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J Biol Chem, Vol. 273, Issue 42, 27741-27748, October 16, 1998
From the The central nervous system expression of
myelin basic protein (MBP) is restricted to oligodendrocytes and is
developmentally regulated; these regulatory features are
transcriptionally mediated. We have previously shown that the proximal
149 nucleotides of the MBP promoter were both necessary and sufficient
to activate the transcription of MBP in cultured oligodendrocytes, but
not in other cell types. Sequences within the distal portion of this promoter, which contains a nuclear factor 1 (NF1) binding site, repressed activation of the MBP promoter in Cos-7 cells, but not in
oligodendrocytes. We now describe a sequence upstream of and partially
overlapping the NF1 site that activates the MBP promoter in
oligodendrocytes, but not in Cos-7 cells. A protein complex binds to
this site, designated MEBA (myelinating glia-enriched DNA binding activity), and is enriched in nuclear
extracts prepared from the brain, oligodendrocytes, and Schwann cells.
The amount of MEBA parallels MBP expression and myelinogenesis in the
developing brain and parallels new MBP expression as purified
oligodendrocytes differentiate. Mutational analyses of binding and
function distinguish MEBA, an activator, from NF1, a repressor of MBP
transcription, and suggest that MEBA consists of at least two proteins.
Because the binding sites of MEBA and NF1 overlap, we suggest that MEBA may either compete with or modify NF1 binding, thereby activating the
MBP promoter in oligodendrocytes.
Myelin basic protein
(MBP)1 is a structural
protein of myelin expressed only by oligodendrocytes or Schwann cells.
In the central nervous system, MBP expression by oligodendrocytes is
required for normal myelinogenesis (1). MBP mRNA expression in the
rodent brain rises approximately 100-fold between birth and 30 days and falls to 40% of the peak expression in the adult. Transcriptional run-on analysis suggests that these regulatory features are primarily transcriptionally mediated (2, 3). In the peripheral nervous system,
MBP expression by Schwann cells is not strictly required for normal
myelinogenesis, although when the amount of basic intracellular domains
contributed by other myelin proteins is reduced, MBP becomes essential
(4). MBP mRNA expression also rises rapidly in the peripheral
nervous system during postnatal development, suggesting that its
regulation is transcriptionally mediated, although this has not been
demonstrated directly by transcriptional run-on analysis.
The transcriptional regulation of the MBP gene in the central nervous
system has been studied extensively, both in vivo and in vitro. The 5'-flanking regions of the mouse MBP promoter
ranging from 9.5 kilobases to 256 nt were sufficient to drive
oligodendrocyte-specific and developmentally regulated expression of
reporter genes in transgenic mice (5-8). Several studies in
vitro have sought to identify specific DNA sequences within the
proximal MBP promoter that might be necessary for cell-specific
expression, but most of these studies were performed using cells that
do not transcribe MBP or nuclear extracts prepared from the brain,
which contains a complicated mixture of cell types (9-11).
We have previously shown that 750 nt of human MBP promoter were
sufficient to activate oligodendrocyte-specific, developmentally regulated expression of lacZ during active myelinogenesis in
transgenic mice (3). In addition, we showed by transient transfection analysis in primary cultures of oligodendrocytes that 750, 420, or 149 nt of human MBP promoter was sufficient to activate a 5-10-fold increase in the expression of the CAT reporter gene in
oligodendrocytes, but not in other cell types (12). We also showed that
the 149-nt region contained two subregions with opposing functional
activities: (a) the proximal 102-nt region activated the
expression of CAT in most cell types; and (b) the more
distal region, from We now describe a DNA sequence that flanks and partially overlaps the
NF1 site, which activates the MBP promoter in oligodendrocytes, but not
in Cos-7 cells. This sequence is bound by a unique set of nuclear
proteins enriched in the brain, oligodendrocytes, and Schwann cells,
designated MEBA (myelinating glia-enriched
binding activity). The appearance of MEBA parallels
both myelination in the developing brain and MBP mRNA expression in
differentiating oligodendrocytes. Mutations within the region of MEBA
and NF1 binding distinguish MEBA from NF1 and suggest that MEBA
consists of at least two proteins. Because these binding sites with
opposing functions overlap, we suggest that MEBA may either compete
with or alter the NF1 binding of the proximal MBP promoter and
derepress the MBP promoter in oligodendrocytes.
Plasmids--
All deletions of the proximal 149-nt MBP promoter
were prepared by a modification of the exonuclease III/mung bean
nuclease technique (Stratagene), as described previously (12).
Transversion mutations spanning from 149 to 110 nt of the MBP promoter
were prepared by polymerase chain reaction-mediated mutagenesis, as described previously (12). All mutations were confirmed by sequence analysis. pRSVZ (13) was a gift from Dr. G. MacGregor (Baylor College
of Medicine, Houston, TX).
Cell Culture and Transfection Analysis--
CG4 cells, a
bipotential glial cell population derived from primary cultures of rat
brain, were cultured as described previously (14). In medium
conditioned by B104 neuroblastoma cells (B+, 30% in Dulbecco's
modified Eagle's medium plus N1 and biotin), these cells resemble
oligodendrocyte precursors; in the absence of B104-conditioned medium
(B
MEBA Derepresses the Proximal Myelin Basic Protein Promoter
in Oligodendrocytes*
,§,,
, and**
DIBIT and Department of Neurology, San
Raffaele Scientific Institute, 20132 Milan, Italy, ¶ Division of
Neuroscience Research, Children's Hospital of Philadelphia,
Philadelphia, Pennsylvania 19104, and Department of Neurology,
Wayne State University School of Medicine, Detroit, Michigan 48201
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
149 to 102 nt, silenced the expression of CAT
in them. Interestingly, the distal region did not silence the
expression of CAT in oligodendrocytes. This distal region contained a
consensus nuclear factor 1 (NF1) site; a deletion that removed half of
the NF1 site activated the expression of CAT in oligodendrocytes. Based
on these results, we hypothesized that a specific alteration of NF1 may
alleviate repression and contribute to the oligodendrocyte-specific
activation of MBP transcription (12).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
), these cells are induced to differentiate into oligodendrocytes.
A1.20 (SV40 T-antigen-transformed oligodendrocytes), Cos-7, HeLa, C6,
HJC, A7, or L cells were cultured in Dulbecco's modified Eagle's
medium + 10% fetal calf serum or cultured as described previously
(12). Rat Schwann cells from the sciatic nerve were purified and
cultured in the presence or absence of 4 µm of forskolin as described
previously (15). Primary oligodendrocyte cultures were prepared as
described by Grinspan et al. (16) and maintained in 2 ng/ml
platelet-derived growth factor for 7 days before transfection.
-galactosidase enzymatic assays and Southern blot
analysis for the transfected plasmid were performed as described previously (12, 17). Autoradiographic signals were quantitated by
densitometry. Relative CAT activity was determined as the ratio of the
percentage of 14C converted into mono- and
di-acetylchloramphenicol products to either the amount of the
transfected plasmid DNA or to the -galactosidase activity. For
example, the experiments shown in Fig. 1 were performed using either
Southern blot analysis for the transfected plasmid or cotransfection
with pRSVZ as controls for transfection efficiency. The changes in
relative CAT activity for the progressive deletions were reproduced
using either control. All transfections were performed in triplicate
and, in most cases, were repeated several times with separate plasmid
preparations. The relative CAT activities were expressed as the
mean ± S.E. for n repetitions.
Electrophoretic Mobility Shift Assay (EMSA)-- 10 g of liver, forebrain, or brainstem (includes cerebellum, from the cervical-medullary junction to the midbrain) were harvested from Sprague-Dawley rats of various ages. After homogenization in buffer containing 2.0 M sucrose, 10 mM HEPES (pH 7.6), 15 mM KCl, 1 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 80 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1% w/v low-fat milk, nuclei were pelleted through a sucrose cushion containing the same buffer without low-fat milk by centrifugation in a SW-28 rotor at 24,000 rpm (75,000 × g) for 60 min at 4 °C. Nuclear extracts were prepared from tissue nuclei or from 50-100 million cultured cells as described previously (12), except that extraction Buffer C contained 420 mM NaCl.
EMSA was performed as described in Ref. 12, with the following modifications: (a) probes were prepared from synthetic double-stranded oligonucleotides containing portions of the proximal human MBP promoter (Fig. 2) by end-labeling with both [
-32P]dCTP and [-32P]dGTP using
Klenow enzyme; (b) competitors included NF1
5'-ATTTTGGCTTGAAGCCAATATG-3', NF1 MBP 5'-AATGGCAGGATGCCCAAA-3', and NS
(nonspecific) 5'-AGCTTGGTACTAGTACCGGTACCGCGGCCGCAGATCTCTGCA-3' and were
used at a 100-fold molar excess; (c) the binding reactions contained 100 mM KCl, 2 µg of
poly(deoxyinosinic-deoxycytidylic acid), 1-4 µg of nuclear protein
in 1 µl, and 60,000 cpm of probe (approximately 0.4 ng of DNA); and
(d) protein-DNA complexes were resolved on low ionic
strength 8% polyacrylamide gels in 4× Tris-acetate EDTA buffer.
DNase Footprint Analysis--
End-labeled sense or antisense
fragments from 187 to 25 nt of the proximal MBP promoter (20,000 cpm of probe corresponding to 1-2 ng of DNA) were used in binding
reactions with increasing amounts (10, 20, and 40 µg) of rat adult
brain and liver nuclear extracts as described previously (18). Briefly,
binding reactions were carried out for 45 min at 4 °C in a 50-µl
reaction volume containing 80 mM KCl, 3 mM
MgCl2, 20% (v/v) glycerol, 20 mM Tris-Cl (pH
7.9), 1 mM dithiothreitol, and 1 µg of
poly(deoxyinosinic-deoxycytidylic acid) and then treated with 300 ng of
DNase I for 90 s at room temperature and stopped by the addition
of EDTA/SDS (20 mM and 0.7%, final concentration). In the
NF1 competition assays, 40 µg of nuclear extract were incubated with
either a 100-fold or a 200-fold molar excess of NF1 competitor
oligonucleotides (see above) for 20 min at 4 °C before the binding
reaction. The samples were phenol-extracted, precipitated, and resolved
on 6% polyacrylamide sequencing gels. Maxam-Gilbert G+A sequence
ladders were generated from the same probes by the standard protocols
(19).
Northern Blot Analysis-- Total RNA was prepared from rat forebrain or brainstem by the method of Chirgwin et al. (20) and from cells by the method of Chomczynski and Sacchi (21). Northern blot analysis of 10 µg of total RNA was performed as described in Ref. 12 using either rat MBP (12), rat glyceraldehyde-3-phosphate dehydrogenase (22), or rat proteolipid protein cDNA (23) as a template to generate probes. All quantitation of autoradiographic signals was performed by densitometry (Molecular Dynamics 300A densitometer).
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Nuclear Proteins Bind Upstream of the NF1 Site and Activate the MBP
Promoter--
We showed previously that the 149 to 102 nt segment
of the MBP promoter containing a consensus NF1 site reduced the
transcription of MBP in most cells, but not in oligodendrocytes (12).
To identify other regulatory elements within this segment, we prepared
a series of MBP promoter-CAT fusion constructs containing progressive
deletions from 149 to 95 nt and performed transient transfection
analysis in either primary cultures of oligodendrocytes, which
transcribe MBP, or Cos-7, HeLa, C6, HJC, A7, or L cells, which do not
(12). The results for oligodendrocytes and Cos-7 cells are shown in Fig. 1. Deletion to 95 nt reduced CAT
levels in all cell types, confirming that the proximal segment
activates the transcription of MBP. Instead, deletion to 116, which
bisects the NF1 site, increased CAT levels in both oligodendrocytes and
Cos-7 cells, suggesting that the NF1 sequence silences MBP
transcription. Deletion from 149 to 128 nt reduced CAT levels
approximately 50% in oligodendrocytes, but not in Cos-7 cells. Similar
results were obtained for the five other cell lines tested. These data
suggest that there is an oligodendrocyte-specific activating sequence
upstream of the NF1 site.
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149 to 105 (probe A) and from 149 to 118
(probe B). The results of this experiment are shown in Fig.
2. EMSA of the nuclear extract prepared
from CG4 oligodendrocytes, which transcribe MBP (see below), with probe
A generates a complex pattern of at least six bands (lanes
10 and 11). This binding was sequence specific, because
the six bands were abolished by competition with excess unlabeled A
oligonucleotide (lane 12). Four of the six bands were
abolished by competition with an oligonucleotide containing either the
consensus NF1 sequence or the MBP NF1 sequence (lanes 13 and
14). Conversely, the two remaining bands were abolished by
competition with oligonucleotide B, which contained a truncated NF1
site and the upstream sequence, whereas the first four NF1-related bands were not competed (lane 15). These results suggest
that there is nuclear protein binding upstream of and distinct from NF1.
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The Upstream DNA Binding Site Overlaps the NF1 Site--
The
partial competition of the doublet by cold NF1 oligonucleotide
suggested that its binding site could contain a portion of the NF1
sequence. In keeping with this, the EMSA of CG4 nuclear extracts using
probe C, which contained the sequence from 149 to 124 nt, revealed
no binding, showing that the 5' portion of the NF1 site is necessary to
generate the doublet (Fig. 3). The specific sequence of the 5' portion of the NF1 site was required for
binding, because EMSA with probe D, which contained a transversion mutation of the nucleotides from 124 to 118 nt, produced a
nonspecific band of a different mobility that was not competed by cold
D oligonucleotide (Fig. 3, lanes 15-17).
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MEBA Is Enriched in Oligodendrocytes and Schwann Cells--
To
analyze the tissue distribution of this binding activity, we performed
an EMSA of nuclear extracts prepared from various tissues and cell
types using probe B. As shown in Fig.
4A, the doublet was present in
the brain, in CG4 B() cells, and in Schwann cells, all of which
transcribe MBP. On the contrary, the doublet was not present in the
liver, A1.20, C6, or Cos-7 cells, which do not transcribe MBP.
Therefore, we designated this binding activity MEBA.
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MEBA Correlates with Oligodendrocyte Differentiation and Myelination-- To see whether MEBA is associated with the induction of MBP expression in the developing brain, we correlated the EMSA of brain nuclear extracts with the Northern blot analysis of total RNA, each of which was prepared from the forebrain of rats of various ages. As shown in Fig. 4B, MEBA was present 2 days after birth but then disappeared during the next week. MEBA reappeared at 10 days after birth and increased in amount over the subsequent 8 weeks. Similarly, MBP mRNA was first detected at 10 days after birth and rose to much higher levels in the 18-day-old and 10-week-old forebrain (Fig. 4D). We then correlated the presence of MEBA and MBP mRNA in the brainstem and forebrain at 5 and 18 days after birth, because MBP mRNA appears first in the brainstem and later in the forebrain, as shown in Fig. 4D. EMSA analysis for MEBA showed the same trend; MEBA levels were significantly higher in the brainstem than in the forebrain at 5 days after birth (see Fig. 4C, lanes 1 and 4, in which 2 µg of nuclear extract produced an unsaturated signal), whereas the levels were more similar in the brainstem and the forebrain at 18 days after birth. Thus, MEBA appears as MBP expression is induced during myelinogenesis in the developing forebrain and brainstem, although the early postnatal appearance of MEBA does not correlate with myelination.
The early postnatal appearance of MEBA in the brain could be explained by a contribution of nuclear proteins from cells other than oligodendrocytes. To directly correlate the appearance of MEBA with MBP expression in pure differentiating oligodendrocytes, we analyzed CG4 oligodendrocytes. CG4 cells model oligodendrocyte differentiation well, because they up-regulate myelin-specific gene expression when cultured in the absence of growth factors, and they form myelin when transplanted into rodent brain (24). The MEBA doublet appeared at much higher levels (Fig. 5A) only in the differentiated CG4 oligodendrocytes, as measured by the up-regulation of MBP and proteolipid protein mRNA levels (Fig. 5B). Thus, in pure oligodendrocytes, the appearance of MEBA and MBP mRNA are strictly correlated.
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) CG4 cells, the upper band of the MEBA doublet is present along
with a lower-intensity lower band (Fig. 5A). In contrast, at
a high dosage of nuclear extract from undifferentiated (B104+) CG4
cells, a more intense lower band is seen, but the upper band is not
present. This suggests that the upper band does not simply appear as a
consequence of high dosage of the lower band.
DNase I Footprint Analysis of MEBA Binding--
To locate the MEBA
binding site, we performed DNase I footprint analysis of the MBP
promoter using brain and liver nuclear extracts. Fig.
6 shows that both brain and liver nuclear
extracts protected the sequence from 132 to 106 nt, an extension of
the NF1 binding site from 124 to 111 nt. Extended footprints have been reported for purified NF1 in several other promoters (see Ref. 25
for example). Instead, hypersensitive sites upstream of the NF1
protection at nucleotides 135 to 136, 143, and 147 to 148
appeared more prominently with brain than with liver nuclear extract,
although no protection upstream of nt 132 could be detected. These
results were confirmed in analysis of the opposite strand (data not
shown). To better reveal MEBA binding, we performed footprint analysis
in the presence of NF1 oligonucleotide competitor. As shown in Fig.
6B, a protection from 133 to 127 nt appeared with the
brain extract, but not with the liver extract, as the NF1 footprint was
diminished by competition. Thus, MEBA binds upstream of the NF1 site as
predicted by the EMSA analysis of oligodendrocyte nuclear extracts.
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Mutational Analysis Correlates MEBA Binding and Function--
In a
further attempt to localize the binding of MEBA, we synthesized a
series of oligonucleotides containing transversion mutations from 149
to 124 nt and investigated the resulting effects on MEBA in an EMSA
of oligodendrocyte nuclear extracts. As shown in Fig.
7, mutations between 146 and 141 nt
or between 132 and 125 nt abolished all sequence-specific binding,
whereas a mutation between 140 and 133 nt did not. However, the
mutation between 140 and 133 nt produced a sequence-specific
binding that shifted with a slower mobility (Fig. 7). Thus, MEBA binds two noncontiguous sequences between 149 and 124 nt.
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124 and 110 nt and performed a
transient transfection analysis in primary cultures of
oligodendrocytes. As shown in Fig. 8, the
wild-type pSN149 produced a relative CAT activity 10-fold above the
promoterless construct p0SNCAT. pSN149A with a mutated NF1
site produced CAT levels 400% higher than that of wild-type pSN149,
confirming that NF1 acts as a repressor of MBP transcription. Instead,
the mutations between 146 and 141 nt or between 132 and 125 nt
reduced the relative CAT levels by 70 or 60%, respectively, as
compared with that of the wild-type pSN149, whereas the mutation
between 140 and 133 nt did not reduce CAT levels significantly.
Thus, the same two noncontiguous sequences upstream of the NF1 site
that bind MEBA are also necessary for the activation of the MBP
promoter in oligodendrocytes.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Silencers play an important role in determining neuron-specific (26-28) and astrocyte-specific (29) gene expression. In this study, we show that silencing may also be an important mechanism for restricting gene expression to oligodendrocytes. We and others have previously shown that short fragments of proximal MBP promoter are sufficient to mediate oligodendrocyte-specific transcription in transgenic mice (3, 7), and that both positively and negatively acting DNA sequences regulate activation of the proximal MBP promoter in primary cultures of oligodendrocytes (12). Here, we showed that an NF1 site in that region silences the MBP promoter in many cell types that do not transcribe MBP. Instead, in oligodendrocytes, we identified an activating sequence upstream of and overlapping the NF1 site in the MBP promoter. A protein complex, MEBA, binds this site and is enriched in nuclear extracts from the brain, oligodendrocytes, and Schwann cells in which MBP is transcribed. Mutational analyses of MEBA correlate its binding and function and suggest that MEBA binds two noncontiguous sequences to activate the MBP promoter. Our results suggest that unlike many neuron-specific genes that are regulated via the reciprocal expression of activators in neurons and repressors in other cells, oligodendrocyte-specific genes such as MBP are regulated by the interaction of activators and repressors expressed simultaneously in oligodendrocytes, whereas the repressors are expressed ubiquitously in other cells. Finally, silencers are also used to restrict gene expression to glia in Drosophila (30). Thus, an emerging body of work suggests that the restriction of gene expression by silencers is a general, evolutionarily conserved mechanism used to differentiate neurons and various glia in the central nervous system.
Association of MEBA with MBP Transcription in Vivo and in Vitro-- We found MEBA in oligodendrocytes and Schwann cells, both of which transcribe MBP and synthesize myelin. In the brain, however, the appearance of MEBA is not perfectly correlated with myelination in development, as we found MEBA in forebrain nuclear extracts at 2 days after birth, when MBP transcription and myelination are minimal. One potential explanation is that cells other than oligodendrocytes contribute nuclear proteins whose binding is similar to that of MEBA. In fact, MEBA appeared robustly in tight correspondence with MBP mRNA as cultured CG4 oligodendrocytes differentiated. Another possibility is that MEBA acts in combination with other transcription factors to activate the MBP promoter, because DNA elements outside of the proximal MBP promoter are required for full activation of MBP transcription during peak myelinogenesis (3). Thus, this combination of factors may correlate with MBP transcription and myelination, even if the presence of its component factors, such as MEBA, does not. Finally, an EMSA of nuclear extracts prepared from cultured Schwann cells also produces bands with the mobility of MEBA. Although we showed previously that DNA sequences in the proximal MBP promoter activate transcription differently in Schwann cells as compared with oligodendrocytes (17), MEBA could play an important role in both. Further identification and purification or cloning of MEBA constituents will create the reagents necessary to clarify these important issues.
MEBA Binds a Novel cis-acting Sequence-- Extensive functional and biochemical studies of the proximal MBP promoter have not previously revealed MEBA or its binding site. However, many of these studies were performed using cells that do not transcribe MBP (reviewed in Ref. 12), and here we show that MEBA is not present in those cells. In the few other functional studies performed in oligodendrocytes, deletional and mutational analysis targeted areas that would not have revealed MEBA (31, 32). Of note, in vitro transcription analysis of the proximal MBP promoter in HeLa nuclear extracts has revealed a potential repressor, Myef-2. However, its target silencer is in a more proximal region of the MBP promoter, and Myef-2 function has not been characterized in oligodendrocytes as compared with cells that do not transcribe MBP (33, 34); therefore, what role, if any, it may play in restricting MBP expression to oligodendrocytes is not clear.
Biochemical analyses of the region from150 to 100 nt of the MBP
promoter include footprint analysis of brain nuclear extracts that
revealed an extended protection upstream of the 124 to 110 nt NF1
site that could represent MEBA (10). However, NF1 footprints typically
extend beyond the consensus TGGN7CCA binding site. For example, the same extended footprint from 130 to 105 nt was produced using purified NF1 (35). In keeping with this, NF1 binding in
other promoters has limited the recognition of nearby binding by other
transcription factors (36). In fact, our footprint analysis in the
presence of excess competitor NF1 oligonucleotide revealed a protection
from 133 to 127 nt with brain nuclear extract, but not with liver
nuclear extract. This provides direct evidence of MEBA binding upstream
of the NF1 site and coincides well with one of the two binding sites
(132 to 125 nt) predicted by the functional and EMSA binding
analysis of transversion mutants (see Figs. 7 and 8). Downstream of the
NF1 site, EMSA analysis of the sequence from 128 to 94 nt has
revealed binding (37). A cDNA encoding for a protein, MRF-1, with
similar nucleotide binding specificity was subsequently identified
(38). However, it is highly unlikely to be a component of MEBA, because
MRF-1 is a single-stranded RNA/DNA-binding protein, and methylation interference analysis identified the majority of nucleotides required for its binding to be downstream of the NF1 site; they are not contained in probe B that we used to identify MEBA. Finally, MEBA is
not simply a variant of NF1, because it is clearly distinct from NF1,
based on the cross-competition data of Fig. 2. That NF1 partially
competes MEBA binding of probe B suggests only that some of the same
sequences are necessary for both NF1 and MEBA binding.
Precise identification of the MEBA binding site is difficult with crude
nuclear extracts, because NF1 binding obscures MEBA binding in
footprint analysis (see above), and MEBA shifts relatively few counts
in EMSA analysis (see Fig. 4, lane 4). For example, EMSA
analysis of two-base transversions from 149 to 116 nt produced variable alterations in MEBA (data not shown). However, the binding site identified by the EMSA analysis of eight nucleotide transversion mutations is a discontinuous, nonpalindromic sequence that suggests binding by distinct proteins. Also, dosage analysis of the bands in the
MEBA doublet show that the upper band is not stoichiometrically related
to the lower band, suggesting that MEBA consists of at least two
proteins, not multimers of one protein (see Fig. 5). Finally, Signal
Scan analysis for previously reported cis-acting sequences
(Ref. 39; http://bimas.dcrt.nih.gov/molbio/signal/) did not reveal an
exact match to the MEBA sequence other than the 5' half of a NF1
binding site. Taken together, these findings suggest that the MEBA
binding site is novel, and that MEBA may comprise a unique combination
of factors.
Model of MEBA and NF1 Interactions--
Our analysis of the MBP
promoter performed in oligodendrocytes is in keeping with and extends
the model proposed by Mikoshiba and colleagues (11) based on in
vitro transcription analysis performed with brain nuclear
extracts. Mouse MBP promoter from 256 to 53 nt (contains the
composite MEBA/NF1 binding site) activated transcription in brain
nuclear extracts and silenced transcription in liver nuclear extracts
when placed upstream of the 53 to +60 nt basal MBP promoter (11).
Moreover, mutation analysis showed that nucleotides CAG from 122 to
119 were required for transcription in brain extracts. Because these
nucleotides lie in the 5' half of the NF1 site, these authors concluded
that NF1 is required for brain-specific activation of the MBP promoter (10). They further identified a novel NF1 isoform in the brain and
speculated that brain-specific NF1 was responsible for brain-specific activation of the MBP promoter (40). However, they also found by
transfection analysis that the sequence containing the NF1 site
silenced the MBP promoter in cells not transcribing MBP (see Fig. 6 in
Ref. 40). Also, we note that these nucleotides are contained in the
MEBA/NF1 overlap sequence that we showed was required for MEBA binding
and function. Therefore, we would modify their model to suggest that
MEBA and NF1 interact at this sequence to activate MBP
transcription.
116 or a transversion mutation of the NF1/MEBA overlap sequence activates the MBP promoter in oligodendrocytes. Because either mutation removes both MEBA and NF1 binding, MEBA may not
be required as a direct activator; rather, MEBA's role may be to
compete NF1 binding and thereby activate the MBP promoter. Cloning of
the genes encoding MEBA proteins and identification of the NF1 isoforms
expressed by oligodendrocytes will be required to test these
hypotheses.
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ACKNOWLEDGEMENTS |
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We thank Sue Shumas for excellent technical assistance and Prof. Edoardo Boncinelli (San Raffaele Scientific Institute, Milano, Italy) for support to C. T., A. P., M. L. F., and L. W.
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FOOTNOTES |
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* This study was supported in part by grants from the Associazione Italiana Sclerosi Multipla (to L. W.), the Istituto Superiore Sanità, Progetto Sclerosi Multipla (to L. W.), and the National Multiple Sclerosis Society (to L. W. and J. K.). C. T. and A. P. contributed equally to this study.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.
§ Supported by an advanced postdoctoral fellowship from the Associazione Italiana Sclerosi Multipla.
** To whom correspondence should be addressed: San Raffaele Scientific Institute, DIBIT, via Olgettina 58, 20132 Milan, Italy. Tel.: 011-39-02-2643-4837; Fax: 011-39-02-2643-4767; E-mail: l.wrabetz{at}hsr.it.
The abbreviations used are: MBP, myelin basic protein; NF1, nuclear factor 1; EMSA, electrophoretic mobility shift assay; nt, nucleotide(s); CAT, chloramphenicol acetyltransferase.
2 L. Wrabetz and J. Kamholz, unpublished results.
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REFERENCES |
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Abstract
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Discussion
References
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