Role for RFX Transcription Factors in Non-neuronal
Cell-specific Inactivation of the Microtubule-associated Protein
MAP1A Promoter*
Atsuo
Nakayama
,
Hideki
Murakami,
Naomi
Maeyama,
Norie
Yamashiro,
Ayako
Sakakibara,
Naoyoshi
Mori, and
Masahide
Takahashi
From the Department of Pathology, Nagoya University School of
Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Aichi,
Japan
Received for publication, September 18, 2002, and in revised form, October 28, 2002
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ABSTRACT |
Microtubule-associated protein MAP1A is expressed
abundantly in mature neurons and is necessary for maintenance of
neuronal morphology and localization of some molecules in association
with the microtubule-based cytoskeleton. Previous studies indicated that its complementary expression together with MAP1B during nervous system development is regulated at the transcriptional level and that
the mouse Map1A gene is transcribed under the
control of 5' and intronic promoters. In this study, we investigated
the regulatory mechanisms that govern the neuronal cell-specific
activation of the MAP1A 5' promoter. We found that two
regulatory factor for X box (RFX) binding sites in exon1 of both the
mouse and human genes are important for effective transcriptional
repression observed only in non-neuronal cells by reporter assays.
Among RFX transcription factor family members, RFX1 and 3 mainly
interact with repressive elements in vitro. Cotransfection
studies indicated that RFX1, which is expressed ubiquitously,
down-regulated the MAP1A 5' promoter activity in
non-neuronal cells. Unexpectedly, RFX3, which is abundantly expressed
in neuronal cells, down-regulated the transactivity as well, when it
was expressed in non-neuronal cells. Both RFX1 and 3 did not
down-regulate the transactivity in neuronal cells. These results
suggest that RFX1 and 3 are pivotal factors in down-regulation of the
MAP1A 5' promoter in non-neuronal cells. The cell
type-specific down-regulation, however, does not depend simply on which
RFX interacts with the elements, but seems to depend on underlying profound mechanisms.
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INTRODUCTION |
MAP1A is a high molecular weight
MAP1 that plays an
important role in determining neuronal morphology and in
determining the balance between plasticity and stability of neuritis
(1, 2). MAP1A and 1B are particularly abundant filamentous MAPs in the brain and possess highly similar structures and functions in their stabilization of microtubule bands. Recent studies have demonstrated that MAP1A interacts with postsynaptic density-93, a
membrane-associated guanylate kinase (3), and is involved in the
general attachment of mRNA to the cytoskeleton (4). Thus, MAP1A
seems to act not only in stabilizing microtubule bands but also in
regulating the distribution of associated molecules as a linker between
them and the microtuble-based cytoskeleton. MAP1B has been shown to link
-aminobutyric acid, type C receptors to microtubule (5), indicating that MAP1A and 1B anchor distinct target molecules to the
cytoskeleton. The different expression patterns of MAP1A and 1B, which
are transcriptionally regulated (6), also support the functional
dichotomy of these two filamentous MAPs. MAP1B is the earliest MAP
expressed during central nervous system development; it is most
highly expressed during the postneonatal period, diminishing as the
animal matures to adulthood (6-9). In contrast, MAP1A expression
increases continually during the postneonatal period and is maintained
at a high level throughout adulthood (6, 8-10).
Multiple promoters, cell type-specific cis-regulatory elements and
interacting homeoproteins have been reported for the MAP1B gene (11-13); little is known, however, about the transcriptional regulation of the MAP1A gene. We previously revealed that
two mRNA species differing at their 5' ends were transcribed from the mouse Map1a gene and that each transcript was regulated
under independent promoters (14). The two alternative transcripts were
expressed principally in the brain, but a short transcript under the
control of an intronic promoter had a broader tissue-specific expression pattern. However, no cis-regulatory elements that confer neuronal cell-specific expression in a reporter assay were identified within about 2 kb 5' to the transcription initiation sites of either
promoter region. In the present study, we further investigate the
regulatory elements necessary for neuronal cell-specific expression of
the MAP1A gene. We present here several lines of evidence
that RFX proteins, which comprise a unique winged-helix transcription family, down-regulate its 5' promoter activity in non-neuronal cells.
RFX homologues in Drosophila and Caenorhabditis
elegans are expressed specifically in subsets of sensory neurons
(15, 16), and the C. elegans RFX homologue is necessary for
the normal function of these cells (15), suggesting that
transactivation of target genes by RFX is crucial for the nervous
system in these species. We propose that mammalian RFX proteins
contribute to the neuronal cell-specific gene expression by their
repressive activity outside the nervous system.
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EXPERIMENTAL PROCEDURES |
RT-PCR--
For expression analysis of the human long isoform of
MAP1A mRNA, 2 µg of RNA isolated from culture cells with an
RNAeasy mini-kit (Qiagen, Hilden, Germany), were primed with random
hexamers and reverse-transcribed with 200 units of SuperScript II
reverse transcriptase (Invitrogen) at 42 °C. PCR was carried out
using the primer pair hU94 (5'-GGCTGGGGCTCCGAAGTCC-3') and hL456
(5'-GCTGCTAAGAAGATGATGAACCTC-3'). PCR with a primer pair for
-actin
(forward: 5'-TCAGAAGGACTCCTATGTGG-3', reverse:
5'-TCTCTTTGATGTCACGCACG-3') was performed in parallel as a control.
Each PCR was carried out using Taq DNA polymerase (Takara
Biomedicals, Tokyo, Japan) for 30 cycles in a GeneAmp 9700 (Applied
Biosystems, Foster City, CA) under the following conditions: denaturing
at 96 °C for 10 s, annealing at 58 °C for 10 s,
extension at 72 °C for 20 s. Some of the amplified products were subcloned into the pGEM T easy vector (Promega, Madison, WI) and
sequenced in both directions with either a Prism Dye primer Sequenase
kit or a BigDye Sequenase kit (Applied Biosystems). The sequence
products were analyzed with a Geneanalyzer 373 and 310 (Applied Biosystems).
Cloning of Human MAP1A Promoter--
Based on the similarity
between the mouse Map1a gene and sequence in the human
chromosome 15 working draft sequence segment (GenBank accession no.
NT010194), we designed primer sets (5' primer:
5'-GGAATGGCTGGAATACTGTT-3', 3' primer: 5'-ACTGCGGTGGAGGGTGTGAC-3') to
amplify putative exon 1 and the 5'-untranslated region of the human
MAP1A gene. Fragments amplified from HeLa cell genomic DNA were subcloned into the pGEM-T vector (Promega) and sequenced.
Luciferase Reporter Constructs--
The mouse Map1a
promoter constructs p1(+23), p1(+57), and p1(+104) were made by
inserting PCR fragments into the pGL3 basic vector (Promega). The PCR
fragments were amplified from a subclone of the mouse Map1a
gene derived from the phage clone N4 described previously (14). A 5'
primer used for all three constructs was designed to add an
XhoI site at the 5' end, and 3' primers specific for
p1(+23), p1(+57), and p1(+104) were designed to add NcoI, HindIII, and XhoI sites, respectively, at the 3'
end. Each amplified fragment was digested with appropriate restriction
enzymes and inserted into the respective sites in the pGL3 basic vector.
The human MAP1A promoter constructs hMP1-2,
hMP1-7, and hMP1-8 were made in a similar way.
PCR fragments were amplified from the human MAP1A gene
subclone described above and were engineered to contain an
XhoI site at the 5' end and a HindIII site at the 3' end. The fragments were introduced between the
XhoI-HindIII sites of the pGL3 basic vector after
restriction enzyme digestion.
Reporter constructs with mutations at the RFX sites, hMP1-8mu1,
hMP1-8mu2, hMP1-8mu3, and hMP1-8mu4, were obtained using a QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, CA). The nucleotide replacements introduced into the parental
hMP1-8 clone are shown in Fig. 4C. Altered MAP1A
gene fragments were sequenced for confirmation, excised, and subcloned
again into the pGL3 basic vector.
The reporter construct, hMP1-2 G4BS, was made by introducing a
PCR-amplified fragment corresponding to 5 tandem repeats of the GAL4
binding site into hMP1-2. The original fragment
containing the 5× GAL4-binding sites was derived from the pFR-Luc
plasmid supplied as a component of the PathDetect® system
(Stratagene) and engineered to contain appropriate restriction sites at
both ends for subcloning.
RFX Expression Constructs--
The human RFX1 and RFX3
expression constructs, pFC R1 and pFC R3, were generated by subcloning
full-length human RFX1 and RFX3 cDNAs into the NotI and
BamHI sites of the pFLAG-CMV-5a vector (Sigma). Full-length
RFX1 and RFX3 cDNA (GenBank accession nos. NM002981 and X76092)
were obtained by PCR from a MARATHON human brain cDNA library
(Clontech) and engineered to contain a
NotI site at the 5' end and a BamHI site at the
3' end. The pFC R1DBD was constructed by inserting a PCR fragment
encoding residues 420-512 of RFX1 into the pFLAG-CMV-5a.
pFC R1-G and pFC R3-G were obtained by inserting a cDNA fragment
encoding the GAL4 DNA-binding domain (DBD) in front of the RFX cDNA
in pFC R1 and pFC R3. The fragment was derived from pAS2-1, the bait
vector in the yeast two-hybrid system
(Clontech). Expected chimeric proteins have
PGGRVG residues between GAL4 DBD and RFX1, and
PGGRPGRSPPSPSPTNNTTIIHSQETI between GAL4 DBD and RFX3. pFC E-G was
constructed by introducing the cDNA fragment encoding GAL4 DBD
alone into the pFLAG-CMV-5a vector.
pEGFP-R1 and pEGFP-R3 were made by inserting the respective cDNAs
into pEGFP-N1 (Clontech) as described above.
All of the PCR fragments described above were sequenced and confirmed
as not harboring any PCR errors. The constructs were introduced into
HeLa cells, and the expression of proteins of the expected sizes was
verified by Western blotting.
Transfection and Luciferase Assay--
Transfection and
luciferase assays were performed as described in our previous report
with minor modification (14). Briefly, each culture cell was plated on
Falcon 12-well tissue culture plates at appropriate densities 16-18 h
before transfection. Transfection was performed using LipofectAMINE and
PLUS Reagent (Invitrogen) according to the manufacturer's
instructions. 0.4 ml of Opti-MEM (Invitrogen) containing 0.3 µg of
the reporter DNA, 0.03 µg of the pRL-TK vector as internal standard
for transfection efficiency, 1.5 µl of LipofectAMINE, and 3 µl of
Plus reagent was applied to one well, in which cells were incubated at
37 °C for 3 h. In cotransfection experiments, 0.2 µg of the
reporter and 0.2 µg of the expression construct, instead of 0.3 µg
of the reporter alone, were applied. After a 3-h incubation, 0.4 ml of
RPMI 1640 medium or Dulbecco's modified Eagle's medium with 20%
fetal bovine serum was added to each well. The medium was replaced with
fresh medium containing 10% fetal bovine serum 24 h after
transfection. Transfected cells were harvested by lysis after an
additional 24 h culture. Cell lysis buffer and reagents used in
the luciferase assay were supplied in a dual luciferase assay kit
(Promega). Luciferase activity was measured by Lumicounter (MicroTech
Niti-on, Saitama, Japan). For each measurement, luciferase activity was normalized by the activity of sea-pansy luciferase derived from the
pRL-TK. Each transfection was performed in triplicate for one
experiment, and at least three independent experiments were conducted.
DNase I Footprinting and Electrophoretic Mobility-shift Assay
(EMSA)--
Nuclear proteins used for DNase I footprinting and EMSA
were prepared as described previously (14). To prepare the probe for
DNase I footprinting, the DNA fragment from
141 to +116 relative to
the transcription initiation nucleotide of exon 1 of the mouse Map1a gene was subcloned into pBlueScript II (Stratagene).
The construct was digested with XhoI and filled in with
Klenow fragment and [
-32P]dCTP to label the 3' end of
the coding strand. The end-labeled DNA was digested with
BamHI, and the excised fragment was gel-purified. Nuclear
proteins and 2000-3000 cpm of the labeled probe were incubated in a
final volume of 100 µl (20 mM Tris, 1 mM
EDTA, 10% glycerol, 0.1% Nonidet P-40, 2 mM
MgCl2, 1 mM dithiothreitol, 2% polyvinyl alcohol, and 1 µg of poly(dI-dC)) at room temperature for 20 min. After the addition of 100 µl of salt mix (10 mM
MgCl2, 5 mM CaCl2), DNase I
digestion was performed on ice for 3 min by adding 10 µl of diluted
DNase I (Takara Biomedicals). Digestion was stopped by adding 200 µl
of stop mix (200 mM NaCl, 40 mM EDTA, 1% SDS, 125 µg/ml tRNA, and 100 µg/ml proteinase K), followed by incubation at 37 °C for 15 min. DNA was extracted, precipitated, resuspended in
denaturing loading dye, and separated on a sequencing gel. The
Maxam-Gilbert sequencing G ladder of the probe was run in parallel as a
molecular size marker.
The oligonucleotides used as EMSA probes and unlabeled competitors were
as follows: 5' RFX, (5'-CGCGGGTGTTTCCATGGAGACCGAGGCC-3'); 3' RFX,
(5'-CGGCGTTGCCATGGAGACAACTGGC-3'); Polyoma virus EP site, (5'-GGCCAGTTGCCTAGCAACTAATAC-3'); 3' RFX mutant1,
(5'-GGCGTTGCCATGCAGTGAACT-3'); and 3' RFX mutant2,
(5'-GGCCATGGCATGGAGACAACT-3'). The above oligonucleotides were annealed
with respective complementary oligonucleotides, and some of them were
labeled with [
-32P]dCTP during a filling-in reaction
using Klenow fragments. The labeled probes were purified with G-25
Quick Spin Columns (Roche Molecular Biochemicals, Mannheim, Germany).
The binding reaction was carried out in a total volume of 16 µl (10 mM HEPES, 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10 mM MgCl2, and
10% glycerol), containing 1 µg of poly(dI-dC) for 20 min at room
temperature. The amounts of nuclear proteins used are indicated in the
legend for Fig. 5. The probes were applied at volumes
corresponding to 10,000-20,000 cpm. The reactions were analyzed by
electrophoresis on 4% polyacrylamide, 22.5 mM Tris borate,
0.5 mM EDTA gels at 200 V for 2-3 h at room
temperature. For supershift assay, the binding reactions were incubated
further with antiserum or preimmune rabbit serum at final dilutions of
1/100 for RFX1, 1/20 for RFX2, and 1/40 for RFX3, for 30 min at room
temperature. Antisera against RFX 1, 2, and 3 were kindly provided by
Walter Reith (University of Geneva, Geneva, Switzerland).
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RESULTS |
Exon1 of the MAP1A Gene Contains Regulatory Elements That Repress
Reporter Gene Expression in Non-neuronal Cells--
Of two 5'
alternative transcripts driven by independent promoters, the expression
of the shorter transcript, regulated by the intronic promoter, was not
highly restricted within neuronal cells (14). Therefore, we focused our
investigation on the 5' promoter that confers brain-specific expression
of the long transcript in mouse tissues and neuronal cell-specific
expression among culture cells. Because we did not find significant
neuronal cell-specific cis elements within a 2.4-kb region 5' to exon 1 (14), we examined a region 3' to the transcription initiation site. We
made several constructs differing in their 3' ends, but all constructs
contained in common the 5' basal promoter consisting of three
Sp1-binding sites and one Y/CCAAT box (Fig.
1A). 3' extension of the
inserted genomic fragment to a part of exon 1 resulted in repression of promoter activity in non-neuronal cells, especially in HeLa cells, whereas it did not significantly affect transactivity in neuronal cells
(Fig. 1B). Further extension of the 3' end had no
significant effects on promoter activity in either neuronal or
non-neuronal cells (data not shown).

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Fig. 1.
Exon 1 of the mouse MAP1A
gene contains cis-elements that repress reporter gene expression
in non-neuronal cells. A, schematic of genomic
fragments inserted into the reporter vector pGL3 Basic. The basal
promoter consists of three Sp1 sites and one Y/CCAAT box. Three
constructs p1(+23), p1(+57), and p1(+104) contain genomic fragments
represented by arrows. The nucleotide number is relative to
the transcription initiation site of exon 1. B, three
non-neuronal cells, NIH3T3, L cell, and HeLa, and three neuronal cells,
Neuro2A, C1300, and TGW, were transfected with the indicated constructs
together with pRL-TK as an internal control for transfection
efficiency. The cells were harvested and assayed for luciferase
activity 48 h after transfection. Promoter activity was normalized
by the sea-pansy luciferase activity derived from pRL-TK, and is given
as -fold activity above the promoterless pGL3 Basic. The values are
averages ± S.E. of at least nine data points. Transfection was
performed in triplicate, and three or more independent transfection
experiments were performed.
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To determine whether the region necessary for the transcriptional
repression in non-neuronal cells was occupied by nuclear proteins, and
to determine the precise nucleotide regions that interact with nuclear
proteins, we performed DNase I footprinting. The result shown in Fig.
2 demonstrates that protected patterns differed slightly from each other depending on the cell type from which
the nuclear proteins were prepared. Two regions spanning from +24 to
+46 and from +68 to +89 relative to the transcription start site of
exon 1, however, were protected in all cases. In addition, multiple
regions of the basal promoter were also protected, confirming our
previous report (14). Data base search by MatInspector V2.2 at
transfac.gbf.de (17) revealed that both of the protected regions
in exon 1 include nucleotide sequences highly similar to the RFX1
binding consensus sequence. Thus, the results presented in Figs. 1 and
2 suggest that exon 1 of the mouse Map1a gene contains repressive elements that confer neuronal cell-specific expression of
the long transcript and that the elements are most likely RFX1 binding
sites, yet they are occupied by nuclear proteins prepared from both
non-neuronal and neuronal cells.

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Fig. 2.
Nuclear proteins from neuronal and
non-neuronal cells bind to RFX sites in exon 1 of the MAP1A
gene. DNase I footprint analysis of a DNA fragment spanning
the region 141 to +116 relative to the transcription initiation site
was performed. The fragment, labeled at the 3' end of the coding
strand, was incubated with either 10 µg or 3 µg of nuclear proteins
from Neuro2a, NIH3T3, TGW, and HepG2 cells. Ten µg of bovine serum
albumin was used as a control. The Maxam-Gilbert sequencing G ladder of
the fragment was run as a molecular size marker (G). Regions
protected from DNase I digestion and DNase I hypersensitive sites are
indicated by open bars and solid triangles,
respectively. A schematic of the genome is presented on the
left. The positions of cis-elements in the promoter,
transcription initiation site (arrow), 3' ends of the
luciferase constructs (open triangle), and two
putative RFX sites in exon1 are indicated.
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RFX-binding Motifs Are Conserved in the Human MAP1A Gene--
To
further investigate the role of the RFX1 binding sites in
MAP1A transcriptional regulation, we searched for conserved
elements in the human MAP1A gene, for which the long
transcript has not yet been identified. We retrieved 20 kb in sequence
5' to, and 5' of the human MAP1A gene from a Homo
sapiens chromosome 15 working draft sequence segment (GenBank
accession no. NT010194). Comparison of the region with the 5' part of
the mouse Map1a gene (Ref. 14, GenBank accession no.
AF182208-11) revealed that the 5' exons are conserved in the human
gene. Segments highly similar to exons 1, 2, and 3 of the mouse gene
were identified about 9.7 kb, 8.4 kb, and 3.0 kb, respectively,
upstream from the previously identified human exon 1 (18). We confirmed
that the segments homologous to mouse exons 1, 2, and 3 were
transcribed as parts of MAP1A mRNA in human brain by PCR from a
human brain cDNA library (data not shown); exon numbers of the
human gene will be presented as they occur in the mouse gene hereafter
in this study (Fig. 3A). We
designed primer sets to obtain a human genomic fragment containing the
MAP1A 5' promoter and exon 1 and amplified a fragment from HeLa cell genomic DNA. Its sequence was completely identical with the
sequence reported in GenBank, and a portion is shown in Fig. 3B aligned with the corresponding mouse genomic sequence.
The basal promoter region containing three Sp1 sites and one Y/CCAAT box is conserved between human and mouse. Both RFX binding motifs in
exon1 are also conserved in the human gene, with minor differences in
the 5' RFX binding motif. Next, we examined by RT-PCR whether the long
isoform of MAP1A mRNA, the 5' part of which consists of
exons 1, 2 and 4, is expressed specifically in human neuronal cells. A
386-bp DNA fragment was amplified with a set of specific primers from
RT products of neuroblastoma mRNA (Fig. 3C,
TGW and LA-N-5). The fragment was confirmed to be
an expected product by sequencing. The long transcript was also
expressed at low levels in A431 epidermoid carcinoma cells and HeLa
adenocarcinoma cells (Fig. 3C). In contrast, amplified
products were hardly observed from HT29 colorectal adenocarcinoma
cells, HepG2 hepatocellular carcinoma cells, and Saos-2 osteosarcoma
cells (Fig. 3C). These results demonstrate that the genomic
structure and RFX-binding motifs in exon 1 are conserved in human, and
the long transcript is expressed preferentially in human neuronal
cells.

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Fig. 3.
5' promoter and RFX sites are conserved in
the human MAP1A gene. A, partial
structure of the human MAP1A gene in chromosome 15 is
presented schematically. Numbers above each exon indicate the
nucleotide position in the human chromosome 15 working draft sequence
segment (GenBank accession no. NT010194). B, nucleotide
sequences of the 5' promoter and a part of exon 1 of the mouse and
human MAP1A gene are aligned. Mouse sequence (m)
is above and human sequence (h) is
below. Putative Sp1-binding sites, a Y/CCAAT box, and
RFX-binding sites are boxed. An angled arrow indicates the
transcription start site confirmed in mouse. Solid and
open triangles indicate the 5' and 3' ends, respectively, of
the examined fragments in the luciferase assay. Broken
underlines represent the segments protected from DNase I
digestion. C, mRNA transcribed from exon 1 is expressed
preferentially in human neuroblastoma cells. RT-PCR products from each
culture cell were analyzed on a 1.5% agarose gel. Primer sequences and
RT-PCR conditions are described under "Experimental Procedures."
Actin bands are shown as a loading control.
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Identification of Two RFX-binding Sites as Repressors of MAP1A Gene
Transcription--
On confirmation that the exon-intron structure and
5' promoter region were conserved in the human gene, further reporter
assays were conducted using human genomic fragments to take advantage of their ability to be examined in a variety of homologous cell lines.
The human MAP1A basal promoter construct,
hMP1-2, efficiently transactivated the reporter gene up to
70- to 300-fold above the promoterless reporter, pGL3 basic in all cell
lines tested except for Saos-2 osteosarcoma cells, in which the
activity was 14- to 20-fold above the pGL3 basic. An additional
potential Sp1 binding site found only in the human gene (Fig.
3B, Sp1(H)) was not required for promoter
activity (data not shown). Like the mouse promoter, the 5' end of exon
1, which contains the two RFX-binding sites, has a remarkable
repressive effect in non-neuronal cells (Fig. 4, A and B).
Promoter activity of a reporter hMP1-8 construct containing
the two RFX sites was 5% or less of that of a basal promoter
construct, hMP1-2 in HeLa, HT29, and A431 cells. Repression was also seen in Saos-2 osteosarcoma and HEK293 embryonic kidney cells.
In contrast to the mouse promoter, however, the two RFX sites acted as
weak repressors in the neuronal cell lines TGW and LA-N-5. To confirm
that the RFX-binding sites are critical for repression, we created
reporters with mutations in the RFX sites. Because an RFX protein can
bind to a half-consensus site as a monomer, albeit less effectively
than as part of a homo- or heterodimers binding to a full palindromic
consensus site (19, 20), mutations were introduced in each of the four
half-sites of the two full palindromic consensus sequences in the
MAP1A gene (Fig. 4C). As shown in Fig.
4D, all mutations resulted in up-regulation of transactivity
compared with the parental reporter hMP1-8 in HeLa, HT29,
and A431 cells, indicating that all the mutations disabled repression.
Among the four RFX-binding half-sites, the 3' half of the 3' RFX,
mutated in hMP1-8mu1, which was 8- to 10-fold more active than the
wild-type in epithelial cells, appears to be most crucial for
repressive activity. This site also seemed responsible for the weak
repression in TGW cells. The 5' half of the 3' RFX-binding site and
either halves of the 5' RFX-binding site were considered necessary for
full repression in epithelial cells. These three mutations had no
significant effects on promoter activity in TGW.

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Fig. 4.
RFX sites are repressive elements highly
effective in non-neuronal cells. A, schematic of human
genomic fragments examined by luciferase assay is shown. Some
cis-elements and RFX sites in exon 1 are indicated. Arrows
represent the region examined by luciferase assay. Fig. 3B
shows precise nucleotide positions. B, five human
non-neuronal cell lines (HeLa, HT-29, A431, HEK293, and Saos-2) and two
human neuronal cell lines (TGW and LA-N-5) were transfected with the
indicated constructs, together with pRL-TK. The cells were harvested
and assayed for luciferase activity 48 h after transfection.
Promoter activity was normalized by sea-pansy luciferase activity.
Average activities of hMP1-2 were set at 100%, and
promoter activity of hMP1-7 and hMP1-8 is
presented as percent activity relative to that of hMP1-2.
The values are averages ± S.E. of at least nine data points.
C, substituted nucleotides in the RFX sites of the parental
clone hMP1-8 are underlined and in
lowercase. One of the four halves of the two palindromic
sites was mutated in each of the following constructs: hMP1-8 mu1,
mu2, mu3, and mu4. D, three non-neuronal cell lines (HeLa,
HT-29, and A431) and one neuronal cell line (TGW) were transfected with
the indicated constructs together with pRL-TK. The cells were harvested
and assayed for luciferase activity 48 h after transfection.
Promoter activity was normalized by sea-pansy luciferase activity.
Promoter activity of mutants is given as an -fold activity above the
average activity of hMP1-8. The values are averages ± S.E. of at least nine data points.
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RFX1 and RFX3 Bind to Repressors in the MAP1A Gene--
The above
reporter assays strongly suggested that the two RFX sites are
repressors acting preferentially in non-neuronal cells. DNase I
footprinting, however, indicated that the RFX sites in the
MAP1A gene were occupied by nuclear proteins from all cell types. Identification of the binding proteins would give an insight into the mechanism by which transcription is regulated differently among cell types. Gel-shift experiments revealed that multiple complexes that differ among cell types interact with the 3' RFX sequence of the MAP1A gene (Fig.
5A). Five retarded bands, A, B, and C, representing relatively slow moving complexes, and D and E,
representing fast moving ones, were identified using TGW nuclear
extract. Bands A, B, and C were obtained using nuclear extracts from
all cells examined, but their relative intensities were variable. Band
E was also observed very faintly in experiments conducted with LA-N-5
and Neuro2A nuclear extracts. These retarded band patterns were
observed as well when the 5' RFX sites in the mouse and human
MAP1A genes, an RFX site in the interleukin 5 receptor
gene, and a polyoma virus EP site, were used as probes (data not
shown). The specificity of complex formation was examined further by
competition experiments. Complexes formed with both HeLa and TGW
nuclear extracts were competed apart by an excess amount of
non-radiolabeled double-stranded oligonucleotide containing the 3' RFX
sequence (Fig. 5B, lane self). Competing
oligonucleotides containing the MAP1A 5' RFX sequence or the
polyoma virus EP sequence diminished complex formation as well, but
less effectively than those encoding the MAP1A 3' RFX
(compare lanes 5'RFX and PyEP with
lane self in Fig. 5B).
Oligonucleotides containing the MAP1A 3' RFX site harboring
the same mutations as were introduced in the reporter assay did not
affect the band patterns, confirming that complex formation depends
critically on the entire core sequence of the RFX site (Fig.
5B, mu1 and mu2).

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Fig. 5.
Each cell expresses unique nuclear protein
complexes that bind to the RFX site of the MAP1A
gene. A, gel shift experiments were performed
with nuclear extracts from the indicated cells and a
32P-labeled 3'-RFX probe. Amounts of nuclear proteins
extracted from each cell line and applied were as follows: 2.5 mg
(TGW), 10 mg (LA-N-5), 2 mg (Nauro2A), 2.5 mg (HeLa), 5 mg (HT-29), 10 mg (A431), 2 mg (HEK293), and 3.5 mg (NIH3T3). The reaction was
performed without nuclear protein ( ) as a negative control. The
binding reaction was electrophoresed on a 4% polyacrylamide 0.25%
TBE gel. B, 10-fold molar excess of unlabeled competitor
oligonucleotide was added to the binding reaction before the
radiolabeled probes were applied. The competing oligonucleotides used
were as follows: unlabeled 3'-RFX probe (self), 3'-RFX
mutant1 (mu1), 3'-RFX mutant2 (mu2), 5'-RFX, and
Polyoma virus EP site (PyEP). The nucleotide sequences of
the competitors are described under "Experimental Procedures." An
experiment without added competitor was performed as a negative control
( ). Two µg of either HeLa or TGW nuclear extract was applied.
C, antiserum against RFX1 (1), RFX2
(2), or RFX3 (3) was added to the reaction after
the DNA-protein complex was formed. Preimmune rabbit serum was added in
each control lane ( ). Other conditions, including the volumes of
nuclear extract, were as in A.
|
|
Next, supershift experiments with specific antibodies were performed to
identify which RFX proteins are involved in the complexes. Among
mammalian RFX proteins, RFX1-RFX3 comprise a subfamily because of
their highly similar target DNA specificities (20). As shown in Fig.
5C, all bands except for band E were supershifted by an anti-RFX1 antibody. The supershifted band ss1 was not thought to
contain a complex derived from band A and B, because it was not
observed when EMSA was conducted using the HeLa cell nuclear extract,
which shifts band A and B intensely. The complex derived by the
anti-RFX1 antibody and band A and B did not seem to enter the gel
efficiently, because high amounts of radioactive complexes were
observed in the loading wells above the gel in the lanes where
anti-RFX1 antibody had been applied (data not shown). Thus, the
supershifted band ss1 was most likely derived from band C. The complex
E band was supershifted by an anti-RFX3 antibody, but the supershifted
band could not be separated clearly from the other bands, as was
reported previously (21). The complex C band was also supershifted by
anti-RFX3 antibody, which was observed most clearly in experiments
using HeLa nuclear extracts. Incubation with anti-RFX2 antibody did not
change the band patterns. These results and previous reports (21, 22)
suggest that complexes A, C, and E contain RFX 1/1, RFX 1/3, and RFX
3/3 homodimers, respectively. The remaining complexes B and D, which
have not been reported previously, appear to contain RFX1, but not RFX3 or 2. Differences between bands A, B, and D are possibly due to various
other cofactors included in the complexes.
Both RFX1 and RFX3 Down-regulate the MAP1A Promoter in Non-neuronal
Cells--
Because EMSA experiments suggested that RFX1 and 3 are the
major proteins binding to the RFX sites in the MAP1A gene,
we examined their functional effects on the promoter activity in
reporter assay. We made RFX1 and RFX3 expression constructs driven by
the CMV promoter. The expression of proteins in expected sizes and appropriate nuclear localizations of EGFP-tagged proteins were confirmed by Western blotting and confocal microscopy, respectively (data not shown). We then examined their effects on the
hMP1-8 reporter activity in non-neuronal and neuronal cells
(Fig. 6A). Overexpression of
RFX1 resulted in down-regulation of the reporter compared with control
in all non-neuronal cells examined (Fig. 6B, pFC
R1). Reporter activity was also down-regulated when RFX 3 was
overexpressed in HT29 and HEK293 cells (Fig. 6B, pFC
R3). However, overexpression of RFX3 did not result in
down-regulation, but slight up-regulation of the reporter in HeLa
cells. Forced expression of the RFX1 DNA binding domain, which was
expected to act as a dominant-negative protein by occupying the RFX
sites, up-regulated transcription 2-fold compared with control in
non-neuronal cells (Fig. 6B, pFC R1DBD). This
further confirmed that the RFX sites in the MAP1A gene
function as repressors in non-neuronal cells. Neither RFX1 nor RFX3
down-regulated transactivity in TGW cells. Although these
overexpression experiments clearly indicate that ubiquitously expressed
RFX1 down-regulates the MAP1A 5' promoter in non-neuronal
cells and not in neuronal cells, the results are ambiguous in terms of
RFX3 function. Therefore, we performed additional cotransfection
studies in which cDNAs encoding GAL4 DBD-RFX chimeric proteins
were introduced (Fig. 6C) in an attempt to minimize the effect of endogenous RFX proteins. In this system, both GAL4 DBD-RFX1 and -RFX3 proteins down-regulated reporter activity in non-neuronal cells (Fig. 6D); the effects were more obvious than in the
simple overexpression study shown in Fig. 6B. The most
effective down-regulation was observed in HeLa cells, in which the
introduction of GAL4 DBD-RFX1 resulted in down-regulation of reporter
activity to 13% of the control. In contrast to the overexpression
experiments, GAL4 DBD-RFX3 also down-regulated reporter activity to
35% of the control in HeLa cells. In two other non-neuronal cell
lines, the rates of down-regulation by RFX1 and 3 were not
significantly different. Again, reporter gene down-regulation was not
observed upon expression of chimeric RFX proteins in TGW. Based on
these experiments, we conclude that both RFX1 and RFX3 down-regulate the MAP1A promoter activity in non-neuronal cells but not in
neuronal cells.

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|
Fig. 6.
Forced expression of RFX1 and RFX3 results in
down-regulation of reporter gene transcription in non-neuronal cells,
but not in a neuronal cell line. A, schematic of a
reporter construct and RFX expression constructs. The coding regions
for the DNA-binding (DBD) and dimerization domains
(DmrD) in RFX1 and RFX3 are indicated. pFC R1DBD contains
only cDNA for the RFX1 DBD. B, HeLa, HT-29, HEK293, and
TGW cells were transfected with a hMP1-8 reporter construct
and the indicated expression constructs. An empty CMV expression
vector, pFC E, was used as a control. The cells were harvested and
assayed for luciferase activity 48 h after transfection. Promoter
activities are presented in relative luciferase units without
normalization, and the values are averages ± S.E. of at least
nine data points. C, schematic of a reporter construct and
expression constructs for GAL4 DNA-binding domain
(GAL4DBD)-RFX chimeras. The reporter construct has 5 tandem
repeats of the GAL4-binding site in front of the MAP1A 5'
basal promoter, followed by the luciferase gene. The expression
constructs contain the fragments for the GAL4DBD-RFX1 chimera (pFC
R1-G) and GAL4DBD-RFX3 chimera (pFC R3-G), and the expected proteins
were confirmed as translated in HeLa cells. D, HeLa, HT-29,
HEK293, and TGW cells were transfected with a hMP1-2 G4B
reporter construct and the indicated expression constructs. A vector,
pFC E-G, containing the fragment for GAL4DBD alone was used as a
control. The cells were harvested and assayed for luciferase activity
48 h after transfection. Promoter activities are presented in
relative luciferase units without normalization, and the values are
averages ± S.E. of at least nine data points.
|
|
 |
DISCUSSION |
In the present study, we have shown that the two RFX-binding sites
in the MAP1A gene act as repressors specifically in
non-neuronal cells. We identified several distinct complexes bound to
the RFX sites sequences in the MAP1A gene and confirmed that
they contained RFX1, RFX3, or both. Forced expression of
dominant-negative RFX protein attenuated the repression in non-neuronal
cells, confirming that they are involved in the in vivo
process. Overexpression of RFX1 and RFX3 and forced expression of Gal4
DBD-RFX chimeric proteins indicated that both RFX1 and RFX3
down-regulate reporter gene expression specifically in non-neuronal
cells. These data strongly suggest that the 5' promoter of the
MAP1A gene is under the control of RFX transcription
proteins and that RFX1 and 3 contribute to the neuronal cell-specific
expression of the gene by their repressive activity in non-neuronal cells.
RFX1, a prototypic mammalian RFX protein, was first cloned as a
transcription factor that interacts with the X-box in the class II
MHC gene (19, 23). Subsequent studies revealed that RFX
family members, of which there are five in human (RFX1-5) and four in
mice (RFX1-3 and RFX5), share a unique winged-helix type DNA-binding
domain (24-26). In addition, one family member in each of several
other eukaryotes
dRFX in Drosophila, DAF-19 in C. elegans, sak1 in Saccharomyces pombe, and Crt1 in
Saccharomyces cerevisiae
has been identified (15, 25,
27-29). Thus, the RFX proteins appear to have been conserved
throughout evolution in a wide variety of species, and their functions
elucidated to date are diverse. Crt1 acts as a repressor in the DNA
damage and replication checkpoint pathways (28). dRFX in
Drosophila and DAF-19 in C. elegans are expressed
in type I sensory neurons and all ciliated sensory neurons,
respectively (15, 16). Loss of daf-19 function causes an
absence of cilia, resulting in severe sensory defects (15). In mammals,
the function of RFX5 has been studied most intensively, because
mutations of the gene coding RFX5 cause bare lymphocyte syndrome, a
type of primary immunodeficiency (26). RFX5 is essential for the
activation of class II MHC transcription as a DNA-binding
component of RFX, a complex binding to the X box (30). Although RFX1
was originally cloned by virtue of its in vitro interaction
with the X box in the class II MHC promoter, its role in the regulation
of class II MHC expression has been unproven. Aside from the class II
MHC gene, several potential target genes that may be under
the control of RFX1 and/or the closely related RFX2 and 3 have been
reported. EF-C or EP sites, partially palindromic sequences to which
RFX1-3 bind, have been found in the enhancers of hepatitis B virus,
polyomavirus, cytomegalovirus, and Epstein-Barr virus (31-34).
RFX-binding sites have been also recognized in some cellular genes,
including the genes for c-Myc (35), proliferating cell nuclear
antigen (36), the ribosomal protein rpL30 (37), collagen
2(I)
(38), and the interleukin 5 receptor
(22). RFX1, with or without
RFX2 and/or 3, binds to these elements in vitro and seems to
regulate their transcription. The physiological significance of
RFX1-3, however, has yet to be determined by knockout analysis.
According to the phylogenetic tree of the RFX DNA-binding domain, the
RFX1-3 subgroup is most closely related to dRFX and DAF-19 (29). X-box
sequences for the RFX1-3 subgroup are very similar to that for DAF-19
(15), whereas they differ from those for RFX4 and RFX5 (20). Thus, the
mammalian RFX1-3 subgroup is expected to play an important role in the
nervous system, like its Drosophila and C. elegans homologues. The present results are the first evidence
that RFX1 and 3 indeed are involved in the cell type-specific
transcription of a gene important for the neuronal function; however,
it seems to be accomplished by their repressive activity in
non-neuronal cells and not by their activity in neuronal cells.
RFX-binding sites in the genes for c-Myc (35), proliferating cell
nuclear antigen (36, 39), and collagen
2(I) (38) are negatively
acting regulatory elements, as in the MAP1A gene, whereas
those in most of the viral promoters (34, 40) and the genes for
ribosomal protein rpL30 (37) and interleukin 5 receptor
(22) are
positively acting regulatory elements. Thus, RFX-binding sites function
differently in a context-dependent manner, possibly
reflecting various interactions between transcriptional activation and
repression domains in RFX proteins and other regulatory factors
recruited by adjacent elements (22, 41). Besides the context of the
regulatory unit, cofactors recruited by the DNA-binding protein are now
considered to be important modulators of the transcriptional control
and often become the determinant of cell and tissue specificity. In
fact, such a cofactor is identified and well characterized in
association with another member of RFX proteins. The class II
transactivator, CIITA, which itself does not bind DNA and is recruited
by RFX5 to the X box, is the determinant for cell-type specificity and
interferon-
inducibility of MHC class II expression (42-44). Thus,
it is tempting to speculate that non-neuronal cell-specific repression
by RFX1 and 3 depends on a corepressor only available in non-neuronal
cells. Unfortunately, little is known about the precise mechanisms of
repression and activation by the RFX1-3 subfamily and the cofactors
associated with RFX1-3. RFX1 has been reported to coexist in a binding
complex with p107, a retinoblastoma-related protein, to the
proliferating cell nuclear antigen promoter, but a direct interaction
between RFX1 and p107 has not been ascertained (36). p107, best known
as the corepressor for E2F-dependent transcription, has
also been reported to associate with Sp1 and repress
Sp1-dependent transcription in reporter assays (45). Because the promoter activity of the MAP1A gene highly
depends on Sp1-binding sites (14), p107 recruitment via RFX proteins may be involved in the down-regulation of the MAP1A
promoter. However, our preliminary experiments failed to detect p107 or other pocket protein family members in HeLa cell nuclear protein complexes bound to RFX sites in the MAP1A gene (data not
shown). Further study of cofactors involved in the DNA-binding
complexes mediated by RFX is necessary to understand the mechanisms
underlying its repressor activity, and such studies may provide insight
about cell specificity as well.
In conclusion, we propose that RFX1 and RFX3 participate in
transcriptional regulation of a gene important for the nervous system.
The MAP1A 5' promoter, which is under their control, is a
robust model system in which non-neuronal cell-specific repressor mechanisms RFX1 and RFX3 can be explored.
 |
ACKNOWLEDGEMENTS |
We thank Dr. W. Reith for anti-RFX1 to 3 rabbit serum and Dr. K. Ando for C1300 neuroblastoma cells.
 |
FOOTNOTES |
*
This work was supported in part by a grant for COE research
from the Ministry of Education, Science, Culture and Sports in Japan.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: Department of
Embryology, Institute for Developmental Research, Aichi Human Service Center, Kamiya-cho, Kasugai, Aichi 480-0392, Japan. Tel.:
81-568-88-0811; Fax: 81-568-88-0829; E-mail:
k46191a@nucc.cc.nagoya-u.ac.jp.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M209574200
 |
ABBREVIATIONS |
The abbreviations used are:
MAP, microtubule-associated protein;
RFX, regulatory factor for X box;
DBD, DNA-binding domain;
CMV, cytomegalovirus;
MHC, major histocompatibility
complex.
 |
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