From the Genetics Unit, Department of Biochemistry,
University of Oxford, South Parks Road, Oxford OX1 3QU, United
Kingdom
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ABSTRACT |
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Dystrobrevin, a dystrophin related protein, was originally
identified from the Torpedo californica electric organ as an
87-kDa phosphoprotein associated with the cytoplasmic face of the
postsynaptic membrane (1, 2). The protein is expressed in the electric organ, skeletal muscle, and brain, and it has been postulated to play a
role in synaptic structure and function, because it copurifies with the
acetylcholine receptors and rapsyn from the electric organ membranes.
The protein is concentrated with the acetylcholine receptors at the
synapse, but it is also found extrasynaptically at the sarcolemma of
both Torpedo electric organ and vertebrate muscle.
Furthermore, dystrobrevin is also found in association with dystrophin
and the 58-kDa syntrophins in the Torpedo electric organ (3,
4).
By contrast to the single known Torpedo dystrobrevin
molecule, cDNAs encoding several different isoforms differing in
their domain content and tissue distribution have been identified in both human and mouse (5-7). The longest isoform, Considerable evidence supports an association of dystrophin and
In view of the similarity between dystrophin and Isolation, Characterization, and Sequencing of Genomic
Clones--
To determine the order and estimate the distance between
the 5'-UTR exons, a BAC (mouse 129/sv) genomic library was screened (Research Genetics, Huntsville, AL) by a PCR approach using
primer sets specific for the 5'-UTR exon A (RnpAf,
5'-GGGGGAAAAGAATCTGACTCTGT-3'; RnpAr, 5'-CTGCTTTTGTAGTTCACGCAC-3'),
exonB (RnpBf, 5'-GTGCGTGCGCGTCCGTGG-3'; RnpBr,
5'-CTCGCCAAACTCTTAGAAGGTG-3'), exonC (RnpCf,
5'-GCATCTGCCAGTGGGACTTC-3'; RnpCr, 5'-CCAGGTACAGCATCCTTTCTTC-3'), exon
D (UTRDf1, 5'-GATAAATAGGATTTACAAGCC-3'; UTRDr,
5'-GAAGAGACAGCATGGACTTT-3'); and coding exon3 (DB3f,
5'-ACATAGAACTCAACGTGGCC-3'; DB3r1, 5'-GTGGATTTGGTGAGTGGTTG-3').
Selected BAC clones, which were positive for more than one exon, were
isolated and further analyzed. To establish a rare-cutter restriction
enzyme map, 0.25 µg of mBAC DNA was digested with MluI,
NotI, SalI, and XhoI and separated on
a 1% agarose gel using the FIGE Mapper Electrophoresis System
(Bio-Rad). After transfer to a nylon membrane (Hybond-N+,
Amersham Pharmacia Biotech), the DNA was probed sequentially with
exon-specific oligonucleotide sequences derived from the UTR exons A-G
and coding exon 1 (UTRAf, 5'-CGGAAGAGTTAGAGGCATGTTG-3'; UTRBf,
5'-GGTGACACAGGCGCCGGTCC-3'; UTRCf, 5'-TGGCTAAATCTGTTCTCCCATG-3'; UTRDf2, 5'-GTCTTCAAATGCCACAGTCAGC-3'; DB1s,
5'-GATTGAAGATAGTGGAAAAAGAGG-3'; UTREf1, UTRFf1, UTRGf1, see Fig.
1). To isolate genomic DNA upstream and downstream of the 5'-UTR exons
E, F, and G, which were detected in a minority of RNA Extraction, Northern Blotting, and RNase Protection--
RNA
extraction, Northern blots, and RNase protection assays were performed
as described previously (22). Genomic regions spanning the most 5'-end
exons A, B, and C, and their adjacent 5'-flanking sequences were
amplified by PCR using the primer sets RnpAf/RnpAr for exon A,
RnpBf/RnpBr for exon B, and RnpCf/RnpCr for exon C, subloned into
pGEM-T, and used to generate cRNA probes.
RT-PCR--
1 µg of denaturated total RNA from mouse tissues
or cultured cells was converted into first strand cDNA with Expand
Reverse Transcriptase (Life Technologies, Inc.). 2 µl of diluted
first strand reactions (1:5 in water) were used in 50 µl of PCR
reactions, performed under standard conditions using Taq DNA
polymerase (Boehringer Mannheim). UTR exon containing transcripts were
amplified using oligonucleotides derived from exons A-G (UTRAf-Gf)
and an oligonucleotide derived from exon 3 (DB3r2,
5'-CGTTCCAAATGTCCACCAGG-3'). Transcripts encoding different
5'-RACE--
5'-RACE was carried out using the 5' AmpliFINDER
RACE kit (CLONTECH). 1 µg of poly(A)+
RNA isolated from mouse tissues was reverse transcribed using a
primer derived from exon 6 (DB6r, 5'-TGCAGAAGAGGCAGCCATACC-3'). The anchor ligated cDNA was then PCR amplified using the
anchor primer and the internal primer derived from exon 3 (DB3r2,).
PCR-products were cloned into pGEM-T vector (Promega), and the largest
were sequenced using Sequenase v2.0 (U. S. Biochemical Corp.) The
sequences of 20 exon A-, B-, or C-containing clones were aligned using
PILE UP (Genetics Computer Group, version 8.0; Madison, WI).
Construction of Promoter-Reporter Fusion
Plasmids--
Mouse genomic DNA and the primer set ProAf/ProAr
(5'-CGCGGATCCTCCAGTGAGGGAAGGCAG-3',
5'-CGCGGATCCCTCTAACTCTTCCGCAG-3') was used to
amplify a 1068-bp product spanning the 858-bp flanking region and the
first 210 bp of exon A. Accordingly, a 666-bp product, including the
528-bp flanking region, and the first 138 bp of exon B and a 1169-bp
product spanning the 994-bp flanking region and the first 175 bp of
exon C were amplified using the primer set ProBf/ProB
(5'-CGAAGATCTGATAGGCAACCACTGCACAC-3', 5'-CGAAGATCTTGCAGGGAGGTGTGGGCC-3') and
ProCf/ProCr
(5'-CGCGGATCCCAGACTGATTGGGACATGTG-3', 5'-CGCGGATCCTTTAGTTCCAGTTCATCTAGAG-3').
After digestion with BamHI or BglII, the
PCR products were subcloned into the BglII site of the pGL-3
basic vector.
Cell Culture, Transfection Procedures, and Luciferase and
Immunoblot Analysis--
Western blots using the primary
antibody 5'-UTR Exons and Alternative Splicing--
We have described
multiple transcripts for mouse
Comparison of the nucleotide sequences of the seven 5'-UTR exons to the
sequences of 5'-RACE products from brain and heart revealed a complex
pattern of alternative splicing. As indicated in Fig. 2B,
exon A was spliced directly to exon 1, the first protein-coding exon,
while both exon B and exon C were separated from exon 1 by a further
exon, exon D. In a minority of RACE clones, one or the other of three
additional 5'-UTR exons, exons E, F, and G, were spliced between exons
B and D. All RACE clones were identical in sequence downstream of exon
1. These results suggest that the different dystrobrevin transcripts
are generated by differential usage of three promoters.
Mapping of Transcriptional Initiation Sites--
Twenty RACE
clones for each type of end-terminal UTR exon (exons A, B, and C) were
sequenced and provide an indication of the location of transcriptional
start sites. The majority of the exon A-positive clones terminated 239 bp upstream of the 3'-end of exon A. By contrast, RACE clones positive
for exon B and exon C ended at several different points, but in each
case there was a cluster of clones. Thus for exon B clones terminated
at nucleotide
To confirm the location of the cap sites for the various
Nucleotide Sequence of Regions Upstream of 5'-UTR Exons A, B, and
C--
To identify putative regulatory promoter elements that flank
the most upstream 5'-UTR exons, we sequenced a 858-, 528-, and 994-bp
upstream of exons A, B, and C, respectively (Fig.
4). In common with other genes with
multiple transcription start sites, no typical TATA or CAAT box was
present in the most distal promoter B region. However, we found the
motif GCTCCC downstream of the ~100-bp multiple start site window,
which is identical to a conserved downstream element defining a new
subclass of RNA-polymerase II promoters (26). Computer-assisted
analysis revealed several putative transription factor binding sites
including Sp1 and Ap2. The promoter C sequence also contained no TATA
box in the first 100 bp, although a CAAT box consensus was observed at
nucleotide Promoter Activities in the 5'-Flanking Regions of Upstream
Exons--
Genomic fragments containing the putative promoter regions
A, B, and C (Fig. 4) were cloned upstream of the luciferase gene. These
constructs were then transiently transfected into the mouse fibroblast
NIH3T3 and mouse myogenic H2K-tsA58 cell lines. All constructs directed
luciferase expression in NIH3T3 cells (Fig. 5A). The level of luciferase
activity produced by the constructs containing sequences of the
5'-flanking regions of exon B and exon C was substantially higher in
forward (Bf and Cf) than in reverse orientation (Br and Cr). The
promoter strength of the construct Bf was comparable with the activity
of the herpes simplex virus thymidine kinase promoter used as a
control. The construct Cf was about 3-fold stronger than this promoter.
The 5'-flanking region of exon A showed orientation-independent
expression in this cell line and was excluded from further
analysis.
To investigate the effect of myoblast differentiation on
In order to determine the endogenous transcription pattern of
To determine whether exon C-containing transcripts encode all three
Western blot analysis of protein extracts prepared from H2K cells at
different time points during myoblast fusion using an antibody against
Tissue-selective Expression of
To determine whether mRNA species encoding the three major
In the present study we have identified multiple promoters that
control the expression of the The In cases in which a promoter switch does not affect the coding region,
as in the The regions upstream of the 5'-UTR exons A and B of the
Our RT-PCR studies clearly indicate that in H2K-tsA58 myoblast the
transcriptional activation of the -Dystrobrevin, the mammalian orthologue of the
Torpedo 87-kDa postsynaptic protein, is a
dystrophin-associated and dystrophin-related protein. Knockout of the
gene in the mouse results in muscular dystrophy. The control of the
-dystrobrevin gene in the various tissues is therefore of interest.
Multiple dystrobrevin isoforms differing in their domain content are
generated by alternative splicing of a single gene. The data presented
here demonstrate that expression of
-dystrobrevin from three
promoters, that are active in a tissue-selective manner, also plays a
role in the function of the protein in different tissues. The most
proximal promoter A is active in brain and to a lesser extent in lung, whereas the most distal promoter B, which possesses several Sp1 binding
sites, is restricted to brain. Promoter C, which contains multiple
consensus myogenic binding sites, is up-regulated during in
vitro myoblast differentiation. Interestingly, the organization and the activity of the
-dystrobrevin promoters is reminiscent of
those in the dystrophin gene. Taken together we suggest that the
multipromoter system, distributed over a region of 270 kilobases at the
5'-end of the
-dystrobrevin gene, has been developed to allow the
regulation of this gene in different cell types and/or different
developmental stages.
INTRODUCTION
Top
Abstract
Introduction
References
-dystrobrevin-1 (94 kDa), has a tyrosine kinase substrate domain similar to the Torpedo protein (2, 5), in addition to a ZZ- (8) and two
predicted
-helical-coiled coil domains (9), which it shares with
-dystrobrevin-2, while
-dystrobrevin-3 has simply the ZZ-domain. The genetic basis of this isoform diversity and additional alternative splicing was resolved by the determination of the genomic organization of the coding region of a single gene on mouse chromosome 18 (6). The
conservation of the genomic organization between dystrophin and
-dystrobrevin is maintained across the homologous
CRCT,1 indicating that both
genes evolved from an ancestral duplication event (10).
-dystrobrevin. Dystrophin and
-dystrobrevin colocalize in skeletal muscle, copurify biochemically, and associate directly in vitro via the coiled-coil region of dystrophin (11-13).
The expression pattern of the
-dystrobrevin gene also closely
parallels that of dystrophin, where a set of diverse isoforms are
generated in vivo by alternative splicing in brain and
muscle. Dystrophin expression in muscle and brain results in three
14-kb transcripts (muscle-type, brain-type, and Purkinje cell-type),
controlled by promoters at the 5'-end of the gene (14, 15). In
addition, a whole family of smaller mRNAs are transcribed from
promoters lying between exons within the rod domain of the gene. The
first of these transcripts designated Dp71 (apodystrophin-1) and
apodystrophin-3, encode the CRCT, or the first part of the CRCT,
respectively, and are expressed predominantly in brain and non-muscle
tissues from a housekeeping like promoter (16, 17). Other smaller transcripts are expressed in a tissue-specific manner, such as in
peripheral nerve (Dp116, Ref. 18), the retina, brain, and cardiac
muscle (Dp260, Ref. 19), and throughout the central nervous system
(Dp140, Ref. 20).
-dystrobrevin, and
the recent evidence that mice null for
-dystrobrevin suffer from
muscular dystrophy and impaired aggregation of acetylcholine receptors
(21), it was of interest to determine the regulation of this gene. Our
preliminary evidence indicated a minimum of four 5'-UTR exons,
suggesting that the regulation of the
-dystrobrevin gene might be as
complex as dystrophin. While in the process of investigating whether
particular
-dystrobrevin isoforms are associated with a specific
5'-UTR region, we identified additional 5'-UTR exon sequences,
consistent with a regulation of expression by multiple promoters. Here
we present evidence that the mouse 5'-region of the gene is composed of
seven 5'-UTR exons covering 270 kb of genomic DNA and demonstrate that
-dystrobrevin is expressed from three promoters that are active in a
tissue-selective manner. Our data suggest that this multipromoter
system of the
-dystrobrevin gene has been developed to allow the
regulation of this gene in different cell types and/or different
developmental stages.
EXPERIMENTAL PROCEDURES
-dystrobrevin
cDNAs, a YAC vectorette library of the YAC clone ICRFy902M0312Q was
screened by PCR using hemi-nested primer sets made from the sequences
of exon E, F, and G (see Fig. 1) and a universal vectorette primer as
described previously (6). Vectorette PCR products were cloned into
pGEM-T vector (Promega) and sequenced using Sp6 and T7 primers. Splice
junction sequences were determined by nucleotide alignment of genomic
sequence to the cDNA sequence using BESTFIT and GAP (Genetics
Computer Group, version 8.0; Madison, WI).
-dystrobrevin isoforms were amplified by PCR using a common
oligonucleotide derived from exon 8 (DB8f,
5'-CCTGAGCTGTGCTTCCAGCCGTG-3') and oligonucleotides specific for
-dystrobrevin-1 (DB21r, 5'-AGGCAGATGCTGAACGGATG-3'),
-dystrobrevin-2 (DB18r, 5'-AGCAATGAGAAGGTCAGCAGGAC-3'), and
-dystrobrevin-3 (DB11r, 5'-TCATGTTATCCATCTAGACGC-3'). PCR
products were detected by Southern hybridization with an
oligonucleotide derived from exon 1 (DB1s) or a 416-bp
NsiI/HindIII fragment of the m32 cDNA clone
(5).
-Galactosidase Assays--
H-2Kb-tsA58 cells (23) were
propagated at 33 °C in Dulbecco's modified Eagle's medium
supplemented with 20% fetal calf serum, 2% chicken embryo extract,
and 20 units/ml mouse recombinant interferon-
(Life Technologies,
Inc.) (growth medium). Myoblast fusion was induced by culturing the
cells in Dulbecco's modified Eagle's medium supplemented with 5%
horse serum and 1% chicken embryo extract (differentiation medium) at
39 °C in 10% CO2. H-2Kb-tsA58 myoblasts at
approximately 40% confluence were transiently transfected with 2 µg
of luciferase transporter construct and 0.5 µg of
pSV-
-galactosidase plasmid (Promega) using Superfect protocol
(Qiagen). The cells to be maintained as myoblasts were incubated in
growth medium throughout the experiment, whereas cells to be
differentiated into myotubes were switched to differentiation medium
after reaching 80% confluence (usually within 24 h). Cells were
harvested 72 h after the start of transfection. NIH3T3 cells were
transfected in the same manner and harvested 60 h after the start
of transfection. Each transfection was performed in triplicate and
repeated twice. Cells were washed twice with phosphate-buffered saline,
harvested into 200 µl of Reporter Lysis Buffer (Promega). Cell
extract (20 µl) was mixed with 100 µl luciferase assay reagent (Promega) and light production quantified using a Turner Designs Model
20 luminometer.
-Galactosidase activity was measured using an enzyme
assay system (Promega) and then analyzed using a spectrophotometer at
420 nm.
-CT-FP (diluted 1:1000) and Horseradish
peroxidase-conjugated secondary antibody, donkey anti-mouse (diluted
1:5000; Jackson ImmunoResearch Laboratories, Inc.) were performed as
described previously (24).
RESULTS
-dystrobrevin that encode three
protein isoforms (5, 6, 11). Some of these transcripts differed in
their 5'-UTRs, suggesting that they may be by alternative splicing of
different 5'-exons, indicative of transriptional regulation by multiple
promoters. To determine the origin of these transcripts and the
mechanism of
-dystrobrevin expression, we characterized genomic
fragments covering the 5'-end of the gene. We have reported previously
the exon-intron boundaries of four UTR exons A-D, derived from
different cDNA clones. Here, 5'-UTR sequences have been extended to
their 5'-ends by sequencing of RACE products of the dystrobrevin
mRNAs from mouse heart and brain. A PCR approach on a YAC
vectorette library of the YAC clone ICRFy902M0312Q positive for the
dystrobrevin gene was used to clone three additional 5'-UTR exons and
determine their exon-intron boundaries (Fig.
1). Exon sizes and splice junctions
sequences of all seven 5'-UTR exons are summarized in Table
I. The splice junctions have the
conserved GT and AG dinucleotides present at the 5' splice donor and 3'
splice acceptor sites (25). The three most upstream exons, exons A, B,
and C, are all of similar size and are larger than the internal exons
D-G. Mapping of the 640-kb YAC clone ICRFy902M0312Q
indicated that the maximum distance between the UTR exons is
approximately 270 kb. To determine the order and estimate the distance
between the UTR exons, a mouse BAC genomic library was screened using
primer pairs for all 5'-UTR exons and the coding exon 3. This resulted
in the isolation of three overlapping BACs, two of which (BAC 343 and
BAC 547) had insert sizes of 170 and 140 kb and contained the first
coding exon, UTR exons A and D. The third BAC, with an insert size of
160 kb, contained the other five UTR exons: exons C, G, E, F, and B. Fig. 2A shows the approximate
positions of the seven UTR exons spanning a distance of approximately
270 kb relative to the 25 coding exons of the gene.
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Fig. 1.
The nucleotide sequence of
the -dystrobrevin 5'-UTR exons E, F, and
G. The exon sequences are in uppercase letters; the
intron sequences are in lowercase letters. Primers used for
YAC vectorette PCR are indicated.
Exon-intron boundaries of the -dystrobrevin 5'-UTR exons
-dystrobrevin gene (6).
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Fig. 2.
Genomic organization and alternative splicing
of -dystrobrevin 5'-UTR exons.
A, long range restriction map of the 5'-end of the
-dystrobrevin gene. The three promoters (arrows) and the
approximate position of exons A-G in the first 270 kb of the
-dystrobrevin gene are shown relative to the 25 coding exons 1-25.
Data on placement of exons 1-25 are derived from previous studies (6).
Restriction sites for MluI, NotI,
SalI, and XhoI are indicated by M,
N, S, and X, respectively. Exons are
shown as open boxes. The positions of the mBAC clones (mBAC
467, mBAC 547, and mBAC 343) are shown above the gene. B,
schematic illustration of the predominant splicing patterns in brain
and muscle. The previously described m24, m21, and m32 transcripts (5)
are generated by alternative usage of exons A, B, C, and D (open
boxes) with lines indicating regions that are spliced out of the
primary transcripts. In a minority of m32 transcripts one or the other
of three internal exons, exons F, E, and G (black boxes),
were found to be spliced between exon B and D and therefore generate
further complexity in the 5'-untranslated region.
95,
110,
121,
128,
134,
142,
148,
197,
and
200, where the 3'-end of the exon is designated as
1. Similarly
transcription initiation sites were identified at nucleotide positions
121,
146,
171,
185, amd
209 for exon C. In all three
populations of RACE clones we did not find any clones that were spliced
within these regions. Moreover, there are no multiple AG sequences to serve as splice acceptor sites.
-dystrobrevin mRNAs, three genomic probes were constructed and
used in the RNase protection assay. Probe A was a 299-bp PCR fragment of exon A and its 5'-flanking region. Using this probe, a major 243-bp
fragment was protected by mRNA from mouse brain, heart, lung, and
skeletal muscle. Additionally, a second product at 170 bp was also
evident, albeit at very low intensity, indicating that this fragment
might reflect a minor transcription start site (Fig.
3). By contrast probe B, a 268-bp PCR
fragment of exon B and its 5'-flanking region, revealed multiple
protected products ranging in size from 223 to 109 bp. None of these
fragments were protected by mRNA from lung or muscle. Using a
258-bp PCR fragment of exon C and its 5'-flanking region revealed major
protected products at 209, 195, 164, and 135 bp. Interestingly, the
intensity of the 135-bp protected fragment was significantly higher in
skeletal muscle than in all the other tissues. This protected fragment probably reflects a preferentially used cap site for
-dystrobrevin mRNAs in skeletal muscle. Weak muscle-specific products at 183 and
175 bp were also detected. In summary, multiple transcription start
sites for all three promoters were detected.
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Fig. 3.
Mapping of transcription initiation sites in
the -dystrobrevin gene. 40 µg of total
RNA from the indicated tissues was hybridized to antisense cRNA probes
shown at the bottom. An RNase protection assay was then
performed as described under "Experimental Procedures." The
following fragments were used as templates to synthesize cRNA probes:
in A a 299-bp PCR fragment of exon A and its 5'-flanking
region; in B a 268-bp fragment of exon B and its 5'-flanking
region; in C a 258-bp fragment of exon C and its 5'-flanking
region; H, heart; L, lung; B, brain,
S, skeletal muscle; Y, yeast tRNA; DNA size
markers are designated by number of base pairs.
337. Interestingly, we identified multiple sequences
within a 370-bp region known to mediate skeletal and cardiac muscle
expression of a number of genes. These include four conserved MEF-1
motifs (27) at
294,
234,
150, and
48, a TGCCTGG motif at
363
(28), and a M-CAT motif at
30 (29), where the most distal cap site is
designated as +1. Examination of the most proximal promoter A sequence
did not reveal a consensus TATA box or binding sites for common
transcription factors such as Sp1 or CAAT factors, and the sequence was
not GC-rich. The major initiation site is located within a
pyrimidine-rich sequence, TTTTGTCAGTCTTTT (cap site
underlined). This sequence is similar to the initiator (Inr) consensus
sequence, 5'-PydPyd-CA-PydPydPydPydPyd, that can direct specific
transcription initiation in TATA less and non-GC-rich promoters (30).
Several putative transcription factor sites, inclucding AP1 and PuF,
were found within the promoter A sequence.
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Fig. 4.
Nucleotide sequence of the
-dystrobrevin gene in regions upstream of exons A
(A), B (B), and C
(C). The positions of the transcription
initiation sites estimated by RNase protection are marked by
black inverted triangles. The most 5'-cap site is designated
as +1 and nucleotides that precede it are indicated by
negative numbers. Putative transcription factor binding
sites are shown. Exon sequences are in lowercase. MED-1 (26)
is shown in a gray box. The initiator sequence (Inr) located
within the major transcription start site of exon A is double
underlined. Tracts of alternating dinucleotide repeats located in
the upstream regions are overlined. Different putative
transcription factor binding sites, including Sp1, Ap1, Ap2, CBF, PuF,
NFkB, TEF-1, and MEF-1, are underlined.
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Fig. 5.
Analysis of the activities of
-dystrobrevin promoter fragments fused to the
luciferase reporter gene in different cell lines. The 528-bp
flanking region and the first 137 bp of exon B and the 994-bp flanking
region and the first 175 bp of exon C were fused to the pGL-3 vector in
forward (Cf and Bf) and reverse orientation
(Cr and Br) and transiently transfected into
NIH3T3 fibroblasts (A) or H2K-tsA58 myoblasts
(B). H2K-tsA58 cells were either allowed to remain myoblasts
or were induced to form myotubes; L-MB, late
H2K-tsA58 myoblasts; MT, differentiated H2K-tsA58 myotubes
(day 2 after switching to differentiation medium). TK refers
to a control plasmid (pXp2luc) containing the herpes simplex virus
thymidine kinase promoter fused to the luciferase gene. pGL3 represents
a pGL3 basic vector without an insert. The luciferase activity of each
construct was normalized in comparison with co-expressed
-galactosidase activity. The relative luciferase activities are
shown as a percentage of the activity of either pXp2luc (in NIH3T3
cells) or Cf constructs (in H2K-tsA58 myoblasts), which were arbitarily
set to 100%. Error bars on graphs and numerical errors on
overall percentages are shown.
-dystrobrevin expression, we transfected the aforementioned
constructs into H2K-tsA58 myoblasts and analyzed luciferase activity in
total cell extracts prepared from H2K-tsA58 muscle cells at different time points during myoblast fusion. The construct Cf exhibited higher
luciferase activity in myotubes than in undifferentiated myoblasts,
with a ~200-fold increase in differentiated H2K-tsA58 myotubes over
that obtained with the promoter construct Bf (Fig. 5B),
which showed similar low levels of activity in myoblasts and myotubes.
Luciferase activities in extracts of cells transfected with a construct
containing the same region in reverse orientation (Cr and Br) and a
promoterless pGL-3 basic vector are shown as negative controls.
-dystrobrevin in the H2K-tsA58 cell line, total RNA was isolated from myoblast cultures at various stages of differentiation. First, the
differential usage of 5'-UTR sequences was investigated by RT-PCR using
UTR exon-specific forward primers and a common reverse primer in exon
3. PCR products were detected by hybridization to an oligonucleotide
probe derived from exon 1. Little or no exon A or exon B containing
transcripts could be detected in this cell line at the different time
points tested (Fig. 6A). By
contrast, levels of exon C-containing transcripts increased steadily
from very low levels at the nonconfluent myoblast stage to higher
levels in cultures containing differentiated multinucleated myotubes. A
370-bp amplification product of utrophin was present at all different
time points at similar levels, indicating that its expression was not
affected during myogenesis (11).
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Fig. 6.
Expression of
-dystrobrevin isoforms in H2K-tsA58 myoblasts.
Total RNA was prepared from H2K-tsA58 muscle cells at different time
points during myoblast differentiation. MB, early
(E) proliferating and late (L) confluent
myoblasts; MT, different time points (in days, d)
during myotube formation. A, autoradiographs of Southern
blots of PCR products (20 cycles), which were amplified from same
amounts of cDNA using forward primers specific for the 5'-UTR exons
A-C and a common reverse primer. An amplification product of utrophin
was used as a positive control. The blot was hybridized with an
internal specific oligonucleotide derived from exon 1. The sizes of the
PCR products in bp are indicated on the right of the figure.
B, autoradiographs of Southern blots of PCR products (20 cycles), which were amplified from same amounts of cDNA using a
common forward primer derived from exon 8 and reverse primers specific
for
-dystrobrevin-1,
-dystrobrevin-2, and
-dystrobrevin-3,
respectively. The blot was hybridized with a radiolabeled 416-bp
NsiI/HindIII restriction fragment of the m32
cDNA clone (5). C, total cell extracts were prepared
from H2K-tsA58 cells at time points indicated above. 20 µg of protein
were separated on 8% SDS-polyacrylamide gels, blotted, and probed with
an antibody against
-dystrobrevin-1.
-dystrobrevin isoforms, we performed RT-PCR using a common forward
primer and isoform-specific reverse primers. Southern blot analysis of
the PCR products using a radiolabeled m32 cDNA probe (5) revealed
single bands for all three dystrobrevin isoforms in proliferating
myoblasts (Fig. 6B). After switching to differentiation
medium, the level of
-dystrobrevin-3 (Fig. 6B,
-db-3) increases and then stabilizes as myoblast fusion
proceeds. In the case of
-dystrobrevin-1 (Fig. 6B,
-db-1) and -2 (Fig. 6B,
-db-2),
an additional upper band appears at increased levels, whereas the lower
band seen in proliferating myoblasts remains at almost at the same
level. This amplification product probably reflects an alternatively
spliced form of
-dystrobrevin-1 and -2 containing the vr3 sequence
(5).
-dystrobrevin-1 detects two proteins of similar relative mobility
(Fig. 6C). Consistent with our detection of
-dystrobrevin-1 mRNA (Fig. 6B,
-db-1),
the protein is found at low levels in undifferentiated H2K-tsA58
myoblasts. After switching to differentiation medium,
-dystrobrevin-1 becomes more abundant and remains expressed at
constant levels as myoblast fusion proceeds. At this time, the
muscle-specific splice variant of
-dystrobrevin-1 containing the vr3
sequence is also detected (5). Similar results were obtained for
-dystrobrevin-2 from expression studies in the myogenic C2C12 cell
line (11). Taken together these results indicate that in the H2K-tsA58
cell line the transcriptional activation of the mouse
-dystrobrevin
gene occurs upon differentiation of myoblasts into multinucleated
myotubes. Promoter C, which contains multiple consensus myogenic
binding sites, is active, whereas promoters A and B are not. However it
is formally possible that a fourth as yet unidentified promoter is
active as well.
-Dystrobrevin mRNAs
Containing Different 5'-UTR Exons--
To examine the expression
pattern of UTRs originating from the three putative dystrobrevin
promoters, RT-PCR on the same amount of total RNA isolated from mouse
brain, lung, skeletal, and cardiac muscle was performed using UTR
exon-specific forward primers and a common reverse primer in exon 3. Fig. 7 indicates that exon A was
predominantly expressed in brain and to a lower extent in lung. By
comparison, exon C was highly expressed in muscle tissues, with
significant but lower expression in brain and lung, and expression of
exon B was only found in the brain. Primers for the exons E, F, and G
produced products of the expected size in brain, confirming that they
are expressed and are not artifacts of the RACE protocol. However,
there was no evidence for the presence of exon F and exon G in
mRNAs derived from exon B transcripts in brain, indicating that
additional promoters might be present within this huge control region
of the
-dystrobrevin gene.
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Fig. 7.
Tissue-selective usage of
-dystrobrevin 5'-UTR exons. Autoradiographs of
Southern blots of PCR products (20 cycles), which were amplified from
same amounts of cDNA using primers specific for 5'-UTR exons A-G
and a common reverse primer derived from exon 3. The blot was
hybridized with a common oligonucleotide derived from exon 1. The sizes
of the PCR products in bp are indicated on the left.
H, heart; L, lung; B, brain,
S, skeletal muscle.
-dystrobrevin isoforms are associated with a particular promoter, we
hybridized 5'-UTR exon-specific probes to multiple tissue Northern blots. The hybridization patterns are summarized in Fig.
8. Hybridization with a common probe
spanning exons 1-6 illustrates the five predominant transcripts
encoding the three major isoforms, which are estimated to be 7.5 and
4.0 kb (
-dystrobrevin-1), 5.0 and 3.6 kb (
-dystrobrevin-2), and
1.7 kb (
-dystrobrevin-3) in size as described previously. The main
difference in the length of these transcripts is due to differential
splicing of three exons containing stop codons and alternative usage of
polyadenylation sites (except for
-dystrobrevin-3). All three UTR
exon probes hybridized to the dystrobrevin-1 and dystrobrevin-2
transcripts in brain. Both exon A and exon C sequences were found in
the full-length dystrobrevin-1 transcript expressed in lung and in a
3.8-kb transcript that has not been assigned to any of the three
isoforms (6). The 1.7-kb transcript corresponding to the recently
described muscle expressed
-dystrobrevin-3 isoform (11) was detected
by the exon C probe in muscle, but also hybridized, albeit weakly, with
the exon A probe in brain and possibly lung. These results indicate
that the promoters are active in a tissue-selective rather than a
tissue-specific manner and that the formation of individual
dystrobrevin variants is independent of promoter activity and
is probably the result of a post-transcriptionally regulated process.
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Fig. 8.
Northern blot analysis of mouse
-dystrobrevin mRNAs containing different 5'-UTR
exons. Approximately 2 µg of poly(A)+ RNA from the
indicated tissues were hybridized consecutively with each of the
following probes shown at the bottom of the figure: a
1.25-kb BglII-HindIII fragment of m32 cDNA
clone, that detects all dystrobrevin isoforms, a 299-bp PCR fragment
specific for 5'-UTR exon A, a 268-bp fragment specific for 5'-UTR exon
B, and a 258-bp PCR fragment specific for 5'-UTR exon C. H,
heart; L, lung; B, brain; S, skeletal
muscle. The major hybridizing transcripts (described in the text) are
indicated on the left, and the sizes of molecular weight
markers in kb are shown to the right.
DISCUSSION
-dystrobrevin gene. The exon-intron arrangement of the unusually long 5'-end of the gene explains the
origin of the different mRNA species that we have characterized (Ref. 5 and Fig. 2B). It is clear from the structure of the gene that both the alternative use of three promoters and the differential splicing of the resulting transcripts is involved in the
generation of the multiple
-dystrobrevin mRNAs. The most distal
brain promoter is separated by 120 kb of genomic sequence from the
muscle promoter. A third promoter located 70 kb further downstream is
separated by another 80 kb of genomic sequence from exon 1, which
contains the translational start site. Remarkably, the region
containing the seven small UTR exons A-G spans 270 kb of genomic DNA.
Considering that the
-dystrobrevin gene is organized into 25 coding
exons contained within a genomic interval of 170 kb, the total size of
the gene can now be estimated as at least 440 kb (6).
-dystrobrevin gene exhibits a structural arrangement similar to
a number of other genes in which multiple promoters generate tissue-specific mRNAs that differ only at their 5'-untranslated region. Four short 5'-noncoding exons of the rat gene for brain-derived neurotrophic factor can be spliced to a common coding exon and are each
regulated by separate promoters (31). These promoters confer
tissue-specific, axotomy- and neuronal activity-induced expression in
transgenic mice (32). Our results show that all three promoters of the
-dystrobrevin gene are active in brain. It is therefore possible
that the
-dystrobrevin transcripts detected in the brain are
transcribed in a region or cell specific manner from these promoters.
Interestingly, the arrangement of the control region of the
-dystrobrevin gene is very similar to that described for the
dystrophin locus, where tissue-specific promoters regulate expression
of full-length dystrophin isofoms (14, 33). One of these promoters,
which is located upstream of the muscle promoter, regulates the
expression of dystrophin in the cortex and hippocampus (34), while a
third promoter, active in brain Purkinje cells, has been identified
between the muscle promoter and the second exon of dystrophin (15).
However, whereas the dystrophin isoforms have different first coding
exons, the dystrobrevin transcripts described utilize the same first
coding exon and ATG.
-dystrobrevin gene, the translation efficiency of the
mRNAs can be affected (35). We found that the long 5'UTRs of the
-dystrobrevin mRNAs contain small partially overlapping upstream
open reading frames that precede the major translation initiation site,
which are highly conserved between human and mouse (data not shown). It
has been suggested that such an arrangement might be particularly
suitable for translational regulation (36). The mouse gene for the
retinoic acid receptor-
2 has a similar complex 5'-UTR exon
organization. Mutation in the small open reading frames of the 5'-UTR
affected expression of the downstream major open reading frame,
resulting in an altered regulation of gene expression in
vivo (37).
-dystrobrevin gene exhibited none of the consensus features that define proximal promoter regions of tissue-specific promoters. The
presence of multiple transcriptional start sites could be expected,
since these regions lack a canonical TATA box-like element conferring a
unique transcription start site (38). This situation is reminiscent of
housekeeping genes (30). Despite the fact that the discovery of
TATA-less promoters is steadily increasing, there is little information
how this multiple selection process occurs. Recently, a protein binding
sequence GCTCCC (MED-1; multiple start
element downstream) was found to be
positionally conserved in a number of promoters that initiate at
multiple unclustered start sites (~100-bp window) and was shown to be
involved in the regulation of this multiple initiation process (26). We
found an identical sequence stretch immediately downstream of the
multiple cap sites in exon B, and therefore the exon B promoter might
be a member of this new subclass of TATA-less promoters.
-dystrobrevin gene occurs upon
differentiation of myoblasts into multinucleated myotubes. Functional
promoter studies show that the 1169 bp promoter C fragment is able to
direct luciferase expression during myoblast differentiation. The
proximal part of this region is composed of cis-acting sequences that
have been implicated in muscle specific expression. Four copies of the
CANNTG motif present in the MyoD target sequence and characteristic of
many muscle-specific regulatory regions (27, 39) and a "M-CAT"
similarity and a TGCCTGG sequence, both of which have been proposed as
muscle-specific motifs (33, 34), are present within a 370-bp region
upstream of the transcription initiation site. Taken together we
suggest that the multipromoter has been developed to allow the
regulation of the
-dystrobrevin gene in different cell types or at
different developmental stages at the level of both transcription and translation.
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ACKNOWLEDGEMENTS |
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We thank Roger Cox for providing PCR pools and BAC clones from the Caltech BAC library, Jenny Morgan for supplying the myogenic H2K-tsA58 cell line, and members of the Genetics laboratory for useful discussions.
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FOOTNOTES |
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* This work was generously supported by grants from the European Communities, the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Wellcome Trust, and the Deutsche Forschungsgemeinschaft (to R. A. N.).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/EMBL Data Bank with accession number(s) AF092287, AF106836, AF106837, AF106838, AF106839, and AF106840.
§ Carried out this work as part of a training program (Training and Mobility of Researchers) financed by the Commission of the European Communities. Present address: Dept. of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, United Kingdom.
¶ Present address: Zeneca Diagnostics, Gadbook Park, Rudheath, Northwich, Cheshire CW9 7RA, United Kingdom.
Present address: Max-Planck-Institut fuer Biochemie, Abteilung
Proteinchemie, D-82152 Martinsried bei Muenchen, Germany.
** Holds a Wellcome Trust Career Development Fellowship.
To whom correspondence should be addressed. Present address:
Dept. of Human Anatomy and Genetics, University of Oxford, South Parks
Road, Oxford OX1 3QX, United Kingdom. Tel.: 44-1865-272179; Fax:
44-1865-272427; E-mail: kay.davies{at}human-anatomy.ox.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are: CRCT, cysteine-rich carboxyl-terminal domain; 5'-UTR, 5'-untranslated region; bp, base pair(s); kb, kilobase(s); YAC, yeast artificial chromosome; RT-PCR, reverse transcriptase-polymerase chain reaction; RACE, rapid amplification of cDNA ends; MED-1, multiple start site element; MEF-1, myocytespecific enhancing factor 1; M-CAT, muscle-CAT heptamer CATTCCT.
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REFERENCES |
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