From the Section on Genomic Structure and Function, Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830
Received for publication, October 20, 2000
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ABSTRACT |
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Hypermethylation of the FMR1 promoter reduces its
transcriptional activity, resulting in the mental retardation and
macroorchidism characteristic of Fragile X syndrome. How exactly
methylation causes transcriptional silencing is not known but is
relevant if current attempts to reactivate the gene are to be
successful. Understanding the effect of methylation requires a better
understanding of the factors responsible for FMR1 gene expression. To
this end we have identified five evolutionarily conserved transcription factor binding sites in this promoter and shown that four of them are
important for transcriptional activity in neuronally derived cells. We
have also shown that USF1, USF2, and Fragile X syndrome is caused by the expansion of a CGG repeat in
the 5'-untranslated region of the fragile X mental retardation (FMR1)
gene (1, 2). This results in hypermethylation of the promoter and
transcriptional silencing (3). The major symptoms of fragile X
syndrome, mental retardation and macroorchidism, are consistent with
the observation that high levels of FMR1 expression occurs in specific
cells in brain and testis (4). The GC-rich human FMR1 promoter lacks a
typical TATA-box and contains several potential Sp1 binding sites as
well as an E-box and putative binding sites for the transcription
factors Because FMR1 knockout mice demonstrate learning deficits and
macroorchidism similar to those seen in fragile X patients (7), and
mice show patterns of temporal and tissue-specific FMR1 expression similar to humans (8), many of the control elements important for FMR1
gene regulation are also likely to be evolutionarily conserved. To
identify conserved promoter elements, we compared the sequence of the
human FMR1 promoter with that from two other primates: Pan
troglodytes (chimpanzee) and Macaca arctoides
(stump-tailed macaque) as well as two more evolutionarily distant
species: Mus domesticus (mouse) and Canis
familiaris (dog). We also examined the activity of promoter
mutations in transient expression assays using a neuronally derived
cell line, PC12, and identified the major transcription factors that
bind the FMR1 promoter in nuclear extracts of brain and testis. We also
demonstrate that binding of one of these factors is abolished by
methylation, and binding of the other two factors is also affected.
This may have implications for therapeutic strategies aimed at
reactivating the gene, since it indicates that methylation of the FMR1
promoter in Fragile X patients does not simply inhibit transcription
via the formation of transcriptionally inactive chromatin, as suggested
from in vivo dimethyl sulfate footprinting in lymphoblasts
(6) and from the presence of deacetylated histones on the FMR1 promoter in individuals with Fragile X syndrome (9).
Isolation of the Mouse fmr1 Promoter--
A 129/SVJ mouse
(M. domesticus) BAC library was screened by polymerase chain
reaction using two primers from exon 1 of the mouse FMR1 gene (Genome
Systems, St. Louis. MI). The resultant clone was subcloned and mapped
using standard procedures. Exon 1 of the mouse FMR1 gene was localized
to a 3.1-kilobase EcoRI fragment that was subcloned into
pZero (Invitrogen, Carlsbad, CA). This clone designated pEco3.3 was
then sequenced using standard procedures. Sequence comparison was done
using the GCG package (Wisconsin Package Version 10.0, Genetics
Computer Group (GCG), Madison, WI). The mouse sequence was scanned
against the GenBankTM data base. The only significant
matches were to the human FMR1 5' end (including
GenBankTM/EBI locus HUMFMR1S). The sequence was
submitted to GenBankTM/EBI (Accession Number:
AF251347).
Amplification, Sequencing, and Computer Analysis of the Promoter
Region from Different Species--
Polymerase chain reaction
amplification of FMR1 promoter from the genomic DNA of chimpanzee
(P. troglodytes), macaque (M. arctoides), and dog
(C. familiaris) was carried out using the ExpandTM
HiFidelity polymerase chain reaction system (Roche Molecular Biochemicals) and the primers Fraxa f
(5'-dAGCCCCGCACTTCCACCACCAGCTCCTCCA-3' from exon 1 and FMRUP2
(5'-dGCNTTCCCGCCNTNCACCAAG-3') homologous to the 5' end of the promoter
that is conserved in mice and humans. The polymerase chain reaction
product was directly sequenced using the Thermosequenase radiolabeled
terminator cycle sequencing kit (U. S. Biochemicals Corp.) according
to the manufacturer's recommendations. The sequences obtained were
aligned initially using MacvectorTM version 5.0.2 (Oxford Molecular
Group, Inc., Campbell, CA), followed by visual inspection. The
transcription factor binding sites in the human sequence were
analyzed using TESS (Transcription Element Search Software, available
on the World Wide Web2). The
sequences were submitted to GenBankTM (accession numbers
AF251349, AF251350, and AF251348 for chimpanzee, macaque, and dog, respectively).
Generation of Reporter Constructs--
A single base insertion
was introduced into the middle of the E-box site in p32.9 (10) using
the QuickChange MutagenesisTM protocol (Stratagene, La Jolla, CA) and
the primer pair 5'-GAACAGCGTTGATCACTGTGACGTGGTTTCAGTGTTTAC-3' and 5'-dGTAAACACTGAAACCACGTCACAGTGATCAACGCTGTTC-3'. The
introduction of the single base change to the resultant plasmid
(pUSFmut) was confirmed by sequencing. A 909-bp fragment from the
original FMR1 promoter in p32.9 and a 910-bp fragment from the mutated
promoter from pUSFmut containing 869 and 870 bases from the human FMR1 promoter, respectively, were cloned into
KpnI-NheI-digested plasmid pGL3-basic (Promega,
Madison, WI), which contains the firefly (Photinus pyralis)
luciferase reporter gene to make pGL-FMR and pGL-USFmut. The 909-bp
wild-type FMR1 promoter was also cloned into
HindIII-SpeI digested plasmid pRL-null (Promega),
which contains the sea pansy (Renilla reniformis)
luciferase-coding sequence, to make the control plasmid pRL-FMR.
Deletions in the promoter were created either by Exonuclease III and S1
nuclease treatment or by digestion with a combination of restriction
enzymes followed by T4 DNA polymerase. Specifically plasmids
p Methylation of Reporter Constructs--
Specific methylation of
the E-box and Cell Culture, Transient Transfections, and Promoter
Assays--
Cells were grown in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 5% fetal calf serum (Life Technologies Inc.), 10% heat-inactivated horse serum (Sigma), and 1×
penicillin-streptomycin (Sigma) at 37 °C and 5% CO2 to ~70% confluence. Culture medium was replaced 18-24 h before the transfection. Ten micrograms of test plasmid DNA together with 10 µg
of the control plasmid pRL-FMR were introduced into ~107
cells by electroporation (300 V, 1180 capacitance; Cell-porater, Life
Technologies). Transfected cells were plated in duplicate on 6-well
plates. After 16 h, the culture medium was replaced with fresh
medium. Cells were collected 42-44 h after transfection and assayed
for luciferase using the Dual-Luciferase® Reporter assay
system (Promega) and a MicroLumat LB 96 P luminometer (Berthold
Systems, Inc. Aliquippa, PA). At least three independent transfections
were performed for each plasmid. The mean of the luciferase activities
was plotted after adjusting for the activity of pRL-FMR.
Transcription Factors, Competitor Oligonucleotides, and
Antibodies--
Oligonucleotides used in the electrophoretic mobility
shift assay are listed in Table I. The consensus binding sites for their cognate transcription factors are underlined. Double-stranded oligonucleotides containing the consensus binding sites for AP1, AP2,
Sp1, OCT1, TFIID, and CREB were obtained from Promega. The top and
bottom strands of two variants of the Preparation of Nuclear Extracts--
Nuclear extracts were
prepared from the brain, testis, and liver of FVB/N mice (The Jackson
Laboratory, Bar Harbor, ME) and from human lymphoblastoid cell lines
(Coriell Cell Repositories, Camden, NJ) by a modification of the method
of Dignam et al. (11). Briefly, the tissues and
lymphoblastoid cells were washed with phosphate-buffered saline and
resuspended in two volumes of ice-cold buffer A (10 mM
HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM
NaCl, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 7 µg/ml calpain inhibitor II)
containing 1 protease inhibitor mixture tablet (CompleteTM mini,
EDTA-free, Roche Molecular Biochemicals, Indianapolis, IN) per
10 ml of buffer A. The cells were lysed by 10 strokes of a Dounce
homogenizer, and the tissues were homogenized using a VIRTIS 45 homogenizer (Virtis company Inc., Gardiner, NY). The lysed cells and
tissues were then centrifuged at 3500 × g for 15 min
to pellet the nuclei. The pellet was spun again at 25,000 × g for 20 min to remove the residual cytosolic material. The
nuclei were resuspended in 3 ml of buffer C/109 cells (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol, 7 µg/ml calpain inhibitor II, and the same protease
inhibitor mixture tablet used previously) and stirred at 4 °C for 30 min. The nuclear debris was removed by centrifugation at 25,000 × g for 30 min. The supernatant was dialyzed against 50 volumes of buffer D (20 mM HEPES, pH 7.9, 20% glycerol,
100 mM NaCl, 0.2 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol) at
4 °C overnight using a Slide-A-Lyzer® cassette
(Pierce). The dialysate was centrifuged at 25,000 × g
for 20 min. The supernatant was quick-frozen in a dry ice/ethanol bath
and stored in aliquots at Electrophoretic Mobility Shift Assays (EMSAs)--
A 200-bp
mouse FMR1 promoter fragment was amplified with primers mba1
(5'-dCTTTAAGCTTTCCCGCCTTTCACCAAG-3'] and mba2
5'-dGCCAAAAGCTTCGCTGCGCCTCCTGTAAA-3'] from pEco3.3. The 202-bp
human FMR1 promoter (
Binding was carried out at 30 °C or 4 °C in 30 µl of reaction
buffer containing 25 mM HEPES, pH 7.5, 5 mM
MgCl2, 2 mM dithiothreitol, 100 mM
NaCl, 0.25 ng of probe, 5 µg of protein, and 1 µg of
poly[dA-dT][dA-dT] for 30 min. A 1000-fold excess of
nonspecific DNA and transcription factor binding
oligodeoxyribonucleotides were included in the reactions as nonspecific
or specific competitors. For antibody supershift assays, 4 µg of
antibody or BSA was incubated with the protein before the addition of
the probe, and reactions were carried out for 40 min. The reactions
were stopped by the addition of 30 µl of 2× gel loading buffer (200 mM Tris, pH 8.8, 10.5% glycerol, 0.002% bromphenol blue).
Reactions were subjected to electrophoresis on a 4% polyacrylamide gel
(60:1, acrylamide:bis) containing 1.6% glycerol in 1×
Tris-glycine-EDTA buffer (50 mM Tris, 380 mM
glycine, 2.1 mM EDTA, pH 8.5). Gels were dried and exposed
to x-ray film.
DNase I Footprinting--
For DNase I footprinting, a
PstI/EcoNI fragment of p32.9 was end-labeled at
EcoN I site by [ Western Blot Analysis--
The nuclear extracts were subjected
to electrophoresis on 10% polyacrylamide gels containing SDS and
electro-blotted to the NitroPure membrane (MSI, Westboro, MA) using
standard procedures. Hybridization with primary antibody was carried
out as specified by the supplier. Hybridization with the secondary
antibody and signal detection were done using ECL Western blotting kit
(Amersham Pharmacia Biotech) as per the manufacturer's recommendation.
Phylogenetic Footprinting of the 5' End of FMR1 Gene Identifies
Five Evolutionarily Conserved Regions--
A 466-bp fragment including
272 bp of sequence upstream of the transcription start site of the
human FMR1 gene has previously been shown to contain all the elements
necessary for the appropriate tissue-specific expression of the FMR1
gene in transgenic mice (12). To define the minimal promoter region
more closely and to identify those transcription factor binding sites
that are evolutionarily conserved and that may therefore be important
for regulation of this gene, we compared the sequences of the 5' end of
the FMR1 gene that we obtained from a number of different mammals.
Fig. 1 shows a dot matrix comparison of
the exon 1-containing portion of the mouse fmr1 gene with
the corresponding region of the human FMR1 sequence
(GenBankTM/EBI locus: HUMFMR1S). The homology between the
two sequences is highest in the first exon, but islands of homology
both 3' and 5' of exon 1 are also seen. The 5' regions fall within the 272-bp previously defined minimal promoter fragment (12), the upstream
border of which is marked by the black arrow in Fig. 1. No
transcription factor binding sites were conserved between mouse and
human 5' of the
We used a primer derived from the conserved 5' boundary of the promoter
and a primer from exon 1 to amplify the FMR1 5' region from chimpanzee,
Stump-tailed macaque, and dog. The sequences were then aligned with the
human and mouse promoter sequences to identify those regions that are
conserved in all five species (Fig. 2).
The four described previously in vivo protein binding sites,
The region containing the dimethyl sulfate hyperreactive G residue
close to the reported transcriptional start site (5) is also well
conserved (the complementary C is marked by an asterisk in
Fig. 2). This region does not show a protein binding footprint in
vivo, at least in cells with low FMR1 activity (5). This hyperreactivity is absent in individuals with fragile X syndrome and is
therefore thought to reflect some DNA structure that is associated with
transcription (5). The transcription factors Zeste (5), AP2 (15), and
CREB (13) have all been suggested to be important for FMR1 regulation.
However the binding sites for these factors were not conserved (shown
in the open boxes Fig. 2).
Mutational Analysis Confirms That the Evolutionarily Conserved
Regions Are Important for Transcriptional Activity--
Mutated
versions of the human FMR1 promoter were assayed in PC12 cells, a
neuronally derived cell line. Fig. 3
shows that single base insertion in the E-box (pGL-USFmut) or the
deletion of 9 bases from the
Deletion of the region that included the CGG repeats in the 1st exon
(p USF1, USF2, and
An excess of unlabeled oligonucleotide containing the E-box consensus
sequence eliminated complexes I and III (Fig. 4, lane 5) as
well as the minor shifted products. An oligonucleotide containing the
consensus sequence for the
USF1 and USF2 were unable to bind the E-box variant with the single
base insertion that was used in the promoter activity studies described
above. Moreover, whereas an oligonucleotide containing the unmutated
E-box was able to eliminate USF binding to the FMR1 promoter when
present in a 10-fold excess, an oligonucleotide containing the mutated
E-box did not eliminate binding completely even when a 1000-fold excess
was used (data not shown).
Sp3 Binds a FMR1 Promoter Subfragment--
Factor binding to the 2 GC boxes in the human promoter has been reported in fibroblasts and
lymphoid cells (5, 6) and in vitro on a 71-bp
promoter-containing fragment in extracts of the neuronally derived cell
line SK-N-SH (15). However, GC-box-specific factors did not bind the
full-length promoter in any of our extracts under a variety of reaction
conditions; unlabeled GC-box oligonucleotides did not alter the EMSA
profile (Fig. 4, lane 6), and antibodies to members of the
Sp1 transcription factor family Sp1, Sp3, and Sp4 did not supershift
complex I, II, or III (Fig. 4, lanes 16, 18, and
19). Antibodies to Egr-1, a related transcription factor that binds to some GC boxes, also had no effect (Fig. 4, lane 17). However, an 82-bp SphI-PmlI fragment
that contained both the conserved GC-boxes but that lacked both the
E-box and the CREB and AP2 Do Not Bind the FMR1 Promoter--
A CREB binding
site that overlaps with the E-box sequence has also been suggested to
be important for FMR1 regulation (13). However, the E-box consensus
oligonucleotide that was able to abolish complex I lacked sequences
corresponding to a CREB site (Table I).
Moreover, oligonucleotides containing the consensus sequence for CREB
binding did not compete out any of the retarded bands (Fig. 4,
lane 7). This is not due to the absence of CREB, because our
extracts contained high levels of full-length CREB (Fig. 4,
inset). Moreover, when a CREB consensus oligonucleotide is
used as a probe with our extracts, a shifted product is seen (data not
shown). We found no evidence for any other factor binding including AP2
(data not shown), which has also been reported to be involved in FMR1
regulation (15). The differences between our data and those previously
reported might reflect differences between the source of the material
used to prepare the nuclear extracts and other experimental conditions.
However, the lack of evolutionary conservation of the CREB and AP2
binding sites and the failure of these factors to bind in brain and
testis extracts suggests that these sites may be less important for
FMR1 expression than previously thought.
A Similar Pattern of Protein Binding Is Seen in Extracts of Liver,
Lymphoblasts, and PC12 Cells--
A similar pattern of three shifted
bands was observed with nuclear extracts from liver, human
lymphoblastoid, and PC12 cell lines (Fig. 4, lanes 2 and
3, and data not shown). However, the two smaller of the
three minor bands indicated by the bracket in the brain and liver
extracts were not seen with the extracts prepared from the cell lines.
These bands may be due to proteolysis during the preparation of the
nuclear extracts from the mouse tissues or specific USF variants that
are only seen in certain cells. Complexes I, II, and III in liver
extracts could be eliminated by competition with E-box and
Testis Extracts Have a Different Pattern of Protein
Binding--
Although a similar pattern of gel mobility shift was seen
for brain, liver, lymphoblasts, and PC12 cells, the pattern of mobility shift in testis extracts was very different. Complex I was absent, and
complex II and a large amount of heterodisperse complex, "complex III," were seen together with a small amount of a novel band (complex IV) with faster mobility (Fig. 4, lane 1). Unlabeled
When the formation of complex III is prevented by the addition of
Methylation of FMR1 Promoter Prevents
Methylation of the E-box also affects the footprint at the We have identified five evolutionarily conserved sites in the
promoter of the FMR1 gene (Fig. 2). These include the four in vivo protein binding sites found in fibroblasts and lymphoid cells (5, 6), namely the E-box, two GC-boxes, and a We have identified three major transcription factors that seem to
positively affect transcription. They are USF1 and USF2, two similar
basic helix-loop-helix/leucine zipper (b/HLH/Z) type transcription
factors that are ubiquitously expressed but have nonetheless been
implicated in the regulation of several tissue-specific as well as
developmentally or metabolically regulated genes (e.g. Refs.
22 and 23). The third positive regulator is a putative bZip factor
known as In liver, the amount of USF protein that binds to the promoter in gel
shift or DNase I footprinting assays is reduced relative to that in
brain and testis, and very little Methylation of the FMR1 promoter in individuals with fragile X syndrome
is correlated with reduced transcription of this gene. In such
individuals no transcription factor binding to the promoter in
vivo is seen (5, 6). This may reflect the binding of proteins such
as MECP2, that recognize methylated CpGs and bind the promoter in a
sequence-nonspecific manner (27). One consequence of this binding is
thought to be the recruitment of histone deacetylases to the region and
subsequent formation of transcriptionally silent chromatin containing a
large proportion of deacetylated histones (27). The FMR1 promoter in
cells from fragile X patients is associated with deacetylated histones,
and reactivation of the promoter using the demethylating agent
5-azacytidine increases transcription (9, 28) and the proportion of
acetylated histones (9). However, treatment of cells from fragile X
patients with the deacetylase inhibitor trichostatin A, which increases
the level of acetylated histones directly, has little (29) or no effect
on reactivation of the gene (9). This has led to the suggestion that
there might be an additional component to the methylation-dependent silencing of this gene (9). We have
shown that methylation significantly reduces Pal/Nrf-1 are the major
transcription factors that bind the promoter in brain and testis
extracts and suggest that elevated levels of these factors account in
part for elevated FMR1 expression in these organs. We also show that
methylation abolishes
Pal/Nrf-1 binding to the promoter and
affects binding of USF1 and USF2 to a lesser degree. Methylation may
therefore inhibit FMR1 transcription not only by recruiting histone
deacetylases but also by blocking transcription factor binding. This
suggests that for efficient reactivation of the FMR1 promoter,
significant demethylation must occur and that current approaches to
gene reactivation using histone deacetylase inhibitors alone may
therefore have limited effect.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Pal/Nrf-1,1 AP2,
AGP/EBP, and Zeste (5). Four regions of protein binding in the
unmethylated promoter of the FMR1 gene have been described by in
vivo dimethyl sulfate footprinting analysis in human
fibroblasts, peripheral lymphocytes, and lymphoblastoid cell lines (5,
6). These footprints correspond to the
-Pal/Nrf-1 site, 2 GC-boxes, and the E-box. However, the transcription factors that interact with
these sites have not yet been identified. Moreover, whereas these
footprints do reflect in vivo interactions, their relevance to the regulation of FMR1 transcription in brain and testis, the two
major affected organs, is unknown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
88/+244, p
+55/+244, and p
447/+244 were generated by
digesting pGL-FMR with BglII and filling in the recessed 3'
ends with
phosphothioate deoxyribonucleotides followed by
digestion with NheI, Exonuclease III, and S1 nuclease according to standard procedures. Plasmids p
131/
123 were made by
digesting pGL-FMR with BssHII followed by ligation.
p
131/
123/USFmut was generated from pGL-USFmut in the same way.
Plasmids p
149/
116 and p
151/
81 were generated by
SphI digestion followed by T4 DNA polymerase treatment and
ligation. Plasmid p
85/
9 was obtained by digestion of pGL-FMR
with PmlI and treatment with Exonuclease III and S1
nuclease. A 6-base pair deletion was made in the Initiator (Inr)-like element in the FMR1 promoter using the QuickChange MutagenesisTM protocol and the primer pair
5'-dGGCCGGGGGTTCGGCCAGGCGCTCAGCTCC-3' and
5'-dGGAGCTGAGCGCCTGGCCGAACCCCCGGCC-3'. This resulted in plasmid p
+5/+10. The deletion was confirmed by sequencing. Similarly, a
four-base pair change was made in the two GC-boxes by QuickChange MutagenesisTM protocol, and the mutation was confirmed by sequencing. Specifically, Sp1 site was mutated using primers
5'-dCACTTGAAGAGAGAGGATCTGGCCGAGGGGCTGAGC-3' and
5'-dGCTCAGCCCCTCGGCCAGATCCTCTCTCTTCAAGTG-3' to get plasmid pS1mut, and
the Sp1-like site was mutated using primers
5'-dGCTGAGCCCGCGGGGGATCTGAACAGCGTTGATCAC-3' and
5'-dGTGATCAACGCTGTTCAGATCCCCCGCGGGCTCAGC-3' to get plasmid pS2mut. The
plasmids pS1mut and pS2mut were digested with BssHII and
ligated to get p
131/
123/S1mut and p
131/
123/S2mut, respectively.
Pal/NRF-1 sites were achieved by in vivo
methylation with PmlI and BssHII, methylases, respectively. This was accomplished using pLG339 as the vector for the
methylase genes. pLG339 containing the BssHIIM gene (pLG339/BssHIIM) was a gift of New England Biolabs, Beverly, MA. A similar clone was
constructed for the PmlI methylase by subcloning the
3.5-kilobase EcoRI-SalI fragment from pEco72
M (a gift of MBI Fermentas Inc.) into pLG339 to
generate pLG339/PmlIM. pGL-FMR was cotransformed into Escherichia
coli along with either pLG339/BssHIIM or pLG339/PmlIM. Methylation
in all cases was confirmed by digestion with PmlI or
BssHII. Since the plasmids obtained by in vivo
methylation are contaminated with the low copy number pLG339
derivative, an unmethylated control was prepared by cotransforming
pGL-FMR with pLG339.
-Pal/Nrf-1 site and that of
consensus E-box and SREBP-1 sites were synthesized by Life
Technologies. The complementary strands were annealed and used without
further purification. Antibodies against USF1, USF2, Max, c-Myc, Sp1,
Sp3, Sp4, and Egr-1 were from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). CREB antibody was from New England BioLabs.
Pal/Nrf-1
antibodies were a gift from Dr Brian Safer (NHLBI, NIH, Bethesda, MD).
80 °C. Protein concentrations were
determined using the Bio-Rad protein assay reagent. Nuclear extracts
from PC12 cell lines were purchased from Promega.
214 to
13) was amplified from plasmid p32.9
with primers hmba1 5'-dCTTTCAAGCTTCCCGCCCTCCACCAAG-3') and hmba2
(5'-dGAACCCAAGCTTCGCTGCGGGTGTAAACA-3'). The amplified fragments
were gel-purified and cloned into the HindIII site of pBS
(SK+). For EMSA and DNase I footprinting experiments, a gel-purified SalI/NotI fragment containing the promoter was
end-labeled at the NotI site using
[
-32P]dCTP and Klenow DNA polymerase (Life
Technologies). The labeled probe was extracted with
phenol/chloroform and purified by passing through a Sephadex G-50
column. The promoter was methylated using SssI methylase
(New England BioLabs). Methylation was verified by digestion with the
methylation-sensitive enzymes PmlI, EagI, and
HhaI (New England BioLabs).
-32P]dGTP and Klenow DNA
polymerase (Life Technologies). Binding reactions were carried out with
1 ng of 3' end-labeled probe and 20 µg of protein in a 100-µl
reaction volume containing poly[dA-dT][dA-dT]. The reactions were
treated with 0.25 units of DNase I in the presence of 10 mM
MgCl2 and 1 mM CaCl2 for 10 min at
30 °C. The reactions were phenol-extracted and ethanol-precipitated
with tRNA as carrier. The sample was dissolved in 100 µl of TE (10 mM Tris-HCl, pH 8.0, 1 mM Na2EDTA)
and butanol-precipitated. The pellet was washed with 70% ethanol,
dried, and resuspended in 10 µl of formamide stop buffer (95%
formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05%
xylene cyanol FF). A 2-µl sample was electrophoresed on a 6%
sequencing gel at 1600 V until the bromphenol blue dye reached the
bottom of the gel. The gel was dried and exposed to x-ray film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Pal/Nrf-1 site, 131 bases 5' of the start of
transcription. This suggests that the minimal FMR1 promoter may only be
131 bp long. The conserved 3' regions may represent regulatory elements
in the first intron, but they have not been studied in any detail to
date.
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Fig. 1.
Dot matrix comparison of 3181 bases of the
mouse fmr1 gene spanning exon 1 with the sequence of
the human FMR1 gene. The gray lines delineate the start
and end of exon 1. The black arrow indicates the 5' end of
the previously defined minimal promoter (12). The circled
region indicates the region of homology between the two sequences
due to the CGG repeat in the 5'-untranslated region of exon 1. The
numbering on the right-hand side of the matrix refers to the
sequence of a mouse 3.1-kilobase EcoRI fragment that
includes exon 1. The numbering at the top of the matrix
refers to that of the human sequence in the GenBankTM entry
GB:HUMFMR1S. The numbering on the left-hand side and along
the bottom of the matrix corresponds to the numbering used for the
alignment of sequences shown in Fig. 2, where +1 corresponds to the
first transcribed base.
Pal/Nrf-1, the 2 Sp1 or GC boxes, and the E-box (sometimes referred to as the c-Myc binding site) (5, 6) are conserved in all five
species (shown in the dark gray boxes in Fig. 2). None of
these sequences had a good TATA-box. However, they all contained a
conserved motif close to the reported start of transcription (13) that
resembles an Initiator (Inr) element. Such elements direct
transcription initiation in certain TATA-less promoters (14).
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Fig. 2.
Sequence alignment of the 5' end of the FMR1
promoter from five different mammalian species. The human sequence
is shown in its entirety with gaps in the alignment shown as
dashes. The bases in the sequence of the remaining four
species that are identical to the human sequence are shown as
dots, with only those bases that differ from the human
sequence shown. The previously identified putative factor binding sites
that are conserved are shown in the dark gray boxes. These
sites correspond to the four protein binding sites that have been
identified in fibroblasts, lymphocytes, and lymphoblastoid cells. The C
complementary to the G at +14 that is dimethyl sulfate-hyperreactive
in vivo is marked by an asterisk. Those
previously identified putative factor binding sites that are not
conserved are shown as the open boxes. The newly identified
conserved Inr element binding site downstream of the start of
transcription is shown in the light gray box. The
sequence of the mouse and human fragments used in all the EMSA
experiments described in this manuscript is shown in
bold.
Pal/Nrf-1 site (p
131/
123) each
reduced the expression of the reporter gene about 5-fold. Both
mutations together (p
131/
123/USFmut) reduced activity almost to
that of a construct containing a deletion of almost all of the FMR1 sequence (p
447/+244). Therefore the upstream boundary of the minimal promoter region is probably close to the 5' end of the
Pal/Nrf-1 site, as suggested by the phylogenetic footprinting. A
deletion that included the
Pal/Nrf-1 site and one of the GC-boxes (p
151/
81) led to an increase in activity over that seen with the
nine-base deletion in the
Pal/Nrf-1 site alone (p
131/
123). Similarly, a deletion that removes both the E-box sequence and its
adjacent Sp1 site (p
85/
9) produced a higher activity than the
mutant with the single base insertion in the E-box. The activity of
this construct was in fact higher than the full-length promoter (pGL-FMR). However, when a 4-base substitution mutation was made in
either of the two GC boxes, the activity was reduced to 75 and 50%,
respectively (pS1mut and pS2mut). This suggests that these two regions
have a positive role in the control of gene expression. The increase in
activity seen for deletion constructs p
151/
81 and p
85/
9
that lack these sites could be due to decreased distance between the
Pal/Nrf-1 site and the transcription initiation site or to the
deletion of additional sequences that contain negative regulators of
FMR1 activity. Constructs containing both the
Pal/Nrf-1 deletion
and the point mutation in the first GC box (p
131/
123/S1mut) had
an activity similar to the
Pal/Nrf-1 deletion alone, suggesting
that there might be some interaction between factors that bind these
two sites. However, a
131/
123:S2mut double mutant showed a slightly
higher activity than the
131/
123 deletion by itself. Why this
should be is not clear at this time but may be related to the
uncovering of secondary transcription factor binding sites that can now
be used. The p
85/
9 construct lacks the TATA-like sequence
upstream of the transcriptional start site. Since deletion of this
sequence did not negatively affect promoter activity, it is possible
that the TATA-like sequence is dispensable. However, although the
initiator-like element is evolutionarily conserved, its deletion had no
effect on the promoter activity in PC12 cells (construct p
+5/+10 in
Fig. 3). Since the region that includes the putative transcriptional
start site is not conserved, it may be that transcription initiation in
the FMR1 promoter occurs via a novel mechanism that involves as yet undefined signals.
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Fig. 3.
Functional analysis of the human FMR1
promoter in PC12 cells. PC12 cells were electroporated with the
various constructs shown on the left-hand side of the figure as
described under "Experimental Procedures." Base substitution
mutations are indicated by the ball and stick. The amounts of
luciferase produced were plotted as percentages of the luciferase
activity of the full-length promoter construct.
+53/+244) produced a small increase in reporter gene activity.
Since this sequence is located within the transcript, it is possible
that this effect is mediated either at the level of transcription,
mRNA stability, or translation. A negative effect of CGG repeats on
translation has been suggested based on the observation that the levels
of FMRP, the protein product of the FMR1 gene, are reduced in
individuals with long premutation alleles (16). However, this effect is
probably only significant at high repeat numbers. A 20-kDa protein has
been shown to bind to CGG repeats and inhibit FMR1 transcription (17).
Deletion of the CGG repeat tract may simply eliminate the effect of
these or similar proteins.
Pal/Nrf-1 Bind to the FMR1 Promoter--
The
200-bp region of both the mouse and human FMR1 promoters, which
contains all the evolutionarily conserved putative transcription factor
binding sites (shown in bold in Fig. 2) was used as a probe for binding
factors in nuclear extracts from mouse brain, testis, and liver and
human lymphoblastoid and PC12 cell lines. EMSA using brain nuclear
extracts and the mouse promoter produced a pattern of three major
retarded bands (complexes I, II, and III, Fig. 4, lane 4). In addition, three
minor shifted products with mobilities slightly faster than complex I
were also seen. All of the bands were specific, since they could be
eliminated by an excess of nonradioactive probe (data not shown) but
not with a variety of other sequences (Fig. 4, lanes 6,
7, and 8). The same pattern of gel-shifted bands
was produced when the human promoter was used (data not shown).
View larger version (49K):
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Fig. 4.
EMSA analysis of the mouse promoter in the
presence of antibodies to different transcription factors. The
EMSA was carried out as described under "Experimental Procedures"
using mouse brain nuclear extracts unless otherwise indicated. Specific
competitor DNA (see Table I for their sequence; lanes 5-9)
or antibody to specific transcription factors (lanes 11-20)
or bovine serum albumin was added where indicated. The location of the
free probe, complexes I, II, and III, and the supershifted complexes
are indicated by the arrows on the right-hand side of the
figure. Inset, Western blot analysis of the levels of
various transcription factors in brain (B), testis
(T), and liver (L). SDS-polyacrylamide gel
electrophoresis and Western blotting of brain, testis, and liver
nuclear extracts were carried out as described under "Experimental
Procedures" using antibodies to c-Myc, CREB, USF1, and USF2 as
indicated.
Pal/Nrf-1 binding site eliminated complex II and III (Fig. 4, lane 9). Complexes II and III
were also eliminated when poly[dI-dC] was present, presumably due to the GC richness of the
Pal/Nrf-1 binding site (data not shown). Antibodies to the E-box-binding proteins USF1 or USF2 supershifted both
complex I and III as well as the minor products, indicating that these
complexes contained both USF1 and USF2 (Fig. 4, lanes 14 and
15). Antibodies to c-Myc or max, two other E-box binding proteins, had no effect on the gel mobility profiles (Fig. 4, lanes 12 and 13). This is not due to the absence
of c-Myc in the extracts since Western blots with c-Myc antibody showed
the presence of large amounts of full-length c-Myc protein (Fig. 4,
inset). Antibodies to
Pal/Nrf-1 shifted both complex II
and complex III (Fig. 4, lane 20), indicating that
Pal/Nrf-1 was involved in both of these complexes.
Pal/Nrf-1 site did produce gel shifted products, one
of which could be eliminated by competition with Sp1 oligonucleotide
and was supershifted by Sp3 antibody (data not shown). It is possible that lack of Sp3 binding to the larger promoter fragment is due to
binding of USF and/or
Pal/Nrf-1. The in vivo binding of
Sp factors in lymphoid cells and fibroblasts might reflect the high concentrations of Sp factors in these cells relative to brain, testis,
and liver (19).
Oligonucleotides used for cold competition in EMSA
Pal/Nrf-1 oligonucleotides as in brain extracts (results not
shown), suggesting that they are similar in composition to those seen
in brain extracts.
Pal/Nrf-1 eliminates complex II and III, and anti-
Pal/Nrf-1
antibody supershifts both complex II and III (data not shown).
Unlabeled E-box oligonucleotide eliminates both complex III and complex
IV, and both of these complexes are supershifted with antibodies to
USF1 or USF2 (data not shown). Thus, complex II and III in testis are
apparently similar to complex II and III in brain and liver, being
comprised of
Pal/Nrf-1, and
Pal/Nrf-1 plus USF proteins,
respectively. Complex IV contains USF1 and USF2 proteins since it is
supershifted by antibodies to both USF1 and USF2. However since there
are no unique testis-specific USF1 or USF2 polypeptides visible on
Western blots of SDS-polyacrylamide gel electrophoresis gels of these extracts (Fig. 4, inset, and data not shown), the rapid
mobility of USF proteins in complex IV may be due to either charge
differences or to the association of USF with a different subset of
proteins in this organ.
Pal/Nrf-1 oligonucleotide, there is little if any increase in
complex I in brain (Fig. 4, compare lanes 1 and
6) and liver or complex IV in testis (data not shown). No
new bands corresponding to complex I are seen in testis extracts either
(data not shown). In contrast, there is an increase in the amount of
complex II seen in brain, testis, and liver extracts when excess E-box
oligonucleotide is present (Fig. 4, compare lanes 4 and
5). One interpretation of these data is that the USF
proteins in complex III do not bind the promoter effectively in the
absence of
Pal/Nrf-1, perhaps because they are complexed with
other proteins or modified in some way and that in testis most of the
USF is of this form.
Pal/Nrf-1
Binding--
Methylation of the FMR1 promoter in fragile X patients
(20) and in reporter constructs (21) greatly reduces the ability of the
promoter to drive transcription. Methylation of the C residues in all
the CpG residues in the promoter by SssI methylase
eliminated complex II and III in gel shift assays done with the human
FMR1 promoter and brain extracts but had only a small effect on the formation of complex I (Fig. 5,
panel A). Consistent with this observation is the fact that
methylation of three CpGs in the
Pal/Nrf-1 site eliminated the
DNase I footprint seen at the
Pal/Nrf-1 site (Fig. 5, panel
B, lane 4), whereas methylation of the single CpG in
the E-box reduced but did not eliminate the DNase I protection at that
site (Fig. 5, panel B, lane 6). Moreover, transfection experiments showed that methylation of the promoter at the
E-box reduced promoter activity by about 20%, whereas methylation of
the
-Pal/Nrf-1 site reduced activity by 55% (Fig. 5C).
This suggests that binding of
Pal/Nrf-1 is sensitive to
methylation and that USF1/USF2 binding is also sensitive but somewhat
less so than
Pal/Nrf-1. The fact that a deletion in the
-Pal/Nrf-1 site which prevents
-Pal/Nrf-1 binding causes a 75%
drop in promoter activity suggests that methylation of this site
reduces the ability of
-Pal/Nrf-1 to drive transcription by more
than 70%.
View larger version (47K):
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Fig. 5.
The effect of methylation on the human FMR1
promoter. A, the effect of methylation on the formation
of complex I, II, and III. The EMSA was carried out as described under
"Experimental Procedures" using mouse brain nuclear extracts and
either unmethylated probe (0) or a probe that has been
completely methylated using SssI methylase
(SssIM). The location of the free probe and the gel-shifted
complexes I, II, and III are indicated by the arrows on the
right-hand side of the figure. B, the effect of methylation
of individual transcription factor binding sites on DNase I
footprinting. Footprinting was carried out as described under
"Experimental Procedures" using mouse brain extracts and either an
unmethylated promoter fragment (lanes 1 and 2, 0)
or promoter fragments that had been methylated with BssHII
methylase (BssH IIM) (lanes 3 and 4)
or PmlI methylase (Pml IM) (lanes 5 and 6). The Pal/Nrf-1 site and the E-box site are
indicated by the square brackets. The numbering
corresponds to the numbering used in Fig. 2. C, the effect
of methylation on promoter activity. The promoter activity of
unmethylated pGL-FMR (0) or the pGL-FMR methylated by
PmlI M or BssHIIM was determined as described
under "Experimental Procedures." The activity is shown in
panel B as a percentage of the activity of the unmethylated
plasmid.
Pal
site (Fig. 5B, lane 6). This suggests that
the USF proteins bind to both the E-box and the
Pal/Nrf-1 site
and/or that there is some interaction between these factors. This
together with the observations that abolishing complex III by the
addition of excess
-Pal/Nrf-1 oligodeoxyribonucleotide does not
increase the amount of complex I that is seen and that mutations in the E-box or the
-Pal/Nrf-1 site both reduce promoter activity to 25%
that of the wild-type promoter supports this idea. We are currently
examining these possibilities in more detail.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Pal/Nrf-1 binding
site. The conservation of these sites among species as evolutionarily
divergent as primates, canids, and rodents, which last shared a common
ancestor 80-120 million years ago, indicates that they are important
for regulation of this gene. That the E-box and the
Pal/Nrf-1
sites together can account for almost all of the transcriptional
activity of the FMR1 promoter in the neuronally derived PC12 cells
supports this contention (Fig. 3). Removing the TATA-like sequence just
upstream of the transcription start site does not abolish gene activity
nor does a deletion of an Initiator-like element close to the reported
transcription start site. Initiation of transcription of the FMR1 gene
may thus involve a novel mechanism whose molecular details remain to be elucidated.
Pal or Nrf-1 (nuclear respiratory factor-1). The term
Pal/Nrf-1 is used here for consistency with the literature and to
avoid confusion with NF-E2-related factor-1, which is also abbreviated
to Nrf-1.
Pal/Nrf-1 has been implicated in the regulation of
metabolic genes in response to cellular proliferation (24). However, it
has strong homology to the Drosophila erect wing gene (ewg) that is required for normal central nervous system
development (24), which may be relevant given the role of FMR1 in
learning and memory. Deletion of the binding sites for these factors
reduces transcriptional activity in a neuronally derived cell line. We have also shown that Sp3, a member of the Sp family of ubiquitously expressed zinc finger factors that bind GC-boxes, binds to the 82-bp
SphI-PmlI mouse promoter subfragment containing
the conserved GC-boxes. Deletions that include one or the other of
these GC-boxes do decrease promoter activity (Fig. 3), suggesting some
contribution of these regions to optimal gene activity.
Pal/Nrf-1 binding is seen at all
(Fig. 4, and data not shown). The high level of USF1 and USF2 in adult
brain (Fig. 4 and Ref. 25) and the high levels of
Pal/Nrf-1 (26)
and the
Pal/Nrf-1-dependent USF variant in adult
testis relative to other organs may account for their relatively high
levels of FMR1 mRNA.
Pal/Nrf-1 binding and also affects binding of USF1 and USF2. Failure of these factors to bind
the methylated promoter could well be this additional component. If
this is true, it would have important implications for the development
of therapies aimed at reactivating FMR1 transcription in patients with
fragile X syndrome.
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ACKNOWLEDGEMENTS |
---|
We thank A. V. Furano, N. Nossal, and R. Owens of the Laboratory of Molecular and Cellular Biology, NIDDK and B. Safer of the Molecular Hematology Branch, NHLBI, National Institutes of Health, for thoughtful reading of this manuscript.
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FOOTNOTES |
---|
* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF251347, AF251348, AF251349, and AF251350.
To whom correspondence should be addressed: Bldg. 8, Rm. 202, National Institutes of Health, 8 Center Dr. MSC 0830, Bethesda, MD
20892-0830. Tel.: 301-496-2189; Fax: 301-402-0053; E-mail: ku@helix.nih.gov.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M009629200
2 Schug, J. and Overton, G. C. (1997) Technical Report CBIL-TR-1997-1001-v0.0 , Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania.
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ABBREVIATIONS |
---|
The abbreviations used are: Nrf, nuclear respiratory factor; CREB, cAMP-response element-binding protein; bp, base pair(s); EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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