From the
The myelin basic protein (MBP) gene is expressed only in
oligodendrocytes and Schwann cells, and expression follows a tightly
regulated developmental time course. Cell type- and developmental
stage-specific expression of the MBP gene appears to be regulated by a
series of cis-acting elements located upstream of the transcription
start site. The proximal element of the MBP regulatory region (MB1),
located between nucleotides -14 and -50, is one of several
elements participating in the programmed expression of MBP. In this
report, we describe the molecular cloning and characterization of
myelin gene expression factor-2 (Myef-2), a protein isolated from mouse
brain that binds specifically to single-stranded DNA derived from the
MB1 element and represses transcription of the MBP gene in transient
transfection assay. Myef-2 mRNA is developmentally regulated in mouse
brain; its peak expression occurs at postnatal day 7, prior to the
onset of MBP expression. The developmental pattern of Myef-2 mRNA
expression coincides with that previously described for SCIP, a POU
domain transcription factor that also represses myelin basic protein
expression. The myef-2 gene maps to mouse chromosome 2. The
relevance of these findings for regulation of MBP gene expression and
oligodendrocyte differentiation is discussed.
Myelin basic protein (MBP)
Functional dissection of the MBP
5`-flanking region has identified multiple elements important for cell
type-specific transcription in transient transfection and cell-free
in vitro transcription
systems
(7, 8, 9) . Distal promoter elements,
between nucleotides -93 and -209 relative to the
transcription start site, stimulate MBP transcription and encompass
binding sites for the CTF/NF1 and thyroid hormone receptor
transcription factors
(10, 11) . A proximal promoter
element of the MBP gene, termed MB1 and located at nucleotides
-14 to -50 relative to the transcription start site, has
been shown to participate in cell type-specific transcription of the
MBP gene (12-14). A developmentally regulated nuclear protein
derived from mouse brain has recently been identified that binds the
MB1 element and stimulates MBP gene transcription
(15) .
It
has long been assumed that promoter and enhancer binding factors
recognize their cognate sites on duplex DNA
(4, 16) ,
although some transcription factors have the capacity to bind in a
sequence-specific manner to single-stranded DNA. The myogenic
determination factor MyoD has been shown to bind specifically to the
muscle-specific enhancer of the mouse creatine kinase M gene, whether
the element is presented in duplex or single-stranded form
(17) .
The estrogen receptor, a nuclear protein that acts as a receptor for
the hormone estradiol and modulates transcription of genes containing
its cognate estrogen responsive element, binds the estrogen responsive
element only when presented in single-stranded form
(18) or in
the presence of an associated single-stranded DNA binding
protein
(19) . Other novel transcription factors have been
identified that possess sequence-specific single-stranded DNA binding
activity, interacting with elements in a number of muscle-specific
genes
(17, 20) and in the regulatory region of the human
neurotropic JC virus
(21) . Perhaps it should be emphasized that
the importance of this kind of interaction in transcription of the
respective genes has been well established.
The promoter regions of
several genes are unusually sensitive to nucleases, indicating that
these regions assume unusual non-B DNA conformations. As first
demonstrated by Weintraub and Groudine
(22) , the enzyme DNase I
preferentially digests genes that are transcriptionally active. In this
respect, analysis of the chicken adult
Since the torsional stress of DNA bending may be relieved by
localized melting of the double helix
(25) , the site-specific
bends observed in the promoter regions of some eukaryotic genes may
indicate the presence of single-stranded DNA in these elements. In this
context, it has been demonstrated that several transcription factors
can bind to bent DNA and/or induce DNA bending after forming
DNA-protein complexes (26-31). Of particular interest is the
recent finding that the general transcription factor TFIID, which binds
to the TATA box of many pol II promoters, has an affinity for its site
on single-stranded DNA and induces localized bending of the DNA
molecule
(27, 32) . Despite its lack of a consensus TATA
motif, the MB1 element has been reported to interact with
TFIID
(13) , leading to the assumption that this element may
exhibit a non-B DNA conformation.
In this study, we have
investigated the potential role of sequence-specific single-stranded
DNA binding proteins in mediating the transcriptional activity of MBP
through the MB1 element. Using the in situ filter detection
method
(33) , we have identified a mouse brain cDNA encoding
Myelin Expression Factor-2 (Myef-2), a protein which binds specifically to the noncoding
strand of the MB1 element of the MBP gene. We have also characterized
the DNA binding activity, tissue distribution, and developmental
regulation of the Myef-2 gene product and assessed the transcriptional
activity of Myef-2 at the molecular level.
For eukaryotic
expression, an initiator methionine codon (AUG) and the proper flanking
sequences
(36) were engineered into a synthetic double-stranded
oligonucleotide (coding strand, 5`-TCGACGCCGCCATGG-3`; noncoding
strand, 5`-AGCTCCATGGCGGCG-3`) and inserted into pBlue-Myef-2 between
the SalI and HindIII sites directly upstream of the
5`-end of the Myef-2 cDNA. The cDNA containing the initiator codon was
excised by digestion with SalI and XbaI and cloned
directly downstream of the cytomegalovirus (CMV) immediate early
promoter to generate the eukaryotic expression vector pCMV-Myef-2. In
pCMV-Myef-2, the initiator codon and vector sequences encode the
heptapeptide MELDIEF, which is fused to the N terminus of the Myef-2
protein.
To further localize the
binding site for Myef-2 within the MB1 element and determine the
specificity of DNA-protein complex, two partially overlapping synthetic
oligonucleotides representing the 5`- (MB1
To examine
the site-dependent repression of MBP gene transcription by Myef-2,
mutations were introduced at the Myef-2 binding site shown in
Fig. 3D (altering 5`-TTGTCC-3` to 5`-GAGTCC-3`) of the
full-length MBP promoter. The mutant plasmid, pMB
The Myef-2 gene product appears to be a repressor of
transcription from the mouse MBP promoter. It is not surprising that
this repressor functions most efficiently in non-glial cells, since
those cells do not normally express the endogenous MBP gene and must,
therefore, contain all the necessary cofactors required for suppression
of MBP expression. Transcriptional repression has been postulated to
occur by a number of mechanisms, including direct competition with
basal transcription factors, inhibition of the binding of activator
proteins to their cognate sites, and ``silencer'' function
analogous to activation by enhancers (Ref. 16, also reviewed in Refs.
51 and 52). Identification of the cofactors present in non-glial cells,
which allow Myef-2 to efficiently suppress MBP expression, will
increase our understanding of transcriptional repression mechanisms in
general.
We cannot exclude the possibility that the full-length
Myef-2 protein has a transcriptional effect different from the one
observed here. In other systems, positive-acting transcription factors
have been shown to suppress transcription when truncated, either by
competing with the wild-type protein for binding sites on DNA
(trans-dominant effect) or by competing for accessory factors by
protein-protein interactions (squelching). However, similarities
between the developmental profile of Myef-2 expression in mouse brain
and that of another MBP transcriptional repressor, SCIP
(49) ,
also called Oct-6 or Tst-1, suggest that this protein may indeed
repress MBP gene expression.
Further hypotheses can be formulated by
comparison with SCIP. SCIP is a POU domain transcription factor that
represses expression of several myelin-specific genes, notably MBP and
P0 glycoprotein
(50) . SCIP evidently participates in maintenance
of the phenotype of the oligodendrocyte progenitor cells, since it is
down-regulated when these cells differentiate into
oligodendrocytes
(53) . The developmental accumulation of SCIP
mimics that of Myef-2, with the level of both proteins peaking prior to
detectable MBP expression in mouse brain tissue
(49) . Whether or
not coordinate regulation of these two repressors of myelin gene
expression plays a role in some common developmental event during
oligodendrocyte differentiation remains to be investigated. Examination
of the molecular mechanism of Myef-2 action may help to decipher this
cellular differentiation pathway. Also of interest is the finding that
SCIP has recently been shown to stimulate transcription of the human
neurotropic JC virus promoter
(54) in direct contrast to its
repressor function on cellular myelin-specific genes. Experiments to
assess the effect of Myef-2 on the JC virus promoter are underway in
our laboratory.
Myef-2 contains the RRM-type nucleic acid binding
motif common to a growing family of proteins that bind single-stranded
DNA and RNA in a sequence-specific manner
(24, 46) . Some
proteins in this class appear to participate in cell type-specific
transcriptional regulation, e.g. CBF, a transcriptional
repressor of the human smooth muscle
The mouse mutations
anx (anorexia), lm (lethal milk), mg (mahogany), pa (pallid), ro (rough), Tsk (tight skin), and we (wellhaarig) map near the Myef-2 gene
(56) . The gene product of only the pa mutation (Ebp4.2) has been identified. Two of the
remaining mutations (anx and lm) display a
neurological phenotype with behavioral abnormalities (summarized in
Ref. 57), thus making Myef2 a potential candidate gene.
However, Myef2 mRNA is expressed in many tissues and may play
a role in biological processes other than myelination. Therefore, a
role for Myef-2 in any of these mutations cannot be excluded. Mutation
of the Myef-2 gene is apparently not responsible for any known
dysmyelinating mouse mutant, since no previously characterized mutant
involving dysmyelination maps to this region of mouse chromosome
2
(57, 58) . Interestingly, the epilepsy (EL) mouse is a
neurologic mouse strain with unusual susceptibility to seizures, and
one of the loci participating in this phenotype, denoted El-2, maps near Mpmv28 on mouse chromosome 2 (40).
Experiments presently in progress in our laboratory include cloning
of a full-length Myef-2 cDNA and characterization of the effect of
Myef-2 on other oligodendrocyte-specific promoters of both cellular and
viral origin. The murine model system allows us to examine the
distribution of Myef-2 in mouse brain at the cellular level. By
mutating the endogenous Myef-2 gene in transgenic mice, we may
determine whether this protein plays a role in myelinogenesis in
vivo. Identifying the nature of the biochemical differences
between glial and non-glial cells, which allow Myef-2 to function more
efficiently in some cell lines than others, will help us to understand
the complex regulatory mechanisms involved in expression of myelin
basic protein and other myelin-specific gene products.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We are very grateful to Dr. A. T. Campagnoni for
kindly providing cDNA expression library and for valuable comments and
helpful discussions. We thank members of the Molecular Neurovirology
Section of the Jefferson Institute of Molecular Medicine for sharing
reagents and for support and helpful suggestions during the course of
this study. We thank Craig Goldstein and Phillip Liaw for technical
assistance. We also thank Drs. Wayne N. Frankel and Jonathan P. Stoye
for providing the Mpmv-28 flanking probe. We are grateful to Dr.
Devjani Chatterjee for assistance with sequence analysis. We thank
Marina Hoffman and Jennifer Gordon for critical reading of this
manuscript and Cynthia Schriver for preparation of this manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a
peripheral membrane protein that is required for maintenance of the
compact multilamellar membrane structure of mature myelin. This protein
is produced only by specialized myelin-forming cells, i.e. oligodendrocytes in the central nervous system and Schwann cells
in the peripheral nervous system (reviewed in Ref. 1). Myelin formation
occurs postnatally in the mouse, such that MBP is first detected at 7
days postnatal, increases dramatically to a peak at 18 days, and
decreases to about 20% of peak levels in the mature animal
(2) .
Steady-state levels of MBP mRNA correspond well with the observed
pattern of protein accumulation
(3) , and direct measurement of
MBP RNA synthesis by nuclear run-on transcription assay demonstrates
that MBP gene expression is determined primarily at the level of
transcript synthesis (4-6).
-globin gene indicates that
its 5`-flanking region is highly sensitive to cleavage by nuclease S1
(23) and by bromoacetaldehyde (24), suggesting that this
promoter region contains unpaired or single-stranded DNA bases.
Library Screening by in Situ Detection of DNA
Binding Activity
The 15-day postnatal mouse brain cDNA
library cloned in gt-11 (kindly provided by Dr. A.T. Campagnoni,
UCLA) was screened as described
(33) for clones encoding DNA
binding proteins. The probe was generated by end labeling of the
single-stranded MB1 oligonucleotide
(5`-TCAGAGGGCCTGTCTTTGAAGGTGTTGTCCTCCCT-3`) using T4 polynucleotide
kinase and [
P]ATP
(34) . Recombinant
phage
(400, 0) were screened with the MB1 probe, and the
Myef-2 cDNA was one of two clones isolated.
Subcloning and DNA Sequencing
Myef-2
phage was grown by the plate lysis method
(34) , and DNA was
purified using commercially available Qiagen columns. Phage DNA was
digested with EcoRI to release a cDNA fragment of
approximately 2.5 kb in size. Plasmid pBlue-Myef-2 was generated by
insertion of the 2.5-kb Myef-2 cDNA into the EcoRI sites of
pBluescript KS (Stratagene). The Myef-2 cDNA was sequenced from
pBlue-Myef-2 using the dideoxy chain termination method
(35) .
Sequencing proceeded from both ends until the sequences overlapped, and
sequences were verified by sequencing of the opposite strand. Sequence
analysis was performed on GCG sequence analysis software through the
Jefferson Cancer Institute shared computer facility.
Expression of Recombinant Myef-2 Protein in Bacteria
and Southwestern Blot Analysis
Maltose-binding/Myef-2
fusion protein and maltose-binding/lacZ fusion protein were
produced in Escherichia coli HB101 using the plasmids
pMAL-Myef-2 and pMAL-cRI, respectively, according to the procedure
described by the supplier (New England BioLabs). For Southwestern blot
analysis, approximately 100 µg of proteins were fractionated on a
10% SDS-polyacrylamide gel, and the proteins were transferred to
nitrocellulose filters by a transblot apparatus (Bio-Rad Laboratories)
using transfer buffer containing 192 mM glycine, 25
mM Tris-base, and 20% methanol for 3 h at 400 mA. The
transferred proteins were denatured in buffer containing 50 mM
Tris (pH 8.0), 7 M guanine HCl, 50 mM NaCl, 1
mM EDTA, 1 mM dithiothreitol for 1 h at room
temperature. The nitrocellulose filters were immersed in 50 mM
Tris (pH 8.0), 100 mM NaCl, 2 mM dithiothreitol, 2
mM EDTA, 0.1% Nonidet P-40, and 0.25% Blotto for 24 h at 4
°C prior to incubation with 10 cpm/100 ng of
P-end-labeled single-stranded probes in TN50 buffer
containing 20 mM Tris (pH 8.0), 1 mM EDTA, and 100
mM NaCl. After incubation for 1 h, filters were washed five
times in TN50 buffer, dried, and exposed 3-5 h at -70
°C.
RNA Isolation and Northern Blot
Analysis
Total RNA was isolated from mouse tissues by the
guanidine isothiocyanate/cesium chloride method
(37) . Total RNA
samples (20 µg/lane) were separated on a formaldehyde-agarose gel,
stained with ethidium bromide, and photographed. Gels were then
transferred to nylon membrane (Hybond N, Amersham Corp.). Myef-2 and
MBP (gift of Jeffrey Green, Frederick Cancer Research and Development
Center, Frederick, MD) cDNA probes were labeled by nick translation
with [P]dCTP, hybridized at a concentration
of 2.5
10
cpm/ml at 65 °C in a solution
containing 5
SSC, 2
Denhardt's solution, 0.1% SDS,
and 0.1 mg/ml salmon sperm DNA, and washed under high stringency
conditions (65 °C, 0.1
SSC, 0.1% SDS). Blots were
quantitated by laser densitometry of autoradiograms.
Chromosomal Localization
To identify
useful restriction fragment length polymorphisms (RFLPs) for mapping
the MYEF2 locus, genomic DNAs from AEJ/Gn and Mus spretus mice were digested with 14 different restriction enzymes and
analyzed by Southern blot hybridization as described
(38) , The
Myef-2 cDNA detected multiple fragments in both species, some of which
were polymorphic. To simplify the RFLP analysis, hybridization with a
253-bp BamHI fragment derived from the 3`-end of the Myef-2
cDNA was performed, detecting a 13-kb HindIII fragment in
M. spretus and a 4.3-kb HindIII fragment in AEJ/Gn.
DNA from N progeny of an (AEJ/Gn
M.
spretus)
AEJ/Gn interspecific backcross
(38) were
subjected to Southern blot hybridization with the Myef-2 probe and
scored for the presence or absence of the 13-kb M. spretus band. Results using the full-length and 253-bp Myef-2 probes were
in complete agreement. RFLP mapping of probes that identify the
B2m, II1a, and Avp loci has been
described
(38) . A probe flanking the MPMV28 integration
site (gift of Wayne N. Frankel, The Jackson Laboratory, Bar Harbor, ME
and Jonathan P. Stoye, NMRC, Mill Hill, London, United Kingdom) was
also used in the interspecific backcross; the MPMV28 locus was
previously mapped in other crosses
(39, 40) . The simple
sequence length polymorphism identified by the D2Mit19 oligomer pair
(41) was mapped by polymerase chain reaction
analysis as described
(42) .
Site-directed Mutagenesis
The 423-bp
HindIII-BamHI DNA fragment of the mouse MBP gene
upstream of the regulatory sequences (-402 to +21) was
cloned into the HindIII-BamHI site of the M13 mP19
and mutagenized as previously described
(43) . The mutagenic
oligonucleotide contained altered nucleotides in the Myef-2 binding
site within the MB1 domain as illustrated in Fig. 3D.
After sequencing of the candidate mutated clones, the
HindIII-BamHI fragment was removed from the
double-stranded phage DNA and placed at 5`-position of the CAT gene in
the pBL-CAT promoter plasmid.
Figure 3:
Binding of Myef-2 to single-stranded DNA
derived from the proximal promoter element (MB1) of the MBP gene.
A, Coomassie Blue staining of protein extracts from
IPTG-treated E. coli containing control pMAL-cRI (lane1) and recombinant pMAL-cRI-Myef-2 (lane2) on 10% SDS-polyacrylamide gel electrophoresis. The
arrow indicates position of the Myef-2 fusion protein.
B, Southwestern analysis of bacterial extracts as shown in
panelA with the probes representing the non-coding
strand of MB1 or its various regions MB1 and MB1
sequences. C, Coomassie Blue staining and Southwestern
analysis of proteins derived from E. coli containing
pMal-cRI-Myef-2, which were grown in the absence (lane1) or presence (lane2) of IPTG. An
arrow indicates the position of Myef-2, and the asterisk points to the unrelated band appearing in both extracts upon
binding to MB1dl
. D, primary structure of the
oligonucleotides used in this study. The underlined nucleotides represent the binding site for Myef-2, and the
dottedunderlinednucleotides indicate the
position of the sequence with similarity to the Myef-2 binding
site.
Transient Transfection and CAT Assay
The
reporter plasmid pMB402 contains mouse MBP promoter/enhancer sequences,
from nucleotide -402 to +21 relative to the transcription
start site, cloned directly upstream of the CAT coding region as
described
(15) . Transfections were performed by the calcium
phosphate precipitation method
(44) . At 48 h post-transfection,
cellular proteins were harvested, and an equal amount of protein
extract was used to analyze for CAT activity. Each experiment was
repeated multiple times to ensure transfection efficiency and
reproducibility of the results.
Molecular Cloning Identifies Myef-2, a Novel DNA
Binding Protein
The proximal cis-acting element of the MBP
gene, termed MB1, directs cell type-specific transcription of a
heterologous promoter in cultured cells and is expressed in a
tissue-specific manner in
vitro(7, 12, 13, 14) . Since
tissue-specific and developmentally regulated DNA binding proteins are
known to interact with the MB1 region
(15, 45) , we set
out to isolate cDNA clones encoding such DNA binding proteins using the
in situ filter detection method. A gt11 cDNA library
prepared from mouse brain RNA was screened with a single-stranded
oligonucleotide corresponding to the noncoding strand of the MB1
element. We chose to use the single-stranded DNA probe since our
preliminary bandshift assay revealed a strong and specific interaction
of nuclear proteins from brain with the critical proximal regulatory
element of MBP encompassing the MB1 sequence. The result of this
screening was isolation of partial Myef-2 cDNA clone. Sequence analysis
of the Myef-2 cDNA (Fig. 1) predicts that it encodes a novel
protein of 436 amino acids, consistent with its apparent molecular mass
in SDS-polyacrylamide gel electrophoresis of 46 kDa (data not shown).
The predicted protein is basic in nature (pI = 9.9).
Figure 1:
Primary sequence of the Myef-2 cDNA.
Sequence determination of the Myef-2 cDNA was by the method of Sanger
et al. (35). Sequences were verified by sequencing of the
second strand. Translation of the Myef-2 cDNA predicts a protein of 435
amino acids, which is shown below the nucleotide
sequences.
The
Myef-2 protein contains two copies of an RNA recognition motif (RRM),
previously shown to be responsible for sequence-specific binding to
both RNA and single-stranded DNA
(24, 46) . Of all
RRM-containing proteins, Myef-2 shares the greatest degree of homology
with heterogeneous ribonucleoprotein M4 (hnRNP M4)
(47) . The
N-terminal (amino acids 1-203) and C-terminal (amino acids
350-435) regions of Myef-2 share some homology with hnRNP M4,
while the central region (amino acids 204-349) is more divergent
(Fig. 2). The N-terminal RRM, denoted RRM1 and found within the
N-terminal homology region (amino acids 78-162), is 87% identical
to the corresponding RRM in hnRNP M4. The C-terminal RRM, denoted RRM2
(amino acid 350-435), shares 70% amino acid sequence identity
with its corresponding sequence in hnRNP M4. RRM1 and RRM2 demonstrate
a lesser degree of homology to RRMs found in a wide variety of RNA and
single-stranded DNA binding proteins
(24, 46) . Sequence
analysis demonstrates that Myef-2 contains no other commonly recognized
DNA binding motifs, such as zinc finger, homeobox, POU, or
helix-loop-helix domains. Presumably, the RRMs are responsible for the
sequence-specific single-stranded DNA binding activity of Myef-2, and
their presence suggests that this protein may also bind RNA.
Figure 2:
Sequence homology between Myef-2 and hnRNP
M4 proteins. Top, schematic diagram of the structure of the
Myef-2 and hnRNP M4 proteins. RRMs in both proteins are shown as
openboxes, and homologous regions are shown with
dottedlines. Sequence homology is greatest within
the RRM1 and RRM2 motifs. Bottom, amino acid sequence
comparison of RRM1 and RRM2 to homologous regions in hnRNP M4 is shown,
and identical residues are marked with lines between the two
sequences. The most highly conserved residues within the previously
characterized RRM consensus are underlined (20).
Myef-2 Binds Specifically to the Noncoding Strand of
the Proximal (MB1) Element of the MBP Promoter
To study the
DNA binding properties of Myef-2, the protein was expressed in E.
coli using the bacterial expression vector pMAL-cRI. This vector
produces the protein of interest as a fusion with maltose binding
protein. The ability of the Myef-2 fusion protein to bind the MB1
sequence was examined by Southwestern blot assay. The control protein
that consists of maltose binding/lacZ fusion was produced in
bacteria containing pMAL-cRI. As shown in Fig. 3, lane2, the maltose binding/Myef-2 fusion protein bound to MB1
oligonucleotide probe appeared in the IPTG-treated cells (panelB). This binding was not observed in the extract
containing the control maltose binding/lacZ fusion protein
(Fig. 3B, lane1), which demonstrates
specificity of MB1-Myef-2 interaction.
) and the 3`-
(MB1
) non-coding sequence of the MB1 (shown in
Fig. 3D) were used as probes in a Southwestern assay. As
shown in Fig. 3B, unlike the MB1
probe,
MB1
showed a significant binding affinity to the Myef-2
fusion protein, suggesting that the critical nucleotides for binding of
the Myef-2 reside in the 12 base pairs of the MB1
that do
not overlap with the MB1
sequences. To further identify the
important nucleotides for binding of Myef-2 to MB1
, we
performed Southwestern blot analysis using MB1dl
probe,
which contains alterations in the pentanucleotides (5`-TGTCCT-3`
5`-atcag-3`) located in the center of the MB1
. As shown in
Fig. 3D, MB1dl
showed insignificant, if any,
binding activity corresponding to Myef-2. The mutant oligonucleotide
showed binding activity to an unrelated bacterial protein produced in
uninduced and induced cells (shown by asterisk in
Fig. 3C, lanes1 and 2).
These observations corroborate our previous competition bandshift assay
data and indicate that the pentanucleotide sequence 5`-TGTCC-3` (as
underlined in Fig. 3D) is the important component of the
Myef-2 binding site within the MB1
(48) . From these
data, we conclude that Myef-2 binds efficiently to specific sequences
located in the non-coding strand of the MB1 regulatory element of the
MBP promoter.
Myef-2 Represses Transcription of the MBP
Gene
We employed transient cotransfection assay to
investigate the possible role of Myef-2 in modulating transcription of
the MBP gene (Fig. 4). Consistent with the restricted tissue
specificity of the MBP promoter, the reporter plasmid pMB402,
containing a 423-bp segment of the MBP promoter, was expressed more
strongly in glial cells (U-87MG, 2.81%; RN22, 2.65%) than in non-glial
3T3 fibroblasts (1.10%) in the absence of exogenous Myef-2. In fact,
even this low level of basal expression in non-glial cells is unusual,
and no CAT activity was detectable in non-glial Hela cells (data not
shown).
Figure 4:
Transcriptional activity of Myef-2.
Transient cotransfection analysis of Myef-2 transcriptional activity in
3T3 mouse fibroblasts (A), U-87MG human glioblastoma cells
(B), and RN22 rat Schwannoma cells (C). Reporter
plasmid pMB402 containing mouse MBP promoter sequences from nucleotides
-402 to +21 relative to the transcription start site was
cotransfected with Myef-2 expression vector pCMV-Myef-2 and/or control
plasmid pCMV. CAT activity is expressed as percent conversion (bargraphs), and errorbars represent S.E.
of three independent experiments performed with different pCMV-Myef-2
plasmid preparations. Representative CAT assay autoradiograms are shown
(insets).
In cotransfection assays, Myef-2 suppressed expression of
the MBP promoter 6.5-fold in 3T3 mouse fibroblasts
(Fig. 4A). A modest (less than 2-fold) yet detectable
decrease was also observed in U-87MG human glioblastoma cells
(Fig. 4B) and RN22 rat Schwannoma cells
(Fig. 4C). Apparently, the decrease seen in glial cells
(U-87MG and RN22) is not greater than the statistical variability of
the experiment. Thus, Myef-2 appears to repress transcription of the
myelin basic protein gene, primarily in non-glial cells.
was introduced either alone or together with the pCMV-Myef-2 into
U-87MG and 3T3 cells. As shown in Fig. 5, expression of Myef-2 in
the transfected cells had no negative effect on the transcriptional
activity of the mutated MBP promoter in U-87MG. Thus, binding of Myef-2
to its target DNA sequence is important for this protein to exert its
regulatory function. Fig. 5also illustrates that in
cotransfection assay, Myef-2 exhibits no negative activity on
transcription of a heterologous simian virus 40 promoter in the
transfected cells. These observations strongly suggest that Myef-2 is a
sequence-specific DNA binding protein that reduces its transcriptional
activity in transfected cells by interacting with its target sequence
on MBP promoter.
Figure 5:
Transcriptional activity of Myef-2 on the
promoters with no Myef-2 binding site. Human glioblastoma cells, U-87MG
(A), and mouse fibroblast cells, 3T3 (B), were
transfected with the reporter plasmid pMB and
pSV-CAT alone or together with Myef-2 expression vector. Cells were
harvested 48 h post-transfection, and CAT assay was performed (59). The
percent conversion of chloramphenicol to its acetylated forms was
determined by liquid scintillation counting of substrate and acetylated
forms. Each experiment was repeated multiple times, and results for a
representative experiment are shown.
Myef-2 mRNA Is Most Abundant in Mouse Brain and Is
Developmentally Regulated
To obtain some clues to the
possible functional role of Myef-2 in transcriptional regulation of the
MBP gene, we compared the expression profile of Myef-2 and MBP during
brain development. Northern blot analysis of total RNA isolated from a
panel of mouse tissues and from mouse brain tissue at different stages
of development indicated that Myef-2 mRNA was expressed at highest
levels in mouse brain (Fig. 6A). Non-neural tissues
expressed 16% (i.e. heart or kidney) to 54% (i.e. spleen) of the levels of Myef-2 mRNA in brain as determined by
laser densitometry of the autoradiograph. In contrast, MBP mRNA was
expressed only in brain tissue (Fig. 6B). Ethidium
bromide staining showed that approximately equal amounts of RNA were
loaded on both gels (Fig. 6C).
Figure 6:
Tissue distribution and developmental
accumulation of Myef-2 mRNA. Northern blot analysis of total RNA (20
µg/lane) derived from brain (lane1), heart
(lane2), kidney (lane3), liver
(lane4), lung (lane5), and spleen
(lane6) of young adult mice and from brain tissue of
mice aged 3 (lane7), 7 (lane8),
14 (lane9), 18 (lane10), 21
(lane11), 30 (lane12), and 60
days (lane13) and from brain tissue of adult
lactating females (lane14). A, tissue
distribution and developmental accumulation of Myef-2, showing a major
band at 3.7 kb. B, Identical blots show tissue distribution
and developmental accumulation of MBP mRNA (2.1 kb). C,
ethidium bromide (EtBr) staining of the gels corresponding to
the blots.
During development,
Myef-2 mRNA levels were maximal in the brain of 7-day-old mice,
decreasing more than 3-fold in 14-day-old mice, and remaining
relatively constant thereafter throughout maturation of the animal
(Fig. 6A). In contrast, MBP mRNA was first detected at
postnatal day 7, increasing dramatically between 7 and 14 days, peaking
at 18 days, and decreasing to a lower level in the adult mouse
(Fig. 6B), in excellent agreement with previous
studies
(2, 3) . Note that the developmental pattern of
Myef-2 mRNA accumulation is very similar to that of
SCIP
(49, 50) , also called Oct 6 or Tst-1, a member of
the POU domain family of transcription factors and a repressor of MBP
transcription.
The myef-2 Gene Maps to Mouse Chromosome
2
The Myef2 genetic locus, encoding the Myef-2
gene product, was localized to mouse chromosome 2 by RFLP and simple
sequence length polymorphism analysis of a panel of murine genomic DNAs
derived from the progeny of an interspecific backcross (Fig. 7).
The Myef-2 probe detected a polymorphic 13-kb HindIII band,
which was scored for mapping of the locus (see ``Materials and
Methods''). Gene order was resolved by minimizing the number of
recombinants between loci along the length of the chromosome. The order
of the loci and the ratio of the number of recombinants to the total
number of N offspring examined are as follows: B2m - 7/190 - Mpmv28 - 1/184 -
(Myef2, Avp, Il1a) - 3/92 - D2Mit19). The
genetic distances between loci in centiMorgans ± S.E. are:
B2m - 3.7 ± 1.4 - Mpmv28 -
0.5 ± 0.5 - Myef2, Avp, Il1a) - 3.3
± 1.7 - D2Mit19. Fig. 5shows the placement
of these genes on the map of mouse chromosome 2. This region of the
mouse chromosome contains genes whose human homologs reside on human
chromosomes 2, 15, and 20. Therefore, the human homolog of the
myef-2 gene likely resides on one of these three chromosomes.
Figure 7:
Genetic linkage map showing the
chromosomal location of the MYEF2 locus. The chromosome on the
left shows the loci typed in the interspecific backcross, with
distances between loci given in centimorgan. The chromosome on the
right shows a partial version of the consensus linkage map of mouse
chromosome 2 (56). MYEF2 is shown in boldfacetype. The chromosomes are aligned at the Il-1a locus. Loci mapped in humans are underlined; locations of
these genes in humans are shown between the
chromosomes.
-actin gene
(20) , and
the polypyrimidine tract binding protein, which binds specifically to
DNA present in a liver-specific enhancer
(16, 55) . Most
other members of this family are RNA binding proteins that are involved
in RNA metabolism (reviewed in Ref. 24). Although HIV-1 Tat protein
provides a model for RNA binding proteins participating in
transcriptional regulation, it is unlikely that Myef-2 represses
transcription through RNA binding since its cognate site is not
transcribed. Probably, Myef-2 interacts with its cognate site in the
MBP promoter DNA sequence to exert its transcriptional effect. We
hypothesize that the MB1 element may adopt some unusual non-B DNA
structure, which facilitates this interaction.
/EMBL Data Bank with accession number(s) U13262.
-D-galactopyranoside.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.