From the Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245-3207
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ABSTRACT |
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The neuronal nicotinic acetylcholine receptor
gene family consists of 11 members, 2-
9 and
2-
4. Three of
the genes, those encoding the
3,
5, and
4 subunits, are
clustered tightly within the genome. These three subunits constitute
the predominant acetylcholine receptor subtype expressed in the
peripheral nervous system. The genomic proximity of the three genes
suggests a regulatory mechanism ensuring their coordinate expression.
However, it is likely that gene-specific regulatory mechanisms are also
functioning because the expression patterns of the three genes,
although similar, are not identical. Previously we identified
regulatory elements within the
4 promoter region and demonstrated
that these elements interact specifically with nuclear proteins. One of
these elements, E1, interacts with the regulatory factor Pur
as well
as three other unidentified DNA-binding proteins with molecular masses of 31, 65, and 114 kDa. Another element, E2, interacts with Sp1 and
Sp3. Because E1 and E2 are immediately adjacent to one another, we
postulated that the proteins that bind to the elements interact to
regulate
4 gene expression. Here we report the identification of the
65-kDa E1-binding protein as heterogeneous nuclear ribonucleoprotein K
and demonstrate that it affects the transactivation of
4 promoter activity by Sp1 and Sp3 differentially.
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INTRODUCTION |
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Key molecular components of synapses are ligand-gated ion channels
that are intimately involved in generating the electrical signals that
underlie information processing within the nervous system. The most
well characterized ion channels are the members of the nicotinic
acetylcholine (nACh)1
receptor family. Sixteen genes encoding members of this family have
been identified, with 11 of them, 2-
9 and
2-
4, being expressed within neuronal populations (1-5). Reconstitution studies have shown that the
2-
6 subunits in combination with either the
2 or
4 subunit can each lead to the formation of functional nACh
receptors. Each of these receptor subtypes has distinct
electrophysiological and pharmacological properties (4, 6). In
contrast, the
7-
9 subunits appear to be able to form homomeric
receptors (7-9). It is likely that the functional diversity exhibited
by the neuronal nACh receptor family results from the differential
expression of these subunits which leads to incorporation of different
subunits into mature receptors. Although the consequences of this
diversity are beginning to be appreciated, an understanding of the
molecular basis governing expression of neuronal nACh receptor subunits remains elusive. However, recent advances indicate that regulation of
the receptor subunit genes at the level of transcription plays a
critical role (see below).
In situ hybridization studies have demonstrated that each of
the nACh receptor subunit genes exhibits distinct temporally and
spatially restricted patterns of expression in the peripheral and
central nervous systems, suggesting that they are most likely regulated
independently (although in some cases there is overlap in expression
patterns which may indicate that such genes share certain regulatory
features, as discussed below; Ref. 2, 3, 10-15). As mentioned above,
it is clear that transcriptional regulation, both positive and
negative, plays a key role in the establishment of the differential
expression patterns of the subunit genes (16-36). We would like to
understand the molecular details of this regulated expression. We have
focused upon characterizing the transcriptional mechanisms involved in
the expression of a cluster of receptor subunit genes, those encoding
the 3,
5, and
4 subunits. This cluster of genes spans
approximately 60 kilobase pairs of the rat genome (see Fig. 1 and Ref.
37). Because the
3,
5, and
4 subunits make up the predominant
nACh receptor subtype expressed in the peripheral nervous system (38,
39), the clustering of their genes raises interesting questions
regarding the regulatory basis of their coexpression. It is certainly
plausible that these genes are expressed coordinately via a set of
common regulatory mechanisms. On the other hand, neither the temporal
nor the spatial patterns of expression of the
3,
5, and
4
subunit genes are completely identical. Although the developmental and
functional implications of these temporal and spatial differences in
gene expression are unclear, they underscore the conclusion that each gene of the cluster has unique aspects to its regulation.
Previously we identified several regulatory elements within the
promoter region of the 4 gene and demonstrated that these elements
interact specifically with nuclear proteins present in extracts
prepared from brain tissue and an established neuronal cell line, SN17
(22, 30). One of these elements, E1, interacts with the transcriptional
regulatory factor Pur
(34) as well as three other unidentified
DNA-binding proteins that we refer to as neuronal
ACh receptor promoter-binding
proteins (NARP). These proteins have molecular masses of 31, 65, and
114 kDa (34). Another element, E2, interacts with the transcription
factors Sp1 and Sp3 (30, 31). An intact E2 is required for
transcriptional activation of the
4 promoter by Sp1 and Sp3 (31).
Because E1 and E2 are immediately adjacent to one another and both are
required for wild type
4 promoter activity, we postulated that the
proteins that bind to the elements interact to regulate
4 gene
expression (31). In this report, we describe the identification of the E1-binding protein NARP65 (34) as heterogeneous nuclear
ribonucleoprotein K (hnRNP K) and demonstrate that it affects the
transactivation of
4 promoter activity by Sp1 and Sp3
differentially.
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EXPERIMENTAL PROCEDURES |
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Protein Purification and Peptide Sequencing-- The E1-binding protein, NARP65, was purified from crude nuclear extracts prepared from bovine brains as described previously (34) with some modifications. Briefly, after partial purification by P11 phosphocellulose chromatography, fractions containing E1 binding activity were pooled, concentrated, and dialyzed for further purification by two rounds of DNA sequence-specific affinity chromatography. Two 20-base complementary oligonucleotides containing the E1 sequence (5'-GACCCTCCCCTCCCCTGTAA-3'; see Fig. 1) were used to make the DNA affinity matrix. Annealed, concatenated oligonucleotides were coupled to cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia Biotech) essentially as described by Kadonaga and Tjian (40). After the DNA sequence-specific affinity chromatography, fractions containing E1 binding activity were pooled, precipitated with acetone, and analyzed on 10% SDS-polyacrylamide gels. After staining with Brilliant Blue R-250 (Fisher Biotech) and destaining, NARP65 was excised, washed with 50% methanol, and subjected to amino acid microsequencing (Harvard Microsequencing Facility) as described previously (41).
Substrate Preparation for Electrophoretic Mobility Shift Assay
(EMSA)--
The E1/E2 oligonucleotides (Fig. 1) used as probes and
competitors in the EMSA were synthesized using an Oligo 1000 DNA
Synthesizer (Beckman). Single-stranded oligonucleotides corresponding
to either the coding or noncoding strand of E1/E2 were labeled
radioactively with [-32P]ATP (NEN Life Science
Products) using T4 polynucleotide kinase (Promega). When
double-stranded probes were used, equal amounts of E1/E2 complementary
oligonucleotides were annealed and then labeled radioactively. The
specific activities of these probes were typically between 5,000 and
15,000 cpm/fmol.
Protein Expression and Preparation of Nuclear Extracts from Transfected Drosophila Cells-- A glutathione S-transferase (GST)-hnRNP K fusion construct was generously provided by Dr. David Levens (National Cancer Institute). Expression and purification of GST and GST-hnRNP K fusion proteins were carried out as described previously (42). Nuclear extracts from transfected Drosophila cells were prepared by the method of Dignam et al. (43) as described previously (44) except that nuclear extracts were dialyzed against binding buffer of composition 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 100 µM ZnCl2.
EMSA-- When using recombinant proteins, EMSAs were performed as described previously (22) with radiolabeled double- or single-stranded oligonucleotides incubated with the indicated amounts of GST or GST-hnRNP K in the presence of 2 µg of nonspecific competitor poly(dI-dC). For competition experiments, unlabeled double- or single-stranded oligonucleotides were preincubated with the recombinant proteins for 15 min before the addition of labeled oligonucleotides. After a 1-h incubation at 4 °C, the reaction mixtures were electrophoresed through 6% native polyacrylamide gels and visualized by autoradiography. When using nuclear extracts as protein sources, EMSAs were performed using a 32P-labeled double-stranded E1/E2 oligonucleotide (Fig. 1) and 3.5 µg of nuclear extracts prepared from transfected SL2 cells. Reaction mixtures containing nuclear extracts, binding buffer, and 2 µg of poly(dI-dC) were preincubated for 5 min at room temperature before the addition of 5 fmol of the end-labeled probe. After addition of the probe, binding reactions were incubated further for 15 min at room temperature. Reaction mixtures were then electrophoresed through 6% native polyacrylamide gels. Radioactivity was detected by autoradiography of the dried gels.
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Expression Constructs-- The Sp1 expression construct pActSp1, containing the Sp1 coding sequence inserted downstream of the Drosophila melanogaster actin 5C promoter, was generously provided by Dr. Ed Seto (University of South Florida; Ref. 45). The Sp3 expression construct pPacUSp3, containing the Sp3 coding sequence inserted downstream of the actin 5C promoter, was kindly provided by Dr. Guntram Suske (Philipps-Universität Marburg, Germany; Ref. 46). The pAct-hnRNP K expression plasmid was constructed by subcloning the mouse hnRNP K coding sequence from pCRII-hnRNP K (the kind gift of Dr. Karol Bomsztyk, University of Washington) into the pAct vector. The EcoRI fragment containing the mouse hnRNP K coding sequence was isolated from pCRII-hnRNP K, treated with the Klenow fragment of DNA polymerase I and ligated subsequently to EcoRV-digested pAct to generate the construct pAct-hnRNP K.
Cell Culture and Transfections--
SN17 cells (47) were
cultured as described previously (22). D. melanogaster
Schneider SL2 cells were obtained from the American Type Culture
Collection and were maintained at room temperature in modified
Schneider's Drosophila medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum and antibiotics. Cells were
seeded at 2 × 106/35-mm culture well immediately
before transfection. DNAs were introduced into the cells by
liposome-mediated transfection using 9 µl of CellFECTIN/sample (Life
Technologies, Inc.) and 0.75 µg of target DNA (pX1B4FHwt, see Fig. 1)
in the absence or presence of 5, 10, or 50 fmol of effector DNA
(pActSp1, pPacUSp3, or pAct-hnRNP K). The target DNA constructs were
also transfected with the vectors pAct and pPacU devoid of Sp1 and Sp3
coding sequences, respectively, as negative controls for luciferase
expression. All transfections included 0.23 µg of a -galactosidase
expression vector, RSV-
Gal, in which expression of the bacterial
lacZ gene is driven by a Rous sarcoma viral promoter. After
a 5-h incubation with the DNA-CellFECTIN complexes, the cells were
overlaid with 0.5 ml of medium containing 30% fetal bovine serum and
incubated for 24 h. The next day, the cells were overlaid with 2 ml of complete growth medium. After another 24 h, the cells were
harvested and assayed for luciferase activity using a commercially
available kit (Promega Corp.). Luciferase values were normalized to
-galactosidase activity, which was measured using a commercially
available kit (Galacto-Light; Tropix, Inc.). For nuclear extract
preparation, SL2 cells were transfected essentially as described above,
except that transfections were carried out in 100-mm culture dishes,
and only the effector DNAs were introduced into the cells (pActSp1,
pPacUSp3, or pAct-hnRNP K).
Western Blotting-- Western blotting was performed essentially as described before (31) with minor modifications. 1 µl of an SN17 cell extract and 10 µg of nuclear extracts isolated from untransfected or transfected SL2 cells were electrophoresed through SDS-polyacrylamide gels (10%). Anti-hnRNP K antibody 3C2 (the kind gift of Dr. Gideon Dreyfuss, University of Pennsylvania School of Medicine; Ref. 48) was used at a 1:1,000 dilution in Blotto. Experiments utilizing anti-Sp1 and anti-Sp3 antibodies were done as described previously (31).
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RESULTS |
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NARP65 Is the Bovine Homolog of hnRNP K--
Our earlier studies
led to the identification of two regulatory elements, E1 and E2,
located between nucleotides 54 and
81 relative to the
4
transcriptional initiation site (Fig. 1; Refs. 21, 22, 30). As
mentioned above, E2 is a binding site for members of the Sp family of
transcriptional regulatory factors (30, 31). Biochemical purification
from bovine brain tissue of nuclear proteins that bind to E1 led to the
identification, in the most highly purified fraction, of four
polypeptides that interact specifically with E1 (34). One of these
proteins, NARP43, was shown by amino acid sequence analysis to be the
transcriptional regulator, Pur
(34). We have now obtained amino acid
sequences of two tryptic peptides of another E1-binding protein,
NARP65. The sequences of these peptides are identical to two regions of the hnRNP K coding sequence (Fig. 2; Ref.
48), thus establishing NARP65 as the bovine homolog of hnRNP K.
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hnRNP K Interacts Specifically with the 4 E1/E2
Region--
Recently hnRNP K has been shown to be a transcriptional
activator, a function that is dependent upon its binding to a CT
element (42, 49). hnRNP K binds both single- and double-stranded
nucleic acids, having a higher affinity for the former (for review, see Ref. 50). As shown in Fig. 1, E1 is characterized by three repeats of
5'-CCCT-3'. To determine whether hnRNP K interacts with the E1/E2
region, EMSAs were carried out using a GST-hnRNP K fusion protein and
both single- and double-stranded E1/E2 probes. As shown in Fig.
3, hnRNP K interacts specifically with
both the double-stranded E1/E2 probe and the upper strand (coding)
E1/E2 probe, but not at all with the lower strand (noncoding) E1/E2 probe. As seen most clearly with the coding strand E1/E2 probe, two
specific protein-DNA complexes are formed, with the most prominent complex migrating the fastest. The presence of two complexes is most
likely a consequence of oligomerization of hnRNP K, as has been
reported previously (62). Competition experiments demonstrated that
unlabeled double-stranded E1/E2 competed very little for binding to the
single-stranded coding E1/E2 probe, whereas unlabeled single-stranded
coding E1/E2 virtually eliminated hnRNP K binding to the
double-stranded probe (Fig. 3). Unlabeled single-stranded noncoding
E1/E2 did not compete for binding to either the single-stranded coding
or double-stranded E1/E2 probes, consistent with the observation that
the single-stranded noncoding E1/E2 probe did not form any specific
complexes with hnRNP K (Fig. 3). These observations indicate that hnRNP
K interacts specifically with the E1/E2 region and that the protein
appears to have a higher affinity for the single-stranded CT-rich
coding E1/E2 probe, as has been reported for hnRNP K binding sites in
other genes (50).
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hnRNP K Is Expressed Highly in SN17 Cells but Not in Drosophila
Cells--
To test the potential functional significance of hnRNP K on
4 promoter activity, it was necessary to identify a suitable cell
line in which to carry out transfection studies. Because high
endogenous levels of hnRNP K may complicate interpretation of
transfection results, it was desirable to identify a cell line that
either does not express hnRNP K or expresses it at very low levels. Two
cell lines that we have used extensively in the past to study
4 gene
expression are the neuronal cell line SN17 and the
Drosophila Schneider SL2 cell line (21, 22, 30, 31). To
determine the expression levels of hnRNP K in these cell lines, Western
blot analysis was carried out. As shown in Fig.
4, SN17 cells express relatively high
levels of hnRNP K, but SL2 cells do not express any detectable hnRNP K
protein. However, when transfected with an expression construct
producing mouse hnRNP K under the control of the Drosophila
actin 5C promoter (see "Experimental Procedures"), SL2 cells
expressed readily detectable levels of exogenous hnRNP K protein (Fig.
4, lane 3). Therefore, we chose SL2 cells to perform
functional analysis of hnRNP K effects on
4 promoter activity.
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hnRNP K Regulates Sp1 and Sp3 Transactivation of the 4 Promoter
Differentially--
To investigate whether hnRNP K has any effect on
the transcriptional activity of the
4 promoter, SL2 cells were
transfected with a wild type
4 promoter/luciferase expression
construct (pX1B4FHwt; see Fig. 1) and with increasing amounts of an
expression construct for hnRNP K (pActMK) or of the parental vector
(pAct) as a control. Surprisingly, given that hnRNP K has been shown to
function as a transcriptional activator by virtue of its interaction
with a 5'-CCCT-3' recognition site similar to that of E1 (50), hnRNP K
had no effect on
4 promoter activity in this assay (Fig.
5).
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DISCUSSION |
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We have begun a molecular dissection of the regulatory processes
governing the transcription of neuronal nACh receptor subunit genes
with a particular emphasis on the rat 4 subunit gene. Four transcriptional regulatory factors have now been identified which interact with the promoter region of the
4 gene. Sp1 and Sp3 interact in a functionally relevant manner with E2 (30, 31), whereas
Pur
and hnRNP K bind to E1 (Ref. 34 and this report). The functional
significance of the Pur
-E1 interaction is currently under
investigation. hnRNP K was identified initially as a component of the
multiprotein heterogeneous nuclear ribonucleoprotein particle (48), a
structure thought to be involved in pre-mRNA processing (51).
Although hnRNP K can bind RNA through conserved K homology (KH)
domains, its precise role in pre-mRNA processing remains obscure.
In addition to binding RNA, hnRNP K has been shown to bind both double-
and single-stranded DNA, having a higher affinity for the latter (50).
Moreover, in vitro, hnRNP K can stimulate transcription by
RNA polymerase II, and in vivo, it can both activate and
repress transcription (52). The functional studies on hnRNP K reported
here yielded some unexpected results. Surprisingly, hnRNP K by itself
did not affect
4 promoter activity in the context of a
transactivation assay using Drosophila cells. However, even more surprisingly were the differential effects of hnRNP K on the
transactivation abilities of Sp1 and Sp3 on
4 promoter activity. In
the absence of hnRNP K, both Sp1 and Sp3 transactivate the
4
promoter, with Sp3 being the more potent activator. When hnRNP K was
included in the transfection, the Sp1-mediated transactivation was
virtually eliminated, even at the lowest concentration of hnRNP K used.
In contrast, Sp3 transactivation of the
4 promoter was repressed
only modestly (approximately 30%) by the same low amount of hnRNP K. Even at the highest concentration of hnRNP K used, Sp3 still
transactivated the
4 promoter 80-fold. Given that all three genes
were transcribed from the Drosophila actin 5C promoter, it
is unlikely that the differential effects of hnRNP K were a consequence
of differential expression of the Sp1 and Sp3 genes. Western blot
analysis was used to determine whether the levels of the Sp1, Sp3, and
hnRNP K proteins were significantly altered by cotransfection of the
various expression constructs. Although Sp1 protein levels were reduced
when cotransfected with either Sp3 or hnRNP K expression constructs, it
seems unlikely that this is the reason for the differential affects of
hnRNP K because cotransfection of Sp3 with Sp1 did not affect the
ability of Sp1 to transactivate the
4 promoter and, in fact, led to
a synergistic activation.
Although the precise mechanism by which hnRNP K exerts its effects on
the Sp factors is unknown, it clearly interferes with Sp1 binding to
the double-stranded E1/E2 oligonucleotide while having a small effect
on Sp3 binding, consistent with the transfection data. Whether these
observations reflect different DNA binding affinities of Sp1 and Sp3 in
the context of the 4 promoter remains to be determined. However, if
this were true, a very simple model would suggest that hnRNP K either
prevents Sp1 from binding to E2 by physically blocking access to the
DNA or displaces bound Sp1 from the DNA. The fact that hnRNP K does not
completely block Sp3 function on the
4 promoter suggests that Sp3
has a higher binding affinity for E2 than Sp1 such that Sp3 is not
displaced from the DNA by hnRNP K. This model obviously highlights
differences in the biochemical properties of Sp1 and Sp3 which in turn,
most likely underlie the functional distinctions between the two
proteins. It is becoming increasingly clear that, despite the
significant structural similarities between Sp1 and Sp3, they can
differ dramatically in terms of transcriptional regulatory properties.
For example, whereas Sp1 has been shown to be a transcriptional
activator, Sp3 can function as either an activator or repressor
depending on the promoter and the cellular context (46, 53). In the case of the
4 promoter, both Sp family members function as
transcriptional activators (Refs. 30 and 31 and this report) and can do
so in at least an additive, if not a synergistic, manner. Thus, the physiological relevance of hnRNP K totally repressing Sp1 function but
not that of Sp3 remains obscure but may be related to one of the many
potential functions of hnRNP K in regulating
4 gene expression (see
below). Alternatively, it may be coupled to the appearance of the
various factors during development. Although the expression patterns of
Sp1, Sp3, and hnRNP K have not been studied extensively in terms of
neuron-specific expression, other than a report describing high levels
of Sp1 expression in embryonic neural tissue of mouse (54), it is
possible that a developmental gradient of expression of the three
factors exists such that Sp1 and hnRNP K are expressed early in
development, and Sp3 is activated later. Interestingly, it has been
demonstrated recently that cell cycle withdrawal and subsequent
neuronal differentiation of the PC12 pheochromocytoma cell line, as
induced by nerve growth factor, is at least in part caused by
transcriptional activation by Sp1 of the genes encoding the cell cycle
control proteins, the cyclin-dependent kinase inhibitor,
p21 WAF1/CIP1, and cyclin D1 (55). Therefore, it is plausible that
during early embryonic development, Sp1-induced expression of
cyclin-dependent kinase inhibitors and cyclin D1 may lead
to premature exit of specific neuronal precursors from the cell cycle.
If hnRNP K is present in sufficient quantities at the same time in
development as Sp1, it may prevent Sp1 from binding to its recognition
sites in neuron-specific genes, particularly those encoding proteins
involved in cell cycle withdrawal, thus allowing, at some level at
least, normal development to proceed.
hnRNP K exhibits a broad array of regulatory functions leading to its
characterization as a "nucleic acid-regulated docking platform"
(50). As mentioned earlier, it interacts with RNA as well as single-
and double-stranded DNA, with single-stranded DNA being the preferred
substrate (50, 56). This latter property has led to the suggestion that
hnRNP K can facilitate transcription indirectly by functioning as an
"architectural" transcription factor (57). This would be done by
hnRNP K creating a single-stranded bubble that would make the DNA more
flexible and thus allow other DNA-binding proteins to align more easily
with the basal transcriptional machinery in a manner that might
otherwise be energetically unfavorable (for a complete discussion of
this potential function of hnRNP K, see Ref. 57). Such a model has been
proposed for the regulation of the c-myc promoter by hnRNP K
(58). Michelotti et al. (58) demonstrated that there are
regions of single-stranded DNA in the c-myc promoter in
cells that actively transcribe the c-myc gene, whereas such
regions are absent in cells that do not express c-myc (58).
One of these single-stranded DNA regions, the CT element, has been
shown to be a binding site for hnRNP K (59). Indeed, hnRNP K was
demonstrated to induce single-stranded DNA conformation at this site;
moreover, opening of the CT element by hnRNP K was augmented in the
presence of the transcriptional suppressor of the sterol regulatory
element, CNBP, a single-stranded DNA-binding protein that interacts
with the purine-rich strand of the CT region (58). Interestingly, in
the context of the 4 promoter, the favored substrate for Pur
,
which also prefers binding to single-stranded DNA, is the lower strand
of E1, thus it is easy to imagine a bubble structure created by the
binding of hnRNP K to the upper strand and Pur
to the lower strand.
How this would lead to differential regulation of Sp1 and Sp3 is
currently being investigated using all four proteins in structural and
functional studies. In the case of the c-myc promoter, in
addition to hnRNP K and CNBP, the CT element can also be bound by Sp1.
However, in contrast to the
4 promoter, Sp1 had a dominant effect
over hnRNP K and CNBP, eliminating a "bubble" they created on the
c-myc promoter (58). Together these observations reflect
further the potential complexity of functional interactions between
hnRNP K and Sp1 family members, indicating that these interactions are dependent on cellular and promoter contexts.
In addition to nucleic acid interactions, hnRNP K binds to a variety of
regulatory proteins, some of which are involved in signal transduction
pathways (such as Src, Fyn, Lyn, and Vav; for review see Ref. 50),
whereas others participate in transcriptional processes. The latter
group of proteins include several that are particularly relevant to the
present study. One of them, TATA-binding protein (TBP), can be
coimmunoprecipitated with hnRNP K from nuclear extracts (42). This is
of interest because the 4 gene promoter does not contain a TATA
sequence, and therefore the coupling of the basal transcriptional
machinery to the
4 promoter must occur independently of TBP binding
to DNA. Protein-protein interactions (e.g. hnRNP K-TBP) may
be one way by which this is achieved. Interestingly, Sp1 and Sp3 have
also been postulated to tether the basal transcriptional machinery to
TATA-less promoters via protein-protein with TBP-associated factors
(60, 61). Thus, three proteins we have shown to bind to regulatory
regions of the TATA-less
4 promoter all have the potential to
interact with the general transcriptional apparatus. We are currently
studying the significance of this observation. Investigation of whether
the remaining E1-binding proteins, NARP31 and NARP114, are relevant to
the transcriptional regulation of the
4 gene awaits their
identification.
In summary, we have identified hnRNP K as a regulatory factor that
interacts with the promoter region of the neuronal nACh receptor 4
subunit gene. Furthermore, we have shown that hnRNP K regulates the
transcriptional effects of Sp1 family members on
4 gene expression
differentially. The repressive effect of hnRNP K on Sp1 transactivation
represents a novel property of this multifunctional protein. The
physiological significance of this phenomenon in the context of the
4 gene has yet to be determined; however, given the important roles
hnRNP K plays in mRNA processing as well as transcription, we
believe the present report provides further evidence that these two
critical biological processes are intimately linked.
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ACKNOWLEDGEMENTS |
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We are very grateful to Karol Bomsztyk, Gideon Dreyfuss, Amy Hreha, David Levens, Dan Schullery, Ed Seto, and Guntram Suske for generously providing the reagents. We also thank Alan Tomkinson for help during the early phases of this work and William Lane, Eric Spooner, and John Neveu for amino acid sequencing.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NS30243 (to P. D. G.) and by the Council for Tobacco Research and the Smokeless Tobacco Research Council, Inc.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.
To whom correspondence should be addressed: Dept. of Molecular
Medicine, Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Dr., San Antonio, TX 78245-3207. Tel.:
210-567-7251; Fax: 210-567-7247; E-mail: gardner{at}uthscsa.edu.
1 The abbreviations used are: nACh, nicotinic acetylcholine; NARP, neuronal ACh receptor promoter-binding protein; hnRNP K, heterogeneous nuclear ribonucleoprotein K; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; KH, K homology; TBP, TATA-binding protein.
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REFERENCES |
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