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J. Biol. Chem., Vol. 275, Issue 27, 20734-20741, July 7, 2000
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,From the Genetic Pharmacology Unit, Experimental Therapeutics Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, October 14, 1999, and in revised form, March 15, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Three-amino acid extension loop (TALE) homeobox
proteins are highly conserved transcription regulators. We report that
two members of this family, Meis2 and TGIF, which frequently have overlapping consensus binding sites on complementary DNA strands in
opposite orientations, can function competitively. For example, in the
D1A gene, which encodes the predominant
dopamine receptor in the striatum, Meis2 and TGIF bind to the activator
sequence ACT ( The three-amino acid loop extension
(TALE)1 superclass of
homeobox proteins is characterized by an extension of three amino acids
between Human Meis1 (myeloid ecotropic viral integration site 1) has
been cloned from BHX-2 myeloid tumors as a novel common oncogenic site
of proviral integration (8). Murine Meis2 was isolated by
DNA cross-hybridization with a murine Meis1 cDNA (9) or with a partial human Meis2 cDNA (10) and was found to be
inducible by retinoic acid during differentiation of P19 embryonal
carcinoma cells (11). Murine Meis2 is highly expressed in
the lateral ganglionic eminence and in the developing striatum
(12).
Human TGIF (5'-TG-3'interacting factor) is known to bind to the
retinoid X receptor (RXR)-responsive element in the cellular retinol-binding protein II promoter, which contains an unusual DNA
target for homeobox proteins. The interactions of TGIF and RXR The interplay of a variety of transcription factors like the
homeoproteins plays an important role in the regulated,
tissue-specific, and developmental expression of eukaryotic genes.
These factors exert their effects on target genes by both activating
and repressing transcription. Such complex regulation is particularly
evident in the brain, where a large number of genes are expressed with unique regional distributions. For example, specificity of the biological effects of dopamine is determined at least in part by the
intricate spatial and temporal regulation of genes encoding its
receptors. To date, five G protein-coupled dopamine receptors encoded
by different genes have been cloned. Based on their homology and
pharmacological criteria, they are classified into two subtypes, D1-like (D1A and D5 or
D1B) and D2-like (D2,
D3, and D4) receptors (14, 15). Among these,
the D1A subtype is the predominant dopamine receptor in the
striatum, the brain region that is innervated by dopaminergic neurons
of the substantia nigra which degenerate in Parkinson's disease (16).
Alterations in the functional state of dopamine receptors in
neuropsychiatric disorders and with chronic dopaminergic therapy are
thought to underlie long term complications such as parkinsonian motor
response fluctuations and tardive dyskinesias (17, 18).
Our previous analysis of the human D1A receptor
gene revealed that the region between nucleotides Cell Culture--
The murine neuroblastoma cell line NS20Y was a
gift from Dr. Marshall Nirenberg (NHLBI, National Institutes of Health,
Bethesda, MD). Human neuroblastoma SK-N-MC and SH-SY5Y, murine
neuroblastoma NB41A3, human hepatoblastoma HepG2, and opossum kidney OK
cells were purchased from ATCC. All cells were grown in Dulbecco's
modified Eagle's medium (Mediatech) supplemented with 10% fetal
bovine serum (BioWhittaker) at 37 °C in a humidified atmosphere of
10% CO2.
Yeast One-hybrid Screening for cDNAs Encoding ACT-binding
Proteins--
The MATCHMAKER One-Hybrid System
(CLONTECH) was used according to the supplier's
protocol. Three tandem repeats of the bp Cloning Human Meis2a-e and TGIF cDNAs and in Vitro
Translation--
The Meis2b cDNA isolated from the
yeast one-hybrid screening was sequenced and oligonucleotide primers
were synthesized to screen a SuperScriptTM human brain cDNA
library using GeneTrapper system (Life Technologies). These PCR primers
were: forward, 5'-AGTATGGGATCCGCTGTCAA-3'; reverse,
5'-GAGCTGCCGTCTCTTTCATC-3'. All positive clones were sequenced, and
five different Meis2 splice variants were identified and
designated pCMV-Meis2a-e, respectively.
The coding region of TGIF cDNA was cloned using RT-PCR
with total RNA from human brain (CLONTECH) using
primers: forward, 5'-CGGGATCCTCCAGAATGAAAGGCAAGAAA-3';
reverse, 5'-GGCTCGAGTTAAGCTGTAAGTTTTGC-3', designed to have
BamHI and XhoI sites (underlined), respectively. The amplified TGIF cDNA was digested with
BamHI and XhoI and subcloned into the respective
sites of pcDNA3.1 (Invitrogen) to generate pcDNA-TGIF.
To construct an expression vector having only the TGIF homeodomain, the
cDNA region covering amino acid residues 31-97 was amplified by
PCR using pcDNA-TGIF as template with primers
5'-AACTGCAGATGGCTGGCTCAGGCAAGAGAAGG-3' (PstI site underlined; inserted ATG codon italicized) and
5'-GAGCTCGAGCATGTCAGGGAGGAGCCTGCG-3' (XhoI site
underlined). The product was digested with PstI and XhoI and ligated into the same sites of pCMV/myc/nuc
(Invitrogen) in frame with the nuclear localization signal of the
vector. The insert in the resultant plasmid pTGIF-HD was confirmed by sequencing.
In vitro transcription/translation (Life Technologies, Inc.)
was carried out with pCMV-Meis2a-e and pcDNA-TGIF in a reaction mixture containing [35S]methionine (Amersham Pharmacia
Biotech) using a TNT coupled reticulocyte lysate system (Promega). The
labeled proteins were then electrophoresed in 10% SDS-PAGE to
determine that they were of the predicted sizes.
Genomic Walking for Identification of Meis2e Exon/Intron
Junction--
Human Genomewalker kit (CLONTECH)
was used to isolate a genomic fragment harboring the exon/intron
junction region of Meis2e. After designing two gene-specific
primers (GSP1 and GSP2) within the exon region of Meis2e,
PCR was performed with GSP1 (5'-CTTCACCTGGTACAGGTGACGATGA-3') and AP1
(adaptor primer 1), followed by a second PCR with GSP2 (5'-TCATGAGAGCATGGCTCTTCCAGCA-3') and AP2 (nested adaptor primer 2).
The second PCR yielded a 1.2-kilobase pair product, which was subcloned
into pCR2.1 (Invitrogen) and sequenced.
RT-PCR Analysis of Meis2 Isoforms--
Mouse Multiple Tissue and
Embryonic Stages cDNA Panel (CLONTECH) was used
to amplify Meis2e isoforms. To separate the Meis2e fragment from other Meis2 isoforms, PCR was performed
with sense primer P1 (5'-GATGATGCAACCTCAACCCAC-3', located between
nucleotides 919 and 939) and antisense primer P2
(5'-ATCCAACACAAAGCTCCC-3', located between nucleotides 1354 and 1372)
(Fig. 2A). PCR products were electrophoresed in a 1.5%
agarose gel, and the 355-bp fragment was subcloned into pGEM-T Easy
Vector (Promega) and sequenced. P2 primer sequence is identical between
human and mouse Meis2 (11). The two base substitutions
(T924C and C936G in primer P1) in the mouse did not affect the outcome
of this PCR with murine tissues. To identify Meis2 isoforms expressed
in SH-SY5Y and SK-N-MC cell lines, RT-PCR was performed using sense
primer P1 paired with either antisense primer P2 or P3
(5'-ATTGGGGGTCCATGTCTT-3', located between nucleotides 1608 and 1625).
These PCR products were electrophoresed in a 3% agarose gel.
Transfections for Transient Expression of CAT
Constructs--
Transfections were carried out by the standard calcium
phosphate co-precipitation method (Invitrogen). Briefly, HepG2, OK, SK-N-MC, and SH-SY5Y cells were plated in 100-mm dishes at a
concentration of 1 × 106 cells/dish and cultured
overnight before transfection. Various plasmids at the indicated
concentrations in each figure were used. Total amount of DNA was kept
constant by adding pcDNA3.1. After transfection periods of 6 h
(OK) or 18 h (SK-N-MC, SH-SY5Y, and HepG2), cells were incubated
in fresh medium for an additional 48 h. CAT protein was quantified
by the CAT ELISA kit (Roche Molecular Biochemicals). Each experiment
was carried out in triplicate.
Generation of GST-Meis2a-e Fusion Proteins and Gel Mobility
Shift Assays--
The coding regions of Meis2a-e were
amplified by PCR using pCMV-Meis2a-e as templates with primers
containing either EcoRI or XhoI sites, and
sequences were confirmed. All five Meis2a-e cDNAs were
subcloned into pGEX4T-1 to produce GST fusion proteins and designated
pGEX-Meis2a-e, respectively. The resultant plasmids were used to
transform Escherichia coli BL21. Fusion proteins of Meis2
isoforms were induced by 0.1 M
isopropyl-1-thio-
For gel mobility shift assays, wild-type and mutant ACT probes (Fig. 1)
were synthesized using an ABI DNA synthesizer. One strand was labeled
with [ In Situ Hybridization--
pCMV-Meis2a was digested with
EcoRI and XhoI, and the Meis2a insert
was subcloned in pGEM-3Zf( Isolation of Human Meis2a-e That Interact with the ACT Sequence in
the D1A Promoter--
To identify transcription factors
that bind to the D1A ACT region, the yeast
one-hybrid screen was performed. A double-stranded oligonucleotide
having three tandem repeats of the ACT sequence (Fig.
1) was subcloned into pHISi and
introduced into yeast cells. The resultant strain was transformed with
a human brain cDNA library. Plasmids prepared from seven positive
colonies that grew on selection medium were transformed into yeast
strain containing the pLacZ-bait construct. One of these seven clones
was found to be a true positive after the lacZ expression test.
Sequence analysis of the positive clone indicated that it is the human
homologue of murine Meis2b. The Meis2b cDNA
insert in the library plasmid was fused in-frame with the GAL4
activation domain.
To obtain full-length Meis2 clones, PCRs were carried out
using a SuperScriptTM human brain cDNA library (Life Technologies) and five splice variants were isolated (Fig.
2A). These clones were
designated Meis2a to Meis2e, analogous to the
nomenclature of murine Meis2 isoforms (11). Human
Meis2a, Meis2b, Meis2c, and
Meis2d use the same exons as in murine Meis2
cDNAs. Meis2a, which includes all exons, and
Meis2b, which lacks exon 3(A), encode isoforms that end at
the first termination codon. On the other hand, Meis2c,
which lacks exon 4, and Meis2d, which lacks both exons 3(A)
and 4, encode isoforms that end at the second termination codon.
Comparison of human and mouse Meis2a and Meis2b sequences revealed
100% amino acid identity. In Meis2c, the two species differ by a
single amino acid at position 439, whereas in Meis2d they differ by two
amino acids at positions 394 and 432. Meis2e, which had not
been identified previously, has the same start codon as that of
Meis2a-d, but an early termination codon TAA within the
homeodomain due to alternative splicing resulting in deletion of exon 1 and a frameshift. Thus, the Meis2e protein has a truncated homeodomain
(Fig. 2, A and B). To exclude the possibility
that Meis2e could be an artifact of library construction, 3'
genomic walking was performed with gene-specific primers to determine the exon-intron boundary in the deleted region. Comparison of genomic
and cDNA sequences indicated that the splice donor site of this
alternatively spliced product obeys the GT-AG rule (data not shown)
(22). The splice acceptor site at this exon has been reported
previously in the mouse (11).
To verify the endogenous expression of the Meis2e splice variant in
biological tissues, PCR was carried out on cDNA obtained from
multiple adult mouse tissues as well as from different embryonic stages
(CLONTECH) using a primer pair designed to separate
Meis2e from the other four Meis2 isoforms (Fig. 2C). In
addition to a doublet band representing 454 bp for variants "a" and
"c" and 433 bp for variants "b" and "d," a 355-bp fragment
was amplified and cloned. Upon sequencing, the latter was confirmed to
represent Meis2e. The relative abundance of Meis2e in different tissues was quite variable with highest level in muscle and lowest levels in
brain, heart, and kidney.
The ability of all five Meis2 cDNA isoforms to encode
proteins was verified by in vitro translation and the
products were analyzed on SDS-PAGE (Fig. 2D). All Meis2a-e
isoforms were expressed and electrophoresed slower than expected from
their calculated molecular weights, probably because of the abundance
of prolines and stretches of acidic residues (9).
GST-Meis2a-d Fusion Proteins Bind Specifically to the
D1A ACT Sequence in Vitro--
To confirm whether Meis2
can indeed bind to the ACT sequence, gel mobility shift assays were
carried out using GST fusion proteins of each human Meis2 isoform (Fig.
3). All GST-Meis2 fusion proteins were
able to shift the target ACT oligonucleotide probe except GST-Meis2e,
which has a truncated homeodomain. This finding indicates that the
homeodomain in Meis2 is essential for consensus sequence recognition
and binding. The specificity of this DNA-protein interaction was
verified by complete inhibition in the presence of cold wild-type ACT
oligonucleotide and by lack of Meis2a-d binding to a mutant ACT probe.
Furthermore, the shifted Meis2a-d bands were supershifted with a GST
polyclonal antibody, indicating that they represent
GST-Meis2 fusion proteins. Interestingly, Meis2a-d showed
two shifted bands (Fig. 3, lanes 2-5), although purified GST-Meis2a-d fusion proteins ran as single major bands of the
expected sizes on SDS-PAGE (data not shown). The latter observations
suggest that Meis2 proteins could form homodimers similar to murine
Meis1 (23).
Meis2a-d Activate Transcription through the D1A ACT
Sequence--
We had previously demonstrated that the
D1A promoter in pCATD1-1197 harboring the ACT
region is expressed in a neuronal cell-specific manner (19). To study
this observation further, Meis2 Northern analysis was
initially carried out using RNA from various cell lines in order to
select appropriate cells for Meis2 co-transfection with
pCATD1-1197. All neuronal cell lines tested (NS20Y, SK-N-MC, NB41A3,
SH-SY5Y) had endogenous Meis2 expression, whereas
non-neuronal cells HepG2 and OK did not (Fig.
4).
To determine whether Meis2 isoforms could increase the activity of
pCATD1-1197 in non-neuronal cells, OK and HepG2 cells were co-transfected with Meis2a-e expression constructs and with
pCATD1-1197 template plasmid. All co-transfected Meis2 isoforms were
able to increase CAT expression significantly in both cell lines,
except Meis2e, which lacks the homeodomain necessary for DNA binding (Fig. 5A). These results
indicate that Meis2 proteins can function as activators of
D1A gene transcription even in non-neuronal
cells. Meis2b could increase CAT activity in HepG2, but not in OK
cells, a finding that remains of unclear significance.
The specificity of the Meis2d effect on pCATD1-1197 was investigated
by co-transfecting OK cells with two additional
D1A promoter-CAT constructs, pCATD1-1154 and
pCATD1-1120, which lack the ACT region (Fig. 5B). This
experiment demonstrated that the Meis2d effect is specific to
pCATD1-1197, which harbors the ACT sequence. Interestingly, although
Meis2e cannot bind to the ACT sequence (Fig. 3), it potently inhibits
Meis2d-induced pCATD1-1197 activation in OK cells (Fig. 5C). The latter finding indicates that the
NH2-terminal region of Meis2 proteins could play a role in
regulating D1A gene transcription in addition to
their homeodomains, which recognize target DNA sequences.
The physiological significance of Meis2-induced activation of
pCATD1-1197 transcription was addressed next by investigating changes
in endogenous D1A mRNA levels in OK cells.
This cell line was chosen because of the absence of Meis2 mRNA
(Fig. 4) to allow interpretation of reconstitution experiments. In
addition, although these cells had no detectable
D1A mRNA in our Northern analysis (Fig. 4),
they have previously been shown to express this dopamine receptor (24),
indicating that they have the basal transcription machinery to express
this gene. Total RNA was prepared from cells transfected with
pCMV-Meis2d or with control vector (pcDNA3.1) and Northern analysis
was performed (Fig. 5D). Meis2d significantly increased the
endogenous D1A message compared with control
vector-transfected cells.
Meis2 and D1A mRNAs Are Co-localized in the
Striatum--
The functional relevance of our findings in cell lines
was further addressed by investigating the cellular co-localization of
Meis2 and D1A messages within the
adult striatum, the brain region in which both genes are highly
expressed (12, 25). Simultaneous hybridization of a digoxigenin-labeled
Meis2a probe and a radiolabeled D1A
probe revealed considerable overlap between the two signals (Fig.
6). While some cells express
Meis2 alone, very few neurons express
D1A alone. This observation could reflect the
important activating function of this nuclear protein on the D1A dopamine receptor gene.
TGIF Binds to the D1A Promoter ACT Sequence and
Competes with Meis2-induced Transactivation--
To study the effect
of Meis2 overexpression on the D1A gene in human
neuronal cell lines, SK-N-MC and SH-SY5Y cells expressing different
amounts of D1A mRNA (Fig. 4) were
co-transfected with all five Meis2 expression constructs and
pCATD1-1197. Compared with the results in SK-N-MC cells, Meis2a-d
induced robust increases of CAT activity in SH-SY5Y cells (Fig.
7A). The minimal changes with
Meis2c and Meis2d in SK-N-MC cells are of unclear biologic significance. The decrease with Meis2e in both cell lines could be
explained by repression of endogenous Meis2 activity.
Since Meis2e is a potent repressor of Meis2d-induced pCATD1-1197
activity (Fig. 5C), the expression levels of
Meis2e were checked in SH-SY5Y and SK-N-MC cells looking for
a clue for the notable inability of Meis2a-d to activate the
D1A promoter in the latter cell line. RT-PCR
with primer pairs P1 and P2, designed to amplify a 355-bp
Meis2e cDNA, failed to detect the expected fragment in
either cell line but could amplify a complex band corresponding to
Meis2a-d (Fig. 7B). In addition, RT-PCR using primers P1 and P3 could amplify each of the Meis2a-d
cDNAs in both cell lines but not Meis2e (Fig.
7C). Thus, the difference between these two cell lines with
respect to Meis2a-d-induced activation of pCATD1-1197 (Fig.
7A) could not be explained by differences in
Meis2e expression since neither cell had this truncated isoform.
To gain further insight into the inability of Meis2a-d to activate
transcription in SK-N-MC cells, careful examination of the ACT sequence
led to the observation that a TGIF consensus sequence actually overlaps
with the Meis2 consensus sequence with the opposite orientation (Fig.
1). Furthermore, TGIF and Meis2 mRNAs are
expressed at different levels in the cell lines tested, with relatively
high TGIF in cells that have low or no
D1A mRNA (Fig. 4). Therefore, to investigate
the role of TGIF in regulating Meis2 effects on the
D1A gene, the TGIF cDNA was
cloned by RT-PCR from a human brain cDNA library and subcloned into
pcDNA3.1 expression vector. The ability of TGIF to bind to the ACT
region was tested by gel mobility shift assay using in vitro
translated protein (Fig. 7D). TGIF indeed bound to the
wild-type ACT probe but not to the mutant ACT probe. Cold competitor
significantly competed off TGIF binding.
The overlapping TGIF and Meis2 binding sites within the
D1A ACT sequence raised the possibility that the
two proteins could compete for binding to the same target. In addition,
TGIF has been known as a transcriptional repressor (13). To address
this hypothesis, first, competitive gel shift assay was performed. In vitro translated TGIF and GST-Meis2a fusion protein were
added to the gel shift reaction mixture and electrophoresed in a 4% native polyacrylamide gel (Fig. 7E). Addition of rising
amounts of Meis2a with a constant amount of TGIF diminished TGIF
binding to DNA. At the same time, presence of TGIF suppressed the
relative intensity of the band shifted by rising amounts of Meis2a.
Second, to investigate whether the in vitro competition of
TGIF for the Meis2 recognition site has a functional impact within
cells, SH-SY5Y cells were co-transfected with expression vectors for
TGIF and for Meis2a and with pCATD1-1197. Co-expression of increasing
amounts of TGIF resulted in a dramatic inhibition of Meis2a-induced
pCATD1-1197 activity in a concentration-dependent manner
(Fig. 7F). Since TGIF has its own repressor domain (26), the
possibility that it blocks Meis2a-induced activation of the
D1A promoter without necessarily displacing
Meis2a was entertained next. pTGIF-HD expressing only the homeodomain
of TGIF fused to a nuclear localization signal significantly repressed
Meis2a-induced activation of pCATD1-1197 in SH-SY5Y cells in a
dose-dependent manner (Fig. 7G), indicating that
TGIF competes with Meis2 function by displacing it from its target DNA.
However, full-length TGIF appeared more potent than its homeodomain
alone in repressing the Meis2 effect.
In our search for transcription factors that regulate the human
D1A dopamine receptor gene, we have identified
two TALE superclass homeobox genes, Meis2 and
TGIF, which share overlapping and complementary common
binding sites in the activator ACT sequence of the
D1A promoter. The yeast one-hybrid screen
employed with the ACT sequence as bait led to the isolation of Meis2
only but not TGIF. The very low expression of TGIF in the adult brain
could explain the latter observation.
The consensus DNA sequence for the murine Meis1 binding site has been
previously characterized (23) and the third helix of the homeodomain
has been shown to be important for its DNA binding (27, 28). Since
murine Meis1 and Meis2 as well as human Meis2 have an identical amino
acid sequence within their homeodomains (11), human Meis2 likely binds
to the same consensus DNA sequence as murine Meis1. Thus, it was not
surprising that we cloned Meis2 with the yeast one-hybrid screen since
the D1A ACT bait contains a consensus sequence
for Meis1 binding, TGACAG, albeit in the opposite orientation relative
to the promoter (Fig. 1).
Alternatively spliced variants that lack the homeodomain have been
recognized in several homeobox genes such as Drosophila bicoid (29), Xenopus XIHbox 2 (30), and Xenopus
Xhox 36 (31), but the precise functional role of these variants is
unclear. In the present study, we identified five alternatively spliced Meis2 isoforms (Meis2a-e) in biological tissues,
one of which, Meis2e, has a truncated homeodomain. In
contrast to Meis2a-d, which transactivate the
D1A promoter in pCATD1-1197 through the ACT
sequence, Meis2e cannot because of its inability to bind to its target
sequence. Furthermore, the homeodomain-lacking Meis2e inhibits
Meis2d-induced transcriptional activation of pCATD1-1197. It, thus,
appears that Meis2e acts as a natural dominant transcriptional repressor of Meis2 proteins. Another homeodomain-lacking protein, CSX1,
has been reported to transactivate a reporter gene (32). These
observations collectively suggest that homeodomain-lacking proteins can
act either as dominant negative regulators or as positive regulators by
a homeodomain-independent mechanism. One of these
homeodomain-independent mechanisms could be protein-protein interactions. For example, the amino termini of Meis1 and Pbx1a are
known to be necessary and sufficient for their dimerization in solution
and their interaction enhances transcriptional activity of
Hoxb2 (33). It is conceivable that the NH2
termini of Meis2 proteins bind to an unidentified factor, and that
Meis2e could interrupt such interactions without binding to the target
DNA sequence. Whether such an interacting factor is a DNA-binding protein or not, Meis2e could act as a dominant negative regulator.
The present study revealed that the consensus sequences for Meis2 and
for TGIF binding sites usually overlap. These sequences are on
complementary DNA strands and in opposite orientations. In the
D1A promoter, for example, the target site for
TGIF is in the same orientation as the promoter whereas that for Meis2 is in the reverse orientation (Fig. 1). Many genes, in fact, have similar overlapped sequences such as CRBPII (34), lactoferrin (35),
complement factor H (36), and myosin heavy chain (37-39). Although the
Meis2 consensus site is in the reverse orientation relative to the
5'-3' orientation of the D1A promoter, Meis2a-d have positive regulatory effects on pCATD1-1197. Our finding is consistent with the recent report that murine Meis1 can activate transcription even if its recognition site has an orientation opposite
that of the promoter (33). The effect of sequence orientation of the
TGIF consensus binding site remains to be investigated.
While Meis2a-d proteins activate transcription of the
D1A gene, TGIF represses it by binding to
complementary DNA sequences. Therefore, the ratio between TGIF and
Meis2 concentrations would be a critical factor for the transcriptional
regulation of their target genes. Our gel shift experiments revealed a
competition for binding to the ACT sequence between Meis2 and TGIF
(Fig. 7E). In addition, our co-transfection experiments
indicated that TGIF or its homeodomain down-regulate Meis2-induced
transactivation of the D1A promoter (Fig. 7,
F and G), supporting the importance of Meis2
displacement from its target DNA. However, full-length TGIF was more
potent than its homeodomain alone, suggesting an additional functional
role of the TGIF repressor domain in modulating this effect. Thus, the
inability of Meis2 to activate the D1A promoter
in SK-N-MC cells (Fig. 7A) may well be due to the high steady-state levels of TGIF expression in this cell line demonstrated by Northern blot analysis (Fig. 4). On the other hand, the relatively low TGIF expression in HepG2 and OK cells could account for the ability
of Meis2 to activate pCATD1-1197 in both cell lines (Fig. 5A), and account for the ability of Meis2 to increase
endogenous D1A message levels in OK cells (Fig.
5D). Furthermore, comparison of mRNA levels in different
cell lines revealed that the presence of more Meis2 relative to TGIF is
a requisite for endogenous D1A expression (Fig. 4). Hence,
our studies provide both in vitro and in vivo
evidence that the balance between Meis2 and TGIF is involved in the
intricate regulation of D1A gene transcription.
During development, Meis2 is highly expressed in the lateral
ganglionic eminence of embryonic day 12.5 mouse telencephalon, suggesting that it can be a marker of striatal neuronal progenitors (12). Similar in situ hybridization studies have shown that the D1A dopamine receptor gene is also highly
expressed in the striatum from day 14 embryos (40). Thus, Meis2
expression begins prior to that of the D1A gene
prenatally supporting the ability of Meis2 to activate the
D1A gene during striatal development. Furthermore, Meis2 and D1A messages
are highly co-localized within the same neurons of the adult striatum
(Fig. 6), providing the anatomic requisite for their functional interaction.
In conclusion, the foregoing observations indicate that truncated
homeoproteins such as Meis2e can function as dominant negative repressors of transcription. Detailed analysis of the spatial and
temporal expression patterns of Meis2e compared with Meis2a-d can shed
light on the mechanism of action of Meis2 proteins. In addition, since
TGIF and Meis2 consensus sites usually overlap on complementary strands
and in opposite orientations, and since TGIF represses Meis2-induced
transactivation, their relative abundance in the cell determines the
expression level of their target genes such as that encoding the
D1A dopamine receptor.
1174 to 1154) and regulate transcription
differentially in a cell type-specific manner. Among the five cloned
splice variants of Meis2, isoforms Meis2a-d activate the
D1A promoter in most cell types tested, whereas
TGIF competes with Meis2 binding to DNA and represses Meis2-induced
transcription activation. Consequently, Meis2 cannot activate the
D1A promoter in a cell that has abundant TGIF
expression. The Meis2 message is highly co-localized with the D1A message in adult striatal neurons,
whereas TGIF is barely detectable in the adult brain. Our observations
provide in vitro and in vivo evidence that
Meis2 and TGIF differentially regulate their target genes. Thus, the
delicate ratio between Meis2 and TGIF expression in a given cell type
determines the cell-specific expression of the
D1A gene. We also found that splice variant Meis2e, which has a truncated homeodomain, cannot bind to the D1A ACT sequence or activate transcription.
However, Meis2e is an effective dominant negative regulator by blocking
Meis2d-induced transcription activation. Thus, truncated homeoproteins
with no DNA binding domains can have important regulatory functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices 1 and 2 within the homeodomain. The genes encoding
these proteins are highly conserved and are present in the common
ancestor of plants, fungi, and animals. Members of this family of
homeoproteins include Meis, Pbx, and TGIF (1). Cooperative function
among TALE family members is critical for transcription regulation
(2-4), and several members have been shown to function as essential
contributors to Hox-mediated developmental programs (5-7).
with
this element occur on overlapping areas and generate a mutually
exclusive binding. In addition, TGIF inhibits 9-cis-retinoic acid-dependent RXR-induced transactivation of this
promoter (13).
1173 and 1154
(ACT) may function for its neural cell-specific expression (19). In
addition, the ACT sequence is highly conserved between human, pig, and
rat D1A genes (20). We now sought to identify
cell- or tissue-specific transcription factors that bind to this
sequence using the yeast one-hybrid screen. We found that human Meis2
and TGIF differentially regulate transcription of the
D1A gene by binding to complementary DNA
sequences within its ACT region.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1173 to 1154 sequence
(ACT) from the human D1A promoter (20) were
ligated into pHISi and pLacZ to generate pHISi-ACT and pLacZ-ACT, respectively, for screening a human brain cDNA library
(CLONTECH). The plasmid from the single positive
blue clone was sequenced, and its homology was analyzed using BLAST.
-D-galactopyranoside and purified using
glutathione-conjugated Sepharose beads (Amersham Pharmacia Biotech).
-32P]ATP and annealed with the cold
complementary strand. Double-stranded, end-labeled DNA probe (20 kcpm/binding reaction; 5 fmol) was incubated with 200 ng of GST-Meis2
fusion proteins in a final volume of 20 µl at room temperature for 30 min. In some experiments, a polyclonal antibody to GST was co-incubated
with GST-Meis2 fusion proteins for 30 min at room temperature prior to
adding the probe. The reaction mixtures were electrophoresed in 4%
polyacrylamide nondenaturing gel in 1× Tris/glycine buffer as
described previously (19).
) to generate pGEM-Meis2a. Antisense probe
was transcribed from the SP6 promoter after linearizing with
EcoRI, and the sense probe was transcribed from the T7
promoter after linearizing with HindIII. Digoxigenin-labeled
RNA probes were transcribed according to the protocol of the kit's
supplier (Roche Molecular Biochemicals) and hydrolyzed to approximately 200-bp fragments by alkaline hydrolysis. [35S]UTP-labeled
D1A probes were transcribed from
pGEM-mD1A that had been linearized with EcoRI
for generation of sense probe from the SP6 promoter or with
HindIII for generation of antisense probe from the T7
promoter using the SP6/T7 transcription kit (Roche Molecular
Biochemicals). Fourteen-µm-thick sections of the mouse striatum were
subjected to simultaneous hybridization with two probes as described
(21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
ACT sequence of the human
D1A promoter. An
oligonucleotide with three tandem copies of the wild-type sequence was
used as bait in the yeast one-hybrid screening. Consensus sequences for
Meis2 and TGIF binding sites are shown in bold, and their
respective orientations are represented by arrows.
Bold nucleotides in mutant ACT replace the Meis2/TGIF
binding sites. Wild-type and mutant sequences were used in gel shift
assays. Nucleotide numbering is relative to the first ATG codon
(41).
View larger version (65K):
[in a new window]
Fig. 2.
Meis2 splice variants and alignment of
homeodomains from Meis2 and TGIF. A, human
Meis2a sequence. Arrowheads indicate known exon
junctions. Since the Meis2 gene is only partially mapped,
the exon numbers shown do not represent their true number in the
genomic organization. The deduced amino acid sequence is shown as
single-letter codes, and the homeodomain is indicated by
parentheses. The first asterisk is the
termination point of Meis2a and Meis2b, and the second
asterisk is the termination point of Meis2c and Meis2d.
Primers P1, P2, and P3 used in PCR in panel C and in Fig. 7
(B and C) are indicated as arrowed
lines above the sequence. B, amino acid sequences
of Meis2 and TGIF homeodomains. Meis2e has an early stop codon
(asterisk) before the 2-helix of the
homeodomain. Bold amino acid residues in TGIF and Meis2a-d indicate
the TALE. C, PCR with primers P1 and P2 using a panel of
cDNAs from mouse multiple adult tissues and embryonic stages. The
355-bp band was confirmed to represent Meis2e by sequencing.
D, in vitro translated Meis2 proteins analyzed by
SDS-PAGE. Meis2a-e are seen as single major bands at 62, 61, 74, 73, and 53 kDa, respectively.
View larger version (84K):
[in a new window]
Fig. 3.
Meis2 proteins bind to the
D1A gene ACT
sequence. GST-Meis2a-e fusion proteins were used in gel shift
assays with ACT probe containing the Meis2 consensus site.
GST-Meis2a-d fusion proteins interacted with the ACT probe
(lanes 2-5), whereas GST-Meis2e or GST did not
(lanes 6 and 1, respectively).
Anti-GST polyclonal antibody supershifted GST-Meis2a-d fusion proteins
and ACT complexes (lanes 7-10). Cold competitor
incubated with GST-Meis2a-d fusion proteins before adding ACT probe
inhibited all DNA-protein interactions (lanes
11-14). Mutant ACT probe used instead of wild-type ACT
failed to bind with GST-Meis2a-d fusion proteins (lanes
15-18). Ab, antibody.
View larger version (65K):
[in a new window]
Fig. 4.
Expression of Meis2,
TGIF, and
D1A mRNAs in various
cell lines by Northern blot analysis. Total RNA was prepared from
each cell line, and 30 µg was loaded on a formaldehyde gel.
32P-Labeled fragments of Meis2a,
TGIF, and D1A cDNAs were used
sequentially as probes. A -actin probe was also used as control to
determine the amount of RNA loaded in each lane.
View larger version (26K):
[in a new window]
Fig. 5.
Transcriptional activity of Meis2
proteins. A, Meis2a-d activate pCATD1-1197 in HepG2
and OK cells. Five µg of Meis2a-e expression plasmids and 5 µg of
pCATD1-1197 were used to co-transfect HepG2 and OK cells using the
calcium phosphate precipitation method, and CAT activity was measured
by ELISA. Meis2a-d significantly increased CAT expression in both cell
lines, whereas Meis2e did not. Data shown are means ± S.E. for
triplicate samples. B, Meis2d-induced transcriptional
activation of the D1A promoter is dependent on
the ACT sequence. 5' Deletion mutants of the D1A
gene 5'-flanking region shown on the left were
co-transfected with pCMV-Meis2d into OK cells. *, p < 0.05. Data shown are means ± S.E. for triplicate samples.
C, Meis2e inhibits Meis2d-induced transactivation of
pCATD1-1197 in a concentration-dependent manner. Rising
amounts of pCMV-Meis2e were used to co-transfect OK cells with fixed
amounts of pCMV-Meis2d and pCATD1-1197. ANOVA, p = 0.03. Data shown are means ± S.E. for triplicate samples.
D, Meis2d activates the endogenous
D1A gene in OK cells. Total RNA was prepared
from OK cells that were transfected with pCMV-Meis2d expression vector,
control vector alone (pcDNA3.1), or with no DNA
(Mock). A 32P-labeled mouse
D1A fragment was used to detect the
D1A message, which is seen only in
Meis2d-transfected cells. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probe was used to demonstrate uniformity of the RNA
amount loaded in each lane.
View larger version (148K):
[in a new window]
Fig. 6.
Colocalization of Meis2 and
D1A expression in the
mouse striatum. A, antisense digoxigenin-labeled
Meis2 and radiolabeled D1A riboprobes
were hybridized simultaneously. Pink indicates
Meis2-positive neurons, and black grains
represent D1A-positive neurons. B,
corresponding sense probes labeled similarly to A indicate
specificity of both signals. Scale bar = 50 µm.
View larger version (25K):
[in a new window]
Fig. 7.
TGIF competes with Meis2 binding and
function. A, Meis2a-d activate pCATD1-1197 in SH-SY5Y
but not in SK-N-MC cells. Five µg of Meis2a-e expression plasmid and
5 µg of pCATD1-1197 were used to co-transfect these two cell lines
using the calcium phosphate precipitation method and CAT activity was
measured by ELISA. Meis2a-d robustly increased the amount of CAT
expression in SH-SY5Y but not in SK-N-MC cells. Data shown are
means ± S.E. for triplicate samples. B, RT-PCR with
primers P1 and P2 using RNA from SH-SY5Y and SK-N-MC cells. Gel
electrophoresis revealed the doublet nature of the amplified band,
representing 454 bp for Meis2a and Meis2c and 433 bp for Meis2b and
Meis2d. A band corresponding to Meis2e could not be amplified.
C, RT-PCR with the same two cell lines using primers P1 and
P3. Four bands representing Meis2a-d were amplified but not Meis2e.
D, TGIF binds to the ACT sequence of the
D1A promoter. In vitro translated
TGIF was used in gel shift assays with ACT probe containing the
Meis2/TGIF consensus sites. TGIF interacted with the wild-type ACT
probe (lane 2) but not with mutant ACT
(lane 4). Cold competitor (100×) totally
inhibited TGIF binding to ACT probe (lane 3).
Control lysate could not bind to the wild-type ACT sequence
(lane 1). E, binding competition
between TGIF and Meis2a. Rising amounts of GST-Meis2a fusion protein
inhibited TGIF binding to the ACT sequence (lanes
1, 3, and 4), and the presence of TGIF
inhibited Meis2a binding (lanes 2 and
4) despite higher amount of Meis2a in lane
4. F, TGIF suppresses Meis2a-induced pCATD1-1197
activation. SH-SY5Y cells were co-transfected with pcDNA-TGIF and
Meis2a expression plasmids and with pCATD1-1197 at the indicated
concentrations. CAT expression was measured 48 h after
transfection. TGIF inhibited Meis2a-induced pCATD1-1197 expression in
a concentration-dependent manner. ANOVA, p = 0.0008. Data shown are means ± S.E. for triplicate samples.
G, TGIF-HD suppresses Meis2a-induced pCATD1-1197
activation. SH-SY5Y cells were co-transfected with pTGIF-HD or
pcDNA-TGIF, pCMV-Meis2a and pCATD1-1197. After a transfection
period of 18 h, cells were incubated in fresh medium for an
additional 48 h and CAT expression was measured by ELISA. Both
TGIF-HD and full-length TGIF significantly inhibited Meis2a-induced
pCATD1-1197 expression. Data shown are means ± S.E. for
triplicate samples. ANOVA, p = 0.0001.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
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) AF178948, AF179896, AF179897, AF179898, and AF179899 (all for human Meis2a-e) and AF179900 (for human TGIF).
Present address: Korea Research Inst. of Bioscience and
Biotechnology, Taejon, 305-333 South Korea.
§ Present address: Dept. of Bioscience and Biotechnology, Silla University, Pusan 617-736, South Korea.
¶ To whom correspondence should be addressed: NINDS, National Institutes of Health, 10 Center Dr., MSC 1406, Bethesda, MD 20892-1406. Tel.: 301-496-7872; Fax: 301-496-6609; E-mail: mouradianm@ninds.nih.gov.
Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M908382199
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
TALE, three-amino
acid extension loop;
RXR, retinoid X receptor;
PCR, polymerase chain
reaction;
RT, reverse transcription;
PAGE, polyacrylamide gel
electrophoresis;
CAT, chloramphenicol acetyltransferase;
ELISA, enzyme-linked immunosorbent assay;
GST, glutathione
S-transferase;
bp, base pair(s);
ANOVA, analysis of
variance;
ACT, activator sequence 1173 to 1154 in the human
D1A gene.
| |
REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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