(Received for publication, June 7, 1994; and in revised form, January 12, 1995)
From the
Analysis of a series of human -myosin heavy chain (MHC)
constructs with progressive deletions in the 5`-flanking region has
localized a strong positive element at positions -298/-277
with a repressor region located immediately upstream at
-332/-300 (Flink, I. L., Edwards, J. G., Bahl, J. J., Liew,
C.-C., Sole, M., and Morkin, E.(1992) J. Biol. Chem. 267,
9917-9924). A 49-base pair restriction fragment containing the
suppressor element was used to screen a cardiac expression library. The
0.65-kilobase pair cDNA identified by this procedure was similar in
sequence, except for the absence of a 21-base pair region encoding
seven amino acids, to cellular nucleic acid-binding protein (CNBP), a
19-kDa zinc finger DNA-binding protein isolated earlier from liver,
which may be involved in negative regulation of cholesterol
biosynthesis (Rajavashisth, T. B., Taylor, A. K., Andalibi, A.,
Svenson, K. L., and Lusis, A. J.(1989) Science 245,
640-643). An additional clone identical to the one originally
found in liver, referred to as CNBP
, was isolated from the cardiac
library by hybridization screening. Gel mobility shift analysis
indicated that CNBP
and CNBP
isoforms preferentially interact
with single-stranded DNA corresponding to the proximal and distal
regions of the suppressor. When cotransfected with a
-MHC reporter
construct, CNBP
repressed activity in a dosage-dependent manner,
whereas repression was not observed with the shorter construct
(CNBP
). Cotransfection of a combination of CNBP
and CNBP
repressed reporter activity to an extent similar to cotransfection with
CNBP
alone, suggesting that CNBP
is not translationally
active under these conditions. The results of RNase protection assays
and genomic sequencing indicated that the
and
isoforms are
formed by alternative use of 5` donor sites within a single exon. These
results suggest that CNBP isoforms may modulate the activity of the
-MHC gene by interaction with a repressor region.
The -myosin heavy chain (MHC) (
)gene is the
major myosin isoenzyme expressed in human slow skeletal muscle and
ventricular myocardium (Liew et al., 1990; Bouvagnet et
al., 1984; Gorza et al., 1984). The
-MHC gene is
expressed in a developmental and muscle-specific manner (Periasamy et al., 1984; Emerson et al., 1987; Mahdavi et
al., 1986; Yu et al., 1989; Lyons et al., 1990),
and in rats and rabbits has been shown to be transcriptionally
down-regulated by thyroid hormone (Lompre et al., 1984;
Everett et al., 1984) (see Morkin(1993) for review). In the
human
-MHC gene, a strong positive cis-regulatory element
has been localized in the 5`-flanking sequences that is required for
high level expression in cultured heart cells (Flink et al.,
1992). A repressor with partial positional dependence is located
immediately upstream (Edwards et al., 1992).
Presently,
little is known about the protein(s) that trans-activate the
repressor element of the -MHC gene or that inhibit transcription
of other contractile protein genes in the heart. In skeletal muscle,
Id, a helix-loop-helix protein that lacks the basic domain necessary
for DNA binding, can associate specifically with MyoD-1 and related
proteins, resulting in attenuation of their ability to bind DNA and trans-activate muscle-specific genes (Benezra et al.,
1990; Evans et al., 1991). Despite considerable effort, no
comparable system of positive and negative regulatory proteins has been
found in cardiac muscle (Thompson et al., 1991).
In earlier
studies of the human -MHC promoter, a region was identified by
DNase I footprinting between positions -278/-296 that
coincided with sequences between positions -274/-300 that
were required for high level expression in primary fetal rat heart cell
cultures (Flink et al., 1992). An additional region was
protected in DNase I footprints using nuclear extracts from rat heart
(-301/-313) and liver (-303/-315), which
corresponded to a suppressor domain. Analysis of a larger series of
deletion constructs and site-specific mutations suggested that there
were proximal (-301/-314) and distal
(-315/-332) negative elements within this region (Edwards et al., 1992). Additionally, the suppressor has been shown to
down-regulate both the thymidine kinase and SV40 promoters when
positioned upstream from these basal promoters. The
-MHC
suppressor region was used in the present study to identify a negative
transcriptional factor. The clone initially characterized was similar
in sequence to the message corresponding to cellular nucleic
acid-binding protein (CNBP) (Rajavashisth et al., 1989),
except for the absence of 21 nucleotides encoding seven amino acids
near the 5` end of the cDNA. Subsequently, a longer cDNA was obtained
from a heart library with a sequence identical to the version
originally found in liver (CNBP
).
The CNBP gene encodes a
19-kDa protein containing seven tandem zinc finger repeats of 14 amino
acids. Each finger region contains the same arrangement of
Cys-X-Cys-X
-His-X
-Cys
residues and has extensive sequence similarity to the finger domains of
retroviral nucleic acid-binding proteins (Covey, 1986). The results of
RNase protection assays and genomic sequence analyses indicate that the
CNBP
and CNBP
isoforms result from variations in mRNA
splicing in which alternative 5` donor sites are utilized within a
single exon. A single copy of the human CNBP gene has been localized to
chromosome 3q13.3-q24 (Lusis et al., 1990).
In
transient assays using primary heart cell cultures, CNBP inhibited
expression of a
-MHC reporter plasmid containing the negative
domain in a dosage-dependent manner, whereas the shorter isoform
(CNBP
) did not repress transcription. These results suggest that
isoforms of CNBP in cardiac muscle may differentially regulate the
transcriptional activity of the
-MHC gene by competing for a
suppressor element. The
and
isoforms of CNBP also are
present in liver and other non-muscle tissues where they may play a
role in modulating the transcriptional activity of non-contractile
protein genes.
The
gene encoding CNBP was isolated by screening a human Fix genomic
library using a 472-bp EcoRI restriction fragment located at
the 5` end of the CNBP
cDNA. A single clone containing the 5` end
of the CNBP gene was isolated. To obtain the 3` end of the CNBP gene, a
BlueSTAR-1 library was screened with a 1028-bp EcoRI
restriction fragment located at the 3`-end of the cDNA. One positive
recombinant was obtained. The DNA insert was converted to a plasmid
subclone by site-specific recombination following in vivo autoexcision utilizing
loxP sites and bacterial host P1 Cre
recombinase. Both strands of the purified phage DNA were directly
sequenced using the Fmol DNA sequencing system (Promega) and the
Applied Biosystems model 373A DNA sequencing system. Sequence
alignments and analyes were carried out using the software package
PC/Gene (IntelliGenetics, Mountain View, CA).
Figure 1:
Comparison of the cDNAs encoding CNBP
isolated from liver (top) and heart (bottom) tissues.
The position of the polyadenylation signal (AATAAA), translation
initiation triplet (ATG), and termination codon (TAA) of
``liver'' CNBP (CNBP) is indicated. The coding and
untranslated regions are indicated by the open and closedboxes, respectively. Nucleotides 206-226 are absent
in CNBP
resulting in the loss of seven amino acids (residues
36-42) in the linker region between the first and second zinc
fingers. The boldlines at the top show the
protection of the
and
products observed in the RNase
protection assay shown in Fig. 3. The shorter, 55-bp protected
fragment, corresponding to CNBP
, could not be visualized on the
gel.
Figure 3:
RNase protection assay of human heart and
liver RNA. Total RNA was prepared from human heart papillary muscle and
liver, and about 4-5 µg were hybridized to antisense probe
encoding CNBP. The purified, full-length probe of 389 bp is shown
in lane1. Lanes2 and 3 demonstrate the two protected bands of 322 bp (CNBP
) and 246
bp (CNBP
) with human papillary muscle and liver RNA, respectively.
Approximately equal amounts of CNBP
and CNBP
mRNA are present
in liver and heart tissues. No protected bands were observed in the
absence of RNA or with yeast RNA (results not
shown).
Using
a 451-bp EcoRI restriction fragment of the CNBP isolate
as a probe, a second clone was obtained from the cardiac cDNA library
by DNA hybridization. This clone contained an insert that was identical
to CNBP
(Fig. 1, top). Northern blot analysis of
RNA from heart, using the same CNBP
EcoRI restriction
fragment as a probe, revealed a single band of 1.5 kilobase pairs,
which was identical to the size of the CNBP mRNA found in liver (results not shown).
Figure 2:
Schematic diagram showing the genomic
structure, alternative splicing, and sequences at exon-intron
boundaries of the human CNBP gene. Boxes indicate exons, and
the thickline indicates intervening sequences. The
isoform is generated by the use of an internal 5` donor site in
exon 2, which results in the fusion, in-frame, of the shortened 5`
portion exon 2 to exon 3. The sequence of the intron/exon junctions
between exons 2 and 3 is indicated at the bottom.
The presence of two alternatively spliced CNBP
mRNA species in human heart was verified by RNase protection analyses.
For this purpose, an antisense riboprobe was constructed spanning the
coding sequence of CNBP from position 151 in exon 2 to position
472 in exon 4 (Fig. 1). This probe contains an additional 67 bp
of Bluescript vector sequence, so that the full-length probe was 389 bp
in length. Fig. 3demonstrates the presence of two major
protected fragments in heart and liver of 322 and 246 bp, respectively,
which corresponds to the predicted sizes of the
and
CNBP
splice variants. The shorter, 55-bp protected fragment from CNBP
could not be visualized on the gel. No protected bands were observed in
the absence of RNA or in the presence of unrelated yeast RNA (results
not shown). Interestingly, the signal intensities of the 322- and the
246-nucleotide fragments were about the same, indicating that similar
amounts of mRNAs encoding the
and
isoforms of CNBP are
present in heart and liver.
Figure 4:
Electrophoretic mobility-shift analysis of P-labeled distal suppressor sense strand
(-331/-311) with heart and liver nuclear extracts. Nuclear
extracts from euthyroid (Eu) normal rabbit heart and liver
were incubated with the distal suppressor sense strand, in the absence
of competitor (lanes1 and 7), a
50-100 fold molar excess of distal suppressor sense strand (lane2), distal suppressor antisense strand (lanes3 and 8), HMG-CoA sense strand (lane4), distal suppressor double-strand (lane5), and nonspecific adenovirus LTR double-strand (lanes6 and 9) ``cold''
competitors. Competition was observed with the distal suppressor sense
and antisense strands and the HMG-CoA sense strands. Arrow indicates the position of the specifically bound major band. Light
exposure of the autoradiogram demonstrates two bands in the position of
the arrow in liver and heart
tissues.
It was of interest to study in more detail the
interaction of expressed CNBP protein with the distal and proximal
suppressor regions by EMSA. Purified CNBP and CNBP
proteins
were found to bind preferentially to single-stranded oligonucleotides
corresponding to the sense strand of the distal element (Fig. 5, left panel, lanes1 and 11), and
sense (Fig. 6, lanes1 and 9) and
antisense strands (Fig. 6, lanes5 and 13) of the proximal element. Binding to each of these cis-elements was competed by the HMG-CoA reductase SRE (Fig. 5, left panel, lanes3 and 13), but not by the adenovirus promoter element (Fig. 5, left panel, lanes4 and 14 and Fig. 6, lanes4, 8, 12, and 15). CNBP
may bind to the repressor with
slightly higher affinity than CNBP
, since an equal amount of
CNBP
protein produced more intense bands by EMSA (Fig. 5, left panel and Fig. 6). Furthermore, mutations in the
SRE-related region of the distal suppressor abolished binding (Fig. 5, right panel). Thus these results indicate that
the CNBP
and CNBP
isoforms interact preferentially with
single-stranded DNA corresponding to the proximal and distal negative
elements of the
-MHC repressor, and they support earlier
conclusions, based upon functional assays, that these elements act
synergistically to repress transcriptional activity (Edwards et
al., 1992).
Figure 5:
Electrophoretic mobility-shift analysis of P-labeled distal suppressor sense, antisense, and
double-stranded DNA (-331/-311) with bacterially expressed
CNBP
and CNBP
proteins. EMSAs were carried out as described
under ``Experimental Procedures'' and in Fig. 4. Left, CNBP
and CNBP
protein interacted only with
single-stranded sense DNA (lanes1 and 11).
Competition was observed with the distal suppressor sense
single-stranded DNA (lanes 2 and 12) and the HMG-CoA
sense strand (lanes3 and 13). No binding
was observed with the antisense (lane5) or distal
suppressor double-stranded DNA (lane8). No
competition was observed with the adenovirus LTR (lanes4 and 14). Arrow indicates the position of the
specifically bound major band. Right, EMSA of mutated distal
suppressor. The wild-type sequence (5`-GGTGGTCGTGG-3`) of the distal
suppressor, which is homologous to the SRE found in the HMG-CoA
reductase gene, was mutated to 5`-TTATTTATATT-3` (underline represents
mutated nucleotides). No binding was observed with expressed CNBP
or CNBP
protein.
Figure 6:
Electrophoretic mobility-shift analysis of P-labeled proximal suppressor sense- and antisense strands
(-314/-301) with bacterially expressed CNBP
and
CNBP
proteins. EMSA assays were carried out as described under
``Experimental Procedures'' and in Fig. 4. Binding was
observed with sense (lanes1 and 9) and
antisense (lanes5 and 13) DNA. Competition
was observed with the proximal suppressor sense (lanes2, 6, 10, and 14) strand. No
competition was observed with double-stranded proximal suppressor (lanes3, 7, and 11) or
the adenovirus LTR (lanes4, 8, 12,
and 15). Arrow indicates the position of the
specifically bound major band.
Figure 7:
Top, comparison of the CAT
activity of p-MHC-332 cotransfected with plasmids expressing
and
isoforms of CNBP in primary cardiomyocyte cultures.
Dosage-dependent repression of p
-MHC-332 CAT activity occurred in
the presence of increasing amounts of a vector (pSG5) expressing
CNBP
(
). Cotransfection with CNBP
in the same vector
resulted in no change in CAT activity (
) compared to transfection
of pSG5 vector alone (
). Results are expressed as percent
activity (means ± S.E.) of p
-MHC-332 in the absence of CNBP
for triplicate determinations in three to six experiments. Bottom, comparison of the CAT activity of wild-type
p
-MHC-332 with constructs containing mutations within the distal
(p
X322) and proximal (p
X305) suppressor regions,
respectively. Cotransfection of CNBP
in combination with CNBP
and p
-MHC-332. Fetal heart cells were transfected with 10 µg
of reporter plasmids (p
-MHC-332, p
X322, p
X305) and 5
µg of CNBP constructs as indicated. LaneA,
p
-MHC-332; laneB, p
X322; laneC, p
X322 and CNBP
; laneD,
p
X305; laneE, p
X305 and CNBP
; laneF, p
-MHC-332 and CNBP
; laneG, p
-MHC-332 and CNBP
and CNBP
. The
activity of p
X322 was increased 310% compared with the activity of
p
-MHC-332, whereas no change was observed with p
X305.
Cotransfection of CNBP
with p
X322 and p
X305 decreased
their activity by
41% and
27% repression, respectively.
Cotransfection of equal amounts of CNBP
with CNBP
and
p
-MHC-332 resulted in
26% decrease in activity. This value is
approximately the same as the activity observed when CNBP
alone
was transfected. Results are expressed as percent activity (means
± S.E.) of p
-MHC-332 in the absence of CNBP for triplicate
determinations in two to three experiments.
To study the activity of the distal and proximal repressor elements
independently, site-specific mutations were designed within these two
regions. In agreement with earlier results (Edwards et al.,
1992), the activity of pX322 (Fig. 7, bottompanel, laneB), which contains a
mutation in the distal element, was
3-fold greater than
p
-MHC-332 (Fig. 7, bottom panel, laneA), indicating disruption of protein-DNA interactions
within the distal element. By contrast, the construct p
X305, which
contains a mutation in the proximal element, did not show a significant
increase in activity (Fig. 7, bottom panel, laneD) compared to p
-MHC-332. These results suggest that
the distal element may be a stronger repressor than the proximal
element. When CNBP
was cotransfected with p
X322,
41%
repression was observed (Fig. 7, bottom panel, lane
C), possibly because of interaction with the wild-type proximal
suppressor element. Cotransfection of p
X305 with CNBP
caused
a slight reduction in activity (
27%) (Fig. 7, bottom
panel, laneE) compared to the activity of
p
X305 alone. Cotransfection of CNBP
in combination with
CNBP
, resulted in a level of reporter activity simlar to
cotransfection with CNBP
alone (lanesF and G). Taken together, these results suggest that both elements
may be required for full silencing activity.
In this report, CNBP has been identified as a trans-acting factor that negatively regulates the -MHC
gene. Two isoforms of CNBP, termed
and
, are shown to be
produced by alternative processing and to interact preferentially with
single-stranded DNA corresponding to a repressor element
(-332/-301) found within the proximal 5`-flanking region of
the human
-MHC gene. RNase protection experiments indicate that
approximately equal amounts of the
and
isoforms are present
in human ventricular myocardium and liver. Interestingly, the two
isoforms appear to have different effects on
-MHC gene
transcription. When increasing amounts of CNBP
were cotransfected
into cultured cardiomyocytes with a
-MHC/CAT reporter plasmid,
activity of the
-MHC gene was decreased in a dosage-dependent
manner, whereas cotransfection of CNBP
did not cause repression.
Additionally, mutations centered within the distal element (p
X322)
increased reporter activity (Fig. 7, bottom panel, laneB), whereas nucleotide changes located within
the proximal region (p
X305) did not appear to affect
-MHC
gene transcription (Fig. 7, bottom panel, laneD). These results suggest that both elements may be
required for full silencing activity. Cotransfection of a combination
of CNBP
and CNBP
repressed reporter activity to a level
similar to CNBP
alone, indicating that CNBP
is not
translationally active under these conditions.
Alternative processing has been shown to generate multiple mRNAs from single pre-mRNA transcripts and is an important mechanism in the regulation of gene expression (Green, 1992; McKeown, 1992). The CNBP isoforms provide an interesting example of a transcriptional regulator with alternatively spliced products that have different trans-activating functions. The splicing process for formation of CNBP isoforms involves selective use of alternative internal 5` donor sites within exon 2. It has been reported earlier that splice site selection can be influenced by both exon sequences and splice site proximity (Eperon et al., 1986; Mardon et al., 1987). In the CNBP gene, the donor site located at the intron/exon junction of exon 2 and the internal donor site within exon 2 contain an identical consensus recognition sequence (Fig. 2); hence, it is not surprising that these two sites are recognized with similar frequency by splicing trans-factors. Another example of an alternative donor site located within an exon is found within the mouse heat shock protein 47 (HSP47) gene (Takechi et al., 1994). In this case, the internal site is selected preferentially at elevated temperatures to produce a spliced mRNA product in which the adjacent downstream exon is skipped.
The three-dimensional solution structure (Lee et
al., 1989) and crystal structure (Pavletich et al., 1991)
of the zinc finger nucleic acid binding motif,
Cys-XCysX
-His-X
His,
which is related to the motif found in CNBP, has been determined. This
motif forms a compact globular domain containing a central zinc ion
tetrahedrally coordinated by Cys/His residues that lie within an
antiparallel
ribbon and an
helix. The zinc fingers bind in
the major groove of B-DNA with residues from the
NH
-terminal portion of the
helix making primary
contacts to the guanine-rich strand of the DNA. Additionally, a
computer simulation of the three-dimensional structure of the finger
domains of CNBP, and their interaction with the sterol regulatory
element, has been described (Kothekar, 1990). According to this model,
each of the seven fingers, including a portion of the linker region
between fingers one and two, contact in succession, a single nucleotide
of the 8-bp SRE. We have confirmed the interaction of CNBP
with
the SRE, and also have shown that the
and
CNBP isoforms
bind preferentially with different affinities to single-stranded
sequences of the proximal and distal negative elements within the
repressor region of the
-MHC promoter ( Fig. 5and Fig. 6). CNBP
lacks seven amino acids at the 3` end of the
linker region involved in DNA contact, which may explain its inability
to repress
-MHC gene expression. Thus repression of transcription
by CNBP may involve a functional domain located between the first and
second zinc fingers that includes amino acid residues 36-42.
The -MHC suppressor, located at positions
-300/-332, is comprised of adjacent proximal and distal
negative cis-acting elements, each of which spans about
10-15 nucleotides. Both negative elements seem to be required for
full suppressor activity (Edwards et al., 1992). CNBP
protein-DNA interactions are somewhat unusual in that both CNBP
and CNBP
isoforms interact preferentially with single-stranded DNA
and that the sequences of the proximal (5`-GTCAGTTCCCTCTC-3`) and
distal (5`-GTGGTCGTG-3`) negative elements are not closely related.
This may indicate that local DNA conformation or unusual secondary
structural features in this region play a role in CNBP binding. The
distal negative element is somewhat homologous to the consensus
sequence (5`-GTG(C/G)GGTG-3`) of the sterol regulatory cis-element (Osborne et al., 1992). The latter
element is found in promoter regions of several genes involved in
cholesterol uptake and synthesis, including the low density lipoprotein
receptor, HMG-CoA synthase, and HMG-CoA reductase genes (Dawson et
al., 1988). Although the precise role of CNBP in regulating the
level of cholesterol is unknown, reduction of plasma cholesterol is
known to be associated with increased levels of CNBP mRNA (Rajavashisth et al., 1989). The interaction of CNBP with SRE is thought to
result in down-regulation of the HMG-CoA reductase gene, thereby
maintaining cholesterol homeostasis. It remains to be established
whether changes in amounts of CNBP isoforms occur in association with
variations in expression of
-MHC mRNA.
The ability of a
transcriptional factor to interact with two unrelated sequences is not
unique to CNBP. Binding to two unrelated sequences has also been
observed with transcriptional enhancer factor I (TEF-1) (Thompson et al., 1991; Flink et al., 1992), a putative
regulator of the -MHC and cardiac troponin-T genes (Mar et
al., 1990; Farrance et al., 1992). TEF-1 binds to the
unrelated GT-IIC and SphI-II enhansons of the SV40 early
promoter (Xiao et al., 1991). The sequence-specific
single-stranded DNA-binding protein, muscle factor 3 (Santoro et
al., 1991), also interacts with multiple elements, including the
CArG motif, E box, and MCAT domains found in the promoters of skeletal
muscle actin, muscle creatine kinase, and cardiac troponin-T genes,
respectively. It is perhaps noteworthy that each of these elements is
known to interact with other transcriptional regulators in addition to
muscle factor 3.
In addition to acting as a repressor, CNBP may play
other roles in regulating gene expression. For example, certain
single-stranded DNA-binding proteins have been shown to be involved in
stabilizing DNA at replication forks (Chase et al., 1986).
CNBP may play a comparable role in maintaining the most favorable DNA
conformation for modulating the transcriptional activity of RNA
polymerase. CNBP interaction clearly requires DNA sequence specificity,
however, because mutations within the -MHC suppressor sequences
completely abolish protein-DNA interactions and eliminate suppression
of reporter constructs (Edwards et al., 1992). A protein found
in Schizosaccharomyces pombe, Byr3, (Xu et al., 1992)
has seven zinc finger domains containing the consensus
CX
CX
HX
C
motif. This protein is able to suppress sporulation defects of ras-1 null diploids and also is required for efficient
conjugation. Interestingly, CNBP is able to substitute for Byr3 in
these functions. Another related protein, Xenopus posterior,
is involved in anterior-posterior axis formation at mid-gastula stage
in the developing embryo (Sato and Sargent, 1991). Further studies will
be required to fully elucidate the role of CNBP in the regulation of
gene expression and cell function within the heart and other tissues.