(Received for publication, December 13, 1995; and in revised form, January 10, 1996)
From the
We have identified a negative cis-acting regulatory element in
the nicotinic acetylcholine receptor -subunit gene's
promoter. This element resides within a previously identified 47-base
pair activity-dependent enhancer. Proteins that bind this region of DNA
were cloned from a
gt11 innervated muscle expression library. Two
cDNAs (MY1 and MY1a) were isolated that encode members of the Y-box
family of transcription factors. MY1/1a RNAs are expressed at
relatively high levels in heart, skeletal muscle, testis, glia, and
specific regions of the central nervous system. MY1/1a are nuclear
proteins that bind specifically to the coding strand of the 47-base
pair enhancer and suppress
-promoter activity in a
sequence-specific manner. These results suggest a novel mechanism of
repression by MY1/1a, which may contribute to the low level expression
of the
-subunit gene in innervated muscle. Finally, the gene
encoding MY1/1a, Yb2, maps to the mid-distal region of mouse
chromosome 6.
Characterizing the molecular mechanisms that regulate the
expression and distribution of synaptic proteins is central to
understanding synapse formation and modification. As a model synapse,
the neuromuscular junction provides an ideal system to study these
intricate mechanisms. Genes encoding muscle nicotinic acetylcholine
receptors (nAChR) ()have been used as probes to delineate
mechanisms by which the presynaptic motor neuron regulates postsynaptic
muscle protein expression. These studies have shown that nerve-induced
muscle electrical activity suppresses expression of the embryonic-type
(
) nAChR genes (reviewed by Hall and Sanes (1993)).
The mechanism mediating this activity-dependent control of gene
expression is not well understood. Recent studies have identified E-box
sequences (CANNTG) in the nAChR gene promoters that are necessary for
activity-dependent regulation of gene expression (Tang et al.,
1994; Bessereau et al., 1994; Su et al., 1995). These
sequences are known to bind helix-loop-helix proteins of the MyoD
family of transcription factors. Myogenin is a strong candidate for
binding to these sequences and mediating developmental stage-specific
and activity-dependent expression, since myogenin knockout mice do not
express embryonic-type - and
-subunit genes (Hasty et
al., 1993). In addition, overexpression of these factors results
in increased nAChR promoter activity in nonmuscle cells (Gilmour et
al., 1991; Prody and Merlie, 1991; Bessereau et al.,
1993; Berberich et al., 1993). However, the
-subunit gene
appears to be unique in that mice lacking myogenin still express this
gene in embryonic muscle fibers (Hasty et al., 1993),
suggesting that other E-box binding proteins may be involved in its
regulation.
Since several other muscle-specific genes have E-box sequences but are not regulated by muscle activity (Chahine et al., 1992; Chahine et al., 1993; Merlie et al., 1994; Gilmour et al., 1995; Su et al., 1995), it is likely that activity-dependent control of gene expression involves additional cis-acting elements that may work in conjunction with the E-box sequences. Identification of these sequences will provide probes for the proteins that bind them.
Using in vivo expression
assays, we have recently identified these additional sequences in the
nAChR -subunit gene promoter. (
)A 47-bp enhancer
(nucleotides -1 to -47 in Chahine et al.(1992))
was identified that contained all the necessary elements to confer
activity-dependent expression onto a heterologous promoter. In addition
to an E-box, this sequence contains a region with similarity to the
SV40 core enhancer, SVCE (Khoury and Gruss, 1983). Point mutations in
either the E-box or SVCE sequence block the ability of the 47-bp
enhancer to activate a heterologous promoter in response to muscle
denervation.
When considering mechanisms mediating
activity-dependent control of nAChR gene expression, it is important to
consider both activation of these genes upon denervation and
suppression of these genes in the innervated state. We report here on a
mutation in the SVCE of the -promoter that results in increased
promoter activity in innervated muscle, suggesting that this sequence
acts as a repressor and may participate in maintaining low levels of
-subunit gene expression following muscle innervation. Using this
DNA sequence as a probe for proteins that may mediate this repression,
we cloned and characterized cDNAs encoding muscle Y-box proteins, MY1
and MY1a, which suppress
-promoter activity. Although these
proteins were identified by their ability to bind to the
-promoter's SVCE sequence, they appear to show a preference
for binding the coding strand of the 47-bp activity-dependent enhancer.
MY1/1a are members of the Y-box family of transcription factors (Wolffe et al., 1992). MY1a lacks 207 nucleotides found in MY1,
suggesting they are derived from the same gene by alternative splicing.
MY1 RNAs are most abundant in skeletal muscle, heart, testis, glia, and
specific central nervous system neurons.
DNA, for injection into muscle, was purified twice on
CsCl gradients. Prior to injection, 150-200 µg of CMV/CAT
along with 150-200 µg of one of the -550 expression
constructs were mixed and ethanol-precipitated twice. The final
precipitate was rinsed two times with 70% ethanol and dried briefly.
This DNA was resuspended at 10 mg/ml in 150 mM NaCl, and
approximately 10 µl was injected slowly into each of four different
locations along the length of the extensor digitorum longus muscle.
Figure 2: Nucleotide and deduced amino acid sequence of cDNA clone encoding MY1. The putative initiator methionine is in bold. The conserved cold shock domain is located between the arrows depicted beneath the sequence. Asterisks beneath the sequence identify a conserved RNA binding domain. The dashed line beneath the sequence identifies residues present in MY1 but deleted in MY1a. Polyadenylation signal sequences are in bold italics in the 3` untranslated region of the sequence.
The CMV/CAT
expression vector harbors the cytomegalovirus (CMV) promoter (Boshart et al., 1985) driving chloramphenicol acetyltransferase (CAT)
expression. The MY1/1a expression constructs contain cDNAs MY1 or MY1a,
subcloned into the pCMV5 vector (Thomsen et al., 1984). The
pXP-321 expression vector harbors the nAChR
-subunit gene
promoter driving luciferase expression and has been described
previously (Walke et al., 1994).
Figure 1:
Scanner linker mutations identify a
suppressor sequence in the nAChR -subunit gene's promoter.
Either wild-type (
-550) or mutant (
-550 slm
-44/-29)
-promoter expression constructs were injected
into innervated and denervated rat extensor digitorum longus muscle to
study their in vivo expression. The expression vectors were
coinjected with CMV/CAT for normalization. One week following DNA
injections, muscles were harvested and luciferase and CAT activities
determined. Bar graphs represent the mean luciferase activity
from 3-4 injections normalized to CAT activity; error bars represent standard error of the mean.
Analysis of the DNA sequences showed that clones MY1 and MY1a are 1847 and 1639 nucleotides long, respectively, excluding their poly(A) tails (Fig. 2). The only difference in DNA sequence between these two clones is that MY1a lacks 207 nucleotides that correspond to nucleotides 736-942 in MY1 (dashed line in Fig. 2). MY1 and MY1a contain open reading frames that encode proteins of 423 and 354 residues, respectively (Fig. 2). The 3` untranslated region of these clones contains two polyadenylation signal sequences, of which the most 3` is followed by a poly(A) tail.
Comparison of the DNA sequence or deduced amino acid sequence with corresponding sequences in the GenBank data base indicate that these two clones are most similar to a family of proteins containing a cold shock domain, referred to as Y-box binding proteins (Wolffe et al., 1992). Within this family of proteins MY1/1a are most similar to dbpA and RYB-a (approx. 81% at the nucleotide level) (Sakura et al., 1988 and Ito et al., 1994).
Comparison of the deduced amino acid sequence of MY1/1a with the Y-box family of binding proteins indicates that the conserved cold shock domain is the most highly conserved region between members of this family of proteins (Wolffe et al., 1992). Within the cold shock domain (amino acids 138-245, encoded by nucleotides 412-735) is a putative RNA binding domain (amino acids 156-163, encoded by nucleotides 466-489) (Landsman, 1992). The predicted isoelectric point for these proteins is 10.8, which is consistent with their high arginine content (approximately 12%). These arginine residues are clustered in the carboxyl half of the protein. In addition, these proteins contain a high percentage of proline residues (12%).
Figure 3:
Tissue distribution of MY1/MY1a RNAs. A, tissue distribution of MY1/MY1a RNAs as determined by RNase
protection assays. Ten micrograms of total RNA was hybridized with 5
10
cpm of MY1 antisense RNA probe 3 (see B) prior to RNase T2 digestion. B, various deleted
antisense RNA probes identify the upper and lower bands in RNase
protection assays as representing MY1 and MY1a, respectively. 1-3 above the lanes represents the probe used in that
particular experiment.
To confirm that the two protected RNA bands do indeed correspond to MY1 and MY1a RNA, probes were generated that contained different amounts of sequence present in MY1 but deleted in MY1a. These probes allowed us to map the RNA sequence that was responsible for generating this doublet RNase protection pattern. The probes used in this experiment are diagrammed in Fig. 3B. It is clear from the RNase protection pattern that the difference in the two protected RNAs result from the 207 nucleotides present in MY1, but lacking in MY1a, since once this region is deleted from the probe a single protected fragment is observed (Fig. 3B).
MY1/1a expression in brain and retina may reflect restricted expression to a few specific cell types or general low level expression throughout these tissues. To investigate this further, we used in situ hybridization to assay for these RNAs in retina and brain (Fig. 4). This analysis showed relatively high levels of MY1/1a RNA in cells of the pia, layer 2 of the cortex, cerebellum, and glia, but lower levels in various neurons located throughout the brain. In layer 2 of the cortex, highest expression was observed in the motor area. The cerebellum showed high expression in all three cell layers and in glia. In the retina, most of the expression is confined to the inner segment of the photoreceptors.
Figure 4: MY1/MY1a RNAs are expressed in neurons and glia of the central nervous system. In situ hybridizations of mouse retina and brain sections following hybridization to a MY1/MY1a probe. A, anterior portion of cortex showing hybridization to neurons that, in more caudal sections, make up layer 2 of the cortex. B, a more caudal section of the cortex showing hybridization to layer 2 (arrows) and on the edge of the tissue, corresponding to cells in the pia. C, cerebellum section showing relatively robust hybridization to scattered neurons in the molecular layer (m), purkinje cells (p), and granule layer neurons (gr). In addition, hybridization is detected in the glia (gl). D, retinal section showing relatively high levels of hybridization to the inner segments of the photoreceptors. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer.
We also surveyed a number of cell lines for MY1/1a expression (Fig. 5). We observed a high level of expression in the muscle C2 and glial C6 cell lines, with relatively lower levels of expression in the NG108 and PC12 cell lines. Interestingly, we were unable to detect any MY1/MY1a RNA in the SK-N-SH cell line. Very low levels were identified in the hepatic HepG2 cell line, although a partially protected band is observed, which may represent an alternatively spliced or related gene product.
Figure 5: MY1/MY1a RNAs are abundantly expressed in muscle and glial cell lines. RNase protection assays were performed by hybridizing 10 µg of isolated RNA with MY1 antisense probe 3 (see Fig. 2B) followed by digestion with RNase T2. Samples were fractionated on 6% polyacrylamide, 8 M urea gels, which were subsequently dried and exposed to x-ray film overnight at room temperature.
Figure 6: MY1/MY1a cDNAs encode nuclear proteins. Nuclear and cytoplasmic proteins were isolated and fractionated on SDS-polyacrylamide gels. Western blots were probed with affinity-purified antibody and signals detected using the enhanced chemiluminescence system (Amersham). Preincubation of antibody in peptide used to generate the antibody resulted in no signal. C, cytoplasm; N, nuclei.
Although MY1 and MY1a are predicted to have molecular masses of 36 and 28 kDa, respectively, it is likely that these highly basic proteins are migrating anomalously on SDS-PAGE. Therefore, based on the identification of a doublet of approximately 32 kDa by Western blot and the absence of this doublet from SK-N-SH cells, we conclude that this doublet represents MY1/1a. The 34- and 45-kDa proteins identified on the Western blot likely represent other members of the Y-box family of proteins that are related to MY1/1a. This antibody cross-reactivity may be expected since the antibody was generated against a peptide whose sequence is in the cold shock domain, which is expected to be conserved among various members of the Y-box family of proteins.
Figure 7:
MY1 overexpression suppresses nAChR
-subunit promoter activity. SK-N-SH and NIH 3T3 cells were
cotransfected with a pCMV expression vector containing or lacking a MY1
cDNA insert, a CMV/CAT expression vector, and the test plasmid, i.e. pXP wild-type nAChR
-promoter expression vector,
550 (left panels) or deletion mutant
550
-52/-5 (right panels). The cells were
harvested and assayed for luciferase and CAT activity 48 h after
transfection. Experiments were repeated a minimum of three times. Bar graphs represent the average of triplicate transfections
normalized to CAT activity; error bars are ± standard
deviation.
The pCMV
vector, without MY1/1a insert, had no effect on -promoter
activity.In addition, MY1/1a did not suppress CMV/CAT or pXP
-321
activity (data not shown). Furthermore, we repeated these experiments
using NIH 3T3 cells, which unlike SK-N-SH cells, endogenously express
low levels of MY1/1a. As shown in Fig. 7, similar results were
obtained in 3T3 cells except that MY1 overexpression resulted in about
a 50% decrease in
-subunit promoter activity.
Interestingly,
compared to wild-type -promoter activity, the deletion mutant
(
550
-52/-5) showed about 1.5-fold higher
activity in 3T3 cells (Fig. 7). This is consistent with the fact
that the deleted sequence (spanning -5 to -52) contains the
repressor element. In SK-N-SH cells, however, the lowered expression of
550
-52/-5, compared to the wild-type
550,
may indicate that the deleted sequence also harbors a neuron-specific
positive regulatory element.
Figure 8: MY1/MY1a recombinant proteins specifically bind pyrimidine-rich single-stranded DNA. Various oligonucleotides were end-labeled and used as probes for MY1/MY1a binding in electrophoretic mobility shift assays. Although the data shown are results obtained with MY1 binding, identical results were obtained when MY1a was used in these experiments. Binding to the double-stranded (ds) probes used 60 and 120 ng of MY1/MY1a recombinant protein, while binding to the single-stranded (ss) probes used 1.2, 3.6, and 10.8 ng of recombinant MY1/MY1a protein. Triangles above the figure represent increasing protein used in that experiment. Following electrophoresis, gels were dried and exposed to x-ray film with intensifying screen at -80 °C overnight. + represents the coding strand, and - represents the non-coding strand.
Oligonucleotide 3 (which contains sequences present in both the SVCE and E-box oligonucleotides) bound MY1/1a better than any of the other single-stranded probes tested (Fig. 8, right panels). A complete shift of the oligonucleotide 3 (+) strand probe was generated with 3.6-10.8 ng of protein, which only caused a partial shift of the SVCE or E-box (+) strand oligonucleotides and the oligonucleotide 3 (-) strand oligonucleotide. In addition, we were able to identify three different complexes generated by binding these proteins to oligonucleotide 3. The increased binding of MY1/1a to either strand of the oligonucleotide 3 probe compared to the corresponding strands of the SVCE or E-box oligonucleotides suggest that the additional sequences found in oligonucleotide 3 facilitate MY1/1a binding.
Figure 9: Genetic map of mouse chromosome 6 near Yb2. The gene encoding MY1/1a (assigned the name Yb2) was mapped in the BSS panel of the Jackson M. spretus backcross(28) . Other genes that were also mapped to this region in this cross are shown. The genetic distance in centimorgans (± standard error) and, in parentheses, the number of recombinant animals over the total number of animals scored are shown. The one recombinant between D6Mit218 and Yb2 was not scored for Kcna1, which places Kcna1 simultaneously on top of D6Mit218 and Yb2. The position of homologous loci to human chromosomes is shown to the right.
We report here the identification of a mutation in the nAChR
-subunit gene's promoter that results in increased promoter
activity in innervated muscle (Fig. 1), suggesting a mechanism
of repression. Proteins participating in this repression were cloned
and found to be members of the Y-box family of nucleic acid-binding
proteins. These proteins are referred to as MY1 and MY1a (muscle Y-box
proteins 1 and 1a) and were shown to decrease
-promoter activity
in a sequence-specific manner (Fig. 7). The fact that these
proteins bind to a previously identified 47-bp activity-dependent
enhancer of the nAChR
-subunit gene (Fig. 8), and require
sequences within this enhancer for their action (Fig. 7),
suggests a role for these proteins in activity-dependent regulation of
the nAChR
-subunit gene.
Y-box binding proteins have historically been defined by their ability to bind to an inverted CCAAT box in DNA. A number of these proteins have recently been cloned from mammalian cDNA libraries and include YB-1, EF1a, MSY1, p50, MUSY-1, and dbpA (Didier et al., 1988; Ozer et al., 1990; Tafuri et al., 1993; Evdokimova et al., 1995; Sakura et al., 1988; Wolffe et al., 1992). These proteins share an 80-amino acid sequence (referred to as the cold shock domain) with each other and with the E. coli cold shock protein, CS 7.4 (Wolffe et al., 1992). The cold shock domain appears to participate in nucleic acid binding (Tafuri and Wolffe, 1992; Bouvet et al., 1995).
The Y-box family of proteins carry out diverse functions ranging from transcriptional to translational controls. For instance, YB-1, a Y-box protein, has recently been shown to repress transcription of human major histocompatibility class II genes (Ting et al., 1994). Some other members of this family have been implicated in activating transcription from the Rous sarcoma virus (Faber and Sealy, 1990), HLA class II (Didier et al., 1988), and the hst gene promoters (Hasan et al., 1994). In these cases the Y-box binding protein recognizes an inverted CCAAT sequence in the double-stranded DNA. Likewise, Y-box proteins from Xenopus laevis, FRGY1 and FRGY2, also are capable of activating transcription from promoters containing an inverted CCAAT sequence (Tafuri and Wolffe, 1992). However, there are also reports that certain Y-box proteins prefer to bind pyrimidine-rich single-stranded DNA (Kolluri et al., 1992). Based on these studies, it has been proposed that these proteins participate in regulating gene expression via binding to single-stranded regions of DNA that are complementary to those participating in an intramolecular DNA triplex structure (Kolluri et al., 1992; Horwitz et al., 1994).
In addition to transcriptional regulation, Y-box proteins MSY1, p50, and FRGY2 also participate in regulating translation by binding and sequestering cytoplasmic RNAs from the translational machinery (Tafuri et al., 1993; Evdokimova et al., 1995; Bouvet and Wolffe, 1994). Therefore, Y-box binding proteins represent a family of proteins with nucleic acid-binding properties that have important implications for DNA and RNA expression.
The Y-box family members we report here,
MY1 and MY1a, are identical except that MY1a lacks 207 nucleotides
found in MY1 at positions 736-942 (Fig. 2). These data
suggest MY1/1a RNAs are derived from the same gene by alternative
splicing. The significance of expressing these two alternatively
spliced forms is not clear. Most tissues examined express both of these
RNAs with similar ratios (Fig. 3). They both suppressed
-promoter activity in transfection experiments (Fig. 7),
and they both bound DNA with a similar affinity (data not shown).
Most interesting were our experiments which showed that MY1/1a
overexpression was able to suppress -promoter activity, while a
mutant
-promoter expression construct containing a deletion
spanning the 47-bp activity-dependent enhancer (
550
-52/-5) completely relieved this suppression (Fig. 7). We chose to use this deletion in the expression
studies since our DNA binding assays suggested that MY1/1a bound best
to the 47-bp enhancer sequence (Fig. 8). These results suggest
that MY1/1a may normally contribute to maintaining a low level of
-promoter activity in innervated muscle, consistent with the high
level of MY1/1a RNAs found in this tissue. However, because we detected
high levels of MY1/1a RNAs in C2 myotubes (Fig. 5) and
denervated muscle (data not shown), it is likely that other
post-transcriptional mechanisms contribute to the regulation of
functional MY1/1a proteins.
It is interesting that MY1/1a binds with
highest affinity to the coding (+) strand of the nAChR
-subunit promoter's 47-bp activity-dependent enhancer. Based
on quantitating the band shifts obtained using single and
double-stranded oligonucleotides and the different amount of protein
used to generate a band shift, we estimate at least a 200-fold
difference in binding affinity between double- and single-stranded SVCE
oligonucleotide. Whether these proteins prefer a particular binding
site could not be determined by the limited number of binding studies
reported here. However, based on the oligonucleotides used in these
studies and the strand preference displayed by MY1/1a, it appears that
these proteins prefer pyrimidine-rich DNA sequences.
Pyrimidine-rich
sequences have been proposed to mediate Y-box family member YB-1
binding to single-stranded DNA of the c-Myc and -globin gene
promoters (Kolluri et al., 1992; Horwitz et al.,
1994). In addition, YB-1 has been reported to promote or stabilize
single-strandedness in the major histocompatibility class II DRA
promoter (MacDonald et al., 1995). However, in this latter
case there is no evidence for this protein preferring pyrimidine-rich
sequences. In addition, these investigators showed that the DNA
sequences responsible for this single-stranded binding activity are
different from those responsible for double-stranded binding (inverted
CCAAT box). These latter studies suggest that YB-1 binding to the DRA
promoter results in single-stranded regions that prevent binding of
other trans-activators necessary for activation of DRA expression.
If MY1/1a acts by a similar mechanism, we predict that binding of
MY1/1a to the -subunit promoter would promote formation of
single-stranded regions and reduce activation by other transcriptional
regulators that normally bind to the double-stranded 47-bp enhancer,
such as the E-box binding MyoD family members. We are currently testing
this possibility.
Furthermore, we have demonstrated that the gene encoding MY1/1a, Yb2, is located in the mid-distal region of mouse chromosome 6. The glial and muscular expression of this gene suggests that it might be involved in neuromuscular diseases. However, the only known mouse mutation that maps into this region is scruffy (Scr), a mutation affecting the coat (Beechey and Searle, 1992), in which MY1/1a is unlikely to be involved. In addition, the genetic mapping to mouse chromosome 6 can be used to predict its homologous location in the human genome. Several genes that map near Yb2 (Elliot and Moore, 1994) map to human 12p13 where the human homologue of Yb2 is thus expected to lie. No human neuromuscular diseases are known to be linked to 12p13. The position of Yb2 is clearly distinct from the position of four unlinked loci, Yb1a-Yb1d, on mouse chromosomes 3, 7, 8, and 9 that represent the genes and possibly pseudogenes of YB-1, a different Y-box protein (Spitkovsky et al., 1992). In contrast to these studies on Yb1, we detected only a single locus for Yb2 and no evidence for related or pseudogenes.
In conclusion, we
report the first characterization of a negative cis-acting DNA element
in the nAChR -subunit gene's promoter that binds Y-box
family members MY1 and MY1a. The fact that the cis-acting element these
proteins bind to is also involved in mediating activity-dependent
regulation of the
-subunit gene suggests that these proteins
participate in this regulation by keeping
-promoter activity low
in innervated muscle. The preferential binding of MY1/1a to the coding
strand of the 47-bp activity dependent enhancer suggests a novel
mechanism of repression that may involve stabilization of
single-stranded DNA regions. Finally, the expression of MY1/1a in the
central nervous system suggests a regulatory role for these proteins
within specific neurons. Whether or not these proteins also serve as
repressors in these neurons remains to be determined.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U22893[GenBank].
The genetic marker for Yb2 has been submitted to the Mouse Genome Database under accession number MGD-CREX-319.