speculate that cells can generate a large number of
GTF3 proteins with distinct DNA binding properties by alternative
splicing and combinatorial association of GTF3 polypeptides.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were implicated in the regulation of fiber
type-specific expression in adult muscle (9-17). In birds and lower
vertebrates, the sonic hedgehog signaling pathway was shown
to be involved in the specification of primary slow myofibers (18-20).
However, the transcription factors and signaling pathways that control
the establishment of slow and fast fiber phenotypes during mammalian
muscle development are not known.
Inr and c-Fos promoter
sequences (29, 30). Largely based on protein sequence, MusTRD1 has been
suggested to bind to the USE B1 enhancer element of the human TnIs gene
via a domain located in its amino-terminal half (12). However, two
lines of evidence indicate that the first two HLH domains of GTF3 are
dispensable for binding to the TnIs BLM; (a) most of the
GTF3 clones we obtained from the yeast one-hybrid screen lack the
sequences encoding R1 and R2, and (b) a partial GTF3 protein
containing the carboxyl-terminal half including R3-R5 forms a complex
with a SURE-derived oligonucleotide probe in electrophoretic mobility
shift assays (EMSAs), whereas a mutant protein encompassing the
amino-terminal half including R1 and R2 does not (11). In that study,
GTF3 cDNAs conforming to either one of two reported human GTF3
transcripts (containing either the long or the short form of exon 19)
were isolated, indicating that sequence variability in the region
between R3 and R4 encoded by this exon does not appreciably affect DNA
binding. However, mice express at least six GTF3 isoforms that differ
more extensively in sequence (Ref. 31 and this paper). Most notably,
both
- and
-GTF3 isoforms contain a sixth HLH domain located
between R5 and the carboxyl terminus, plus an additional 27 amino acids between R4 and R5 encoded by mouse exon 23. Their expression pattern and functional properties are not known.
2,
3, and
2) are reported here for the first time. We furthermore demonstrate that GTF3 proteins
can form dimers via the NH2-terminal leucine zipper (LZ) motif, suggesting that cells can generate a large number of GTF3 transcription factor complexes with potentially different properties and functions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-arabinofuranoside was added to the cultures at day 4 to enrich for myotubes.
12 was described previously (11). All PCR products were generated using a
mix of proofreading (Tgo) and Taq polymerase
(Expand High Fidelity PCR system, Roche). hGTF3.R4 was generated from
hGTF3
1-3 template DNA and subcloned directly into expression
plasmid pCMVSport2 (Invitrogen). All other constructs were amplified
from YIH clone 81 template DNA (11) and subcloned into shuttle vector
pGemT (Promega) for sequence verification before being transferred to pCMVSport2. Gene-specific coding oligonucleotides for hGTF3
1-3, hGTF3
1-4, hGTF3
12C, and hGTF3
124 included a SalI
linker, Kozak consensus sequences, and a translation initiation codon (underlined).
1-3 and hGTF3
1-4--
Primer sequences were
5'-GTC GAC GCC ACC
ATG GGC TTT CAA GAA AAT TAT GAC GC-3' and hGTF3
1-4,
5'-GTC GAC GCC ACC
ATG GAT GAG GAT GAT GCC ACC AGA C-3', respectively. T7 was
used as antisense oligonucleotide. GTF3 inserts in pGemT were released
with SalI and NotI and cloned between the
corresponding sites of pCMV-Sport2.
12C--
Coding oligonucleotide was 5'-GTC
GAC GCC ACC ATG GAT TCT
GGT TAT GGG ATG GAG ATG-3', and antisense oligonucleotide was hGTF3
C (5'-GAA TTC TAT GCA AAG GGT TGG AGC TGG-3').
124--
Coding oligonucleotide was same as for
hGTF3
12C and hGTF3
12 (see above); antisense oligonucleotide was
5'-GAT GAG TCC TTG GAA AGG CGC GTC ATA ATT TTC TTG AAA GCC-3'.
Following ligation to pGemT, a SalI-EcoNI
fragment was inserted between the corresponding sites of
hGTF3
12.
125--
Coding oligonucleotide sequence was 5'-ACC AGG
CCT TTC CAA GGA CTC ATC GCA GAA ATC TGC AAT GAT GCC AAG GTG-3';
antisense primer was T7. Following ligation to pGemT, a StuI
fragment was released and used to replace the corresponding
StuI fragment in hGTF3
12.
4--
A SapI fragment including the
sequences flanking the deletion was released from hGTF3
124 and used
to replace the corresponding sequence in full-length hGTF3.
1-3
template DNA using a cytomegalovirus promoter coding oligonucleotide
(5'-GGT GAC ACT ATA GAA GGT ACG CCT GC-3') and noncoding
oligonucleotide 5'-CCT ACA AGC TTA CTT TGG GAT GAG TCC TTG GAA AGG-3'.
The PCR product was digested with SalI and
HindIII and ligated to pCMV-Sport2.
and -
splice isoforms were mGTF3+2346s, 5'-AGC AAC CCA GGC TCG GTA ATC ATT GAA GG-3' (coding) and mGTF3+3446r, 5'-CCT TTA GTC TTT TGA GTT GAG GTC CTG-3' (noncoding). Cycling parameters were 95 °C/4-min initial denaturation; 35 cycles of 92 °C/30 s, 64 °C/45 s, 72 °C/1 min; last extension step of
72 °C/10 min. PCR reactions were purified and concentrated on
MinElute DNA purification columns (Qiagen) and ligated to
pCRScriptTM-Cam (Stratagene). Inserts were sequenced and
aligned to GTF3 sequences already deposited with GenBankTM
using BLAST (35). Using mGTF3
in pCMV-Sport2 as backbone (IMAGE clone 555547; GenBankTM accession no. AA111609 (Ref. 36)),
alternatively spliced GTF3
and GTF3
sequences were inserted
between the internal BstEII site and vector-derived
HindIII or NotI sites, depending on the orientation of the GTF3 insert in the shuttle vector.
.
3-6 is described elsewhere (11). The amino-terminal
deletion mGTF3
.
LZ was made by PCR-amplifying the GTF3
cDNA
with a coding oligonucleotide located downstream of the leucine zipper
motif (5'-T CCA GTC GAC GCC ACC ATG CAG TCA GAC TTC CTC AGG
TTC TGC-3') and that includes a SalI linker, Kozak sequence,
and translation initiation codon (underlined). T7 was used as antisense
oligonucleotide. The PCR product was digested with SalI and
EagI and inserted between the SalI and
NotI sites of pCMV-Sport2. A 229-bp SacII
fragment including the new translation start site was then excised from
this intermediate and used to replace the corresponding sequence
in GTF3
. The Pk epitope (Ref. 37; also referred to as "V5")
was added on to full-length mGTF3
, mGTF3
.
3-6, and
mGTF3
.
LZ to generate respective affinity-tagged derivatives.
Corresponding constructs were made by inserting a double-stranded
oligonucleotide coding for this epitope into the NcoI site
that overlaps with the translation initiation codon
(CCATGG) in GTF3. The sequence of this oligonucleotide (excluding NcoI linker arms) was 5'-GAA GGT AAG CCT ATC CCT
AAC CCT CTC CTC GGT CTC GAT TCT ACG AG-3'. Mouse versions of human GTF3
1-3 were made by amplifying the coding sequence of the various GTF3 splice variants downstream of HLH repeat 3 using coding
oligonucleotide 5'-GTC GAC GCC
ACC ATG AAG AGA CAG GGC CTT CAA G-3';
SalI linker, Kozak sequence, and translation initiation
codon are underlined) and T7 as the noncoding oligonucleotide. The
resulting PCR products were digested with
SalI-BstEII and inserted between the
corresponding sites of mGTF3 full-length isoform cDNAs in
pCMV-Sport2.
-isoform) in pCMV-Sport6 was obtained as
an Expressed Sequence Tag clone (accession no: AW912318; I.M.A.G.E.
clone 3157775) from Incyte Genomics.
and -
Isoform Expression
/
splice variants was
determined by a combination of RT-PCR and subsequent hybridization analysis. Mouse total RNA (2 µg) from adult soleus and EDL muscles, as well as from whole brain, was reverse transcribed and PCR-amplified with oligonucleotides mGTF3+2346s and mGTF3+3446r using the conditions described in the previous section. Parallel PCR reactions were subjected to 18, 19, and 20 cycles to ensure that PCR product growth
was in the logarithmic phase. PCR products were electrophoresed on a
1.5% agarose gel and blotted onto a charged nylon membrane. For GTF3
isoforms
,
2, and
3, membranes were
hybridized at 50 °C in 6× SSC, 1% SDS, 1 mM EDTA (1×
SSC = 150 mM NaCl, 15 mM sodium citrate,
pH 7.0) with the following [
-32P]-labeled
oligonucleotide probes:
, 5'-CC AAA GCC TG/A AAC CAA ATT-3';
2, 5'-CC AAA GCC TG/G ACA TGA AGC-3';
3,
5'-CC AAA GCC TG/A TGA GGA TGA-3' ("/" indicates the boundary
between exon 22 common to all isoforms and the isoform-specific
adjacent sequences). Following a stringent wash (2× SSC, 1% SDS at
45 °C for
/
3 and 40 °C for
2),
the membrane was exposed to a Storm® phosphorimager screen
(Amersham Biosciences). Specificity was monitored, and
hybridization signals were normalized, by including known quantities of
BstEII-HindIII cDNA fragments of
-
3 isoforms on the same membrane. For GTF3 isoforms
and
2, and to quantify total
- and
-PCR
products, membranes were hybridized with a [
-32P]dCTP-labeled
BstEII-HindIII restriction fragment of mouse
GTF3
2 excised from pCMV-Sport2. To normalize for
variability related to RNA input and reverse transcription, aliquots of
RT samples were amplified in parallel with oligonucleotides specific
for a 352-bp fragment of the mouse ribosomal protein L7 transcript (L7s, 5'-AGA TGT ACC GCA CTG AGA TCC-3'; L7a, 5'-ACT TAC CAA GAG ACC
GAG CAA-3'; Ref. 38). L7 PCR products were electrophoresed and blotted
as described above and hybridized against a
[
-32P]dCTP-labeled cDNA probe derived from the L7
PCR product. Signals were quantified with the phosphorimager and used
to normalize corresponding values for GTF3 splice isoforms.
842/
815 (5'-TAC CGG ATT AAC ATA GCA GGC ATT GTC T-3'). In some
cases, the shorter probe, SURE
844/
827 (5'-GCT ACC GGA TTA ACA
TAG-3') was used (11). Probes were generated with
[
-32P]ATP (6,000 Ci/mmol, Amersham Biosciences) and T4
polynucleotide kinase and purified on acrylamide gels. Binding
reactions were performed in a 10-µl total volume using 1.5 µl of
in vitro-translated GTF3 proteins, 1 µg of poly(dI-dC),
and 32P-labeled probe (15,000 cpm). Binding buffer
composition was: 20 mM HEPES, pH 7.9, 50 mM
KCl, 4 mM MgCl2, 4% Ficoll, 5% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol. 50 ng of
single-stranded oligonucleotide SURE 832/808 (5'-ACA TAG CAG GCA TTG
TCT TTC TCT G-3') was included to reduce nonspecific binding of
proteins present in the reticulocyte lysate. Reactions were incubated
at room temperature for 20 min, and DNA-protein complexes were resolved
by electrophoresis at 4 °C on 5% polyacrylamide gels in 0.5× TBE
buffer. Gels were dried, visualized by autoradiography, and quantified
on the phosphorimager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
842 and
815 of the rat TnI SURE
(11). To ensure that all GTF3 proteins were properly translated, parallel reactions spiked with [35S]methionine were
loaded on a 4-20% gradient SDS-PAGE and autoradiographed (data not
shown). EMSAs were performed with unlabeled proteins synthesized from
the different GTF3 constructs shown in Fig. 1.
View larger version (16K):
[in a new window]
Fig. 1.
Human GTF3 constructs used in this
study. Figure is a schematic representation of full-length and
partial human GTF3 expression constructs used for mapping the DNA
binding domain. All constructs were derived from a cDNA
corresponding to a GTF3 isoform (GenBankTM accession no.
AAF19786) that contains the long form of exon 19 (amino acids
656-671). Boxes represent HLH domains (denoted as R1-R5).
The leucine zipper motif (LZ, amino acids 32-55) at the
NH2 terminus (hexagon) and a nuclear
localization signal (N, amino acids 884-889) at the
carboxyl terminus (circle) are also shown.
Numbers in italics before and
after constructs or construct segments indicate amino acid
positions of start and stop signals as well as internal junction sites
relative to full-length GTF3.
12, lane 3) or three HLH domains (hGTF3
1-3, lane 4) interacted strongly with the probe. In contrast, no
shift was observed when R4 was removed in addition to the first three repeats (hGTF3
1-4; lanes 5 and 14). Thus, a
region between R3 and R4 is necessary for GTF3 to bind to the BLM.
Next, we deleted specific regions in the carboxyl-terminal half of GTF3
within the context of hGTF3
12. A mutant protein that lacks the
carboxyl terminus downstream of R5 efficiently formed a complex with
the probe (hGTF3
12C, lane 6), indicating that this
region, which includes a serine-rich stretch and the nuclear
localization signal (26), is not required for DNA binding. Likewise, a
weaker but significant signal was observed with a protein that lacks
HLH domain 5 (hGTF3
125, lane 7). Next, we removed R4 both
in the context of hGTF3
12 as well as the full-length protein
(lanes 8 and 9). No specific shift was detected
in either one of these reactions, demonstrating that R4 is required for
DNA binding. Finally, we tested the ability of R4 to bind the BLM by
itself, and indeed a strong shift was obtained with this GTF3 protein fragment. In conclusion, we have identified HLH domain 4 as being necessary and sufficient to mediate the interaction between GTF3 and
the BLM of the TnI SURE.
View larger version (43K):
[in a new window]
Fig. 2.
GTF3 binds to the BLM of the TnI SURE via HLH
domain 4. The interaction between in vitro
translated human GTF3 proteins and the TnIs BLM was tested in EMSAs.
A, a representative experiment with various truncated GTF3
proteins. An oligonucleotide spanning the region between 842 and
815 of the TnI SURE was used as probe in lanes
1-10. A shorter probe encompassing the sequence between
844 and
827 was used in lane 11. Full-length
and partial GTF3 proteins were translated from expression plasmids
containing cDNAs for hGTF3 (lane 2),
hGTF3
12 (lane 3), hGTF3
1-3
(lane 4), hGTF3
1-4 (lane
5), hGTF3
12C (lane 6), hGTF3
125
(lanes 7 and 11), hGTF3
124
(lane 8), hGTF3
4 (lane
9), and hGTF3.R4 (lane 10), and added
to the reactions where indicated. Reticulocyte lysate containing the
parental expression vector served as negative control to identify
nonspecific complexes (lane 1) as indicated by
open arrowheads. See "Results" for an
interpretation of multiple bands visible in lanes
3, 4, 7, and 11. This
autoradiogram was exposed for 16 h. Lanes
12-14 show longer exposures (2 days) of lanes
1, 2, and 5 to demonstrate specific
binding of full-length GTF3. B, relative affinities of GTF3
proteins for the BLM. Values were obtained as follows: affinities were
expressed as the percentage of bound probe relative to total probe
count. Resulting numbers were corrected for protein molarity using
[35S]methionine proteins translated in parallel (see
"Experimental Procedures") and normalized to full-length
GTF3.
125 and the
842/
815 probe may represent
an artifact because a stronger shift is obtained with the same protein
when a shorter probe encompassing the sequences between
844 and
827
of the TnI SURE (lane 11) is used. It is therefore
conceivable that the proximity between the carboxyl terminus and R3-R4
in this mutant negatively affects binding to the long probe. We also
noticed that GTF3 proteins encoded by constructs
12,
1-3, and
125 gave rise to additional weak low mobility complexes. The
carboxyl terminus of GTF3 is the only region present in these proteins
but absent from
12C and R4, both of which gave rise to a single
band. Sequences within this region could therefore mediate the
formation of these higher order complexes that may or may not include
other lysate proteins in addition to GTF3.
12 compared with full-length
GTF3 and ~10-fold higher for all other binding-competent GTF3
proteins. hGTF3
125 was not included for reasons discussed above. In
conclusion, we find that the avidity of DNA-protein interaction
increases significantly in the absence of NH2-terminal
sequences, suggesting an inhibitory effect of this region on DNA binding.
and -
Splice Variants from Muscle and
Non-muscle Tissues--
In initial attempts the
-isoform of mouse
GTF3, and truncated versions thereof, failed to produce a complex with
the BLM in EMSAs. We therefore explored the possibility that additional GTF3 isoforms are expressed in rodent skeletal muscle that might interact with the SURE. The mouse GTF3 gene has been reported to give
rise to two additional (
- and
-) isoforms, both of which differ
from GTF3
, and from each other, in sequences carboxyl-terminal of
the DNA-binding domain R4 (31). Human GTF3 most closely resembles the
mouse
-isoform. To determine whether
- and
-isoforms are expressed in skeletal muscle tissue, we employed RT-PCR to amplify the
3' end of the coding region of mouse GTF3. Although all previously known splice variants were reported to share sequences upstream of the
BstEII site we used to clone the PCR fragments into the parental mGTF3
expression vector, we cannot rule out that splice events upstream of the analyzed sequence contribute to the variability of GTF3 transcripts. We used a coding primer that recognizes all three
known GTF3 splice isoforms and an antisense primer that binds to a
sequence downstream of the translation termination codon of
- and
-isoforms. A representative gel is shown in Fig. 3A. GTF3 cDNA fragments
were amplified from RNA extracted from DIV10 rat primary myotube
cultures (PM), pooled RNA from embryonic and early postnatal
mouse hindlimbs (E15, E18, P7; HL), as well as RNA isolated
from various adult slow and fast muscles (S-D). RNAs from
heart, brain, and testis were included as non-skeletal muscle tissues
(H-T).
View larger version (48K):
[in a new window]
Fig. 3.
Rodent tissues express multiple GTF3 splice
isoforms. A, representative gel showing PCR products
for GTF3 splice variants. Total RNA from cultured myogenic cells as
well as from various muscle and non-muscle tissues was used to reverse
transcribe and PCR-amplify 3'-coding sequences of GTF3 and -
transcripts. RNAs tested in lanes 3-9 were from
adult mouse tissues. PM, primary rat myotube culture
(DIV10); HL, pool of perinatal mouse hindlimbs (E15, E18,
and P7); S, soleus; E, EDL; G,
gastrocnemius; D, diaphragm; H, heart;
B, brain; T, testis; c, control PCR on
testis RNA not subjected to reverse transcription; m,
X174-HaeIII digest. The identities of four bands
corresponding to alternative GTF3 transcripts are shown on the
right. B, schematic illustration of the exon
organization of mouse GTF3 splice variants. Exons are
numbered on top. Filled
boxes represent coding exons; open
boxes depict noncoding portions of exon 31 (GTF3
and -
variants) and of exon 30b (GTF3
). Alternatively spliced exons 23 and
exon 26-28 are shown in gray. The optionally spliced 5'
half in exon 23 is highlighted.
and -
isoforms (1100- and 710-bp PCR fragments,
respectively), as well as sequences for three previously unknown splice
variants (1079/1019/791 bp). The exon organization of GTF3 splice
isoforms, deduced from comparison of cDNA and genomic sequences, is
illustrated in Fig. 3B. Like GTF3
, the 1079- and the
1019-bp fragments included exons 26-28 that code for R6 and were
therefore designated GTF3
2 and -
3,
respectively. GTF3 isoforms
and
2 did not resolve
because of their similar size. The 791-bp PCR product lacked exons
26-28 and was named GTF3
2 to indicate its similarity
with GTF3
. The difference between GTF3
and
GTF3
2/
3, and between GTF3
and
GTF3
2, can be attributed to differential usage of exon
23. A shortened form of this exon is generated in GTF3
2
by splicing of exon 22 to a cryptic site in exon 23, 21 nucleotides
downstream of its 5'-boundary. The alternative splice junction
(ACGACCACGAAG) has the consensus AG dinucleotide but lacks the
pyrimidine-rich upstream sequence commonly found in obligatory 3'
splice sites. Like the
-isoform, GTF3
3 lacks exon 23 altogether, whereas it is included in GTF3
2.
/
2-derived signals
compared with GTF3
3 appeared to be variable in different
tissues. GTF3
/
2 fragments were more abundant in
testis, whereas a fairly even distribution or a moderate bias toward
GTF3
3 was seen with RNA from cultured myotubes and
muscle tissues including heart, as well as with RNA from whole brain.
We then asked whether certain isoforms might be differentially
expressed between muscles that transcribe the TnIs gene and those that
do not. We tested RNA from the slow soleus and fast EDL muscles, and
included RNA from brain as a non-muscle tissue. In the mouse, ~60%
of all muscle fibers in the soleus are slow-twitch, whereas the EDL
almost exclusively comprises fast-twitch fibers in which the TnIs gene
is not expressed. We used semiquantitative RT-PCR to test for a
correlation between the relative abundance of
- and
-isoform-derived PCR products and the slow fiber type-specific
expression of the TnIs gene. Because of the similarities in size and
sequence of
- and
- splice variants, we employed the conditions
used above for RT and subsequent PCR, assuming that all isoforms were
amplified with comparable efficiencies. Reactions were subjected to a
low number of PCR cycles to ensure that product growth was captured in
the logarithmic phase, and quantified by radioactive hybridization (for
details, see "Experimental Procedures"). The result of this analysis is summarized in Table I.
Quantitative analysis of mouse GTF3/
-isoform expression
- and
-isoforms was similar between
soleus and EDL muscles, whereas an ~5-fold higher expression level
was observed with RNA from whole brain tissue. This in agreement with
the finding that GTF3 transcript levels in the mouse are low in adult
skeletal muscle compared with other tissues, and with the lack of a
bias toward either slow- or fast-twitch muscles (11). In skeletal
muscle and brain, GTF3
3 was the most abundant isoform,
ranging from 46 to 62% of total GTF3
/
transcripts, followed by
GTF3
(27-39%). In all three tissues, expression of GTF3
2 and of both
-isoforms was low, and together
accounted for less than 15% of the total
/
transcripts. The
distribution of isoforms in soleus and EDL was not significantly
different. Taken together, we conclude that GTF3
3 and
GTF3
isoforms predominate in skeletal muscle, and that the
uniformity of GTF3
/
isoform expression pattern in slow and fast
muscles does not support the idea that the expression of any particular
isoform is correlated with the expression pattern of the TnIs gene.
. We used in vitro translated proteins
and the
842/
815 oligonucleotide probe to assess their DNA binding
properties in EMSAs. To allow for cross-comparison of binding
affinities, reactions were normalized for protein molarity (see
"Experimental Procedures"). As shown in Fig.
4 (left half), no
specific shifts were detected after a 36-h exposure in reactions that
used full-length GTF3 proteins, except for GTF3
that produced a very
weak specific shift (lanes 2-7). This result is in
agreement with the poor binding of full-length human GTF3 to the BLM
probe (see Fig. 2). Next, NH2-terminally truncated versions
that lacked the first three HLH domains (
1-3) were generated from
all variants to determine whether the removal of these sequences
enhances the affinity of GTF3 proteins for the BLM (Fig. 4,
right half).
1-3 versions of GTF3
and
-
2 isoforms interacted avidly with the probe
(lanes 12 and 13). A modest shift was obtained
with GTF3
3 (lane 10). Very weak shifts
(gray arrowheads) not present in the reticulocyte
lysate control (lane 1) were visible with GTF3
, -
2, and -
isoforms (lanes 8, 9,
and 11). Their mobilities, compared with the predominant
complexes obtained with
-,
2-, and
3-isoforms, were aberrant and therefore are not likely
to represent bona fide interactions. The different signal
intensities of specific shifts obtained with the various isoforms
reflect intrinsic differences in the affinity of GTF3 proteins for the
BLM, and are not the result of variability in translation efficiency
because reactions were normalized for protein molarity (see above).
View larger version (89K):
[in a new window]
Fig. 4.
Mouse GTF3 isoforms bind differentially to
the TnI SURE BLM. EMSA shows complex formation between
842/
815 oligonucleotide probe and in vitro translated
mouse GTF3 splice variants. For each splice variant, full-length
proteins (lanes 2-7) as well as amino-terminal deletion
mutants (
1-3; lanes 8-13) were added as indicated. To
allow for direct comparison of binding avidities, the amount of protein
added to each reaction was normalized among full-length proteins and
among truncated mutants, respectively. The black
arrowhead indicates weak but specific complexes obtained
with full-length GTF3
. Gray arrowheads
indicate complexes in lanes 8, 9, and
11 that run aberrantly compared with adjacent lanes.
Open arrowheads indicate complexes that resulted
from nonspecific interactions between the probe and components of the
reticulocyte lysate.
and
-
2 isoforms from modestly binding GTF3
3
is the absence of exons 26-28 (see Fig. 3B), suggesting
that the presence of R6 encoded by these exons interferes with DNA
binding. A second variable appears to be the presence or absence of
amino acids encoded by exon 23. The GTF3
isoform lacks this exon and
forms a complex that is stronger than that obtained with its exon
23-containing counterpart GTF3
2. Likewise, the presence
of the long or the short form of exon 23 in amino-terminal truncations
of GTF3
and -
2 significantly reduces their affinity
for the BLM compared with GTF3
3. We conclude that
multiple GTF3 isoforms are expressed in muscle that exhibit
differential binding properties for the TnI SURE enhancer. In
particular, HLH domain 6 encoded by exons 26-28 and, to a lesser
extent, sequences encoded by exon 23 modulate their affinity for the
BLM.
.
3-6) and either full-length mGTF3
or a mutant
merely lacking the LZ motif (Fig.
5A). The epitope-tagged bait
protein was either mGTF3
.
3-6 or full-length mGTF3 (Fig.
5B). The expression of bait and prey proteins was confirmed
in direct Western blots of nuclear protein extracts prepared from
transfected cells. Bait proteins were immunoprecipitated with an
antibody directed against the Pk epitope (37), and pull-downs were
probed with antibodies against GTF3 (top row) and
Pk (bottom row). As shown in Fig. 5B, the untagged full-length mGTF3
was readily co-immunoprecipitated along with the mGTF3
.
3-6(Pk) bait protein, indicating that they formed a stable complex. Conversely, when the affinity tag was added on
to full-length GTF3, this protein effectively co-precipitated untagged
mGTF3
.
3-6 (right). In contrast, the
LZ mutant was not pulled down using the mGTF3
.
3-6(Pk) bait protein. Taken together, these results demonstrate that GTF3
polypeptides interact with each other via the leucine zipper domain.
View larger version (42K):
[in a new window]
Fig. 5.
The leucine zipper motif is a homomerization
domain. A, schematic overview of mouse GTF3 proteins
used to study the role of the leucine zipper. Respective expression
constructs were based on the cDNA for GTF3 . Functional domains
are depicted as in Fig. 1. An amino-terminal Pk epitope was added on to
full-length mGTF3
and mGTF3
.
3-6 constructs to allow for
co-immunoprecipitation of GTF3 protein complexes and for
immunofluorescence detection using the Pk antibody (see below).
B, co-immunoprecipitation of full-length and partial mouse
GTF3 proteins expressed in HEK293 cells. Affinity-tagged proteins
(Bait) were either mGTF3
.
3-6 (left and
center) or full-length mGTF3
(right). Untagged GTF3 proteins (Prey) were
full-length mGTF3
(left), mGTF3
.
LZ
(center), or mGTF3
.
3-6 (right). 100 µg
of nuclear proteins were immunoprecipitated using the Pk antibody and
probed with a polyclonal GTF3 antibody (top
panel). A second identical blot was probed with anti-Pk
antibody (bottom panel). The presence of bait and
prey proteins in nuclear extracts was confirmed by direct Western
blotting (W) using 10 µg of nuclear protein.
Immunoprecipitated GTF3 proteins are shown as indicated
(IP). C, nuclear translocation in C2C8 myoblasts
of nuclear localization signal-deficient mGTF3
.
3-6
protein by co-expression of full-length mGTF3. Cellular distribution of
affinity-tagged proteins mGTF3
(a, a'),
mGTF3
.
LZ (b, b') or mGTF3
.
3-6
(c, c') is shown in top
panel. Cells co-transfected with constructs expressing
affinity-tagged mGTF3
.
3-6 and untagged mGTF3
(d,
d') or mGTF3
.
LZ (e, e') are
shown in bottom panel. All cells were treated
with anti-Pk antibody, detected with an Alexa 488 secondary antibody
(a-e) and stained with nuclear dye Hoechst 33258 to
visualize nuclei (overlay, a'-e').
.
3-6, which lacks a bona fide nuclear
localization signal located near the very carboxyl terminus of GTF3
(26), would remain in the cytosol unless co-expressed with full-length GTF3. The underlying assumption was that the truncated bait protein would be translocated to the nuclear compartment through association with full-length GTF3. As shown in Fig. 5C, double labeling
with Hoechst 33258 demonstrated that both full-length GTF3
and
GTF3
.
LZ predominantly located to the nucleus, although on
occasion cells were found that showed cytoplasmic staining
(a and a'; b and b'). In
contrast, mGTF3
.
3-6 protein distributed evenly between cytosol and the nucleus (c and c'). Next, cells were
co-transfected with Pk-tagged mGTF3
.
3-6 and either untagged
full-length mGTF3
or mGTF3
.
LZ (panels d and
e). In agreement with results from co-immunoprecipitation experiments (see above), mGTF3
.
3-6(Pk) was predominantly located in the nucleus of most transfected cells when co-expressed with full-length GTF3
(d and d'), but not with
GTF3
.
LZ (e and e'). Substituting mGTF3
and -
isoforms for mGTF3
in this assay, as well as in the
pull-down assay described above, yielded similar results (data not
shown). In conclusion, both biochemical and in situ evidence
indicate that GTF3 polypeptides can interact with each other, and that
this homomeric affinity can be attributed to the amino-terminal leucine
zipper motif.
.
3-6(Pk) can pull down TFII-I (
-isoform) in
HEK293 cells co-transfected with constructs expressing these proteins
(Fig. 6). In contrast to previous
experiments showing interaction between the bait and GTF3 proteins, we
were unable to immunoprecipitate TFII-I with mGTF3
.
3-6(Pk). This result indicates that GTF3 and TFII-I do not form stable complexes that
can be immunologically purified.
View larger version (49K):
[in a new window]
Fig. 6.
TFII-I does not co-immunoprecipitate with
GTF3. HEK293 cells were co-transfected with
constructs expressing human TFII-I as prey and Pk-tagged mouse
mGTF3 .
3-6 as bait. Nuclear extracts were prepared and
immunoprecipitated as described in Fig. 5. Western blots of nuclear
extracts (W) and pulled-down proteins (IP) were
probed with antibodies against TFII-I (top) or the Pk
epitope (bottom).
View larger version (13K):
[in a new window]
Fig. 7.
TFII-I co-localizes with GTF3 to the
nucleus. Subcellular localization of endogenous GTF3 and TFII-I
proteins was assessed in C2C8 cells. a, GTF3 protein
detected with a polyclonal anti-GTF3 antibody (1:100) and visualized
with an Alexa488 secondary antibody. b, TFII-I protein
detected with a monoclonal primary antibody (1:25) and a Cy3 secondary
antibody. c, overlay of GTF3 and TFII-I immunofluorescence.
Yellow indicates areas where both proteins co-localize.
d, A Hoechst 33258 stain was included to visualize
nuclei.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-3 and the BLM (data not shown). Additionally, our
previous yeast one-hybrid screens of human skeletal muscle cDNA
libraries that used the TnIs BLM as bait never yielded TFII-I encoding
clones, even though TFII-I is clearly expressed in skeletal muscle
tissues (29).
, which in preliminary EMSA experiments bound very poorly if at
all to the TnIs BLM, were expressed in skeletal muscle. Using
oligonucleotides that flank the sequences that are alternatively spliced in GTF3
and -
isoforms, we demonstrated their expression in all muscles analyzed, including fast- and slow-twitch muscles like
the EDL and the soleus, respectively. In addition, we also identified
three novel splice isoforms, GTF3
2, -
3,
and -
2, that arise from differential usage of exon 23 and exons 26-28. Because our screen was not designed to exhaustively
identify all splice variants generated from the GTF3 locus, it is
likely that additional splice variants will be identified in the
future. Moreover, although all previously known splice variants were
reported to share sequences upstream of the BstEII site we
used to clone the PCR fragments into the parental mGTF3
expression
vector, we cannot rule out that splice events upstream of the analyzed
sequence contribute to GTF3 isoform variability. The banding patterns
of PCR products generated with our primer set revealed a quite
stereotypical isoform expression profile in developing and adult
skeletal muscles, and in non-muscle tissues. A quantitative analysis of
the relative abundance of GTF3
/
splice isoforms in soleus and the
EDL muscles corroborated that their distribution is quite similar in
slow and fast fiber types. This finding argues against the idea that the differential expression of distinct isoforms in different fiber
types may contribute to the slow fiber type-specific expression of the
TnIs gene. However, as discussed above, this statement does not
preclude the possibility that other yet unidentified isoforms may exert
this function.
, lacking both exon 23 and
exons 26-28, binds most avidly. It is followed by
GTF3
2, which lacks exons 26-28 but includes exon 23, and GTF3
3, which contains exon 26-28 but lacks exon 23. No detectable binding to the BLM was obtained with GTF3
,
-
2, and -
isoforms, which contain both exons 23 and
exons 26-28. Human GTF3 proteins are most closely resembled by mouse
GTF3
, which is in agreement with the fact that both
1-3 variants
derived from these proteins bound avidly to the BLM. Mouse exons 26-28
encode for HLH domain 6 that is identical in sequence to HLH domain 5 (27). It is therefore possible that the close proximity of a second HLH
domain to the DNA binding domain R4 destabilizes DNA-protein
interaction or masks the DNA-binding surface. Exon 23 codes for 27 amino acids that are located COOH-terminal of R4. Because 75% of these
residues are charged or polar, it is likely that this region is exposed on the surface of the protein. No recognizable motifs were detected in
a ScanProsite protein pattern search. One possible effect of these
residues may be that they destabilize protein-DNA interaction by
altering the relative orientation of flanking domains including R4.
) can interact with GTF3, although the immediate
area flanking the LZ is invariable in these isoforms (45). The lack of
a direct interaction between GTF3 and TFII-I would be in general
agreement with a model recently proposed, in which GTF3 expression
negatively regulated the nuclear residency of TFII-I by competing for a
limiting factor that is required by both GTF3 and TFII-I to translocate
to the nucleus (39). However, in C2C8 myoblasts and myotubes,
endogenous GTF3 and TFII-I proteins co-reside in the nucleus. In
contrast, mature rat primary myotube cultures show a nuclear exclusion
of GTF3 protein (data not shown). This phenomenon is currently under
investigation because it might provide insights into how GTF3 functions
as a transcription factor in skeletal muscle. Taken together, we do not
find evidence in myogenic cells that supports the notion of a
competition of GTF3 and TFII-I interaction for nuclear residency.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Steve Kinsey for technical assistance and Dr. Remmert for help with co-immunoprecipitation experiments.
![]() |
FOOTNOTES |
---|
* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY149688 (GTF32), AY149689
(GTF3
3), and AY149690 (GTF3
2).
To whom correspondence should be addressed. Tel.: 301-496-3298;
Fax: 301-496-9939; E-mail: buonanno@helix.nih.gov.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M209361200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GTF3, general transcription factor 3; SURE, slow upstream regulatory element; FBS, fetal bovine serum; EDL, extensor digitorum longus; HLH, helix-loop-helix; bHLH, basic helix-loop-helix; BLM, bicoid-like motif; TFII-I, general transcription factor II-I; WS, Williams syndrome; LZ, leucine zipper; EMSA, electrophoretic mobility shift assay; DIV, days in vitro; RT, reverse transcription.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Donoghue, M. J., Alvarez, J. D., Merlie, J. P., and Sanes, J. R. (1991) J. Cell Biol. 115, 423-434[Abstract] |
2. | Shield, M. A., Haugen, H. S., Clegg, C. H., and Hauschka, S. D. (1996) Mol. Cell. Biol. 16, 5058-5068[Abstract] |
3. | Banerjee-Basu, S., and Buonanno, A. (1993) Mol. Cell. Biol. 13, 7019-7028[Abstract] |
4. | Hallauer, P. L., Hastings, K. E., and Peterson, A. C. (1988) Mol. Cell. Biol. 8, 5072-5079[Medline] [Order article via Infotrieve] |
5. | Salminen, M., Maire, P., Concordet, J. P., Moch, C., Porteu, A., Kahn, A., and Daegelen, D. (1994) Mol. Cell. Biol. 14, 6797-6808[Abstract] |
6. |
McCarthy, J. J.,
Fox, A. M.,
Tsika, G. L.,
Gao, L.,
and Tsika, R. W.
(1997)
Am. J. Physiol.
272,
R1552-R1561 |
7. | Jerkovic, R., Vitadello, M., Kelly, R., Buckingham, M., and Schiaffino, S. J. (1997) Muscle Res. Cell Motil. 18, 369-373[CrossRef] |
8. |
Lupa-Kimball, V. A.,
and Esser, K. A.
(1998)
Am. J. Physiol.
274,
C229-C235 |
9. |
Serrano, A. L.,
Murgia, M.,
Pallafacchina, G.,
Calabria, E.,
Coniglio, P.,
Lomo, T.,
and Schiaffino, S.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
13108-13113 |
10. |
Torgan, C. E.,
and Daniels, M. P.
(2001)
Mol. Biol. Cell
12,
1499-1508 |
11. |
Calvo, S.,
Vullhorst, D.,
Venepally, P.,
Cheng, J.,
Karavanova, I.,
and Buonanno, A.
(2001)
Mol. Cell. Biol.
21,
8490-8503 |
12. |
O'Mahoney, J. V.,
Guven, K. L.,
Lin, J.,
Joya, J. E.,
Robinson, C. S.,
Wade, R. P.,
and Hardeman, E. C.
(1998)
Mol. Cell. Biol.
18,
6641-6652 |
13. | Murgia, M., Serrano, A. L., Calabria, E., Pallafacchina, G., Lomo, T., and Schiaffino, S. (2000) Nat. Cell Biol. 2, 142-147[CrossRef][Medline] [Order article via Infotrieve] |
14. | Spitz, F., Salminen, M., Demignon, J., Kahn, A., Daegelen, D., and Maire, P. (1997) Mol. Cell. Biol. 17, 656-666[Abstract] |
15. |
Wu, H.,
Naya, F. J.,
McKinsey, T. A.,
Mercer, B.,
Shelton, J. M.,
Chin, E. R.,
Simard, A. R.,
Michel, R. N.,
Bassel-Duby, R.,
Olson, E. N.,
and Williams, R. S.
(2000)
EMBO J.
19,
1963-1973 |
16. |
Chin, E. R.,
Olson, E. N.,
Richardson, J. A.,
Yang, Q.,
Humphries, C.,
Shelton, J. M.,
Wu, H.,
Zhu, W.,
Bassel-Duby, R.,
and Williams, R. S.
(1998)
Genes Dev.
12,
2499-2509 |
17. | Lin, J., Wu, H., Tarr, P. T., Zhang, C. Y., Wu, Z., Boss, O., Michael, L. F., Puigserver, P., Isotani, E., Olson, E. N., Lowell, B. B., Bassel-Duby, R., and Spiegelman, B. M. (2002) Nature 418, 797-801[CrossRef][Medline] [Order article via Infotrieve] |
18. | Cann, G. M., Lee, J. W., and Stockdale, F. E. (1999) Anat. Embryol. 200, 239-252[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Blagden, C. S.,
Currie, P. D.,
Ingham, P. W.,
and Hughes, S. M.
(1997)
Genes Dev.
11,
2163-2175 |
20. |
Du, S. J.,
Devoto, S. H.,
Westerfield, M.,
and Moon, R. T.
(1997)
J. Cell Biol.
139,
145-156 |
21. | Zhu, L., Lyons, G. E., Juhasz, O., Joya, J. E., Hardeman, E. C., and Wade, R. (1995) Dev. Biol. 169, 487-503[CrossRef][Medline] [Order article via Infotrieve] |
22. | Nakayama, M., Stauffer, J., Cheng, J., Banerjee-Basu, S., Wawrousek, E., and Buonanno, A. (1996) Mol. Cell. Biol. 16, 2408-2417[Abstract] |
23. | Tassabehji, M., Carette, M., Wilmot, C., Donnai, D., Read, A. P., and Metcalfe, K. (1999) Eur. J. Hum. Genet. 7, 737-747[Medline] [Order article via Infotrieve] |
24. | Franke, Y., Peoples, R. J., and Francke, U. (1999) Cytogenet. Cell Genet. 86, 296-304[CrossRef][Medline] [Order article via Infotrieve] |
25. | Osborne, L. R., Campbell, T., Daradich, A., Scherer, S. W., and Tsui, L. C. (1999) Genomics 57, 279-284[CrossRef][Medline] [Order article via Infotrieve] |
26. | Yan, X., Zhao, X., Qian, M., Guo, N., Gong, X., and Zhu, X. (2000) Biochem. J. 345, 749-757[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Bayarsaihan, D.,
and Ruddle, F. H.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7342-7347 |
28. | Roy, A. L. (2001) Gene (Amst.) 274, 1-13[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Roy, A. L.,
Du, H.,
Gregor, P. D.,
Novina, C. D.,
Martinez, E.,
and Roeder, R. G.
(1997)
EMBO J.
16,
7091-7104 |
30. |
Cheriyath, V.,
and Roy, A. L.
(2001)
J. Biol. Chem.
276,
8377-8383 |
31. | Bayarsaihan, D., Dunai, J., Greally, J. M., Kawasaki, K., Sumiyama, K., Enkhmandakh, B., Shimizu, N., and Ruddle, F. H. (2002) Genomics 79, 137-143[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Francke, U.
(1999)
Hum. Mol. Genet.
8,
1947-1954 |
33. | Merla, G., Ucla, C., Guipponi, M., and Reymond, A. (2002) Hum. Genet. 110, 429-438[CrossRef][Medline] [Order article via Infotrieve] |
34. | Emerson, C. P., and Sweeney, H. L. (1997) Methods in Cell Biology: Methods in Muscle Biology , Vol. 52 , pp. 85-116, Academic Press, San Diego |
35. | Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve] |
36. | Lennon, G., Auffray, C., Polymeropoulos, M., and Soares, M. B. (1996) Genomics 33, 151-152[CrossRef][Medline] [Order article via Infotrieve] |
37. | Southern, J. A., Young, D. F., Heaney, F., Baumgartner, W. K., and Randall, R. E. (1991) J. Gen. Virol. 72, 1551-1557[Abstract] |
38. | Krowczynska, A. M., Coutts, M., Makrides, S., and Brawerman, G. (1989) Nucleic Acids Res. 17, 6408[Medline] [Order article via Infotrieve] |
39. |
Tussie-Luna, M. I.,
Bayarsaihan, D.,
Ruddle, F. H.,
and Roy, A. L.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
7789-7794 |
40. |
Atchley, W. R.,
Wollenberg, K. R.,
Fitch, W. M.,
Terhalle, W.,
and Dress, A. W.
(2000)
Mol. Biol. Evol.
17,
164-178 |
41. | Roy, A. L., Malik, S., Meisterernst, M., and Roeder, R. G. (1993) Nature 365, 355-359[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Sepulveda, J. L.,
Belaguli, N.,
Nigam, V.,
Chen, C. Y.,
Nemer, M.,
and Schwartz, R. J.
(1998)
Mol. Cell. Biol.
18,
3405-3415 |
43. | Jonsen, M. D., Petersen, J. M., Xu, Q. P., and Graves, B. J. (1996) Mol. Cell. Biol. 16, 2065-2073[Abstract] |
44. | Hata, A., Lo, R. S., Wotton, D., Lagna, G., and Massague, J. (1997) Nature 388, 82-87[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Cheriyath, V.,
and Roy, A. L.
(2000)
J. Biol. Chem.
275,
26300-26308 |