From the Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Stage-specific activator protein (SSAP) is the transcription factor responsible for the activation of the sea urchin late H1 gene at the mid-blastula stage of embryogenesis. SSAP contains an extremely potent transcription activation domain that functions 4-5-fold better than VP16 in a variety of mammalian cell lines. We used the two-hybrid screening technique to identify human cDNAs from an HL60 cell-derived cDNA library that encode proteins that interact with the transcription activation domain of SSAP. One of these cDNAs encodes ZFM1, a protein previously identified at the locus linked to multiple endocrine neoplasia type 1 (MEN1) and as presplicing factor SF1. Functional assays establish the ZFM1 protein as a transcriptional repressor. ZFM1 protein represses Gal4-GQC-mediated transcription, and this activity requires both a repression domain found in the N-terminal 137 amino acids of the protein, as well as a GQC interaction region. The physiological significance of repression mediated by ZFM1 comes from the ability of its specific repression domain to function when fused to Gal4 and tethered to promoters containing Gal4 binding sites. The activity is unique in that activated but not basal transcription levels are affected.
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INTRODUCTION |
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Different families of histone genes in sea urchins are expressed
with distinct temporal patterns during early embryogenesis, making this
system ideal for studying mechanisms of temporal gene expression. In
recent years, much progress has been made in studying the expression of
late H1 histone subtype genes (1-4). The late H1- gene is
transcribed at low levels until it is transcriptionally activated at
the mid-blastula stage, and reaches its peak level of expression in
24-h late-blastula stage embryos. The correct temporal activation of
late H1 gene expression is determined by an enhancer element located
220-280 base pairs upstream in its promoter region (1). Stage-specific
activator protein (SSAP)1 is
a 43-kDa polypeptide that can specifically bind to this enhancer (2).
Early in development, SSAP is present as a monomer; however, it
undergoes a posttranslational modification at about 12 h after fertilization, and dimerization coincides with the activation of late
H1 gene (2). Synthetic SSAP mRNA injected into zygotes transactivates reporter genes containing SSAP binding sites (3). Significantly, this transactivation also occurs in a temporal-specific manner.
The DNA binding activity of SSAP maps to its N-terminal 180 amino acids (3). This domain contains two RNA recognition motifs, which recognize both double-stranded and single-stranded DNA in a sequence-specific manner (3). In addition to this novel DNA binding domain, SSAP contains an extremely potent transcription activation domain consisting of amino acids 181-404 (4). This activation domain consists of a central glycine/glutamine-rich sequence and a C-terminal region rich in serine/threonine and basic amino acids (referred to as the GQC domain). In a variety of mammalian cell lines, Gal4-GQC fusion protein can transactivate the expression of Gal4-responsive reporter genes as much as 4-5-fold better than Gal4-VP16 (4). This activity requires the presence of both the central G/Q-rich domain as well as the C-terminal domain. The GQ region alone cannot activate transcription, whereas the C-terminal region has minimal activity (4). The activation domain of a transcription factor may promote transcription at several different steps during the formation of a preinitiation complex by recruiting basal transcription factors or during promoter clearance and elongation (5-7). To do this, the activators must interact with multiple targets in the transcriptional apparatus. These targets include not only basal factors in the transcription machinery, but also various co-activators or adaptors, which facilitate interactions between activator and basal transcription factors or RNA polymerase II (8, 9). In vitro binding assays have shown that the GQC domain can physically interact with several basal transcription factors of RNA polymerase II, including TBP, TFIIB, TFIIF74, and dTAFII110 (4). On the other hand, the GQC domain has self-squelching activity and the ability to squelch VP16- and E1A-driven reporter genes. This suggests that the activation domains of SSAP, VP16, and E1a share some common targets necessary for maximal transcription. It is believed that these targets may not be basal transcription factors, instead, they may be specific adaptors or coactivator proteins (4). Although the ability of the activation domain of SSAP to drive such high levels of transcription may be explained by stronger association with common targets, it is also possible that it may interact with unique protein targets not shared by VP16 or E1a. It has been well documented that different classes of activation domains may interact with distinct coactivators or adaptors to stimulate transcription (8). For example, the glutamine-rich activation domain of SP1 binds specifically to TAFII110 (10, 11), whereas the acidic activation domain of VP16 cannot stimulate transcription without interaction with TAFII40 (12). Similarly, a group of transcription factors require p300/CBP coactivator family members for maximal activity, whereas other transcription factors act independently of this regulatory network (13-15).
In this paper, a two-hybrid screening technique is employed to identify human protein(s) that can interact with the activation domain of SSAP. The rationale for this screen comes from the extremely potent activation domain used as a "bait" in this study. We believed that the GQC activation domain would interact with interesting transcriptional mediators. This paper describes the properties of one SSAP-interacting protein, ZFM1. ZFM1 was previously cloned as a nuclear protein at a locus linked to multiple endocrine neoplasia type 1 (16). It was also identified as a presplicing factor SF1 (17). Here, we show that ZFM1 functions as a transcription repressor.
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EXPERIMENTAL PROCEDURES |
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Plasmids-- To generate pBTM116-GQC as bait for two-hybrid screening, an EcoRI fragment derived from pSG424-GQC (4), which contains the entire GQC domain (amino acids 181-404), was ligated into pBTM116 vector cut with EcoRI. The resulting construct was sequenced to verify that the GQC domain was cloned in the correct orientation and in frame with the LexA DNA binding domain.
To create pGAD424-ZFM1-E for yeast two-hybrid assays, two primers (5'-GGG GTC GAC CAG ACC ACA TGG CGA CCG GAG CGA AC-3') and (5'-CCC GTC GAC TCA CTT GTC ATC GTC GTC CTT GTA GTC CCA ATG GGC GCG GAA AGT-3') were used in a PCR reaction to amplify ZFM1-E from pBluescript SK phagemid DNA. This PCR product was cut by SalI and ligated into the SalI site of pGAD424 to fuse ZFM1-E in frame with the Gal4 activation domain. To generate pCR3.1-ZFM1-A expression construct for functional assays, two PCR fragments that correspond to N-terminal (between nucleotide 383-1607) and C-terminal (between nucleotide 836 and 2251) regions of ZFM1-A, respectively, were created. The N-terminal piece was amplified using template ZFM1-E in pBluescript SK and two primers (5'-GGG GTC GAC CCG CCA CCA TGG CGA CCG GAG CGA AC-3' and 5'-CAT GCC CAC CTG TCA GGC-3'), and the C-terminal piece was amplified using template CE9 and two primers (5'-TTT GTG GGG CTG CTC ATC-3' and 5'-GTC GAC TCA CTT GTC ATC GTC GTC CTT GTA GTC TGG CTC GGG CCA TCG C-3'). A unique EcoRI site at nucleotide position 1489 of ZFM1-A was utilized to join these fragments in the correct reading frame. The two non-overlapping portions were joined together and ligated to pCR3.1 vector (Invitrogen) in a three-piece ligation reaction. In the resulting plasmid, pCR3.1-ZFM1-A, the expression of full-length ZFM1-A in mammalian cells is under the control of cytomegalovirus promoter, and there is a FLAG-tag in frame with the C-terminal amino acid of ZFM1-A. To make pGAD424-ZFM1-A and pSG424-ZFM1-A, ZFM1-A fragment was released from pCR3.1-ZFM1-A by SalI digestion and then cloned into the SalI site of pGAD424 and pSG424, respectively. To create pCR3.1-ZFM1-(1-320), this region of ZFM1-A was amplified by PCR using two primers (5'-GGG GTC GAC CCG CCA CCA TGG CGA CCG GAG CGA AC-3' and 5'-GAC GCG TCG ACT CAC TTG TCA TCG TCG TCC TTG TAG TCC AGT TCA GCC ATG AGG GA-3'). This PCR fragment is cloned in pCR3.1 and the expression of ZFM1-(1-320) with a C-terminal FLAG-tag is under the control of cytomegalovirus promoter in the resulting plasmid. pGAD424-ZFM1-(1-320) and pSG424-ZFM1-(1-320) were created in a manner similar to that for ZFM1-A on these vectors by utilizing the same SalI site. To clone pGEX-KG-ZFM1-(321-478) for bacterial expression of GST fusion protein, ZFM1-(321-478) was amplified from template pCR3.1-ZFM1-A using primer 5'-GCG GGA TCC GTC TAG AGG GTG AAG CAC CTG TC-3' and primer 5'-GCG GGA TCC AAG CTT ACA TCA TGC CCA TAG GTG-3'. The resulting DNA fragment was digested with XbaI and HindIII and ligated into XbaI/HindIII-digested pGEX-KG. To generate various deletion constructs of ZFM1 for repression domain mapping, PCR product corresponding to defined region of ZFM1 was amplified using pCR3.1-ZFM1-A DNA as template and two primers, which span this region and contain appropriate cloning sites at the ends. Resulting PCR fragments were digested and ligated into pSG424 to make fusion proteins with Gal4-(1-147). The regions of ZFM1 contained in each of these constructs are designated by the amino acids given in parentheses.Two-hybrid Screening--
The HL60 library, a generous gift from
Dr. G. Kalpana, was divided into six different pools with a total of
1.12 × 106 individual clones (18). To screen this
library, pBTM116-GQC plasmid was first transformed into the yeast L40
strain (19). Then the DNA from either an individual library pool or
combinations of several pools was transformed using the lithium-acetate
method (20, 21), followed by 16 h of incubation in selective
medium lacking Ura, Trp, and Leu at 30 °C to allow the expression of the HIS3 reporter gene. A fraction of the transformants were
then plated on selective medium lacking Ura, Trp, Leu, Lys, and His but
with 50 mM 3-aminotriazole (3-AT). A total of 2.85 × 107 transformants were screened from these six pools of
HL60 library in three sets of experiments. After the plates were
incubated at 30 °C for 3-5 days, 38 His+ colonies were
chosen for further study. All of them quickly turned dark blue when
they were tested for -galactosidase activity using a filter assay
(22). Plasmid DNA was recovered from most of these His+
lacZ+ colonies and electrotransformed into
Escherichia coli XL1 blue for propogation. The false
positives were then eliminated using genetic tests, in which each
positive plasmid is retransformed into L40 either alone, with pBTM116,
with pBTM116-GQC, with pBTM116-Lamin or with another irrelevant bait. A
plasmid was considered as true positive if it could activate the
expression of LacZ reporter in L40 only when it was cotransformed with
pBTM116-GQC but not in other combinations. Of the 38 His+
lacZ+ colonies, 18 were true positives. The cDNA
inserts from most of these plasmids were sequenced and used in
GenBankTM BLAST searches.
HeLa Cell cDNA Library Screening--
ZFM1-(321-484)
fragment was amplified from its parental yeast clone by PCR and labeled
as a probe by random primer labeling (Amersham Life Science). This
probe was used to screen a -ZAP HeLa cell cDNA library. The
phage from positive clones were then converted to pBluescript SK
phagemid by in vivo excision. The cDNA inserts on the
phagemid were analyzed by restriction enzyme digestion and
sequenced.
In Vitro GST Pull-down Experiments--
All GST fusion proteins,
GST-ZFM1-(321-478), GST-GQC, and GST alone were expressed in E. coli BL(21) by inducing with 0.1 mM
isopropyl-1-thio--D-galactopyranoside and coupled to
glutathione-Sepharose beads according to the manufacturer's
instructions and Ref. 4. In vitro translated GQC domain and
ZFM1-E were synthesized using a coupled transcription-translation
system (TNT lysate, Promega). GST pull-down experiments were conducted
according to Defalco and Childs (4).
Mammalian Cell Culture, Transfection, and Chloramphenicol
Acetyltransferase (CAT) Assays--
HepG2 cells are maintained in
Dulbecco's modified Eagle's medium/Ham's F-12 medium, supplemented
with 10% fetal bovine serum. The cells were transfected using
Lipofectin method as described by the manufacturer (Life Technologies,
Inc.). In a typical transfection experiment, DNA mixtures contain 2 µg of CAT reporter plasmid and 1-2 µg of pGK--gal control
plasmid. pCL-neo (Promega) is used as carrier to make sure that each
transfection mixture contained the same amount of total DNA. Cells were
harvested 48 h after transfection and lysed by the freeze-thaw
method (4). Transfection efficiencies were normalized among all the
samples according to
-galactosidase activity expressed from control
plasmid. Normalized extracts were used in CAT assays (23). The
expression of CAT activity in a given sample was quantitated using a
PhosphorImager (Molecular Dynamics). Each transfection for a given
experiment was repeated at least three times.
Western Blots-- To check the expression of transfected ZFM1 in HepG2 cells, normalized extract from freeze-thaw transfected cells used for CAT assays were resolved on 10% SDS-PAGE. The expression of transfected ZFM1 protein was detected using M2 antibody, which recognizes the FLAG-tag on its C terminus.
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RESULTS |
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Identification of ZFM1 as an SSAP-interacting Protein--
To
identify protein(s) that interact with the GQC activation domain, we
employed a modified version of the yeast two-hybrid system. The GQC
transcription activation domain of SSAP from amino acids 181 to 404 (4)
was fused in frame with the LexA DNA binding domain in pBTM116, and the
resulting fusion protein was used as bait. When LexA-GQC was
transformed into the yeast L40 strain, the resulting transformants can
grow on plates lacking histidine. -Galactosidase filter assays yield
light blue colonies due to the GQC-mediated activation of His3 and LacZ
reporters containing upstream LexA-binding sites. Therefore, this bait
alone can function as a weak transcriptional activator in yeast (Table
I). The GQC domain functions as an
extremely potent activation domain in mammalian cells (4) and in sea
urchins (3), but it was questionable if it would function in yeast due
to its amino acid content. Yeast do not support "standard"
glutamine-rich activators (24), and most yeast activation domains are
acidic (25). The GQC activation domain has a region rich in glycine and
glutamine (GQ) and a C-terminal region (C) rich in serine, threonine,
and basic amino acids (4). We also made two constructs that
individually express LexA fusions to either the GQ or the C regions. As
expected from the result in mammalian cells, neither LexA-GQ nor LexA-C
can produce
-Galactosidase activity by driving LacZ gene
expression in yeast. For this reason, we decided to use the intact
activation domain as bait and to inhibit growth of the parent strain
used for our screen by addition of the competitive inhibitor of the
histidine synthetase 3-AT to the medium. The background growth of
LexA-GQC transformants can be eliminated by adding as little as 5 mM 3-AT in the medium. Therefore, we modified the original
two-hybrid selection technique by screening library on plates
containing 50 or 100 mM of 3-AT to suppress growth of cells
not containing true interacting proteins.
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ZFM1 Binds to the GQC Domain in Vitro-- We used GST pull-down assays to confirm the direct association of ZFM1 and GQC. ZFM1-(321-478), which covers most of the region of ZFM1-A that binds to GQC in yeast was fused in frame with glutathione S-transferase to express the GST-ZFM1-(321-478) chimeric protein in bacteria. Radiolabeled in vitro translated GQC was incubated with glutathione-Sepharose beads coupled with either GST-ZFM1-(321-478) or GST alone. After extensive washing, proteins retained on the beads are eluted and separated by SDS-PAGE. The GQC domain specifically associated with GST-ZFM1-(321-478) but not with GST alone (Fig. 2A). In parallel, we did the reverse experiment. In vitro translated radiolabeled ZFM1-E was incubated with GST-GQC protein. As in the previous experiment, ZFM1-E can bind to GST-GQC but not to GST alone (Fig. 2B).
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Overexpression of ZFM1-A in Mammalian Cells Represses the Transactivation of Reporter Constructs Driven by Gal4-GQC-- The GQC domain can function as a potent transcriptional activation domain in a variety of mammalian cell lines (4). To assess the consequences of an interaction between GQC and ZFM1, we asked whether overexpression of ZFM1 can modulate the transcriptional activity of the GQC domain. When pCR3.1-ZFM1-A is transfected into HepG2 cells, we detected a 70-80-kDa protein band that corresponds to the size predicted as ZFM1-A in Western blots using M2 antibody. Moreover, the level of expression of tagged ZFM1-A correlates well with the amount of DNA transfected (Fig. 3C). The open reading frame of ZFM1 contains a potential nuclear localization signal near its N terminus (16). We confirmed the nuclear localization of ZFM1 protein by immunostaining and Western blot analysis of the nuclear and cytoplasmic fractions of HepG2 cell extract (data not shown). When increasing amounts of pCR3.1-ZFM1-A are cotransfected into HepG2 cells along with pSG424-GQC and G5E1BCAT (a CAT reporter with five Gal4 DNA binding sites upstream in its promoter), we observed that the reporter expression is repressed by expression of ZFM1-A in a dose-dependent manner (Fig. 3A). At the highest levels of ZFM1-A, transcription driven by Gal4-GQC is repressed by 4-5-fold compared with cells containing endogenous levels of ZFM1 variants. In Fig. 3, we used a level of Gal4-GQC that is within the linear range of a titration curve of Gal4-GQC activator (4). Higher levels of Gal4-GQC exhibit self-squelching, whereas lower levels give linear decreases in activation levels (4). We observe very similar 4-5-fold repression when different amount of Gal4-GQC (from 10 ng to 5 µg) are cotransfected with increasing amounts of pCR3.1-ZFM1-A (data not shown). The reporter gene used in this experiment, G5E1BCAT, has a minimal TATA-containing promoter. Without GAL4-GQC-activated transcription, we asked if the minimal activity of the reporter alone could be repressed by overexpression of ZFM1-A. The observation that the expression of the reporter alone cannot be repressed similarly indicates that repression of transcription from this reporter requires the presence of the Gal4-GQC protein (Fig. 3B).
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ZFM1 Functions as a Transcriptional Repressor when Directly Bound to the TK Promoter but Not a Basal Promoter-- The protein-protein interaction between ZFM1 and the GQC transcription activation domain raises the possibility that ZFM1 might inhibit transcription simply by sterically blocking contacts between the GQC domain and target molecules in the preinitiation complex. Alternatively, ZFM1 could function as an active repressor by directly contacting protein(s) within the transcription apparatus. To directly test the idea that ZFM1 is an active repressor, we fused full-length ZFM1-A in frame with the Gal4 DNA binding domain (pSG424-ZFM1-A) and cotransfected this expression vector with CAT reporter plasmids containing the thymidine kinase promoter plus zero, one, or five upstream Gal4 binding sites. We observed that as in the previous experiment, CAT expression is marginally repressed (less than 2-fold) when the G0TKCAT reporter was cotransfected with increasing amounts of Gal4-ZFM1-A (Fig. 5A). When a single Gal4 binding site is included, we observe that Gal4-(1-147) alone has a weak activation domain that results in about 50-75% more activity than the TK promoter alone. This activity can be repressed by Gal4-ZFM1-A to 40-50% of the endogenous activity of the TK promoter. This represents a 2-4-fold repression depending on whether one accounts for the small activation potential of Gal4-(1-147). However, when G5TKCAT is used as reporter, CAT expression is dramatically repressed. At the highest levels of Gal4-ZFM1-A tested, there is a greater than 10-fold reduction in CAT activity. These results clearly demonstrate that ZFM1 can function as a transcriptional repressor when tethered to the TK promoter by the Gal4 DNA binding domain. The marginal repression observed using TK without the Gal4 upstream activating sequence implies that overexpression of ZFM1 in this case has little if any effect via inhibition of splicing of CAT transcripts. We also tested the repression activity of Gal4-ZFM1-A fusion protein in other cell lines. We observed nearly identical titration curves in HeLa, U2OS, and NIH3T3 cells, indicating that the repression of activated transcription is not cell line-specific (data not shown).
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The Repression Domain on ZFM1 Maps to Its N-terminal Domain-- To characterize the regions on ZFM1 required for its repression function, a number of constructs were made in which different regions of ZFM1-A were fused in frame with the Gal4 DNA binding domain (Fig. 6A). We also tested the splicing variant, Gal4-ZFM1-E, for repressor activity. This ZFM1 isoform represses TK promoter activity about 2-fold better than Gal4-ZFM1-A. We then utilized a number of constructs to further localize the amino acid sequences with repressor activity. Gal4-ZFM1-(1-79) and Gal4-ZFM1-(80-528), which span the ABC and D regions of Toda et al. (16), respectively, contain repression activity that is nearly as potent as full-length protein. Additional overlapping deletion constructs within this region confirmed that the repression domain maps to the first 137 amino acids of ZFM1-A just N-terminal to the hnRNP K homology domain. Although there are many splicing variants of ZFM1, the open reading frames of all isotypes but one initiate from the same methionine and contain the entire repression domain (16, 17). Toda et al. (16) reported a novel cDNA encoding what they refer to as the HDEF protein. This subtype contains a novel noncoding H exon spliced to the D exon at nucleotide 619. This protein is predicted to initiate translation at an internal methionine (Met-116) of the ZFM1-A subtype at nucleotide 728. Although we do not have cDNAs encoding this subtype, we would predict that it may not function as a transcriptional repressor. Perhaps regulated expression of this subtype acts as a dominant negative inhibitor of other ZFM1 subtypes or it has a role in splicing. The first 137 amino acids of ZFM1-A, which constitute the repression domain, are rich in charged amino acids (34.3% charged residues) (Fig. 6B). This is a characteristic of several well characterized transcriptional repressors, such as proteins containing Krüppel-associated boxes and E4BP4 (27, 28).
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The Repression Effect of ZFM1 on GQC-mediated Transactivation Depends on the Presence of the GQC Interaction Region on ZFM1-- Our results suggest that two separate regions on ZFM1-A are involved in its ability to repress GQC-mediated transactivation: an N-terminal repression domain and a GQC interaction region in the central portion of the protein just beyond the hnRNP K homology domain and zinc knuckle motifs (Fig. 1). To assess whether the GQC interaction region is critical to mediate repression, we also tested a construct expressing a variant of ZFM1-A, which lacks the region responsible for interaction with GQC but retains the entire repression domain (ZFM1-(1-320)). As expected, ZFM1-(1-320) failed to interact with the GQC domain in the yeast two-hybrid assay (Table I). When increasing amounts of pCR3.1-ZFM1-(1-320) were cotransfected into HepG2 cells along with pSG424-GQC and G5E1BCAT, we did not observe any repression (Fig. 7A). We do not believe that this is due to poor expression of transfected ZFM1-(1-320) because we detected an overexpressed 40-kDa protein band corresponding to transfected ZFM1-(1-320) on Western blots probed with M2 antibody (Fig. 7B). Moreover, ZFM1-(1-320) fused with the Gal4 DNA binding domain (pSG424-ZFM1-(1-320)) could still repress G5TKCAT expression (Fig. 6A) and ZFM1-(1-320) contained the nuclear localization signal. These results strongly suggest that the repression effect of ZFM1-A on GQC-mediated transactivation depends on the presence of an intact GQC interaction region on ZFM1-A. The association of ZFM1-A and the GQC domain recruits ZFM1-A to the proximity of the promoter where the N-terminal repression domain may function.
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DISCUSSION |
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The GQC region (amino acids 181-404) of the transcription factor SSAP of sea urchins functions as an extremely potent activation domain in a variety of mammalian cell lines. In most lines tested, Gal4-GQC activates reporter genes 4-5-fold better than Gal4-VP16 (4). It belongs to a class of transcription activation domain rich in glycine, glutamine, serine, threonine, and basic amino acids. This activation domain most resembles those found in the proteins EWS and TLS, which are involved in translocations in many Ewing's sarcoma and liposarcoma tumors (26, 29). To better understand the mechanisms by which the GQC domain activates transcription to such high levels, we attempted to identify targets that it interacts with in the cell nucleus. We utilized the yeast two-hybrid approach and isolated several human proteins that can specifically interact with this domain. We believe that among these interacting proteins are interesting transcriptional mediators that may positively or negatively regulate the activity of the GQC domain. ZFM1, which was repeatedly picked during our screening of an HL60 cell-derived cDNA library is one such example. ZFM1 protein can interact with the GQC domain in in vitro GST pull-down experiment, and it contains several structural motifs that are characteristic of proteins involved in transcription. It contains a nuclear localization signal in its N terminus. It also contains in its N-terminal half an hnRNP K homology domain and a zinc knuckle motif, which are involved in binding to nucleic acids. Although ZFM1 has been shown to bind to RNA nonspecifically (17), it does not exclude the possibility that ZFM1 may also bind to DNA. The hnRNP K protein is an example of a protein with just such a dual function in RNA metabolism and in transcription. At least one splice variant of hnRNP K binds to the single-stranded CT element of the c-Myc promoter and functions both in vivo and in vitro as a transcriptional activator (30). In its C-terminal half, ZFM1 contains a hydrophobic proline-rich domain, which is characteristic of the activation domain for some transcription factors (31). Within this proline-rich domain, ZFM1 shows significant homology to part of Wilms' tumor suppressor gene product (WT1) and early growth response 2 proteins (EGR2) (16). Both WT1 and EGR2 are presumed to be transcription factors (32, 33). Similar to transcription activators, some classes of transcription repressors can have a modular structure with a transferable repression domain. The repression domain of ZFM1 belongs to a group of repression domains characterized by charged amino acids. A human leucine zipper family protein E4BP4 and proteins containing Krüppel-associated boxes are included in this group (27, 28).
Also contained within the proline-rich domain of ZFM1 is a PPLP motif recognized by the WW domain of the formin binding protein FBP11 and overlapping it a site recognized by the Abl Src homology 3 domain (34). The two motifs are found in all splice variants of ZFM1 including ZFM1-E, which suggests that ZFM1 is regulated by FBP11 and Abl. ZFM1 was selected during expression screening for FBP11-binding proteins and subsequently shown to bind Abl with somewhat lower affinity (34). Among the other ligands binding to the WW domain of FBP11 are the transcriptional regulator ATBF1, methyl-CpG DNA-binding protein (WBP10), a trithorax-related protein (WBP7), the HLH-like protein NDPP1 (WBP8), and SRPK1 (a serine/threonine kinase involved in phosphorylation or SR pre-mRNA splicing factors). All of these are nuclear proteins of varied function and suggest that FBP11 could modulate either the transcriptional repressor or pre-mRNA splicing activity of ZFM1.
It appears that ZFM1 is not a global repressor. Overexpression of ZFM1 has little effect on transcription driven by the TK promoter, which is regulated by CCAAT-binding proteins and SP1 (35). It also does not exert any effect on the SV40 early promoter (36) and, by inference, the transcriptional activators binding to them. However, when tethered to the TK promoter as a Gal4 fusion protein, ZFM1 can inhibit transcription. ZFM1 does not apparently function through direct interactions with general transcription factors, and, in this regard, it is a unique class of repressor protein. Several well known active repressors have been shown to function through interaction with basal transcription factors. For example, the repression activity of unliganded thyroid hormone receptor correlates to its binding to TBP and TFIIB (37, 38). Dr1 functions as repressor by interacting with TBP and thus excluding TFIIB from entering the preinitiation complex (39, 40). The evidence that ZFM1 functions differently is that it cannot repress a minimal promoter which contains only a TATA box, initiator element, and upstream Gal4 upstream activating sequence (Fig. 5B). If the repression activity of ZFM1 does have promoter selectivity, it is likely therefore that ZFM1 exerts its repression activity not by contacting TBP, TFIIB, or TFIIF74, the known general transcription factor targets of in vitro interactions with GQC (4), but perhaps by contacting and inhibiting either a TAFII protein component of TFIID or an as yet unknown coactivator of transcription that coordinates the activity of a family of activator proteins in which GQC is a member. Perhaps some of the other genes isolated in our two-hybrid screen encode this molecule.
Kramer et al. (17) have recently isolated a mammalian splicing factor SF1, which is identical to ZFM1. SF1 is one of several proteins that is required for the formation of a presplicing complex in the early stage of the process in the splicing of nuclear pre-mRNA. Recombinant SF1 protein expressed from baculovirus in insect cells can stimulate pre-splicing complex formation in a dose-dependent manner in in vitro experiments. However, we do not think that the repression activity of ZFM1 we observed is due to inhibition of splicing of reporter CAT gene pre-mRNA for the following reasons. First, overexpression of ZFM1-A only represses transcription of reporters driven by Gal4-GQC but does not significantly affect identical CAT genes driven by TK or SV40 promoters. If the repression effect is due to splicing, we should observe the same relative decrease in expression regardless of the promoter driving CAT expression. Second, the potential of ZFM1 to function as a transcriptional repressor depends on its binding to the promoter region. Gal4-ZFM1-A will inhibit CAT expression dramatically when it is cotransfected with a CAT reporter with five Gal4 binding sites upstream in its promoter (G5TKCAT). In contrast, Gal4-ZFM1-A only marginally reduces CAT expression when cotransfected with a CAT reporter with no Gal4-binding site (G0TKCAT). If ZFM1 only functions in splicing, we should observe the same degree of repression regardless of how many Gal4 binding sites are present in the promoter region of the CAT reporter. Therefore, we strongly believe that ZFM1 functions in transcription as a repressor, and we speculate that ZFM1 may have a dual role, functioning in the splicing process as well.
More and more evidence has supported the idea that mRNA splicing
and processing occurs cotranscriptionally (41-44). ZFM1 maybe an
important factor which links transcription and RNA splicing. Proteins
with dual roles in both transcription control and splicing have already
been identified. In addition to the previously mentioned hnRNP K
protein is WT1, a tumor suppressor gene product involved in Wilms'
tumor (45). WT1 has two isoforms, which differ by the presence of a
3-amino acid KTS insertion between zinc fingers 3 and 4. The KTS form
has been shown to bind to DNA and function as a transcription repressor
in transient transfection assays. In contrast, the +KTS form has been
shown to associate with splicing factors by confocal microscopy and
immunoprecipitation experiments. Similarly, it is an intriguing
possibility that the different isoforms of ZFM1 may participate in
either transcription or splicing. The subcellular localization of
individual ZFM1 isoforms under various metabolic conditions should be
carefully investigated and compared with other transcription factors
and components of the splicing machinery. We detect at least eight
different protein species in HepG2 cells using our polyclonal antibody
against the N terminus of ZFM1-A (data not shown) and at least seven
PCR products are generated from HeLa RNA amplified using primers
spanning exons 12 and 14B (17). Most of the splice variants of ZFM1
have distinct C-terminal amino acid sequences. We currently have no
functional role(s) for the C-terminal domain of ZFM1. Perhaps it is
important for recruiting additional proteins to either transcription or splicing complexes. Our future experiments will be aimed at
understanding the mechanism of ZFM1 induced repression of activated
transcription and the cellular targets it uses to lower transcription
from certain classes of activators.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Sternglanz for yeast vectors and L40 strain, Dr. G. V. Kalpana for the HL60 cDNA library, Richard Torres for help with two-hybrid screening, Dr. C. P. Yang for mammalian cell lines and help with tissue culture, Dr. A. J. Berk for CAT reporters, and Dr. Tatsushi Toda for CE9 cDNA. We are grateful to members of the Childs laboratory for helpful discussion and comments on the manuscript.
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FOOTNOTES |
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* 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.
Supported by National Institutes of Health Grant GM30333. To whom
correspondence should be addressed: Dept. of Molecular Genetics, Albert
Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3569; Fax: 718-430-8778; E-mail: childs{at}aecom.yu.edu.
1 The abbreviations used are: SSAP, stage-specific activator protein; PCR, polymerase chain reaction; 3-AT, 3-aminotriazole; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; TK, thymidine kinase; TBP, TATA-binding protein; TF, transcription factor; hnRNP, heterogeneous nuclear ribonucleoprotein.
2 D. Zhang and G. Childs, manuscript in preparation.
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REFERENCES |
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