 1993 Oxford University Press
Human Molecular Genetics, 1996, Vol. 5, No. 1
115–121
Novel proteins with binding specificity for DNA CTG
repeats and RNA CUG repeats: implications for
myotonic dystrophy
L. T. Timchenko, N. A. Timchenko1, C. T. Caskey2 and R. Roberts*
Department of Medicine, Section of Cardiology, Baylor College of Medicine, Houston, TX 77030, USA,
1Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA and 2Merck Research
Laboratories, Sumneytown Pike, West Point, PA 19486, USA
Received September 6, 1995; Revised and Accepted October 21, 1995
While an unstable CTG triplet repeat expansion is responsible for myotonic dystrophy, the mechanism
whereby this genetic defect induces the disease remains
unknown. To detect proteins binding to CTG triplet repeats, we performed bandshift analysis using as probes
double-stranded DNA fragments having CTG repeats
[ds(CTG)6–10] and single-stranded oligonucleotides having CTG repeats ss(CTG)8 or RNA CUG triplet repeats
(CUG)8. The source of protein was nuclear and cytoplasmic extracts of HeLa cells, fibroblasts and myotubes. Proteins binding to the double-stranded DNA
repeat [ds(CTG)6–10], were inhibited by nonlabeled
ds(CTG)6–10, but not by a non-specific DNA fragment
(USF/AD-ML). Another protein binding to ssCTG probe
and RNA CUG probe was inhibited by nonlabeled (CTG)8
and (CUG)8. Nonlabeled oligos with different triplet repeat sequences, ss(CAG)8 or ss(CGG)8, did not inhibit
binding to the ss(CTG)8 probe. However, when labeled as
probes, the (CAG)8 and (CGG)8 bound to proteins distinct from the CTG proteins and binding was inhibited by
nonlabeled (CAG)8 or (CGG)8 respectively. The protein
binding only to the RNA repeat (CUG)8 was inhibited by
nonlabeled (CUG)8 but not by nonlabeled single- or
double-stranded CTG repeats. Furthermore, the CUG-BP
exhibited no binding to an RNA oligonucleotide of triplet
repeats of the same length but having a different
sequence, CGG. The CUG binding protein was localized
to the cytoplasm, whereas dsDNA binding proteins were
localized to the nuclear extract. Thus, several trinucleotide binding proteins exist and their specificity is determined by the triplet sequence. The novel protein,
CUG-BP, is particularly interesting since it binds to triplet
repeats known to be present in myotonin protein kinase
mRNA which is responsible for myotonic dystrophy.
*To whom correspondence should be addressed
INTRODUCTION
Extensive expansion of a triplet of base pairs repeated in tandem in
the human genome has been shown to be responsible for several
neuromuscular diseases (1–13). In some of these disorders, loss of
gene expression suggests triplet repeat expansion affects the
efficiency of transcription (14–16), or translation (17). The
chromosomal locus l9q, genetically linked to myotonic dystrophy,
has a region of extensive repeats of the triplet CTG (6–11). The
number of repeats correlates with both the presence and severity of
disease (18–22). The extended triplet repeat region is 3′ to the
alleged responsible gene, myotonin protein kinase (Mt-PK). Results
of studies assessing the expression of Mt-PK in patients with disease
are inconclusive, some showing decreased expression of Mt-PK
mRNA (23–28), others increased Mt-PK mRNA expression (29).
The molecular weight of Mt-PK was shown initially to be 52–55
kDa (23,28), however, recently we identified a full-length isoform
of Mt-PK with a molecular weight of 72 kDa (30) which
corresponds to the size of a protein translated from the first AUG
codon in the frame and possesses serine specific kinase activity (30).
Whiting et al. have also described the existence of high molecular
weights Mt-PK isoforms (31). Wang et al. demonstrated increased
efficiency of nucleosome formation at the region of CTG triplet
expansion (32,33) which may repress transcription. The role of the
triplet repeat in transcription has been further questioned by the data
of Krahe et al. (34). It has also been suggested that the CTG repeat
expansion in patients with DM dramatically alters the ability of the
mutant mRNA to be processed into poly(A)+ mRNA (35) or results
in abnormal transfer of Mt-PK transcripts from nucleus to the
cytoplasm. This derives from the observation that increased levels
of the enlarged Mt-PK transcript are present in the nucleus of cells
from DM patients (36). Contradictory data on steady-state levels of
Mt-PK mRNA has provided the impetus to explore the possibility
that the CTG expansion exerts its dilatory effects not on the Mt-PK
but on the gene immediately upstream of Mt-PK (37).
In our attempts to explore other mechanisms whereby expanded triplet repeats may induce disease, we identified a group
of proteins that exhibit binding specific for CTG triplet repeats
whether they be double- or single-stranded. In addition, an RNA
binding protein (CUG-BP) was identified in the cytoplasm of
116
Human Molecular Genetics, 1996, Vol. 5, No. 1
HeLa cells which was specific for the RNA sequence, (CUG)8,
and did not bind to single- or double-stranded DNA CTG repeats.
There are at least two previous reports of triplet DNA binding
proteins (38,39). In this study we have identified several other
novel trinucleotide binding proteins and, in particular, a novel
protein which binds specifically to the RNA triplet repeat
(CUG)8. The latter protein has particular implications for the
pathogenesis of myotonic dystrophy.
RESULTS
Identification of double stranded (ds) CTG binding
proteins
Plasmid DNA containing the myotonin protein kinase cDNA
sequence (pRMK) and human genomic DNA were amplified to
generate double-stranded CTG repeats as probes (Fig. 1A). The
DNA fragment from plasmid pRMK had six (CTG) repeats,
referred to as dsCTG-1 and that from human genomic DNA
(normal allele) had 10 (CTG) repeats referred to as dsCTG-2.
These fragments were labeled with 32P and incubated with
nuclear extracts from HeLa cells. Separation by electrophoretic
mobility shift assay showed two major DNA–protein complexes
(dsI and dsII) and one minor complex (dsIII) bound to each probe
(Fig. 1B and C). Detection of complex dsIII required prolonged
exposure. The prior addition of 100-fold excess of nonlabeled
DNA fragments dsCTG-1 and dsCTG-2 competitively inhibited
the formation of the DNA-proteins complexes indicating that the
protein binding was specific for dsCTG-1 and dsCTG-2 (Fig.
1B). We also used double-stranded USF/Ad-ML DNA (40) as a
potential competitor. An addition of 100-fold excess of USF/AdML did not affect the binding of nuclear proteins to dsCTG-1
(data not shown). Nuclear protein extracts from human fibroblasts, myotubes and HeLa cells were incubated with the dsCTG-l
probe and bandshift assays showed (Fig. 1C) complexes dsI and
dsII of similar intensity in each preparation. Formation of dsIII
complex was registered only in HeLa nuclear extracts. Extracts
from the cytoplasm of HeLa cells exhibited no binding to the
dsCTG-1 probe (data not shown).
The molecular weight of the dsCTG binding proteins, determined by Southwestern analysis, were 21, 34, 37, 45 and 65 kDa
(Fig. 2A). Thus, five proteins were observed despite only three
DNA complexes observed with bandshift analysis using the same
DNA probe (Fig. 1). To verify the results of the Southwestern
analysis, dsCTG binding activity was determined for the protein
fractions with different molecular weights after separation by
PAGE (Denaturation/Elution technique). The proteins eluted
from each piece of the membrane were analyzed in a bandshift
assay (Fig. 2B). Specific complexes dsI and dsII were formed by
proteins with molecular weights of 60–69 kDa (Fraction #3, Fig.
2B). The binding activity of these proteins was significantly
reduced presumably due to denaturation. The other possibility for
decreased binding is the DNA protein complex is composed of
more than one protein. The two proteins with molecular weights
of 37 (Fraction #5) and 45 (Fraction #4) kDa participated in the
formation of the complex dsIII (Fig. 2B). We were unable to
determine dsCTG binding activity to the 21 and 34 kDa proteins
by the Denaturation/Elution technique.
Figure 1. Identification of dsCTG binding proteins. (A) Electrophoretic analysis
of dsCTG-1 and dsCTG-2 DNA fragments generated by PCR from plasmid DNA
pRMK and genomic DNA. PCR with vector DNA was carried out as a control for
nonspecific products (control). The positions of dsCTG-l and dsCTG-2 are
indicated on the right. (B) Interaction of nuclear proteins from HeLa cells with
dsCTG-1 and dsCTG-2 probes. DNA fragments were isolated from the gel,
end-labeled and incubated with increased amount of HeLa NE. Nonlabeled
competitor was added before the addition of radioactive probe. Positions of
DNA/protein complexes are shown on the left. NS is a nonspecific complex (no
self competition). (C) Bandshift analysis of nuclear extracts prepared from HeLa
cells, human fibroblasts and myotubes. Ten micrograms of NEs were incubated
with dsCTG-l probe and loaded on native 5% polyacrylamide gel.
Identification of single-stranded CTG triplet repeat
binding proteins
To determine if there are proteins that bind to single stranded (ss)
DNA repeats, a synthetic DNA oligonucleotide made of CTG
repeats was radiolabeled and used as a probe for bandshift
analysis. Given that (CTG)8 represents GC rich DNA (Fig. 3A),
two triplet repeat sequences [ss(CAG)8 and ss(CGG)8] were
selected as probes to determine whether binding reflects specific
interaction with the CTG sequence or simply binding to GC rich
regions. Two DNA/protein complexes (major ssI and minor ssII)
were observed after incubation of the ss(CTG)8 probe with whole
cell HeLa protein extracts (Fig. 3B). Binding was inhibited by the
addition of nonlabeled ssCTG DNA (30 ng) indicating binding
was specific (Fig. 3B). The same preparation of whole HeLa cell
protein extracts incubated with labeled ss(CAG)8 and ss(CGG)8
resulted in the formation of two (CAG)8 DNA/protein complexes
(Fig. 3B) and four (CGG)8 DNA/protein complexes respectively
(Fig. 3B). Unlabelled ss(CGG)8 and (CAG)8 completely inhibited binding indicating specificity (data not shown).
Comparison of the mobilities of the ss(CTG)8 binding proteins
with the (CAG)8 and (CGG)8 binding proteins demonstrated the
protein participating in the formation of complex ssI is unique and
will be referred to as a single-strand CTG-repeat recognizing
protein (ssCRRP). The protein forming the complex ssII most
likely recognized the GCN region of the probe, because identical
complexes were observed with CAG and CGG probes (Fig. 3B).
The ssCRRP did not interact with ss(CAG)8 probe, but did
recognize the ss(CGG)8 oligonucleotide but with low affinity.
The affinity of ssCRRP for ss(CTG)8 versus that of ss(CGG)8
probe was assessed in a competitive assay. The addition of 10 ng
117
Human Acids
Molecular
Genetics,
Vol.No.
5, No.
Nucleic
Research,
1994,1996,
Vol. 22,
1 1
Figure 2. Estimation of molecular weights of dsCTG binding proteins. (A) The
results of Southwestern analysis are shown in panel A. One hundred
micrograms of HeLa nuclear extract were loaded on 0.1% SDS-12% PAGE,
transferred onto nitrocellulose membrane and incubated with [32P]dsCTG-l
probe under conditions described in Materials and Methods. The molecular
weight as indicated by the control markers (M) are indicated on the left of panel
A. The extract exhibited five bands despite only three bands observed with
bandshift analysis in panel B. The molecular weight varied from 21 to 65 kDa.
(B) The results of bandshift analysis of the various proteins after elution. HeLa
NE was fractionated by 0.1% SDS-12% PAGE, transferred onto membrane and
eluted from the slices with distinct molecular weights as indicated above.
Numerals I, II and III indicated to the left in Panel B refer to DNA protein
complex I, II and III respectively. The band indicated by NS was shown to be
nonspecific (NS) by lack of competitive inhibition. No refers to the control
probe without extract, NE refers to the total nuclear extract of HeLa while
numbers 2–9 refer to different fractions of the total NE. The dsCTG-I referred
to at the bottom is the probe used in all of these experiments. The protein
forming complex dsI and dsII exhibited a molecular weight in the range of
60–69 kDa. The proteins forming complexes dsIII had molecular weights of 37
and 45 kDa. The proteins with molecular weights of 21 and 34 exhibited no
binding to the dsCTG-I probe after elution presumably due to denaturation.
of ss(CTG)8 significantly reduced the binding of the labeled
probe to ssCRRP (Fig. 3C). On the contrary, the addition of 10 or
30 ng of nonlabeled ss(CGG)8 did not affect binding, even 100 ng
reduced the binding only slightly of ssCRRP to the ss(CTG)8
probe (Fig. 3C). Thus, the results of the bandshift competitive
experiments indicate the ssCRRP protein binds to the ssCTG
repeat, but does not interact with the other GC rich repeat probes.
Attempts to determine the molecular weight of the ssCRRP by
Southwestern analysis and Denaturation/Elution technique were
unsuccessful. One possibility is that the various components must
be combined to give the ssCRRP binding activity. We tested this
possibility with fractions containing proteins with different
molecular weights as determined by the Denaturation/Elution
procedure. In this experiment whole cell extract of HeLa cells was
used. Bandshift analysis of fractions #5 (34–45 kDa) and #6
(28–34 kDa) did not display ssCRRP activity, when analyzed
separately, but mixing of these fractions resulted in recovery of
ssCRRP binding activity (Fig. 4A). These results suggest that the
ssCRRP protein consists of two subunits with different molecular
weights or another protein is required for activation of ssCRRP.
Identification of RNA triplet repeat binding proteins
A synthetic RNA oligonucleotide (CUG)8 was labeled by the kinase
method and used as a probe for bandshift analysis with HeLa whole
cell extract. Two RNA/protein complexes (Fig. 5A) were detected
by bandshift analysis after incubation of the (CUG)8 probe with the
117
Figure 3. Identification of single-stranded CTG repeat recognizing proteins. (A)
The CTG, CAG and CGG triplet repeats share common GC dinucleotide repeat.
Black boxes show GC repeat. (B) Two proteins bind specifically to ss(CTG)8 probe.
Ten micrograms of HeLa whole cell extract were incubated with radioactive
single-stranded probes (indicated on the bottom) and analyzed by bandshift assay.
Positions of specific complexes ssI and ssII are indicated. (C) Bandshift-competition assay. Twenty micrograms of HeLa whole cell protein extract were incubated
with ss(CTG)8 probe in the presence of increased amount (indicated on the top) of
nonlabeled ss(CTG)8 or ss(CGG)8 competitors and analyzed by bandshift assay.
HeLa whole cell extract. The addition of 100 ng of nonlabeled RNA
(CUG)8 completely inhibited binding of the labeled probe to either
complex (Fig. 5A). One of the RNA/protein complexes migrated
with a mobility similar to the ssCTG/ssCRRP complex. Incubation
of the extract with nonlabeled ss(CTG)8 or nonlabeled (CUG)8
inhibited the formation of this complex indicating that the same
protein is involved in the formation of DNA/protein and RNA/protein complexes (Fig. 4B and Fig. 6B). Thus, the specificity of
ssCRRP for DNA (CTG) repeats and for RNA (CUG) repeats was
confirmed with both ss(CTG)8 and (CUG)8 probes. The other
RNA/protein complex with different mobility than that of the
(CUG)8/(ssCRRP) complex was inhibited by the nonlabeled
(CUG)8 oligo and was not inhibited by the nonlabeled DNA (CTG)8
oligo (Fig. 6B). The protein with exclusive binding specificity to
CUG triplet repeat was referred to as CUG-BP. To further determine
the specificity of CUG-BP we selected a triplet repeat RNA oligo
having a different sequence, namely, (CGG)8. The addition of
100-fold excess of nonlabeled (CGG)8 RNA competitor did not
affect the binding of CUG binding protein (CUG-BP) to the (CUG)8
probe, indicating specific binding (data not shown). Results shown
in Figure 5B indicate the binding activity of ssCRRP was observed
preferentially in cytoplasmic preparations with minimal binding
detected in the nuclear extract. In contrast, the RNA CUG-BP
complex showed high intensity in the cytoplasmic extract (Fig. 5C),
but was not detectable in the nuclear extract. To verify the purity of
the separation of nuclei from cytoplasm, the probe, USF/Ad-ML
specific for the nuclear upstream stimulatory factor (USF) (40) was
incubated with the same nuclear and cytoplasm preparations. USF
binding was detected as expected only in the nuclear extract and not
in the cytoplasm (Fig. 5D), confirming good separation of nuclear
118
Human Molecular Genetics, 1996, Vol. 5, No. 1
Figure 6. Estimation of molecular weight of CUG-BP. (A) HeLa cytoplasm was
fractionated by 0.1% SDS-12% PAGE and transferred onto nitrocellulose.
Proteins were eluted from the fractions with different molecular weights and
analyzed by the bandshift assay. CUG-binding activity was detected only in the
fraction #4. (B) CUG-BP binds to RNA (CUG)8 repeat but does not bind to
DNA (CTG)8 repeat. Fraction #4 containing purified CUG-BP was incubated
with (CUG)8 probe and analyzed by bandshift. 100-fold excess of nonlabeled
ss(CTG)8 or RNA (CUG)8 competitors was added before probe addition.
Figure 4. The ssCRRP interacts with both DNA CTG and RNA CUG triplet
repeat. (A) Binding activity of ssCRRP is registered after mixing of proteins with
distinct molecular weights. HeLa whole cell extract was fractionated by
Denaturation/Elution technique and 5 µl of different protein fractions (Fig. 2A)
were incubated with ssCTG probe and analyzed by bandshift assay. Positions of
DNA/protein complexes are shown on the left. Number of protein fractions are
indicated on the top of the gel. NS is nonspecific complex (no self competition).
(B) The ssCRRP binds to RNA (CUG) triplet repeat. HeLa proteins were
incubated with ss(CTG)8 probe in the presence of ss(CTG)8 DNA or CUG RNA
competitors (100-fold excess) and analyzed on a 8% native acrylamide gel.
6A). Analysis showed the purified CUG-BP protein fraction
interacted with the RNA (CUG)8 probe, but not with the DNA
(CTG) probe and binding to the RNA probe was inhibited by
nonlabeled (CUG)8 but not by nonlabeled DNA (CTG)8 (Fig. 6B)
or RNA (CGG)8 probe (Table 1).
It is reasonable to speculate that the binding proteins may be
necessary for proper spliceosomes to form or for preservation of
certain sequences for cellular transport or localization. The
diverse clinical manifestations of myotonic dystrophy indicate
multiple organs are involved (muscle, eye, brain and testes) and
the absence of a phenotype with elimination of the Mt-PK gene
(42) is in keeping with an abnormality of more diverse origin. An
abnormality of a triplet binding protein could affect the mRNA
in any or all organs. The protein exhibiting specific binding to the
RNA CUG triplet repeat was found in all cells assessed including
fibroblast, myotubules and HeLa cells (data not shown). A defect
in the DNA CTG triplet repeat protein, could also have diverse
effects due to impaired transcription.
DISCUSSION
Figure 5. Identification of RNA (CUG)8 binding proteins. (A) Two proteins
from HeLa cells bind specifically to RNA (CUG)8 probe. Twenty micrograms
of HeLa whole cell extract were incubated with labeled RNA (CUG)8 probe and
analyzed by bandshift assay. Positions of ssCRRP/CUG and CUG-BP/CUG
complexes are indicated. (B) ssCRRP and CUG-BP are localized preferentially
in the cytoplasm. HeLa whole cell extract (20 µg), NE (10 µg) and cytoplasm
(30 µg) were incubated with radioactive ss(CTG)8 probe, RNA (CUG)8 probe
or with USF/Ad-ML probe (specific for USF) and analyzed by bandshift assay.
and cytoplasmic fractions. Thus, the proteins binding to the (CUG)8
RNA probe are localized to the cytoplasm.
The molecular weight of the CUG-BP determined by the
Denaturation/Elution technique ranged from ∼40 to 50 kDa (Fig.
We have identified and partially characterized several proteins
that specifically recognize double- and single-stranded DNA
CTG repeats and RNA CUG repeats. We also identified proteins
that specifically bind to single-stranded DNA triplet repeats of
CAG and CGG. The binding specificity for each protein whether
it was for DNA or RNA triplet repeats was determined in
competitive binding experiments using several-fold excess of the
corresponding nonlabeled triplet repeat fragment. The main
properties of these proteins are summarized in Table 1. The CUG
binding protein (CUG-BP) was unique in that it exhibited no
binding to single- or double-stranded DNA CTG repeats or to
RNA triplet repeats with a different sequence as indicated by the
lack of binding to the CGG probe. The single-stranded DNA
CTG-binding protein (ssCRRP) and the RNA CUG binding
protein (CUG-BP) were localized primarily in the cytoplasm
while the proteins binding to the double-stranded DNA probes
were detected primarily in the nuclear extract.
119
Human Acids
Molecular
Genetics,
Vol.No.
5, No.
Nucleic
Research,
1994,1996,
Vol. 22,
1 1
119
Table 1. Main properties of the CTG/CUG binding proteins
Proteins
Localization
Binding sites
cyto
NE
dsCTG
ssCTG
ssCAG
ssCGG
RNA(CUG)
RNA(CGG)
dsCTG-BPs
–
+
+++
–
–
+/–
–
–
ssCRRP
+
–/+
–
+++
–
–/+
+++
–
CUG-BP
+
–
–
–
n/d
n/d
+++
–
+++, high level of affinity.
+/– and –/+, some affinity.
n/d, not tested.
Dinucleotide and trinucleotide sequences repeated in tandem
referred to as microsatellites occur every 5000–10 000 bp throughout the human genome and because of the marked variation in the
number of repeats in each satellite are used almost exclusively today
as markers in genetic linkage analysis to map chromosomal loci
(41). The recent finding that extended repeats can induce several
neuromuscular and degenerative diseases (1–13) has intensified the
research for the inherent dysfunction. Myotonic dystrophy is
particularly puzzling in that the triplet repeat occurs in the 3′
non-protein coding region of the gene (6–11). The existence of
proteins specific for trinucleotide repeats particularly the novel
proteins (ssCRRP and CUG-BP) which bind the RNA CUG repeats
sequence found in the mRNA of the gene for myotonic dystrophy
has obvious implications. Reports suggest the mRNA of the
myotonin kinase gene is transcribed (23–29), however, one
hypothesis is that the 3′ end of the mature myotonin protein kinase
mRNA is improperly processed (35). It is reasonable to speculate
that the (CUG) RNA binding proteins may be needed for stability
in the processing of the mRNA or for the attachment of the poly A
tail. Since ssCRRP exhibited dual binding activity to ssCTG and
RNA CUG repeats, we speculate that it may be involved in two
possible events: (i) ssCRRP, found primarily in the cytoplasm, may
participate in translation or the regulation of Mt-PK mRNA stability,
or (ii) under some conditions ssCRRP, since a minimal amount was
also detected in the nuclear extract, may translocate to nuclei and
play some role in the transcription of Mt-PK gene or in the
processing of the pre-mRNA transcript.
Whether the CTG or CUG binding proteins play a role in the
pathogenesis of myotonic dystrophy is unknown but it now
becomes important to characterize and determine the function of
these specific proteins. Two previous reports have described
binding proteins to DNA triplet repeat sequences (38,39). We
confirm the previous reports showing proteins with specific
binding for the DNA repeat sequences of CAG, CGG and have
described several other novel proteins. The novel protein,
CUG-BP, is particularly interesting since it binds specifically and
exclusively to the RNA triplet repeat, CUG, which is the triplet
repeat present in multiple copies alleged to be responsible for
myotonic dystrophy. It is highly likely that these proteins have
specific functions and evolved in conjunction with the evolution
of these trinucleotide tandem repeats. It is reasonable to speculate
that disease or alteration in function will occur if these proteins
are in some way mutated or impaired. A new class of proteins are
now available to be explored both in terms of their possible
functional role in transcription and/or translation and their
potential role of inducing diseases or altered physiological states
as a result of mutations.
MATERIALS AND METHODS
Materials
The following DNA oligonucleotides were used as profiles
5′-CTGCTGCTGCTGCTGCTGCTGCTG-3′,
ss(CTG)8,
ss(CAG)8, 5′-CAGCAGCAGCAGCAGCAGCAGCAG-3′ and
ss(CGG)8, 5′-CGGCGGCGGCGGCGGCGGCGGCGG-3′. The
RNA oligonucleotides were synthesized in the Nucleic Acids Core
Laboratory of the Department of Molecular and Human Genetics,
Baylor College of Medicine or in Oligo’s Etc. Double-stranded
DNA fragments dsCTG-1 and dsCTG-2 were synthesized by PCR
from plasmid pRMK or human genomic DNA respectively with
primers 5′-GCTCGAAGGGTCCTTGTAGCCGGGAATG-3′ and
5′-GAAAGAAAGAAATGGTGCTGTGATCCCCC-3’.
Preparation of whole cell protein extracts (WCE),
nuclear extracts (NE) and cytoplasm (cyto)
HeLa and fibroblasts cultured cells were grown in MM medium
as previously described (43). Human myotubes were grown as
previously recommended (44). To prepare the protein extracts,
cells were washed twice with PBS, pelleted at 1000 g and
resuspended in a solution of 50 mM Tris-HCl, pH 7.5, 0.1 mM
EDTA, 1 mM dithiotreitol (DTT), 10 mM MgCl2, 20% glycerol,
0.1 M KCl, 1% Triton X-100, leupeptine (0.5 µg/ml) and
pepstatine (20 µg/ml). The cells were incubated on ice for 5 min
in this buffer and centrifuged for 5 min at 10 000 g at 4C. The
supernatant (whole cell protein extract) was collected and stored
at –80C.
Nuclear extracts were prepared as described (45). Cells were
washed twice with PBS, pelleted and resuspended in the buffer of
10 mM Tris HCl, pH 7.6, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM
DTT. After 15 min of incubation on ice cells were homogenized
by pulling them through a 23-gauge needle (eight strokes) and
nuclei were pelleted at 10 000 g. The nuclei were resuspended in
a buffer composed of 20 mM Tris-HCl, pH 7.6, 25% sucrose,
0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT and
incubated for 30 min on ice. Nuclear extract (NE) was collected
after centrifugation at 10 000 g and used immediately.
Bandshift analysis
The dsCTG-l and dsCTG-2 DNA fragments were generated by
PCR, purified by agarose gel and labeled by T4 DNA kinase with
[32P-gamma]ATP. Single-stranded DNA or RNA oligonucleotides were labeled by T4 polynucleotide kinase with gamma
[32P]ATP to a high specific activity. The binding reactions were
120
Human Molecular Genetics, 1996, Vol. 5, No. 1
performed in 10 µl mixture containing 0.1–0.5 ng of 32P-labeled
DNA or RNA probe, 10 µg of NE or 20–40 µg of WCE, 2 µg of
poly (dI-dC), 20 mM Tris-HCl, pH 7.6, 100 mM KCl, 5 mM
MgCl2, 5 mM DTT and 10% glycerol. In the experiments with the
(CUG)8 and (CGG)8 RNA probes, we used 2 µg of total RNA
isolated from HeLa cells as a nonspecific competitor. For
single-stranded DNA probes, poly (dI-dC) was heated at 95C for
10 min and incubated for 5 min on ice prior to use in the binding
reaction. To determine binding specificity, 100-fold excess of
nonlabeled probe was added to the binding reaction following the
mixtures were incubated for 30 min at room temperature.
Protein/DNA or protein/RNA complexes were separated from
the free probe by 6 or 8% polyacrylamide gel as described (45).
Southwestern analysis
Nuclear extracts (100 µg) were electrophoresed on 0.1%
SDS-12% polyacrylamide gel (45) and proteins were transferred
to a nitrocellulose membrane (NitroPure, MSI). The membrane
was blocked by prebinding in a solution of 5% dry milk, 10 mM
Tris-HCl, pH 7.6 and 1 mM DTT for 1 h at room temperature.
Binding was performed in a solution of 10 mM Tris-HCl, pH 7.6,
10 mM MgCl2, 100 mM KCl, 1 mM DTT, 0.25% dry milk,
0.1 mM EDTA with 106 c.p.m. of 32P-labeled oligonucleotide/
ml. After incubation for 1 h at room temperature, the membrane
was washed with the binding buffer that did not contain
32P-labeled probe.
Fractionation of the proteins by electrophoresis, elution
and bandshift analysis of protein fractions
(denaturation/elution technique)
Fractionation of proteins was performed as described (46). One
hundred micrograms of total protein extract were resolved by
0.1% SDS-12% polyacrylamide gel electrophoresis. After
transfer to nitrocellulose (NitroPure) the proteins were fractionated by cutting the membrane across the lane into pieces that
represent ∼5–10 kDa each. Proteins from each piece of the
membrane were eluted overnight in 100 µl of renaturation buffer:
20 mM HEPES, pH 7.9, 50 mM KCl, 5 mM MgCl2, 10%
glycerol, 1% Triton X 100 and 0.1 mg/ml BSA (fraction V) at
4C. The eluted protein fractions were collected and stored at
–80C. Five microliters of each fraction were used in the
bandshift assay. Mixed fractions analysis was performed by
combining of 5 µl from each of two fractions in a final volume of
20 µl.
ACKNOWLEDGEMENTS
We greatly appreciate the editorial assistance of Dr Linda
Bachinski and the secretarial assistance of Debora Weaver and
Esther Yeager in the preparation of this manuscript and figures.
This work is supported in part by grants from the National Heart,
Lung and Blood Institute, Specialized Centers of Research
(P50-HL54313-01), the National Institutes of Health Training
Center in Molecular Cardiology (T32-HL07706) and the American Heart Association, Bugher Foundation Center for Molecular
Biology (86-2216).
REFERENCES
1. Fu, Y., Kuhl, D.P., Pizzuti, A., Pieretti, M., Sutcliffe, J.S., Richards, S.,
Verkerk, A.J., Holden, J.J., Fenwick, R.G., Jr, Warren, S.T. and Caskey, C.T.
(1991) Variation of the CGG repeat at the fragile X site results in genetic
instability: Resolution of the Sherman paradox. Cell, 67, 1047–1058.
2. Verkerk, A.J.M.H., Pieretti, M., Sutcliffe, J.S., et al. (1991) Identification of
gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster
region exhibiting length variation in fragile X syndrome. Cell, 65, 905–914.
3. La Spada, A.R., Wilson, E.M., Lubahn, D.B., Harding, A.E. and Fischbeck,
K.H. (1991) Androgen receptor gene mutations in X-linked spinal and bulbar
muscular atrophy. Nature, 352, 77–79.
4. Edwards, A., Hammond, H.A., Jin, L., Caskey, C.T. and Chakraborty, R.
(1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in
four human population groups. Genomics, 12, 241–253.
5. Huntington’s Disease Collaborative Research Group (1993) A novel gene
containing a trinucleotide repeat that is expanded and unstable on
Huntington’s disease chromosome. Cell, 72, 971–983.
6. Harley, H.G., Brook, J.D., Rundle, S.A., Crow, S., Reardon, W., Buckler, A.J.,
Harper, P.S., Housman, D.E. and Shaw, D.J. (1992) Expansion of an unstable
DNA region and phenotypic variation in myotonic dystrophy. Nature, 355,
545–546.
7. Buxton, J., Shelbourne, P., Davies, J., Jones, C., Van Tongeren, T., Aslanidis,
C., de Jong, P., Jansen, G., Anvret, M. and Riley, B. (1992) Detection of an
unstable fragment of DNA specific to individuals with myotonic dystrophy.
Nature, 355, 547–548.
8. Aslanidis, C., Jansen, G., Amemiya, C., Shutler, G., Mahadevan, M.,
Tsilfidis, C., Chen, C., Alleman, J., Wormskamp, N.G. and Vooijs, M. (1992)
Cloning of the essential myotonic dystrophy region and mapping of the
putative defect. Nature, 355, 548–551.
9. Brook, J.D., McCurrack, M.E., Harley, H.G., et al. (1992) Molecular basis of
myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′ end
of a transcript encoding a protein kinase family member. Cell, 68, 799–808.
10. Fu, Y., Pizzuti, A., Fenwick, R.G., Jr., King, J., Rajnarayan, S., Dunne, P.W.,
Dubel, J., Nasser, G.A., Ashizawa, T., de Jong, P., Wieringa, B., Korneluk,
R.G., Perryman, M.B., Epstein, H.F. and Caskey, C.T. (1992) An unstable
triplet repeat in a gene related to myotonic muscular dystrophy. Science, 255,
1256–1258.
11. Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen,
G., Neville, C., Narang, M., Barcelo, J. and OHoy, K. (1992) Myotonic
dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of
the gene. Science, 255, 1253–1255.
12. Orr, H.T., Chung, M., Banfi, S., Kwiatkowski, T.J., Jr., Servadio, A., Beaudet,
A.L., McCall, A.E., Duvick, L.A., Ranum, L.P.W. and Zoghbi, H.Y. (1993)
Expansion of unstable trinucleotide CAG repeat in spinocerebellar ataxia
type-I. Nature Genet., 4, 221–226.
13. Koide, R., Ikeuchi, T., Onodera, O., Tanaka, H., Igarashi, S., Endo, K.,
Takahashi, H., Kondo, R., Ishikawa, A., Hayashi, T., Saito, M., Tomoda, A.,
Miike, T., Naito, H., Ikuta, F. and Tsuji, S. (1994) Unstable expansion of CAG
repeat in hereditary denatatorubral-pallidoluysian atrophy (DRPLA). Nature
Genet., 6, 9–13.
14. Pieretti, M., Zhang, F., Fu, Y., Warren, S.T., Oostra, B.A., Caskey, C.T. and
Nelson, D.L. (1991) Absence of expression of the FMR-1 gene in fragile X
syndrome. Cell, 66, 817–822.
15. Sutcliffe, J.S., Nelson, D.L., Zhang, F., Caskey, C.T., Saxe, D. and Warren,
S.T. (1992) DNA methylation represses FMR-1 transcription in fragile X
syndrome. Hum. Mol. Genet., 1, 397–400.
16. Knight, S.J.L., Flannry, A.V., Hirst, M.C., et al. (1993) Trinucleotide repeat
amplification and hypermethylation of a CpG island in FRAXE mental
retardation. Cell, 74, 127–134.
17. Feng, Y., Zhang, F., Lokey, L.K., Chastain, J.L., Lakkis, L., Eberhart, D. and
Warren, S.T. (1995) Translational supression by trinucleotide repeat expansion at FMR-1. Science, 268, 731–734.
18. Ashizawa, T., Dubel, J.R., Dunne, P.W., et al. (1992) Anticipation in
myotonic dystrophy. II. Complex relationships between clinical findings and
structure of the GCT repeat. Neurology, 42, 1877–1883.
19. Redman, J.B., Fenwick, R.G., Jr., Fu, Y.H., Pizzuti, A. and Caskey, C.T.
(1993) Relationship between parental trinucleotide GCT repeat length and
severity of myotonic dystrophy in offspring. JAMA, 269, 1960–1965.
20. Tsilfidis, C., MacKenzie, A.E., Mettler, G., Barcelo, J.K. and Korneluk, R.G.
(1992) Correlation between CTG trinucleotide repeat length and frequency of
severe congential myotonic dystrophy. Nature Genet., 1, 192–195.
121
Human Acids
Molecular
Genetics,
Vol.No.
5, No.
Nucleic
Research,
1994,1996,
Vol. 22,
1 1
21. Hunter, A., Tsilfidis, C., Mettler, G., Jacob, P., Mahadevan, M., Surh, L. and
Korneluck, R. (1992) The correlation of age onset with CTG trinucleotide
repeat amplification in myotonic dystrophy. J. Med. Genet., 29, 774–779.
22. Lavedan, C., Hofmann-Radvanyi, H., Shelourne, P., Rabes, J., Duros, C.,
Savoy, D., Dehaupas, I., Lucke, S., Johnson, K. and Junien, C. (1993)
Myotonic dystrophy: Size- and sex-dependent dynamics of CTG meiotic
instability and somatic mosaicism. Am. J. Hum. Genet., 52, 875–883.
23. Fu, Y., Friedman, D.L., Richards, S., Pearlman, J.A., Gibbs, R. A., Pizzuti, A.,
Ashizawa, T., Perryman, M.B., Scarlato, G., Fenwick, R.G., Jr. and Caskey,
C.T. (1993) Decreased expression of myotonin-protein kinase mRNA and
protein in adult form of myotonic dystrophy. Science, 260, 235–238.
24. Novelli, G., Gennarelli, M., Fattorini, C., Zelano, G., Dallapiccola, B.,
Pizzuti, A. and Caskey, C.T. (1993) Failure in detecting mRNA transcripts
from the mutated allele in myotonic dystrophy muscle. Biochem. Mol. Biol.
Int., 29, 291–297.
25. Hofmann-Radvanyi, H., Lavedan, C., Rabes, J., Savoy, D., Duros, C.,
Johnson, K. and Junien, C. (1993) Myotonic dystrophy: Absence of CTG
enlarge transcript in congenital forms and low expression of the normal allele.
Hum. Mol. Genet., 2, 1263–1266.
26. Carango, P., Noble, J.E., Marks, H.G. and Funange, V.L. (1993) Absence of
myotonic dystrophy protein kinase (DMPK) mRNA as a result of a triplet
expansion in myotonic dystrophy. Genomics, 18, 340–348.
27. Hofmann-Radvanyi, H., Junien, C. (1993) Myotonic dystrophy: Over-expression or/and under expression? A critical review on a controversial point.
Neuromusc. Disord., 3, 497–501.
28. Koga, R., Nakao, Y., Kurano, Y., Tsukahara, T., Nakamura, A., Ishiura, S.,
Nonaka, I. and Arahata, K. (1994) Decreased myotonic-protein kinase in the
skeletal and cardiac muscles in myotonic dystrophy. Biochem. Biophys. Res.
Commun., 202, 577–585.
29. Sabourin, L.A., Mahadevan, M.S., Narang, M., Lee, D.S.C., Surh, L.C. and
Korneluk, R.G. (1993) Effect of myotonic dystrophy (DM) mutation on
mRNA levels of the DM gene. Nature Genet., 4, 233–238.
30. Timchenko, L., Nastainczyk, W., Schneider, T., Patel, B., Hofman, F. and
Caskey, C.T. (1995) Full-length myotonin-protein kinase (72 kDa) displays
serine kinase activity. Proc. Natl. Acad. Sci. USA, 92, 5366–5370.
31. Whiting, E.J., Waring, J.D., Tamai, K., Somerville, M.J., Hincke, M., Staines,
W.A., Ikeda, J. and Korneluk, R.G. (1995) Characterization of myotonic
protein kinase (DMK) protein in human and rodent muscle and central
nervous tissue. Hum. Mol. Genet., 4, 1063–1072.
32. Wang, Y., Amirhaeri, S., Kang, S., Wells, R.D. and Griffith, J.D. (1994)
Preferential nucleosome assembly at DNA triplet repeats from myotonic
dystrophy gene. Science, 265, 669–671.
33. Wang, Y. and Griffith, J. (1995) Expanded CTG triplet blocks from the
myotonic dystrophy gene create the strongest known natural nucleosome
positioning elements. Genomics, 25, 570–573.
121
34. Krahe, R., Ashizawa, T., Abbruzzese, C., Roeder, E., Carango, P., Giacenelli,
M., Funanage, V.L. and Siciliano, M.J. (1995) Effect of myotonic dystrophy
trinucleotide repeat expansion on DMPK transcription and processing.
Genomics, 28, 1–14.
35. Wang, J., Pegoraro, E., Menegazzo, E., Gennarelli, M., Hoop, R.C., Angelini,
C. and Hoffman, E.P. (1995) Myotonic dystrophy: evidence for a possible
dominant-negative RNA mutation. Hum. Mol. Genet., 4, 599–606.
36. Taneja, K.L., McCurrach, M., Schalling, M., Housman, D. and Singer, R.H.
(1995) Foci of trinucleotide repeat transcripts in nuclei of myotonic
dystrophy cells and tissues. J. Cell. Biol., 128, 995–1002.
37. Jansen, G., Bachner, D., Coerwinkel, M., Wormsamp, N., Hameister, H. and
Wieringa, B. (1995) Structural organization and develomental expression
pattern of the mouse WD-repeat gene DMR-N9 immediately upstream of the
myotonic dystrophy locus. Hum. Mol. Genet., 4, 843–852.
38. Yano-Yanagisawa, H., Li, Y., Wang, H. and Kohwi, Y. (1995) Single-stranded
DNA binding proteins isolated from mouse brain recognize specific
trinucleotide repeat sequences in vitro. Nucleic Acids Res., 23, 2654–2660.
39. Richards, R.I., Holman, K., Yu, S. and Sutherland, G.R. (1993) Fragile X
syndrome unstable element, p(CCG)n and other simple tandem repeat
sequences are binding sites for specific nuclear proteins. Hum. Mol. Genet., 2,
1429–1435.
40. Meier, J.L., Luo, X., Sawadogo, M. and Straus, S.E. (1994) The cellular
transcription factor USF cooperates with varicella-zoster virus immediateearly protein 62 to symmetrically activate a bidirectional viral promoter. Mol.
Cell. Biol., 14, 6896–6906.
41. Weber, J.L., May, P.E. (1989) Abundant class of human DNA polymorphisms
which can be typed using the polymerase chain reaction. Am. J. Hum. Genet.,
44, 388–396.
42. Reddy, S., Rayburn, H., Davis, B., Balice-Gordon, R.J., Cros, D. and
Housman, D. (1994) Anonymous Targeted Disruption of a Murine Homologue of the Myotonic Dystrophy Kinase. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, pp. 259.
43. Wilson, D.R., Juan, T.S., Wilde, M.D., Fey, G.H. and Darlington, G.J. (1990)
A 58-base-pair region of the human C3 gene cofers synergistic inducibility by
interleukin-1 and interleukin-6. Mol. Cell. Biol., 10, 6181–6191.
44. Blau, H.M. and Webster, C. (1981) Isolation and characterization of human
muscle cells. Proc. Natl. Acad. Sci. USA, 78, 5623–5627.
45. Timchenko, N., Wilson, D.R., Taylor, L.R., Abdelsayer, D., Wilde, M.D.,
Sawadogo, M. and Darlington, G.J. (1995) Autoregulation of the human
C/EBPa gene by stimulation of upstream stimulatory factor binding. Mol.
Cell. Biol., 15, 1192–1202.
46. O′Brien, R.M., Lucas, P.C., Yamasaki, T., Noisin, E.L. and Granner, D.K.
(1994) Potential convergence of insulin and cAMP signal transduction
systems at the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter
through CCAAT/enhancer binding protein (C/EBP). J. Biol. Chem., 269, 30
419–30 428.