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INTRODUCTION |
Sp1 is the founding member of a family of five transcription
factors, Sp1-5, that govern the expression of a wide variety of
mammalian genes (for review, see Ref. 1). Sp1 encodes a ubiquitously
expressed nuclear phosphoprotein that has been divided into five
sub-domains based upon their respective functions (2, 3). The Sp1
trans-activation domain is composed of three sub-domains termed A-C, each of which is capable of stimulating transcription if
tethered to DNA via a DNA-binding domain. Sub-domains A and B are
composed by serine- and threonine-rich regions as well as glutamine-rich regions. The glutamine-rich portions of A and B are
believed to be required for trans-activation, whereas the function(s) of the serine/threonine-rich sub-regions is(are) less well
understood. Domain C carries a number of charged amino acids and weakly
stimulates transcription in the absence of domains A or B. Carboxyl-terminal to the domain C is a region featuring three
Cys2-His2 zinc "fingers" required for
sequence-specific DNA binding to GC-rich promoter elements. A
carboxyl-terminal domain, termed D, facilitates protein multimerization
and is essential for synergistic trans-activation of
promoters with multiple Sp-binding sites. Sp1 associates with a large
number of transcription-associated proteins, including components of
the basal transcription complex (e.g.
hTAFII130/dTAFII110 and hTAFII55;
Refs. 4-7), sequence-specific DNA-binding proteins (e.g.
E2F, YY1, p53, and AP-2; Refs. 8-13), and transcriptional regulators
(e.g. p107, HDAC-1, and VHL-1; Refs. 14-16). As might be
expected given the variety of proteins with which it interacts,
protein-binding sites have been identified throughout Sp1. For example,
hTAFII130/dTAFII110 interact with Sp1 via its
trans-activation domain; hTAFII55 binds the Sp1
zinc fingers, and E2F requires the zinc finger and D sub-domains
of Sp1 for protein-protein interactions.
A wide variety of extracellular stimuli have been shown to induce gene
expression via discrete promoter elements bound by Sp1 and Sp3
(17-29). Moreover, subtle mutations that negate the association of
Sp1/Sp3 with their cognate binding sites completely block the induction
of such genes by their respective inducing agents. Although GC-rich
elements within the promoters of many Sp1/Sp3-regulated genes have been
identified and their necessity for induced transcription has been
noted, it remains largely unclear how extracellular stimuli activate
Sp1/Sp3-dependent genes. For example, in most instances
treatment of cells with inducing agents does not lead to consistent
alterations in (i) the abundance or subcellular localization of
Sp1/Sp3, (ii) the affinity of Sp1/Sp3 for DNA, (iii) the formation of
Sp1/Sp3 multimers, nor (iv) the post-translational modification
Sp1/Sp3. Instead, extracellular stimuli may activate
Sp-dependent genes via alterations in protein-protein interactions. For example, transforming growth factor-
induces p15Ink4B transcription by catalyzing the formation of protein
complexes between Sp1 and members of the Smad family of transcription
factors (30). Whether regulated interactions between Sp family members and other factors account for the induced transcription of additional Sp-dependent genes remains to be determined.
Several years ago we identified two novel Sp3-derived proteins, termed
M1 and M2, that arise by internal translational initiation within the
region of Sp3 mRNA that encodes the Sp3 B domain (31). Sp3, M1, and
M2 appear to be expressed in all mammalian cells and tissues at
approximately equivalent levels independent of growth status or
induction by extracellular stimuli. In contrast to full-length Sp3, M1
and M2 function as potent repressors of Sp-mediated transcription, and
Sp3 is at least 10-fold more sensitive to M1/M2-mediated repression
than is Sp1. Given that Sp3 encodes proteins with opposing activities,
we reasoned that understanding their differential regulation may shed
light on mechanisms governing the activity of Sp-dependent
promoters. To understand further the mechanism(s) by which M1/M2
repress transcription, we prepared a panel of M2 proteins carrying a
limited number of random amino acid substitutions and examined their
capacity to function as transcriptional regulators of three
Sp-regulated promoters: DHFR, p21, and
MDR-1. Additionally, we examined each of these mutated proteins for their capacity to bind DNA, to form multimeric complexes with Sp family members, and to bind components of the basal
transcription complex. These studies have resulted in the following
observations. 1) Random mutagenesis generated a panel of mutated M2
proteins that carry "loss-of-function" and "gain-of-function"
mutations. 2) Many amino acid substitutions affect M2-mediated
repression in a promoter-specific fashion. 3) DNA binding activity and
the capacity to multimerize are not required or sufficient for
M2-mediated repression. 4) The minimal region required for
transcriptional repression by M2 consists of 93 amino acids of the B
domain. 5) Several components of the TAFII complex bind
Sp1, Sp3, and M2 in vitro. 6) The binding of two
TAFII1 proteins,
TAFII70 and TAFII40, is compromised in several
M2 mutants. We conclude from these observations that M1/M2-mediated
repression occurs at least in part via the titration of one or more
transcription factors that may be required in a promoter-specific fashion.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
C-33A cells were obtained from the American
Type Culture Collection (ATCC; Manassas, VA), and Drosophila
Schneider line-2 (SL2) cells were a gift of Dr. Cheaptip Benyajati
(University of Rochester, Rochester, NY). C-33A and SL2 cells were
cultured as described previously (31-33).
Antisera--
Rabbit anti-Sp3 was prepared against a GST fusion
protein containing the amino-terminal 300 amino acids of Sp3, and its
preparation and characterization have been described (31).
Affinity-purified mouse anti-HA.11 antibody was obtained from a
commercial supplier (Covance Research Products, Richmond, CA).
Expression and Reporter Plasmids--
pPacSp1 was obtained from
Dr. Robert Tjian (University of California, Berkeley; see Ref. 3).
pPacSp3, pPacM1, pPacM2, pBSK-Sp3/flu, pCR-M1/flu, and pCR-M2/flu were
prepared as described (31, 33). Constructions employed for in
vitro translation of human and Drosophila TAFII proteins were obtained from Dr. Robert Tjian.
DHFR-CAT has been described (34). To generate a
DHFR-luciferase construct, DHFR-CAT, 5'
(5'-GGAGATCTAGCGCGCGGCTGTACTAC-3') and 3' primers (5'-GGAAGCTTGCAGCCTGTACGCTGTGC-3'), and the PCR were employed to
amplify DHFR promoter sequences. A resulting 175-bp promoter fragment was subcloned in plasmid pRL (Promega, Inc., Madison, WI).
pgpLuc-B carries a 1320-bp portion of the human MDR-1
promoter and was obtained from Dr. Kathleen Scotto (Memorial
Sloan-Kettering Cancer Center, NY; see Ref. 35). p21P93-S carries a
44-bp portion of the human p21 promoter and was obtained
from Dr. Xiao-Fan Wang (Duke University Medical Center; see Ref.
19).
PCR-mediated Mutagenesis and DNA Sequencing--
Mutagenesis of
M2 was performed using methods described by Zaccolo et
al. (44). Reactions employed pGEX-M2 (see below) as template,
5' (5'-CCTGACTTCATGTTGTATGAC-3') and 3' primers
(5'-CAGTCACGATGAATTCTCGAGAATCCCTAGCTAGCGTAATCTG-3'), and
Taq polymerase (Invitrogen). Three PCR cycles (92 °C for
1 min, 55 °C for 1 min, and 72 °C for 2 min) were performed in a 20-µl reaction containing 4 ng of linearized template, 0.5 µM primers, 500 µM each dNTP, and 500 µM dPTP (Amersham Biosciences). An aliquot of this
reaction was subsequently employed as template for an additional 22 rounds of amplification in the absence of dPTP. PCR products were
digested with DpnI, purified, and cloned in
pCR-BluntII-TOPO. Expression plasmids carrying mutated M2
cDNAs were prepared by subcloning inserts from
pCR-BluntII-TOPO into pPac (3). Sequencing of M2 mutants
was performed by the North Carolina State University sequencing
facility using a PerkinElmer Life Sciences AB1377 sequenator or
Sequenase version 2.0 DNA polymerase following a protocol supplied by
the manufacturer (Amersham Biosciences).
Bacterial and Baculovirus Expression Constructs--
pGEX-Sp1, a
bacterial expression construct that carries the Sp1 coding region fused
in-frame with glutathione S-transferase (GST), has been
described (36). pGEX-M2 was prepared by subcloning the M2 cDNA
carried by pCR-M2/flu in pGEX-2TK (Amersham Biosciences). GST-Sp3 was prepared using pBSK-Sp3/flu, 5'
(5'-GGGGGATCCGCCACCATGAATTCCGGGCCATCGCCG-3'), and 3' primers
(5'-GGAATTCCTCCATTGTCTCATTTCCAG-3'), and the PCR. A 2142-bp amplified
fragment carrying the entire Sp3 cDNA was subcloned in pGEX-2TK.
pGEX-FSH15 has been described previously (36). To create GST expression
plasmids carrying M2 cDNAs terminating at amino acids 103, 197, or
353, M2 cDNAs were amplified from pPacM2 using a 5' primer
(5'-GGGGGATCCATGGATAGTTCAGACAATTCA-3'), one of three 3' primers
(5'-CTACTAGACTCCTTGAAGTTG-3', 5'-CTAACCAAGTGTGAGGGTTTC-3', or
5'-TCAGTTAACAAACAAAAGGGCG-3'), and Taq polymerase. Amplified M2 cDNAs carrying premature termination codons were subcloned in
pGEX-2TK. Mutated M2 GST fusion proteins were prepared by amplification from pPacM2 plasmids using 5' (5'-GGGGGATCCATGGATAGTTCAGACAATTCA-3') and 3' primers (5'-GGAATTCCTCCATTGTCTCATTTCCAG-3') and
Taq polymerase. Amplified cDNAs were subcloned in
pGEX-2TK. Baculovirus stocks encoding Sp1, Sp3, M1, and M2 were
prepared using the PCR, appropriate primers, and pCMV4-Sp1/flu (37),
pBSK-Sp3/flu, pCR-M1/flu, and pCR-M2/flu as substrates. Amplified
cDNAs were subcloned in pFASTBacHTA and used to prepare virus
stocks according to methods supplied by the manufacturer (Invitrogen).
Transient Transfections--
Transient transfections for
transcription assays were performed by calcium phosphate precipitation
as described (31, 33). Cell extracts were prepared for analysis 48 h after transfection. The Dual-Luciferase Reporter Assay System
(Promega, Inc.) was employed to quantify luciferase activity precisely
as recommended by the manufacturer. Luminescence was detected in a
Lumat LB 9507 luminometer (EG & G Berthold, Bad Wildbad, Germany), and
results were normalized against total cell protein concentration. To
prepare Drosophila SL2 extracts for Western blotting or
protein/DNA binding assays, transient transfections were performed
using SuperFect Transfection Reagent (Qiagen Inc., Hilden, Germany).
Cell extracts were prepared 48 h after transfection.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared using methods described by Lee et al. (38).
Oligonucleotide Probes--
Oligonucleotides were synthesized on
an automated DNA synthesizer, deprotected, and partially purified
through Sephadex G-25 spin columns. Radiolabeled probes for standard
protein/DNA binding assays were prepared from the following
oligonucleotides and their complements: GT box (39),
5'-AGCTTCCGTTGGGGTGTGGCTTCACGTCGA-3'; p21 (19),
5'-GAGCCGGGGTCCCGCCTCCTTGAGGCGGGCCC-3'; and MDR-1 (35),
5'-CAGGAACAGCGCCGGGGCGTGGGCTAGC-3'.
For quantitative protein/DNA binding assays, six double-stranded
60-mers were synthesized each carrying a single promoter-derived Sp-binding site flanked by common nucleotide sequences. The
promoter-derived sequences utilized for these experiments are as
follows: p21, 5'-CCCGCCTCCT-3'; MDR-1, 5'-CGCCGGGGCGTGGGC-3'; DHFR-1,
5'-AGGGCGTGGC-3'; DHFR-2, 5'-GAGGCGGGGC-3'; DHFR-3, 5'-GAGGCGGAGT-3';
and DHFR-4, 5'-TGGGCGGGGC-3'. Annealed and complementary
oligonucleotides were radiolabeled and purified as described previously
(31, 32).
Protein/DNA Binding Assays--
Protein/DNA
binding assays were performed as described previously (31, 32), and
complexes were visualized by autoradiography. For quantitative
protein/DNA binding assays, whole cell protein extracts prepared from
baculovirus-infected Sf9 cells were incubated with a
radiolabeled probe derived from the c-fos gene
(5'-CCCTTGCGCCACCCCTCT-3'; see Ref. 32), and the resulting
protein-DNA complexes were quantified in situ using an
InstantImager (Packard Instrument Co.). Volumes of these extracts that
led to half-maximal binding of this probe were then employed in assays
performed in triplicate with Sp-binding sites derived from the
DHFR, p21, and MDR-1 genes and
quantified in situ.
Western Blotting--
Whole cell or nuclear extracts were
resolved on denaturing polyacrylamide gels and transferred to
nitrocellulose using a semi-dry transfer apparatus. Nitrocellulose
filters were incubated with 5% milk in TBS-T (2.42 g/liter Tris, 8 g/liter NaCl, pH 7.6, 1 ml/liter Tween 20) from 1 h to overnight.
Primary antibodies were diluted in TBS-T (anti-Sp3 at 1:2000 and
anti-HA.11 at 1:1000), incubated with filters for 1 h at room
temperature, and washed with TBS-T. Filters were incubated with
horseradish peroxidase-conjugated secondary antibodies diluted in TBS-T
(anti-mouse, 1:10,000, Amersham Biosciences, or anti-rabbit, 1:40,000,
Invitrogen) for 1 h at room temperature with gentle agitation and
washed in TBS-T, and antigen-antibody complexes were detected using ECL
Western blotting Detection Reagents (Amersham Biosciences).
In Vitro Transcription/Translation--
In
vitro transcribed/translated proteins were produced using a
coupled reticulocyte lysate system (TNT; Promega, Inc.) with T3 or T7
RNA polymerase and L-[35S]methionine
(Tran35S-label; ICN). pBSK-Sp1/flu, pBSK-Sp3/flu,
pCR-M2/flu, PCR-amplified mutated M2 cDNAs, and TAFII
constructs were employed as templates for these reactions. Mutated M2
cDNAs in pPac were amplified and prepared for in vitro
transcription/translation using a 5' T7 promoter-containing primer, a
3' primer, and the PCR.
In Vitro Protein/Protein Binding
Assays--
Protein binding assays were performed as described (40).
GST bead-bound proteins were resolved on denaturing polyacrylamide gels
and visualized by autoradiography.
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RESULTS |
The DHFR, p21, and MDR-1 Promoters Possess Distinct Sensitivities
to Sp-mediated Trans-activation and Repression--
We have shown
previously that Sp1 and Sp3 stimulate transcription of the
DHFR promoter and that Sp1/Sp3-mediated transcription is
repressed by two isoforms of Sp3, termed M1 and M2, that arise via
internal translational initiation (31, 33, 37). To determine whether
these results were likely to reflect a general mechanism of
transcriptional regulation, we extended our analyses to the p21 and MDR-1 promoters, two well characterized
Sp-dependent promoters of physiologic and therapeutic
interest. The p21-luciferase construct employed in these
studies includes an Sp protein-binding site that is required for
transcriptional stimulation by transforming growth factor-, calcium,
or sodium butyrate, as well as a second Sp protein-binding site that is
also required for induction by sodium butyrate (19, 23, 41). The
MDR-1-luciferase construct employed includes an Sp
protein-binding site that is required for induction by serum or the
c-Raf kinase (42, 43).
As illustrated in Fig. 1A, the
ectopic expression of Sp3 in Drosophila SL2 cells stimulated
transcription of the DHFR, p21, and
MDR-1 promoters to varying degrees. p21
transcription was stimulated 40-fold more effectively than
MDR-1 and 12-fold more than the DHFR promoter.
Analogous results were obtained for activation of each of these
promoters by Sp1 (data not shown). Consistent with results reported
previously for the DHFR promoter, co-expression of M1 or M2
with Sp1 or Sp3 resulted in repression of p21 and MDR-1 transcription (Fig. 1B and data not shown).
Interestingly, the sensitivity of each promoter to M1/M2-mediated
repression was noted to be promoter-specific. The promoter most acutely
sensitive to activation by Sp1/Sp3, p21, exhibited the least
sensitivity to M1/M2-mediated repression. In turn, a promoter that is
only modestly activated by Sp1/Sp3, MDR-1, exhibited the
greatest sensitivity to M1/M2-mediated repression. Sensitivity of
DHFR to Sp-mediated activation and repression was noted to
fall between these two extremes. Given the results presented in Fig. 1,
we conclude that intrinsic differences between Sp-dependent
promoters influence the degrees to which they are activated by Sp1/Sp3
and repressed by M1/M2.
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Fig. 1.
Relative fold activation and repression of
the p21, DHFR, and MDR-1
promoters by Sp3 isoforms. A, relative fold
activation of the p21, DHFR, and MDR-1
promoters by Sp3. Drosophila SL2 cells were transiently
transfected with pPac-Sp3/flu and reporter constructs, and luciferase
activity was quantified 48 h later. Levels of
trans-activation obtained from 5 to 21 independent plates of
transfected cells were averaged, and fold activation of the
p21 and DHFR promoters was determined relative to
that of MDR-1 (set equal to 1.0). B, relative
fold repression of the p21, DHFR, and
MDR-1 promoters by M2. Drosophila SL2 cells were
transiently transfected with constant amounts of pPac-Sp3/flu, a 2- (DHFR and MDR-1) or 4-fold (p21) molar
excess of pPac-M2/flu, and luciferase reporter constructs. Luciferase
activity was quantified 48 h later from 5 to 21 independent plates
of transfected cells, and mean levels of repression were calculated in
comparison with plates transfected without M2, and fold repression of
the DHFR and MDR-1 promoters was determined
relative to that of p21 (set equal to 1.0).
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Trans-activation and Repression of DHFR, p21, and MDR-1
Transcription in Vivo Is Not Directly Correlated with the Capacity of
Sp Proteins to Bind to DNA in Vitro--
Because Sp proteins activated
and repressed the DHFR, p21, and MDR-1
to varying degrees, we wished to determine whether these effects might
be accounted for by inherent differences in their capacity to bind DNA.
For example, the relative sensitivity of p21 to Sp3-mediated
transcription and insensitivity to M2-mediated repression might be
explained if Sp3 and M2 bind the p21 promoter with
substantially different affinities. To address this issue, we performed
a series of quantitative protein/DNA binding assays using radiolabeled
DNA probes carrying Sp-binding sites from the DHFR,
p21, and MDR-1 promoters. Recombinant baculovirus
stocks encoding Sp1, Sp3, M1, or M2 proteins were prepared; Sf9
cells were infected with these viruses, and protein extracts from
infected cells were normalized for DNA binding activity using a well
characterized Sp protein-binding site derived from the mouse
c-fos promoter. The volume of each protein extract that
bound 50% of the c-fos probe was then employed in
protein/DNA binding assays with six Sp protein-binding sites derived
from the DHFR, p21, and MDR-1 promoters. Each assay was performed in triplicate and quantitated in situ. As shown in Table I,
Sp proteins bound to each probe, and the capacity of a given Sp protein
to bind these DNAs varied over a 3-fold range. Interestingly, little
correlation was noted between the relative capacity of Sp proteins to
bind these DNAs in vitro and the efficiencies with which
they activate or repress transcription in vivo. For example,
Sp1 and Sp3 bound the Sp-binding site derived from the MDR-1
promoter more efficiently than a site derived from the p21
promoter, yet MDR-1 is activated significantly less
efficiently by both proteins in vivo. Similarly, Sp
protein-binding sites derived from all three promoters were bound
equivalently by M2, yet their sensitivity to M2-mediated repression
varies over a 13-fold range in vivo. We conclude from these
studies that the relative capacity of Sp proteins to stimulate or
repress transcription is not directly correlated with the efficiency
with which they interact with their cognate promoter binding sites
in vitro.
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Table I
Relative binding of recombinant Sp proteins to oligonucleotides
carrying Sp-binding sites derived from three Sp-dependent
promoters
Protein extracts were prepared from Sf9 cells infected with
baculovirus stocks encoding Sp family members, and the volume of each
extract required to bind 50% of a radiolabeled oligonucleotide probe
derived from the c-fos promoter was determined. This volume
of cell extract was later employed in protein/DNA binding assays with
oligonucleotides derived from the DHFR, p21, and
MDR-1 promoters. Binding assays were performed in triplicate
and quantified in situ. DNA binding activities for each
protein were normalized to the amount of binding activity detected for
each protein on the p21 oligonucleotide.
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Mutated M2 Proteins Exhibit Promoter-specific Alterations in Their
Capacities to Repress Transcription--
Based on the data presented
in Table I, the relative sensitivity of the DHFR,
p21, and MDR-1 promoters to M1/M2-mediated repression does not appear to be determined by the intrinsic capacity of M1/M2 to bind DNA. To confirm these data and as a first step toward
defining the mechanism(s) governing M1/M2-mediated repression, we
reasoned that amino acids required for transcriptional repression might
be identified via a functional analysis of proteins carrying random
amino acid substitutions or deletions. We predicted that the analysis
of such mutants should reveal whether previously characterized
functions of Sp proteins, such as DNA binding activity and/or
multimerization, are required for transcriptional repression. Consequently, a limited number of amino acid substitutions were introduced into M2 using a PCR-mediated technique for random
mutagenesis in which dPTP is substituted for dCTP (44). Expression
plasmids carrying mutated M2 cDNAs were transiently expressed in
Drosophila SL2 cells, and cell extracts were analyzed for
the expression of M2 protein by Western blotting with anti-Sp3
antiserum. Western blots of whole cell extracts resulted in the
identification of 102 independent M2 mutants that give rise to stable
proteins varying in size from that of wild-type M2 (78 kDa) to ~30
kDa (Table II and data not shown).
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Table II
Biochemical and functional characteristics of mutated M2 proteins
The functional properties of M2 mutants were established in
transcription, protein/DNA, and protein/protein binding assays.
Introduced mutations were determined by DNA sequencing.
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To characterize the capacity of mutated M2 proteins to repress
Sp-mediated transcription, a series of co-transfection experiments was
performed with Sp3 and a DHFR-luciferase reporter construct. As might be expected, of the 102 mutated M2 proteins analyzed for their
capacity to repress DHFR transcription, the vast majority (74 mutants, 73%) functioned akin to wild-type M2. Of the remaining M2
mutants, repression by nearly half (12 mutants, 43%) was found to be
severely debilitated relative to wild-type M2; such mutants have less
than one-tenth the activity of wild-type M2. The remaining mutants (16 mutants; 57%) exhibited varying capacities to block DHFR
transcription, ranging from one-third to one-tenth the activity of
wild-type M2. To extend this analysis, a panel of 35 mutants was
assembled and analyzed for their capacity to repress p21- and MDR-1 transcription. This panel included all mutants
that lacked the capacity or showed diminished capacity to repress
DHFR transcription as well as seven randomly selected
mutants that repressed DHFR akin to wild-type M2.
Interestingly, although the majority of the 35 mutants examined are
partially or completely incapable of repressing DHFR, their
activities on these additional promoters were quite variable (Table
II). Three examples illustrate this point as follows. (i) Mutant 16 has
a greatly diminished capacity to repress DHFR transcription
but represses p21 and MDR-1 to levels akin to
wild-type M2. (ii) Mutant 42 is as defective as is mutant 16 when
examined as a repressor of DHFR transcription, yet it
represses p21 more efficiently than wild-type M2 and has a
diminished capacity to repress MDR-1. (iii) Finally, mutant 158 exhibits wild-type activity on the DHFR and
p21 promoters yet is 7-fold more active than wild-type M2 on
the MDR-1 promoter. These results suggest that this panel of
mutated M2 proteins has sustained loss-of-function mutations,
i.e. alterations that diminish potency as repressors
relative to wild-type M2, as well as gain-of-function mutations,
i.e. alterations that render particular mutants more potent
than wild-type M2 as transcriptional repressors. It is worth noting,
however, that none of the mutated M2 proteins we examined acquired the
capacity to stimulate transcription of the DHFR,
p21, or MDR-1 promoters. To extend these
findings, two approaches were undertaken. First, 27 M2 mutants were
sequenced in their entirety and compared with the sequence of wild-type
M2. Second, a series of biochemical experiments was performed to
determine whether additional functional properties of Sp family
members, such as DNA and protein binding activity, were affected by the introduced mutations.
Sequence Analysis of M2 Mutants Indicates That a 93-Amino Acid
Region within the M2 B Domain Is Required for Transcriptional
Repression--
To identify domains and amino acids required for
M2-mediated transcriptional repression, 27 of the 35 mutants listed in
Table II were subjected to automated DNA sequencing. As anticipated, the majority (98%) of the mutations generated by incorporation of dPTP
are transitions: almost 40% are A 
G mutations, G A and
T C mutations are 23 and 21%, respectively, and 15% of the mutations are C T. The remainder of the mutations are
transversions; two G C mutations were identified, and one mutation
each of C A, A C, and C G was noted. Based on previously
published results we had anticipated that a limited incorporation of
dPTP would introduce an average of 12 nucleotide changes per M2
cDNA, yielding on average seven amino acid changes per protein.
Sequencing of resulting clones revealed that each M2 cDNA carries
an average of 15 nucleotide substitutions per molecule, resulting in an
average of 10 amino acid changes per M2 protein. Importantly, mutations were found to be distributed throughout the M2 cDNA (data not shown).
Because the transcriptional repression properties of many of the clones
selected for sequencing are altered relative to wild-type M2, we
predicted that amino acids of M2 required for repression might be
mutated more frequently on average than those required for other
functions. Indeed, a number of M2 amino acid positions were mutated at
an above-average frequency; 23 amino acid positions were each mutated
in two independent clones, and 8 amino acid positions were each mutated
in three independent clones (Fig. 2).
Consistent with the notion that these frequently mutated amino acids
may play a role in transcriptional repression, 16 of 19 mutants that
carry mutations at one or more of these mutational "hotspots" are
essentially inactive as repressors on one or more of the promoters
examined. As indicated in Fig. 2, the vast majority of these mutational
"hotspots" map outside of domains that harbor carboxyl-terminal
functions such as DNA binding and protein multimerization. Instead,
two-thirds of the amino acids mutated in three independent clones and
more than half of the amino acids mutated in two independent clones are
located within the BS/T and BQ sub-domains,
regions that account for only 19% of the amino acids that compose M2.
It is worth mentioning that the frequency of mutants carrying mutations
within amino-terminal hotspots is identical to full-length and nonsense
codon-containing M2 mutants. Because the majority of the mutational
hotspots identified in M2 are located within the B domain, these data
suggest that one or more functions encoded by this region are required
for transcriptional repression. This supposition would appear to be
supported by biochemical analyses of M2 mutants that carry
"nonsense" mutations (see below).
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Fig. 2.
Distribution of mutational hot spots in 27 M2
mutants. Amino acids mutated in two or three independent M2
mutants are illustrated above a schematic representation of the domain
structure of M2. Amino acid positions demarcating each domain are
indicated relative to the initiation of M2 translation.
BS/T, serine- and threonine-rich sub-domain;
BQ, glutamine-rich sub-domain; C, C
domain; Zn, zinc finger domain; D, D
domain.
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Twelve of 27 sequenced mutants were found to carry nonsense mutations
within the M2 B or C domains or within the amino terminus of the zinc
"finger" region. Yet approximately half of the truncated proteins
elaborated by these mutants were quite active as transcriptional repressors when analyzed on the p21 or MDR-1
promoters (Table II). For example, translation of mutants 42 and 57 terminates within the amino-terminal portion of the M2 BQ
domain (amino acids 82 and 94, respectively), yet each are capable of
repressing Sp-mediated transcription of the p21 or
MDR-1 promoters. Based on these results, we conclude that
the minimal region of M2 required for transcriptional repression of the
promoters examined includes amino acids 1-93 (corresponding to amino
acids 235-328 of Sp3). This minimal region for transcriptional
repression spans the serine- and threonine-rich sub-domain and 25 amino
acids of the glutamine-rich sub-domain of the M2 B region.
Although the data described thus far indicate that residues within the
B portion of the M2 trans-activation domain appear to be
required for M2-mediated transcriptional repression, amino acids within
other domains are likely to play significant roles in determining the
degree to which a given promoter is repressed. For example, mutant 122 represses DHFR transcription akin to wild-type M2 but is
9-fold more effective as a repressor of p21 transcription. Similarly, mutant 158 represses DHFR and p21
transcription akin to wild-type M2 but is 7-fold more active as a
repressor of MDR-1. As might be predicted given their potent
activities as transcriptional repressors, each of these mutants carries
a wild-type M2 B domain. Yet each of these mutants carries mutations
that dramatically alter their activity as transcriptional repressors.
Translation of mutant 122 terminates within its zinc finger region, and
mutant 122 also carries single amino acid substitutions within the C and zinc finger regions. Mutant 158 is wild-type in length and carries
single amino acid substitutions within its C and zinc finger regions
distinct from those carried by mutant 122. Because mutations outside of
the M2 B domain can alter the capacity of mutated M2 proteins to
repress particular Sp-dependent promoters, we conclude that
such mutations affect regions of M2 that may determine
promoter-specific interactions. This conclusion encouraged us to
determine whether such mutations alter the capacity of mutated M2
proteins to bind DNA or form multimeric protein complexes.
DNA Binding Activity Is Not Required for M2-mediated
Repression--
To determine whether carboxyl-terminal M2 mutations
affect the capacity to bind DNA, mutated M2 proteins were characterized for their DNA binding activity via an in vitro protein/DNA
binding (gel shift) assay. Wild-type or mutated M2 proteins were
expressed ectopically in Drosophila SL2 cells, and nuclear
extracts prepared from transfected cells were incubated with a
radiolabeled high affinity Sp protein-binding site (GT box) that has
been characterized in detail (39). Mutated M2 proteins examined include
(i) mutants competent to repress transcription of the DHFR,
p21, and/or MDR-1 promoters, e.g.
mutants 3, 16, 68, and (ii) mutants with little or no capacity for
repression of Sp-mediated transcription, e.g. mutants 15 and
128. To ensure that equivalent levels of wild-type and mutated M2
proteins were assayed, protein expression was monitored via Western
blotting of nuclear extracts (Fig.
3A and data not shown). As
illustrated in Fig. 3B, although some mutated M2 proteins retain the capacity to bind a high affinity Sp-binding site, many mutated M2 proteins exhibited undetectable or greatly reduced DNA
binding activity relative to wild-type M2. As indicated in Table II,
many mutated M2 proteins that exhibit little or no in vitro
DNA binding activity are quite competent to repress Sp-mediated transcription in vivo. For example, mutant 16 exhibits
little or no DNA binding activity in vitro, yet this mutant
represses the p21 and MDR-1 promoters as
efficiently as wild-type M2. Similarly, mutant 68 gives rise to little
or no protein-DNA complexes in vitro yet is competent to
repress p21 akin to wild-type M2. Finally, three mutants,
42, 46, and 122, that repress p21 transcription more
potently than wild-type M2 also exhibit little or no DNA binding
activity in vitro. It is also quite apparent from the data
in Table II that the capacity to bind DNA is not sufficient to repress
transcription. For example, mutants 38, 172, 189, 229, and 259 each
bind DNA efficiently, yet their capacity to repression transcription is
severely impaired.
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Fig. 3.
Wild-type and mutated M2 protein expression
and DNA binding activity in nuclear extracts prepared from transfected
Drosophila SL2 cells. A, Western blot
of nuclear extracts probed with anti-HA antibody 12CA5. Nuclear
extracts were prepared from SL2 cells transfected with wild-type
(WT) or mutated M2 constructs. Mutant numbers are indicated
at top. B, DNA binding activity of wild-type and
mutated M2 proteins. A radiolabeled GT box probe was incubated with the
above nuclear extracts, a nuclear extract prepared from untransfected
SL2 cells (No DNA), and a nuclear extract prepared from
human C-33A cervical carcinoma cells (Control). Protein-DNA
complexes generated by Sp3, Sp1, and M1/M2 are indicated at the
left. Mutant numbers are indicated at top.
C, DNA binding activity of wild-type and mutated M2
proteins. Radiolabeled probes derived from the p21 (p21) and
MDR-1 (MD) promoters as well as a GT box (GT)
probe were incubated with above nuclear extracts, a nuclear extract
prepared from untransfected SL2 cells (No DNA), and a
nuclear extract prepared from human C-33A cervical carcinoma cells
(Control). Mutant numbers are indicated at top.
Brackets to the left and right
indicate protein-DNA complexes generated by endogenous
Drosophila proteins.
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To ensure that these in vitro protein/DNA binding results
are not specific to the Sp-binding site examined, similar assays were
performed using radiolabeled probes carrying well characterized Sp
protein-binding sites derived from the p21 and
MDR-1 promoters. Consistent with results for the GT box
probe, many mutated M2 proteins that have lost the capacity to bind
Sp-binding sites from the p21 or MDR-1 promoters
in vitro retain activity as transcriptional repressors of
these same promoters in vivo (Fig. 3C and Table II). Additionally, for those mutated proteins that retain the capacity
to bind DNA little correlation was noted between their relative
activity as DNA-binding proteins and their capacity to repress
transcription (Table II). We conclude from these results that DNA
binding activity, at least as revealed by in vitro
protein/DNA binding assays, is not required for M2-mediated
transcriptional repression.
Protein Multimerization Is Not Required for M2-mediated
Repression--
The Sp1 D domain is located immediately downstream of
the DNA-binding domain, and this 80-amino acid region has been shown to
be essential for protein multimerization and synergistic
trans-activation of promoters carrying multiple Sp-binding
sites (2). Although Sp3 has yet to be shown to form multimers in
vitro or in vivo, all Sp3 isoforms carry a region
carboxyl-terminal to their DNA-binding domains that is similar in size
and amino acid composition with the Sp1 D domain. Should Sp3 isoforms
be competent to form multimers in vivo, we reasoned that the
formation of multimeric complexes containing M1 and/or M2 proteins
might "poison" such complexes and thus account for Sp3-mediated
transcriptional repression. In keeping with this reasoning, we
hypothesized that alterations in transcriptional repression by mutated
M2 proteins might reflect their increased or decreased capacity to join
such multimeric protein complexes. Accordingly, an in vitro
protein/protein binding assay was developed, and wild-type and mutated
M2 proteins were examined for their capacity to multimerize with
wild-type M2 protein.
To begin these studies, a bacterial fusion protein was prepared that
links GST with wild-type M2 protein. GST-M2 protein harvested from
bacteria was bound to glutathione-agarose beads and incubated with
radiolabeled in vitro translated Sp1 or Sp3. As negative controls for these binding studies, radiolabeled Sp1 and Sp3 proteins were also incubated with GST alone as well as GST-FSH15, an irrelevant GST fusion protein. Consistent with the notion that M2 can associate with Sp proteins, Sp1 and all isoforms of Sp3 bound to GST-M2 but not
to GST or GST-FSH15 (Fig. 4A).
To extend these studies, wild-type or mutated M2 proteins were
radiolabeled via in vitro translation and were assayed
similarly. As shown in Fig. 4, B and C, wild-type
M2 and mutants such as 3, 15, and 16 bound GST-M2 in vitro,
whereas mutants such as 20 and 60 have lost this function. Consistent
with the hypothesis that the Sp3 D domain may facilitate multimerization, it is worth noting that mutants 20 and 60 carry premature "stop" codons that eliminate 322 and 283 carboxyl-terminal amino acids, respectively, including their D domains.
Similar to results for DNA binding activity, the capacity of M2
proteins to multimerize, at least in vitro, is not required
for repression of Sp-mediated trans-activation (Table II).
For example, mutant 42 does not bind GST-M2, and although its capacity
for repressing DHFR or MDR-1 is compromised, it
is 3-fold more active than wild-type M2 as a repressor of
p21. Mutant 57 does not bind GST-M2 but represses MDR-1 as efficiently as wild-type M2, although it has a
diminished capacity for repression of p21 and does not
repress DHFR. Finally, mutant 103 does not bind GST-M2 but
retains at least some capacity to repress Sp-mediated transcription of
DHFR and p21. In summary, in vitro
multimerization and DNA binding activity do not appear to be required
or sufficient for M2-mediated transcriptional repression. Although we
believe it is highly likely that these functional defects are also
manifest in vivo, it is worth noting that we cannot discount
the possibility that the intracellular milieu may completely or
partially ameliorate these functional deficiencies.
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Fig. 4.
Formation of multimeric protein complexes by
wild-type and mutated M2 proteins. A, interaction of
Sp1 and Sp3 with GST fusion proteins. Radiolabeled and in
vitro translated Sp1 (right panel) and Sp3 (left
panel) proteins were incubated with glutathione-agarose beads
bound to GST-M2 (M2), GST-FSH15 (FSH), or GST
(GST) alone, and radiolabeled bead-bound proteins were
resolved by electrophoresis. In vitro translated proteins
are identified at left. B, in vitro
translation of wild-type and mutated M2 proteins. Wild-type
(WT) and mutated M2 proteins were radiolabeled via in
vitro translation and resolved by electrophoresis. Mutant numbers
are indicated at top. C, interaction of wild-type
and mutated M2 proteins with GST-M2. Radiolabeled in vitro
translates shown in B were incubated with GST-M2, and
bead-bound proteins were resolved by electrophoresis. Mutant numbers
are indicated at top.
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M2 Binds TATA Box-binding Protein-associated Factors, and
Interactions with TAFII70 and TAFII40 Are
Disrupted or Diminished in Some M2 Mutants--
Because M2-mediated
repression does not require DNA binding activity or multimerization, we
reasoned that interactions between M2 and one or more cellular proteins
might account for repression of Sp-mediated transcription. Given that
the C, zinc finger, and D domains are not absolutely required for
transcriptional repression, Sp-binding proteins that interact with
carboxyl-terminal portions of Sp proteins, such as E2F and AP-2, were
considered to be unlikely candidates (8-10). Instead, components of
the basal transcription complex, such as TATA box-binding
protein-associated factors (TAFIIs), presented themselves
as attractive candidates because (i) TAFIIs have been shown
to participate in Sp-mediated trans-activation, and (ii) one
member of the TAFII complex (TAFII110)
interacts with Sp1 via glutamine-rich domains, including a hydrophobic
region that is closely conserved with an analogous portion of the B
domain of Sp3, M1, and M2 (7).
To examine interactions between Sp family members and TAFII
proteins, a series of in vitro protein/protein binding
assays were performed that combined GST fusions prepared with Sp family members and radiolabeled in vitro translated
TAFII proteins (TAFII30
, -30, -32, -40, -70, -110, -150, and -250). To ensure that these assays would reflect
differences in the affinity of particular TAFIIs for
GST-bound proteins, equivalent amounts of bacterially expressed GST
fusion proteins were bound to beads. As illustrated in Fig.
5A, some TAFII
proteins (30, 30, and 32) exhibited little or no capacity to bind
Sp family members, whereas other TAFII proteins (40, 70, 110, 150, and 250) bound readily and specifically. Some differences in
the binding of TAFII proteins were noted between Sp family
members. For example, TAFII250 and TAFII70
appear to bind Sp3 and M2 more efficiently than Sp1 (Fig.
5A).
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Fig. 5.
Characterization of protein complexes between
Sp family members and TAFII subunits. A,
protein complexes between Sp family members and TAFII
subunits. Radiolabeled and in vitro translated
TAFII subunits were incubated with glutathione-agarose
beads bound to equivalent amounts of GST (lane 2), GST-Sp1
(lane 3), GST-Sp3 (lane 4), or GST-M2 (lane
5). For comparison, lane 1 contains 10% of the
radiolabeled protein applied to beads in each of lanes 2-5.
Bead-bound proteins were resolved by electrophoresis through acrylamide
gels. B, summary of M2 amino acids required for interaction
with TAFII subunits. A schematic diagram of the sub-domain
structure of M2 and amino acids that demarcate these sub-domains are
illustrated at the upper left. Below this diagram
is illustrated wild-type M2 and the structure of three truncated M2
derivatives ( 353, 197, and 103) employed for mapping
experiments. + indicates binding akin to wild-type M2;  indicates no
binding activity; +/ indicates partial binding activity relative to
wild-type M2. C, protein complexes between TAFII
subunits and wild-type or truncated M2 derivatives. Radiolabeled and
in vitro translated TAFII subunits were
incubated with glutathione-agarose beads bound to equivalent amounts of
GST (lane 1), GST-M2 (lane 2), GST-M2353
(lane 3), GST-M2197 (lane 4), or GST-M2103
(lane 5). Bead-bound proteins were resolved by
electrophoresis through acrylamide gels.
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Because previous reports that have mapped interactions of
TAFIIs with Sp family members have limited their analyses
to Sp1, we performed a series of protein/protein binding assays that
mapped regions of M2 required for interactions with TAFIIs
40, 70, 110, 150, and 250. As shown in Fig. 5B, three
truncated GST-M2 fusion proteins were employed for these studies as
follows: (i) a wild-type M2 protein whose translation terminates within
the amino terminus of BQ (amino acid 103; 103), (ii) a
wild-type M2 protein whose translation terminates at the carboxyl
terminus of BQ (amino acid 197; 197), and (iii) a
wild-type M2 protein whose translation terminates within the second
zinc finger (amino acid 353; 353). Equivalent amounts of GST alone,
GST-M2, and each truncated GST-M2 fusion protein were employed in
in vitro protein/protein-binding reactions with radiolabeled
in vitro translated TAFII proteins. As shown in
Fig. 5C, TAFII250 bound full-length M2 and
353 but did not interact with 197 or 103. Binding by
TAFII250 to 353 was greatly diminished relative to M2,
and thus amino acids at the extreme carboxyl terminus of M2 appear to
be required for efficient interactions with TAFII250.
Similar results were noted for TAFII150, as binding was
restricted to full-length GST-M2 indicating that the carboxyl-terminal
portion of the M2 zinc finger and D domains (amino acids 353-479) is
required for interactions. TAFII110 bound full-length M2,
353, and 197 but not 103. Thus, at least one binding site for
TAFII110 resides within the M2 BQ domain
between amino acids 103 and 197. This result is entirely consistent
with previous reports that identified a binding site for
TAFII110 within a hydrophobic region of Sp1 that is
conserved in M2 (amino acids 159-174; see Ref. 7). Finally,
TAFII70 and -40 bound 353, 197, and 103 as
efficiently as M2, and thus at least one binding site for these
proteins maps to amino acids 1-103, corresponding to the
BS/T and amino-terminal BQ sub-domains. In
summary, these binding assays revealed that several TAFII
proteins are capable of interacting specifically with Sp family members
in vitro and that binding sites for TAFs are distributed throughout M2. It is worth mentioning that our mapping results identify
the minimal regions of M2 required for association with TAFII proteins and that additional binding sites may also exist.
Should physical interactions between M2 and one or more
TAFIIs play a role in M2-mediated transcriptional
repression, one would predict that mutants exhibiting altered patterns
of repression should also exhibit altered TAFII binding
activity. As a first step toward determining whether M2 mutations
affect TAFII interactions, a set of mutated M2 proteins
were fused to GST and examined in an in vitro
protein/protein binding assay with in vitro translated TAFII proteins. As illustrated in Fig.
6, TAFII110 bound each mutated M2 protein examined as well as wild-type M2, whereas the binding of TAFII70 and TAFII40 was diminished
for a subset of these same mutants. Mutants 68, 138, 158, and 172 showed a significant decrease in their capacity to bind
TAFII70 in vitro, and the capacity of mutant 138 to bind TAFII40 was also diminished. Consistent with
mapping results presented in Fig. 5, some mutants with diminished capacity to bind TAFII70 or TAFII40 carry
mutations within regions of M2 required for interactions with these
proteins. For example, mutants 68 and 138 exhibit diminished capacities
to bind TAFII70 and TAFII40, and these mutants
carry amino acid substitutions within three mutational hotspots located
within the minimal region (amino acids 1-103) of the M2 B domain
required for binding by TAFII70 and TAFII40.
Interestingly, mutants 158 and 172 do not carry mutations within the
minimal region of M2 required for binding TAFII70 and
TAFII40. Instead, these mutants carry mutations within the
C and/or zinc finger sub-domains perhaps indicating the positions of
additional binding sites for these TAFII proteins. We
conclude from results presented in Fig. 6 that mutations carried by
some M2 mutants disrupt interactions with specific TAFII
proteins. Whether the disruption of these protein interactions
contributes to the novel patterns of transcriptional repression
exhibited by these mutants remains to be determined.
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Fig. 6.
Characterization of protein complexes between
TAFII subunits and wild-type or mutated M2 proteins.
Radiolabeled and in vitro translated TAFII
subunits were incubated with glutathione-agarose beads bound to
equivalent amounts of GST (GST), wild-type GST-M2
(WT), or M2 mutants fused to GST. Mutant numbers are
indicated at top. Bead-bound proteins were resolved by
electrophoresis through acrylamide gels.
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DISCUSSION |
Transcription of a wide variety of mammalian genes has been shown
to be stimulated by Sp1 and Sp3 in transient expression assays.
Additionally, the integrity of many promoter elements bound by Sp1 and
Sp3 has been shown to be necessary for cell cycle-regulated and induced
transcription. Given their functional similarities, common target
genes, and ubiquitous expression, Sp1 and Sp3 might be viewed as
entirely redundant transcriptional regulators. Yet this supposition is
challenged by the finding that Sp3 encodes functionally distinct
proteins and by studies of nullizygous animals lacking Sp1 or Sp3
function. It is abundantly clear that in addition to a transcriptional
activator, Sp3 encodes two transcriptional repressors that we have
termed M1 and M2. Experiments reported here were undertaken to identify
Sp3 amino acids and functions required for transcriptional repression
and to begin to determine the mechanism(s) by which
Sp-dependent genes are repressed. As a consequence of these
studies, we have established that DNA binding activity and the capacity
to form multimeric protein complexes are not required for
transcriptional repression. Instead repression requires a discrete
portion of the Sp3 B domain (amino acids 235-328) that includes
serine/threonine-rich and glutamine-rich amino acids. Our studies also
indicate that mutations carried by M2 that disrupt transcriptional
repression of one Sp-dependent promoter do not necessarily
block repression of others, suggesting that transcriptional repression
may require promoter-specific interactions. Finally, five
TAFII proteins were found to bind M2 in vitro,
and we have identified several M2 mutants whose interaction with two
TAFII proteins, TAFII70 and
TAFII40, is compromised. Taken together, our results
strongly suggest that Sp3-mediated transcriptional repression proceeds,
at least in part, via the titration of one or more promoter-specific
trans-acting factors.
Although Sp1 is an alternatively spliced gene, Sp3 is unique among Sp
family members in its utilization of internal translational initiation
to synthesize multiple proteins (31, 45). Unlike other transcription
factors, such as C/EBP, that utilize internal translational
initiation to produce functionally distinct proteins, the relative
abundance of Sp3 isoforms does not vary as a function of cell
proliferative index or signal transduction. Indeed, the abundance of
Sp3, M1, and M2 appears to remain constant in all cells, and thus
regulation of Sp3-dependent genes is likely to be dictated
by one or more post-translational mechanisms. Two potential mechanisms
that appear to be ruled out by our studies are competition between Sp3
isoforms for promoter occupancy and the "poisoning" of
transcriptionally active Sp multimers by M1 and/or M2. Our survey of
mutated M2 proteins identified many that lack the capacity to bind DNA
or to multimerize, yet these same mutants were competent to block
transcription of at least some of the promoters examined. Indeed, some
of these mutants were more potent repressors of transcription than
wild-type M2. Instead, the identification of the amino-terminal 93 amino acids of M2 as the minimal region required for repression
strongly suggests that Sp3-mediated repression proceeds via competition
for one or more key trans-acting proteins that are required
for promoter activity. This 93 amino acid region harbors the entirety
of the serine/threonine-rich region carried by M2 as well as the first 25 amino acids of the glutamine-rich domain. Other than serving as
sites of post-translational modification, little is known about the
precise contributions of serine/threonine-rich regions to the functions
of Sp family members. The primary amino acid sequences of these
sub-domains are very poorly conserved between Sp family members. Yet
the amino acid sequence of any given family member is highly conserved
across animal species, suggesting that these regions are functionally
important. When analyzed for their capacity to function as
trans-activation domains, serine/threonine-rich regions
exhibit little or no activity on their own although they can regulate
transcription when linked to the glutamine-rich domains of Sp family
members (36, 45). Given these results and our findings that (i) many
hotspot mutations map to the M2 BS/T region and (ii) at
least two TAFII proteins, TAFII70 and
TAFII40, are likely to bind M2 via this region, we
speculate that serine/threonine-rich domains may regulate Sp-mediated
transcription via physical interactions with components of the basal
transcription complex. Consistent with the notion that
TAFII70 and TAFII40 may be physiologically relevant targets of M2 function, several M2 mutants carry mutations that perturb these physical interactions. Moreover, N-CoR, yet another
transcriptional repressor, has been shown to require contacts with
TAFII70 and -40 to inhibit transcription (46). Based on our
results, we propose that Sp1/Sp3 and M1/M2 may compete for interactions
with proteins such as TAFII70 and 40, and it is this competition that helps determine the expression level of a given Sp-dependent gene. We predict that extracellular signals
that induce Sp-mediated transcription may do so in part by relieving this competition, perhaps by favoring interactions between Sp1/Sp3 and
proteins such as TAFII70 and -40. In apparent accord with this proposal, we have shown that several TAFII proteins
bind Sp1 and Sp3 in addition to M2 and some TAFII proteins,
such as TAFII250 and TAFII70, bind
differentially to Sp family members. Whether TAFII70 and
-40 are relevant physiological targets for Sp family members remains
to be determined.
,
TOP
ABSTRACT
INTRODUCTION
Present address: NIEHS, Laboratory of Molecular Carcinogenesis,
National Institutes of Health, Research Triangle Park, NC 27709.
