From the
YY1 is a zinc finger transcription factor with unusual
structural and functional features. In a yeast two-hybrid screen, two
cellular proteins, cyclophilin A (CyPA) and FK506-binding protein 12
(FKBP12), interacted with YY1. These interactions are specific and also
occur in mammalian cells. Cyclosporin A and FK506 efficiently disrupt
the YY1-CyPA and YY1-FKBP12 interactions. Overexpression of human CyPA
and FKBP12 have different effects on YY1-regulated transcription with
these effects being promoter-dependent. These results suggest that
immunophilins may be mediators in the functional role of YY1.
YY1 is a zinc finger transcription factor that binds to a
variety of cis DNA elements located in viral and cellular
promoters. It has been implicated as a positive regulatory factor that
binds to the ribosomal protein L30 and L32 gene promoters(1) ,
the B19 parvovirus P6 promoter(2) , the transcriptional
regulatory region of LINE-1(3, 4) , an intracisternal
A-particle upstream element(5) , the Surf-1/Surf-2
promoter(6) , and the c-myc promoter(7) .
Repression of transcription by YY1 was observed in the long terminal
repeat of the Moloney murine leukemia virus(8) , the skeletal
muscle
In the immunoglobulin heavy chain
enhancer, YY1 can act as a repressor or an activator(22) ; and,
in the human papillomavirus type 18 promoter, a switch region
determines the positive and negative action of YY1(23) . In
addition, while YY1 stimulates transcription by its ability to bind to
the adeno-associated virus P5 and the cytochrome c oxidase
subunit Vb initiators(24, 25) , it inhibits
transcription from the human immunodeficiency virus type 1 initiator
(26).
YY1 was first purified from HeLa cell nuclear extracts by
sequence-specific DNA affinity chromatography and analyzed by protein
microsequencing(21) . cDNA encoding YY1 was obtained using
probes generated from microsequencing information. The binding property
of YY1 was also used as a means to screen expression libraries for cDNA
clones encoding it(1, 8, 22) . Functional
studies of YY1 included mutation of its binding sites in a number of
promoters or placing YY1-binding sites upstream of a variety of natural
or synthetic promoters. Studies of cloned YY1 included the general
strategies of expressing YY1 transiently in tissue culture cells or
adding purified YY1 to an in vitro transcription extract and
assaying the transcription from reporter gene promoters containing
YY1-binding sites. So far, such approaches have demonstrated that YY1
activates and represses transcription of many different promoters, but
little is known about its mechanism of action.
The identification
and characterization of proteins in a cell with which a given protein
interacts is often helpful for understanding the function of that
protein. Previously, we and others have used the yeast two-hybrid
screen to identify cellular proteins that interact with YY1. Among the
clones analyzed were genes encoding nucleolar phosphoprotein B23 (27) and the oncoprotein c-Myc(28) . Both the nucleolar
phosphoprotein B23 and c-Myc can relieve YY1-induced transcriptional
repression. In addition, c-Myc inhibits the activator functions of YY1.
These results suggest that a possible mechanism for transcription
regulation by YY1 is by interaction with these two cellular proteins.
We have now sequenced and characterized additional clones from our
two-hybrid screen for encoded proteins capable of binding to YY1. Two
proteins were identified, cyclophilin A (CyPA)
In this report, we present evidence that YY1 interacts
specifically with CyPA and FKBP12 in both yeast and mammalian cells,
suggesting that YY1 may be a common natural ligand for both
immunophilins. Consistent with the ability of these immunophilins to
interact with YY1, overexpression of CyPA or FKBP12 alters the
transcription activity of YY1.
pGH289 (obtained from Gary Hayward, Johns Hopkins University)
contains the full-length VP16 driven by the CMV promoter; pGal4DBD-CyPA
was constructed by taking the human CyPA cDNA, an EcoRI
fragment from pGCyPA(43) , and ligating it to the EcoRI
site of pSG424. pGal4DBD-FKBP was constructed by first isolating an EcoRI fragment from pJDB-GAP-FKBP-A (57) and subcloning
it into pGEM3Z (Promega) and then using EcoRI and XbaI to isolate the FKBP12 cDNA and subcloning the final
fragment into pSG424. pGal4AD-YY1 was constructed by first subcloning
the Gal4 AD from pGAD424 (52) into pcDNAI/Amp (Invitrogen) and
then fusing the YY1 cDNA in-frame with the activation domain downstream
from the CMV promoter. pGal4AD-YY1
Fig. 1C shows that neither the Gal4 DBD-CyPA fusion or the Gal4 AD-YY1
fusion significantly activated the reporter gene (lanes2 and 4), but the two proteins together activated CAT
activity 10-fold (lane3). A deletion mutation of
amino acids 332-414 in YY1 did not have any effect on the
interaction (lane5). Similarly, a Gal4 DBD-FKBP12
fusion and the Gal4 AD-YY1 fusion together activated the CAT reporter
gene (lane8). However, unlike the YY1-CyPA
interaction, deletion of amino acids 332-414 in YY1 reduced the
interaction of YY1-FKBP12 significantly (compare lanes8 and 9). These data confirm that YY1 binds CyPA and FKBP12
and further suggest that the YY1 carboxyl-terminal domain is required
for its interaction with FKBP12 but not with CyPA.
As with all
experiments presented in this paper, in place of using an internal
control (which often gets activated or repressed by YY1, CyPA, or
FKBP12), each experiment shown is representative of at least three (and
sometimes as many as 20) similar experiments, and all results were
reproduced with more than one transfection reagent and DNA preparation.
Our screen with the yeast two-hybrid system for cDNA-encoding
proteins that interact with transcription factor YY1 has revealed
interactions with a class of proteins known as immunophilins. We
demonstrated that the proteins encoded by two clones, identified as
CyPA and FKBP12, interact with YY1 and do so in a highly specific
manner. Our data indicate that YY1 and either CyPA or FKBP12 interact
to mediate transcriptional activation independent of the reporter gene,
cell type, the activation, or the DNA-binding domains. Furthermore,
YY1, CyPA, or FKBP12 do not interact with several unrelated proteins.
Interaction between YY1 and CyPA can be disrupted by the
immunosuppressive drug CsA. Similarly, YY1-FKBP12 interaction can be
disrupted by FK506. Previous studies have shown that CsA and FK506
appear to block the nuclear transport of cytoplasmic transcription
factors, primarily NFAT, thereby inhibiting the activation of cytokine
transcription(51, 66, 67, 68) . CsA and
FK506 also inhibit transcription mediated by AP-3, Oct-1, and, to a
lesser extent, NF-
Although CyP
and FKBP are the only well characterized immunophilins, other members
of this family are known to exist (reviewed in Refs. 29-40 and
46). The fact that we have only isolated CyPA and FKBP12 in the
two-hybrid screen does not exclude the possibility that YY1 may
interact with other members of the immunophilin family.
Our finding
that different domains of YY1 may be required for interaction with CyPA
and FKBP12 is intriguing. While the COOH-terminal 83 amino acids of YY1
are important for YY1-FKBP12 interaction, this region is dispensable
for YY1-CyPA interaction. This COOH-terminal domain of YY1 has
previously been implicated in transcriptional repression (21) and binding to the adenovirus E1A protein(70) . One
can imagine that perhaps YY1-FKBP12 forms a transcriptional repression
complex, and an association between YY1 and E1A results in a
transcriptional activation complex. Overexpression of E1A then would
favor the formation of YY1-E1A over the YY1-FKBP12 complex, thereby
resulting in transcriptional activation.
We were surprised to find
that while overexpression of FKBP12 repressed transcription of an SV40
promoter/enhancer containing YY1-binding sites, activated transcription
was observed when FKBP12 was overexpressed in a Gal4-YY1 assay with the
thymidine kinase promoter. This suggests that one cellular factor may
mediate opposite effects by YY1, and the hypothesis that the
positive/negative regulation by YY1 is achieved through its ability to
interact with positive/negative cellular factors may be oversimplified.
Unlike YY1-E1A(70) , YY1-Sp1(71, 72) , and
YY1-B23 (27) complexes, attempts to show YY1-CyPA or YY1-FKBP12
interactions in vitro have been unsuccessful (data not shown).
It is conceivable that YY1 binds CyPA and FKBP12 only in the presence
of a third cellular protein, and this accessory protein is absent or
non-functional in our in vitro binding assays. Alternatively,
perhaps interaction between YY1 and CyPA and FKBP12 requires
modification of YY1 and/or the immunophilins, and this modification did
not occur in vitro. Further experiments will allow us to
address these questions.
Our findings that CyPA and FKBP12 bind YY1
adds to the growing list of cellular proteins that interact with YY1.
This includes transcription factor Sp1(71, 72) , the
oncoprotein c-Myc(28) , the nucleolar phosphoprotein
B23(27) , TAF
We thank Tim Durfee, Gary Hayward, Stephen Goff,
Nobuhiro Takahashi, and Lan Bo Chen for plasmids, Sandoz
Pharmaceuticals Corp. for cyclosporin A, Fujisawa USA, Inc. for FK506,
Charles Zuker for discussion on cyclophilin and FK506-binding protein,
and Paul D. Gardner and Steve Britt for critical reading of the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-actin promoter(9, 10) , the c-fos promoter(10, 11) , the
-casein gene
promoter(12, 13) , the human cytomegalovirus major
immediate early enhancer/promoter(14) , the rat serum amyloid A1
gene promoter(15) , the human interferon-
promoter(16) , the
and
silencers(17, 18, 19) , and the Epstein-Barr
virus BZLF1 gene promoter (20). Similar repression was reproduced when
YY1-binding sites were placed upstream of a minimal promoter or the
SV40 promoter/enhancer (21).
(
)and FK506-binding protein 12 (FKBP12). CyPs and FKBPs
comprise two families of ubiquitous and often abundant proteins
conserved from prokaryotes to eukaryotes (reviewed in Refs.
29-40). CyPA was first identified and purified from bovine spleen
on the basis of its high affinity for cyclosporin A (CsA)(41) .
It is an abundant, cytosolic protein of 18 kDa found in all tissues in
eukaryotic cells. It possesses rotamase activity, which enables it to
catalyze the cis-trans isomerization of peptide bonds
involving a prolyl residue, and might facilitate protein
folding(42) . It has been shown that CyPA binds the human
immunodeficiency virus type 1 (HIV-1) Gag protein and is specifically
incorporated into HIV-1 virion
particles(43, 44, 45) . Although it has no
sequence homology with CyPs, FKBPs are also abundant cytosolic proteins
that possess cis-trans peptidyl-prolyl isomerase activities
that are inhibited by FK506 but not by CsA (reviewed in Refs. 34 and
46). FKBP12 has been shown to interact with the type I receptors of the
transforming growth factor-
family(47) , and two
FKBP-related proteins are associated with progesterone receptor
complexes(48) . Also, FKBP25 is associated with
nucleolin(49) , a major nucleolar phosphoprotein in
exponentially growing cells. Both CyPA-CsA and FKBP12-FK506 complexes
prevent T-cell response to antigen, bind and modulate activity of the
protein phosphatase calcineurin(50) , and prevent nuclear import
of a subunit of NFAT(51) , a T-cell activation transcription
factor.
Plasmids
The following plasmids have previously
been described: pAS-YY1(27) , which encodes a Gal4 DNA-binding
domain (DBD)-human YY1 fusion protein; pAS-Rb2(52) , which
encodes a Gal4 DBD-retinoblastoma fusion protein; pAS-SNF1(52) ,
which encodes a Gal4 DBD-yeast SNF1 fusion protein; Y14(27) ,
which encodes a Gal4 activation domain (AD)-CyPA fusion protein;
Y20(27) , which encodes a Gal4 AD-FKBP fusion protein;
pG5E1BCAT(53) , which contains five Gal4-binding sites upstream
of the adenovirus E1B TATA box and the chloramphenicol
acetyltransferase (CAT) reporter gene; pGal4-RB(52) , which
contains the DBD (amino acids 1-147) of the Gal4 protein fused to
the retinoblastoma protein under the control of the simian virus 40
(SV40) early promoter/enhancer; pYY1/VP16(27) , which expresses
the full-length YY1 protein fused to the acidic activating domain of
the herpes simplex virus-1 VP16 (amino acids 413-490);
pHCMV-LAP348(54) , which contains a modified lac repressor gene fused to the VP16 acidic activation domain under
the control of the human cytomegalovirus (CMV) immediate early
promoter; pP5-60SVECAT and pP5-60(mt2)SVECAT(21) ,
which carry wild-type or mutant YY1-binding sites 5` to the SV40
enhancer and CAT gene; pSG424(55) , which contains the Gal4 DBD
under the control of the SV40 promoter/enhancer; pGal4-YY1(21) ,
which contains the Gal4 DBD fused to YY1 cDNA; and
pGal4TKCAT(21) , which contains five Gal4-binding sites upstream
of the thymidine kinase TATA box in plasmid pBLCAT2(56) .
is identical to pGal4AD-YY1,
with the exception that nucleotides 1236-1513 were deleted from
the YY1 cDNA, resulting in expression of a Gal4 fusion with YY1 amino
acids 1-331. pGal4DBD-LR was constructed by placing the human
laminin receptor (amino acids 27-295) (58) downstream of
and in-frame with the Gal4 DBD. pCMV-CyPA was constructed by ligation
of an EcoRI fragment from pGCyPA into pcDNAI/Amp. pCMV-FKBP
was similarly constructed by taking an EcoRI fragment from
pJDB-GAP-FKBP-A and subcloning it into pcDNAI/Amp. pSG5-YY1 was
constructed by taking pYY1/VP16 and digesting it with BglII
and then religating the plasmid to remove the VP16 sequence. All
recombinants were verified by dideoxy sequencing.
Yeast Two-hybrid Interaction Experiments
To assay
systematically the pairwise interactions, library-derived Y14 or Y20
plasmids were transformed into Y153 alone or Y153 harboring pAS-YY1,
pAS-Rb2, or pAS-SNF1 as described(27, 52) .
Transformants were assayed for the presence of -galactosidase
activity using the colony filter lift method(59) .
DNA Transfections and CAT Assays
HeLa cells were
grown on 60-mm tissue culture dishes in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and
penicillin/streptomycin. Transfections were performed using the calcium
phosphate method(60) . Forty-eight hours after transfection,
cells were lysed by repeated freeze/thaw cycles and extracts assayed
for CAT activity by thin-layer chromatography (61) and
quantified with the PhosphorImager (Molecular Dynamics). To determine
the effects of CsA and FK506 on YY1-immunophilin interaction, different
amounts of CsA or FK506 (1 mg/ml stock in 50% phosphate-buffered saline
and ethanol) were added to the culture media 4-8 h after
transfection.
Y14 and Y20 Are Identical to CyPA and
FKBP12
Previously, we have used the yeast two-hybrid screen to
detect mouse cDNAs coding for products that interact with
YY1(27) . Of approximately 10 transformants, 14
clones showed specificity in binding to YY1 but not Rb or the yeast
SNF1 protein. Preliminary DNA sequence analysis revealed eight novel
sequences and six that were highly homologous to known sequences. Of
the six known sequences, we have now completely sequenced two
additional cDNA inserts designated Y14 and Y20. The Y14 clone is
identical to the previously published mouse cyclophilin cDNA sequence
(nucleotides 289-679; Ref. 62 (GenBank accession number X52803)).
The insert in this clone begins at the 84th codon of CyPA and continues
through the open reading frame past the 3`-noncoding sequences of the
gene. The Y20 clone is identical to the previously published mouse
FK506-binding protein (nucleotides 50-849; Ref. 63 (GenBank
accession number X60203)). The insert in Y20 begins in the
5`-untranslated portion of the FKBP RNA 47 nucleotides before the
initiator methionine and continues through the entire open reading
frame past the noncoding sequences at the 3`-end of the gene.
Confirmation That Y14 and Y20 Are Dependent on YY1 Hybrid
Expression
To confirm that the phenotype observed in the
original screen was reproducible and dependent on the YY1 hybrid, Y14
and Y20 plasmids were recovered and transformed into Y153 either alone
or with test fusions in pAS1. As shown in Fig. 1A,
transformants were assayed and -galactosidase activities were
positive only in the presence of pAS-YY1.
Figure 1:
CyPA and FKBP12 can interact
specifically with YY1. A, assessing the dependence of the Gal4
DBD-YY1 fusion for transcriptional activation by Y14 and Y20 clones
isolated in the yeast two-hybrid screen. Colony filter lift methods
were performed for each combination. B, schematic drawing of
plasmids used in HeLa cell transfection assays. C,
transfection assays showing that CyPA and FKBP12 can target different
YY1-activation domain fusion proteins to a promoter. Using the calcium
phosphate method, HeLa cells were cotransfected with 5 µg each of
pGal4DBD-CyPA or pGal4DBD-FKBP, pGal4AD-YY1 or pGal4AD-YY1, and
pG5E1BCAT, as indicated. All transfections were normalized to equal
amounts of DNA with parental expression vectors. Forty-eight hours
after transfection, cells were collected and CAT activity determined in
1-h reactions. Three independent experiments yielded consistent
results. The synthetic promoter in pG5E1BCAT contains only a TATA box
and Gal4-binding sites with no upstream activators and, therefore,
gives no basal activity and does not respond to YY1, CyPA, or
FKBP12.
CyPA or FKBP12 Fused to the Gal4 DNA-binding Domain Can
Support Transcriptional Activation Mediated by a YY1-Gal4 Activation
Domain Fusion Protein
If a true interaction exists between YY1
and CyPA or FKBP12, this interaction should occur independent of the
Gal4 DBD and the Gal4 AD. Specifically, activation of a reporter gene
should still occur if the two Gal4 domains are switched. In addition,
since YY1 is a mammalian transcription factor, it is important to
confirm that this interaction occurs in mammalian cells. To address
this issue, we fused human CyPA or FKBP12 to the Gal4 DBD,
Gal4(1-147), and tested, in HeLa cell cotransfection assays, the
ability of each Gal4 fusion protein to support Gal4 AD-YY1-mediated
transcriptional activation using reporter constructs that contained
Gal4-binding sites (Fig. 1B).
Additional Evidence for Interaction between YY1 and CyPA
or FKBP12
If the ability of Gal4 DBD-CyPA or Gal4 DBD-FKBP12 to
support Gal4 AD-YY1 is specific, then other non-related Gal4 DBD fusion
proteins should not support a YY1-AD fusion response. Furthermore, a
Gal4 DBD-CyPA or Gal4 DBD-FKBP12 should not activate transcription in
the presence of other non-related AD fusion proteins. In particular, we
found that a Gal4 DBD-CyPA or Gal4 DBD-FKBP12 can synergize with a YY1
protein fused to the AD of VP16 (Fig. 2, B, lane4 and C, lane4), but neither
the Gal4 DBD-RB nor Gal4 DBD-LR can activate transcription with
YY1-VP16AD (Fig. 2, B, lanes6 and 8 and C, lanes6 and 8).
Also, a Lac I-VP16AD fusion or an entire VP16 protein cannot
stimulate transcription with Gal4 DBD-CyPA or with Gal4 DBD-FKBP12 (Fig. 2, B, lanes10 and 12 and C, lane10). Taken together, these
results argue that the interaction between YY1 and CyPA or FKBP12 is
highly specific.
Figure 2:
CyPA-
or FKBP12-mediated transcriptional activation by activation domain-YY1
fusion is highly specific. A, schematic drawing of plasmids
used in transfection assays. B and C, HeLa cells were
cotransfected with 5 µg each of the indicated plasmids. All
transfections were normalized to equal amounts of DNA with parental
expression vectors. Transfection and CAT assays were done as described
under ``Materials and Methods'' and for Fig. 1, except that
smaller amount of extracts and a lower reaction time were used to keep
the assay linear. The results are the mean ± S.D. from at least
three separate transfections.
CsA and FK506 Inhibit CyPA-YY1 and FKBP12-YY1
Interactions, Respectively
The immunosuppressive drug CsA, a
cyclic undecapeptide of fungal origin, has been shown to bind CyPA with
high affinity (reviewed in Refs. 29-40). This binding results in
an inhibition of peptidyl-prolyl cis-trans isomerase activity.
FK506 is a macrolide (cyclic ester) that is structurally distinct from
CsA but also blocks T-cell activation in the same way as CsA (reviewed
in Refs. 34 and 46). FK506 does not bind to CyPA, but it does bind to
FKBP12. Both CsA-CyPA and FK506-FKBP12 bind to and inhibit protein
phosphatase 2B, the cellular target of calcineurin. To determine
whether CsA has any effect on YY1-CyPA interaction, we transfected
pGal4DBD-CyPA, pYY1/VP16, and pG5E1BCAT into HeLa cells and added
different amounts of CsA into the media. As shown in Fig. 3A, CsA inhibited the YY1-CyPA interaction in a
dose-dependent manner. Similarly, FK506 inhibited the interaction
between YY1 and FKBP12 (Fig. 3B). In both of these
experiments, the concentrations of CsA and FK506 are below the cellular
toxicity levels(64, 65) .
Figure 3:
CsA and FK506 block binding of YY1 to CyPA
and FKBP12, respectively. Transfections of pGal4DBD-CyPA (A and C), pGal4DBD-FKBP (B and C),
pGal4DBD-VP16 (C), pYY1/VP16 (A-C),
and pG5E1BCAT (A-C) into HeLa cells and CAT assays were
performed as described for Fig. 2. CsA or FK506 was added to the
culture medium 4-8 h after
transfection.
To be certain that the
inhibition of interaction observed is not due to a general inhibitory
effect of the two drugs, we repeated the transfection experiments but
switched the two drugs. Specifically, we transfected HeLa cells with
pG5E1BCAT, pGal4DBD-CyPA, and pYY1/VP16, followed by treatment with
1000 ng/ml FK506 but not CsA. As expected, FK506 did not affect
CyPA-YY1 interaction (Fig. 3C, lane9). Similarly, CsA did not affect FKBP-YY1 interaction
because cells treated with 500 ng/ml CsA and transfected with
pG5E1BCAT, pGal4DBD-FKBP, and pYY1/VP16 did not result in a reduction
of CAT activity (Fig. 3C, lane5).
Furthermore, neither 500 ng/ml CsA nor 1000 ng/ml FK506 inhibited
activation of pG5E1BCAT by Gal4 DBD-VP16 (Fig. 3C, lanes1-3). These two sets of experiments,
then, strongly argue that inhibition of CyPA-YY1 interaction by CsA and
inhibition of FKBP-YY1 interaction by FK506 is not due to a generally
nonspecific effect of the two drugs.
CyPA and FKBP12 Enhance Transcriptional Repression
Mediated by YY1-binding Sites
To determine the effects of CyPA
and FKBP12 on YY1-related transcription, plasmids
pP5-60(mt2)SVECAT and pP5-60SVECAT were transfected into
HeLa cells separately in the presence or absence of another plasmid
expressing either CyPA or FKBP12. In agreement with our earlier
study(21) , the presence of YY1-binding sites 5` to the
transcriptional control region of the SV40 promoter/enhancer repressed
transcription (Fig. 4, compare lanes1 and 4). Interestingly, overexpression of CyPA but not FKBP12
caused an increase in CAT activity in the absence of YY1-binding sites
(compare lane1 to lanes2 and 3). However, in the presence of YY1-binding sites, both CyPA
and FKBP12 repressed transcription (compare lane4 to lanes5 and 6). Subtracting the effects of
CyPA and FKBP12 on the SV40 promoter/enhancer, there is a 4- and
20-fold reduction in CAT activity, respectively. In summary,
overexpression of CyPA and FKBP12 represses transcription from the SV40
promoter/enhancer containing YY1-binding sites.
Figure 4:
Overexpression of CyPA or FKBP12 can
repress transcription from YY1-binding sites. HeLa cells were
cotransfected with 5 µg each of pP5-60(mt2)SVECAT (lanes
1-3) or pP5-60SVECAT (lanes 4-6) and
pCMV-CyPA or pCMV-FKBP, as indicated. All transfections and CAT assays
were done as described for Fig. 1 and under ``Materials and
Methods.''
FKBP12 Can Reverse Transcriptional Repression by
YY1
Previously, we have shown that a Gal4 DBD-YY1 fusion protein
represses transcription when directed to a promoter containing
Gal4-binding sites and that the adenovirus E1A or nucleolar
phosphoprotein B23 can relieve the repression(21, 27) .
To determine the effects of FKBP12 on YY1-induced transcriptional
repression, we targeted YY1 to a promoter containing Gal4-binding sites
and cotransfected a plasmid expressing FKBP12. As shown in Fig. 5B (lane3), the addition of a
plasmid that expressed FKBP12 relieved the repression by YY1 and
further activated CAT expression. This is somewhat surprising given the
fact that in the previous experiment overexpression of FKBP12 can
further repress a YY1-binding site (Fig. 4).
Figure 5:
FKBP12 can relieve YY1-induced
transcriptional repression. A, schematic drawing of plasmids
used in transfection assays. B, HeLa cells were cotransfected
with 5 µg each of plasmids pSG424 expressing Gal4 DBD, pGal4-YY1
expressing Gal4 DBD-YY1, pCMV-FKBP expressing FKBP12, and pGal4TKCAT as
indicated. All transfections and CAT assays were done as described for
Fig. 1 and under ``Materials and
Methods.''
B(69) . We have identified, with gel
shift assays, at least one YY1-binding site in the interleukin-3
promoter,
(
)but whether YY1 regulates
interleukin-3 transcription and whether CsA or FK506 has any effect on
transcription mediated by YY1 remains to be determined.
55(73) , basal transcription
factors TATA-binding protein and TFIIB(73, 74) ,
(
)and at least eight other novel proteins(27) .
As increasingly sophisticated techniques to identify transcription
factor-associated proteins become available, we predict there will
probably be even more proteins that can be shown to bind YY1. We
believe the next step in determining the mechanism and cellular
function of YY1 is to determine which YY1-associated proteins are
physiologically relevant. Current studies in our laboratory are focused
upon this important issue.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.