From the
Institut für Zellbiologie
(Tumorforschung), Universitätsklinikum Essen, Virchowstrasse 173, D-45122
Essen, Germany and **Institut für
Molekularbiologie und Tumorforschung, Philipps Universität Marburg,
D-35033 Marburg, Germany
Received for publication, July 18, 2002 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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A first picture how Myc family proteins might regulate gene expression has emerged through a number of experiments in recent years. All Myc family members contain a transactivation domain (TAD)1 at the amino terminus and a basic region directly followed by a basic helix loop helix-leucine zipper motif (b-HLH-LZip) at its carboxyl terminus (810). The b-HLH-LZip domain is responsible for DNA binding and heterodimerization with partner proteins (810). One of the known proteins that interact with Myc family members through this domain is the Max protein that also has a b-HLH-LZip domain (912) (reviewed in Ref. 13). The Myc-Max complex specifically binds to E-box sequences with a preference for the sequence CACGTG (9, 10). The TAD of Myc contains two regions that are highly conserved in sequence among Myc family members and are termed Myc box I and II. Both homology regions appear indispensable for an optimal transcriptional transactivation (14).
In addition to its activities as a transcriptional transactivator, high levels of Myc expression have been correlated with the down-regulation of a number of genes. The mechanisms for a direct transcriptional repression by Myc are less well understood but thought to be independent of E-box-mediated activation (1517). Data obtained to date suggest that Myc-mediated repression is the result of an interference with proteins binding to the initiator element (INR), which is part of the core promoter (1821). In this model, Myc negatively interferes with proteins as YY1, TFII-I, or Miz-1 (1822), which transactivate target gene expression through INR binding. For instance, Miz-1-driven expression of INR-dependent reporter genes could be abrogated by Myc.
To be effective as a transcriptional transactivator, the Myc-Max complex
must recruit proteins with the ability to modify chromatin in order to allow
installment and progression of the basic transcription machinery (reviewed in
Refs. 1 and
2). Indeed, both histone
acetyltransferases and ATP-dependent chromatin remodeling enzymes (SWI/SNF)
have been reported to associate with the Myc protein
(23,
24). It has been shown that
TRRAP, a large protein that interacts with the N terminus of Myc, is part of a
multiprotein complex with histone acetyltransferase activity
(2528).
Moreover, several other proteins are known today that interact with the Myc
TAD and may be involved in the regulation of its activity: p107, Bin-1, and
MM-1
(2931).
Of particular interest in this respect is MM-1, which requires Myc box II
sequences for binding to Myc. Most recently, it was demonstrated that MM-1 can
bind to the transcriptional co-repressor Tif-1, which is known to
recruit the chromatin-associated factors HP-1 and histone deacetylases. This
suggested a mechanism for Myc-mediated transcriptional repression
(29,
31,
32) that involves
E-box-dependent gene regulation.
In many cases, the isolation of Myc regulators has relied on yeast interaction cloning strategies (33). Although the conventional yeast two-hybrid system has been very powerful in the isolation of protein-protein interaction partners in a number of cases, its use with transcription factors is limited, because their transcriptional activation domains strongly interfere with the transcriptional selection mechanism of the system itself. In our present study, we have used a novel yeast interaction cloning strategy, the Ras recruitment system (RRS) (3438), to identify proteins that bind to the TAD of Myc. The RRS selection mechanism is independent of transcriptional regulation and relies on the direct activation of the mitogenic Ras pathway in yeast cells (37). In the RRS, the sequence that is used as a bait to isolate a partner protein is cloned as a fusion protein with the human Ha-Ras protein (37). The expression library that is to be screened with this "bait" encodes fusion proteins with the Src myristoylation signal, which will lead to attachment of these fusion proteins to the plasma membrane. If protein-protein interaction takes place, the bait-Ras fusion is recruited to the membrane and activates Ras-dependent signal transduction. This allows growth of a yeast mutant that is defective in this pathway due to a mutation in the endogenous Ras guanyl exchange factor Cdc25. Since no transactivation is needed to select for positive yeast clones, an endogenous transactivation property of the "bait" does not interfere with the selection process (37).
Here we have used the 262 N-terminal amino acids of c-Myc as a bait in the RRS, and we were able to isolate a novel KRAB box-containing zinc finger protein, Krim-1, that binds to the Myc TAD and can negatively influence Myc functions.
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EXPERIMENTAL PROCEDURES |
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Yes2-derived PlasmidsHuman c-Fos was fused to Src myristoylation signals (M-Fos).
ADNS-derived PlasmidsHuman c-Myc (amino acids 1262; ADNS-Myc2625'Ras) and human c-Jun leucine zipper (amino acids 249331; JZ-5'Sos) were fused to Ras (amino acids 1185) at the amino terminus.
cDNA LibraryDouble-stranded cDNA was generated from poly(A)+ RNA of the GC rat pituitary cell line using random primer adapters and the ZAP-cDNA synthesis kit (Stratagene). The cDNA was directionally ligated with Yes2 plasmid containing the Src myristoylation sequences. The library represents 2 x 106 primary transformants. To reduce the number of isolated Ras-containing clones from the library, the strain used for screening contained an expression plasmid with mammalian GAP (Ras GTPase-activating protein) (36).
Ras Recruitment Screening and Mutational Analysis
RRS Library Screening with c-Myc262 BaitRas recruitment
screening was performed essentially as described
(37). Briefly, for the
construction of the Myc262-RRS bait plasmid the transactivation domain of
c-Myc corresponding to amino acids 1262 was amplified by PCR from a
human c-Myc cDNA. This fragment was subcloned in frame into
HindIII/SmaI-cut plasmid pADNS-Ras(mut), containing
Ha-Ras(L61) cDNA lacking the CAAX-farnesylation signal. Yeast transformation
and manipulation was performed as described
(36,
37). We used a cDNA expression
library (34), constructed from
mRNA of the GC cell line (rat pituitary tumor). To generate a bait strain, the
Myc262 bait plasmid was cotransformed with the plasmid mGAP
(36) into the yeast strain
cdc25-2 and grown at 25 °C on minimal medium lacking
leucine and tryptophan. A starter culture from a single colony of this bait
strain was grown in 100 ml of minimal medium, and then 2x 200 ml of YPAD
medium was inoculated to A600 = 0.4 and then grown at 25
°C for 4 h. Then 60 µg of library plasmid DNA were transformed into the
bait strain and grown at 25 °C on agar plates with minimal medium lacking
leucine, tryptophan, and uracil for 34 days. For selection of clones
with interacting protein pairs, cells were replica-plated to agar plates with
galactose minimal medium (leu, trp, ura) and grown at 36
°C for 6 days. These clones were streaked to glucose minimal medium
(leu, trp, ura), incubated at 25 °C for 34
days, and then tested for their dependence on expression from the
galactose-inducible library plasmid. Colonies were replica-plated to agar
plates containing galactose or glucose (both plates leu, trp,
ura) and grown for 34 days at 36 °C. Clones that showed
glucose-repressible and galactose-inducible growth were used further for the
isolation of the library plasmid. To test dependence on the bait, the isolated
library plasmids were reintroduced into the cdc25-2 yeast
strain in combinations with either the original screening bait or empty or a
heterologous bait. One library clone was identified to confer growth in a
Myc262 bait-specific manner at 36 °C.
Ras Recruitment Screening with Krim-1 1177 as a BaitFor RRS screening, a fragment with the N-terminal amino acids 1177 of Krim-1 was amplified by PCR (primers: Krim bait-T (5'-GCATAAGCTTGCCGCCATGGAGCCAGTGACCTTTG-3') and Krim bait-B (5'-ATACCCGGGATCGGAGGTGAAACTCCTAGATGCTTCG-3'); the Krim bait-B primer mutates an endogenous HindIII site silently). This fragment was inserted via HindIII/SmaI into the RRS bait vector pADNS-Ras-(mut) followed by introduction of this Krim-177-Ras bait plasmid and the mGAP expression plasmid into the yeast cdc25-2 strain. This bait strain was used for RRS screening of the GC cell cDNA expression library as described above.
RRS Test of Internal Deletions of the Myc262 BaitA series
of CMV expression plasmids with internal deletions of c-Myc ("Mbox
I" (4563); "large T/E1A"
(
109126); "E1A + Mbox II" (
104136);
"Mbox II" (
128143)) and serine substitution mutants
(residues 58 and 62 changed to Asp58/Asp62 or
Ala58/Ala62) was used to obtain N-terminal fragments
corresponding to c-Myc amino acids 1262. These N-terminal fragments
were amplified by PCR (primers: Myc-H3-ATG
(5'-TAGAAGCTTACCATGCCCCTCAACGTTAGCTTC-3') and Myc262-Sma-B
(5'-TATCCCGGGGATTTCTTCCTCATCTTCTTG-3')) and inserted as
Hin-dIII/SmaI fragments into the RRS bait vector
pADNS-Ras(mut) cut with the same restrictases. Each of these bait constructs
was introduced with the Krim-1 cDNA library clone into cdc25-2 cells and
tested for activation of the RRS at 36 °C.
RACE Amplification
First strand cDNA preparationFor preparation of first
strand cDNA, 5 µg of total RNA (from rat testis, bone marrow, or intestine)
and 50 pmol of primer (for 5'-RACE, 5'-AAGGTCTCCTTCATCACGTC; for
3'-RACE dT17 adapter primer, 5'-GACTCGAGTCGACATCGA-T17) was mixed
in 8.5 µl of reverse transcription buffer, and heated to 70 °C for 10
min. Then dithiothreitol, RNasin (20 units; Promega), and 200 units of reverse
transcriptase (Superscript II; Invitrogen) was added in the recommended
buffer. After incubation at 49 °C (1 h), reverse transcriptase was
heat-inactivated (70 °C; 15 min) and incubated with 2 units of RNase H
(Invitrogen) at 37 °C for 20 min. This first strand cDNA preparation was
purified on Qiaquick PCR purification spin columns (Qiagen, Hilden, Germany)
and eluted in 50 µl of elution buffer. Only for 5'-RACE, this first
strand cDNA was dA-tailed using 1 mM dATP, 15 units of terminal
deoxynucleotide transferase (Life Technologies) in a 75-µl volume (37
°C; 30 min) and then heat-inactivated (70 °C; 10 min).
5'-RACE AmplificationThe missing 5' region of the Krim-1 cDNA was obtained from dA-tailed cDNA by two-step amplification ("Synergy" Taq polymerase; Genecraft) using the common dT17 adapter primer and a nested pair of Krim-1-specific primers (step 1, 5'-AAGGTCTCCTTCATCACGTC; step 2, 5'-GAATCCAACATAGTCCACTC). The PCR products were purified after separation by agarose gel electrophoresis, blunted, and subcloned. The 3'-RACE products were obtained using dT17 adapter-primed cDNA as template. After two-step amplification using a common dT17 adapter primer and a pair of nested Krim-1-specific primers (step 1, 5'-CCTATTCCAGTGCTTGCTAT; step 2, 5'-CTTTCCCCAGATCCCTTCAC), the resulting PCR products were subcloned as described above. To generate full-length Krim-1A and -1B cDNAs, the insert of the Krim-1 library clone was assembled with the corresponding 3'-RACE fragments by ligation of partial HindIII-digested DNA fragments.
Expression Analysis by Real Time PCR
Total RNA was isolated from various rat tissues by homogenization of the
corresponding tissues in isothiocyanate buffer and subsequent column
purification (RNeasy; Qiagen, Hilden, Germany) according to the manufacturer's
protocol. Then 4.5 µg of RNA were treated with 5 units of DNase (RNase-free
DNase; Roche Applied Science) in 25 µl of first strand buffer for 20 min
(25 °C). DNase digestion was terminated by the addition of 0.8 µl of 75
mM EDTA, and then 2 µl of random primer (hexamer; 200 ng/µl;
Roche Applied Science) was added and immediately incubated at 70 °C for 10
min. This RNA was divided in two 12.5-µl samples for subsequent reverse
transcription (with and without reverse transcriptase). To each sample, 2.5
µl of first strand buffer (5x; Promega), 0.5 µl of 60
mM MgCl2, 1 µl of 1 mM dNTPs, 0.5 µl of
RNasin (20 units/µl; Promega), and 2 µl of H2O was added and
mixed, followed by the addition of 1 µl of reverse transcriptase (Moloney
murine leukemia virus reverse transcriptase point mutant; 200 units/µl;
Promega) to one sample and 1 µl of H2O to the other as negative
control and subsequent incubation for 1 h at 49 °C. Samples were
heat-treated (70 °C, 10 min) and digested with 1 µl of RNase H (1
unit/µl; TaKaRa) at 37 °C for 30 min. Real time PCR was performed using
a GeneAmp 5700 SDS thermal cycler (PerkinElmer Life Sciences) and SYBR Green
Taq polymerase mix (SYBR Green PCR Master Mix; PerkinElmer Life
Sciences). Each amplification reaction contained 10 µl of SYBR Green Master
Mix, 1 µl of cDNA, 0.5 µl of each primer (25 pmol/µl GAPDH primers or
100 pmol/µl Krim-1 primers), and 8 µl of H2O. Primer
sequences were as follows: Krim-SQ4B
(5'-GTGTCTCTCCGTTTTCCTTCATACC-3'), Krim-Y5-426T
(5'-GAACTTCTTTGCGAAGACATGAAC-3'), rGA-PDH-T
(5'-ATCCGTTGTGGATCTGACAT-3'), and rGAPDH-B (5'-ACCTG
GTGCTCAGTGTAGCC-3'). For amplification, the following cycling parameters
were used: initial denaturation at 95 °C for 10 min and then 42 cycles
with 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. After
each experiment, the amplification product was analyzed by thermal
dissociation for the generation of a single product.
Immunoprecipitation
COS-7 cells or HeLa cells were plated on 90-mm dishes and transiently
transfected with 12 µg of SR-HA-Krim and SP-c-Myc plasmids using 20 µl
of the lipofection reagent RotifectTM (Rotilab) or with the calcium
phosphate technique. Cells were refed with complete medium after 6 h and were
harvested after another 18 h. Cells were washed with PBS and then scraped off
the plate in 400 µl of low salt lysis buffer (20 mM Hepes, pH
6.8, 5 mM KCl, 5 mM MgCl2, 0.5% Nonidet P-40,
0.1% sodium deoxycholate, and protease inhibitors). Then the cells were
completely disrupted by Dounce homogenization (30 strokes), centrifuged for 5
min (microcentrifuge; 3600 rpm, corresponding to 1000 x g). The
cytosolic supernatant was discarded, and the remaining crude nuclear pellet
was extracted with high salt lysis buffer (500 mM NaCl in low salt
lysis buffer), and incubated for 10 min on ice. After adding an equal amount
of low salt lysis buffer, the lysate was cleared by centrifugation at 13,000
rpm (microcentrifuge; 4 °C for 15 min). For immunoprecipitation, 8 µl
of -c-Myc polyclonal antibody (C-19; Santa Cruz) or 8 µl of
-Cdk4 polyclonal antibody (H-22; Santa Cruz) and 15 µl of protein
A-agarose (50% slurry previously blocked in bovine serum albumin) was added to
the lysate and rocked for 1 h at 4 °C. Immune complexes were recovered by
centrifugation (2500 rpm; microcentrifuge; 4 °C) and washed four times
with 1.3 ml of medium salt lysis buffer (250 mM NaCl in low salt
lysis buffer). Then recovered proteins were separated on 10%
SDS-polyacrylamide gels and analyzed by Western blotting using monoclonal
-HA antibody (12CA5).
In Vitro Protein Binding Assay
For the preparation of affinity-purified glutathione S-transferase
fusion proteins, 400-ml cultures of Escherichia coli carrying the
plasmids pGST-Myc 1262, pGST-Jun 1223 (kind gift from M. Karin,
University of California San Diego), and pGEX4T2 (Promega) were grown to an
A600 = 0.5, induced by the addition of
isopropyl-1-thio--D-galactopyranoside to an end concentration
of 0.2 mM, and grown for another 3 h. Cells were harvested by
centrifugation (GSA, 4500 rpm, 4 °C), and the cell pellet was resuspended
in 10 ml of NETN buffer (20 mM Tris-Cl, pH 7.5, 100 mM
NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 2 mM dithiothreitol,
and protease inhibitors). After the addition of 30 µlof lysozyme (10
mg/ml), cells were incubated on ice for 45 min and sonicated (6 x 15 s
with 30-s incubation on ice), and the lysate was cleared by centrifugation
(JA-20; 9500 rpm; 4 °C). To the supernatant, 200 µlof GSH-agarose beads
(50% slurry; Sigma) was added and incubated on a rocker platform for at least
4 h. GSH-agarose beads with bound proteins were washed three times with
NETN-buffer. Protein concentration on GSH-beads was normalized to 20 µg of
protein in 15 µl of GSH-beads by the addition of plain GSH-slurry. For
in vitro binding reactions, 15 µlof GSH-agarose beads (with 20
µg of bound protein) was mixed with 400 µl of binding buffer (50
mM Tris-Cl, pH 8.0, 200 mM NaCl, 1 mM EDTA,
0.5% Nonidet P-40, 0.2 mg/ml bovine serum albumin), and 2.5 µl of
[35S]methionine-labeled in vitro translated Krim-1 protein
(2 µg of YM-Y5A Library-Plasmid; TNT® coupled reticulocyte lysate
system; Promega). The binding reaction was incubated on a rocker platform for
1.5 h (4 °C) and washed five times with binding buffer. Bound proteins
were analyzed by 12% SDS-polyacrylamide gel electrophoresis and
autoradiography.
Mammalian Two-hybrid Assay
Expression vectors that code for fusion proteins between the DNA binding
domain of Gal4 and the protein of interest (i.e. c-Myc or Krim-1)
were cotransfected with vectors, allowing the expression of a putative partner
protein or subdomains of this protein fused to a protein that is able to
transactivate transcription (here the transactivation domain of the VP16
protein from herpes simplex virus) as well as reporter gene constructs into
COS-7 cells. Cells were seeded at 50,000/well into a 12-well plate and
transfected with a total amount of 1 µg of DNA. The reporter gene construct
contained a cassette with four Gal4 DNA binding sites 5' of a thymidine
kinase minimal promoter driving the luciferase gene. After transfection, cells
were washed with PBS and lysed in 100 µl of 1% Triton X-100, 1
mM dithiothreitol. 10 µl of the lysate was analyzed for
luciferase activity as previously described
(3941).
-Fold induction was calculated by dividing the values obtained with the
individual mutants by the value obtained with the Gal4-DBD construct alone.
Mean values were calculated from at least three (mostly four or five)
independent experiments with triplicate measurements each.
Subcellular Localization
Subcellular FractionationA series of C-terminal deletion
clones of Krim-1 was generated, which contained three N-terminal fused
hemagglutinin (HA) tags for immunodetection. Deletions were generated by PCR
using the plasmid SK-HA-Krim-1A as template and a T7 promoter primer as a
common 5' primer. The extent of the C-terminal deletion was specified by
the used Krim-specific 3'-primers ("1-spacer" (aa
1184), Y5184BHSNot:
5'-TGTGCGGCCGCTAGGATCCGAAAGTTCCCTCATAATCG-3'; "1-Zn1"
(aa 1214), Y5214BH:
5'-ACGGGATCCTTGAATGTGACTTCTCTCA-3'; "1-Zn2" (aa
1242), Y5242BH: 5'-AGGGGATCCACCAGTGTGGATTCTCTCA-3';
"1-Zn3" (aa 1270), Y5270BH:
5'-CCCGGATCCTCCAGTGTGAGTCCTCTCA-3'; 1-Zn5' (aa 1344),
Y5344BHSNot:
5'-ATCGCGGCCGCTAGGATCCGTATGCAGAACATTTAAACG-3'). The deleted Krim-1
fragments were inserted as N-terminal HA-tagged fragments into a
KpnI/BamHI-cut pcDNA3 vector with a stop codon 3' of
the BamHI site. For the expression of the fusion proteins, the
corresponding plasmid (13 µg) was introduced into COS-7 cells (10-cm dish)
by lipofection with 20 µl of Rotifect reagent (Roth, Germany) according to
the manufacturer's protocol. Cells were dislodged from the plates by short
trypsin digestion and washed with phosphate-buffered saline. Cytoplasmic
extracts were prepared by careful resuspension of the cells in 1000 µl of
CE buffer (20 mM HEPES buffer, pH 7.8, 10 mM KCl, 0.2
mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 1 mM
dithiothreitol, proteinase inhibitors), incubation on ice for 10 min, and
centrifugation (microcentrifuge, 10,000 rpm, 20 s). The supernatant contained
cytoplasmic extract. The pellet was resuspended in 1000 µl of CE buffer and
sedimented as above. This wash step was repeated. Nuclear extract was prepared
by resuspending the washed pellet in 400 µl of NE buffer (CE buffer with
500 mM NaCl), incubation for 10 min at 4 °C and subsequent
agitation on a roller platform (4 °C) for another 10 min. The nuclear
lysate was cleared by centrifugation (microcentrifuge, 14,000 rpm, 15 min, 4
°C), and the pellet was discarded. An equal amount of the obtained
extracts was separated by SDS-polyacrylamide gel electrophoresis and analyzed
by Western blotting using -HA monoclonal antibody. Cells were seeded on
glass coverslips in 12-well plates, and after 12 h they were transfected by
calcium phosphate precipitation with 500 ng of GFP fusion construct. After 1.5
days, the cells were rinsed twice with PBS, fixed for 10 min in methanol, and
rinsed carefully twice with PBS. Then cells were stained for 20 min with PBS
containing propidium iodide and RNase A (250 µg/ml) and then rinsed three
times with PBS. Coverslips with cells were sealed with ProLong Antifade
(Molecular Probes, Inc., Eugene, OR) and analyzed by laser-scanning microscopy
(LSM510; Zeiss).
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RESULTS |
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Krim-1 Is a Ubiquitously Expressed Nuclear ProteinRNA from various rat tissues was extracted for quantitative PCR with primers specific for Krim-1A and GAPDH. The Krim-1 expression levels were calculated after normalization over the values obtained for GAPDH. Relative to the Krim-1 levels found in thymus (arbitrarily set to 1), all other organs tested express lower levels except spleen, which contained the highest level (Fig. 2a). Using a multiple tissue Northern blot (Stratagene) and a probe covering rat sequences from the spacer region of Krim-1 between the KRAB domains and the zinc finger domains, we detected two transcripts at around 2.4 and 2.7 kb (Fig. 2b). Both mRNA species were present in all tissues, but both showed highest levels in spleen (Fig. 2b, lane 3). A probe specific for Krim-1A covering sequences deleted in Krim-1B revealed that the major 2.4-kb RNA species is specific for the longer isoform of Krim-1 (Fig. 2b). A transcript specific for Krim-1B was not readily detected. The longest cDNAs that were obtained for Krim-1A and Krim-1B were 2113 and 1862 nt, respectively, suggesting that both still lack sequences presumably in the 3'-untranslated region. Interrogating the Celera data base and the NCBI-based expressed sequence tag libraries revealed the existence of a murine cDNA clone with the potential to encode a protein of 534 amino acids with over 94% sequence identity to the rat Krim-1A (Fig. 2c). The zinc finger region was entirely conserved, with no amino acid exchange, and the KRAB domain contained only one altered amino acid, indicating that this so far anonymous cDNA encodes the bona fide murine Krim-1 homologue (Fig. 2c). Although the rat Krim-1 cDNAs lacked a stop codon upstream of the AUG translation initiation triplet, the murine cDNA contained a stop codon 51 nt 5' of its AUG, suggesting that the 533-aa Krim-1A and the 451-aa Krim-1B molecules indeed represent the full-length proteins, which adds 6 amino acids to the N terminus of the originally identified sequence (see Fig. 1). According to the Celera data base, the murine Krim-1A gene is located on chromosome 19, and the coding region spreads over four exons, of which the second encodes the entire KRAB domain (Fig. 2d). In rats, the 5'- and 3'-untranslated regions are identical in Krim-1A and Krim-1B, suggesting that both mRNA molecules originate from the same genomic locus and very probably are generated by alternative splicing, although the splice site is located in an exon (Fig. 2d). The closest human homologue of Krim-1A that is known so far is the KRAB box containing protein Kox 13 with 41% sequence identity and an as yet unannotated sequence with 47% sequence identity on the amino acid level.
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To test the subcellular localization of the Krim-1 proteins, the coding
sequences were inserted into an expression vector that allowed the production
as GFP fusion proteins under the control of a CMV promoter. Transient
transfection into COS-7 cells demonstrated that both Krim-1A and Krim-1B
reside in the nucleus (Fig.
3a). In the next step, we constructed expression vectors
for the production of HA-tagged deletion mutants of Krim-1A and transfected
them into NIH 3T3 cells. Cytoplasmic and nuclear extracts were prepared after
transfection, separated by SDS-PAGE, and transferred to nitrocellulose
membranes. Immunoblot analysis with an -HA antibody demonstrated that
only the full-length Krim-1A is entirely located in the nucleus
(Fig. 3b,
arrowheads). Whereas Krim-1B is mostly nuclear similar to Krim-1A, a
small percentage of the Krim-1B protein can be found in the cytoplasm
(Fig. 3b,
arrowheads). In contrast, mutants that contain the KRAB box domain,
the spacer region, and the first three zinc fingers are predominantly
cytoplasmic. Only when regions containing zinc fingers 4 and 5 are present do
the proteins show significant transport into the nucleus
(Fig. 3b,
arrowheads), suggesting that the C-terminal zinc finger domains are
critical for nuclear localization.
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The Krim-1 Protein Interacts with Sequences Covering the Homology Region Myc Box IITo find further support for the genetic evidence described above that suggests an interaction between Krim-1 and c-Myc, biochemical assays were performed. First, the sequences coding for the c-Myc N-terminal 262 amino acids were appended to the glutathione S-transferase (GST) coding sequence and expressed as a fusion protein that binds avidly to glutathione-agarose beads. Radioactively labeled Krim-1 proteins that span the region of the isolated library clone (aa 1427), the full-length Krim-1 protein without KRAB box domain (aa 65527), and two truncated Krim-1 mutants (aa 105527 and aa 171527) were generated by a coupled in vitro transcription/translation reaction (Fig. 4a). GST-Myc (aa 1262) protein and as a control GST alone and GST-Jun (aa 1223) containing the first 223 amino acids of c-Jun bound to glutathione-agarose beads were mixed with radiolabeled Krim-1A proteins and were washed extensively. Labeled proteins bound to immobilized GST fusion proteins were detected by SDS-polyacrylamide gel electrophoresis. Whereas radiolabeled Krim-1A 1427 and Krim-1A 65527 could be specifically precipitated with GST-Myc (262 amino acids), no signal was detected with GST alone or with GST-Jun (223 amino acids) (Fig. 4a). The truncated proteins Krim-1A 105527 and 171527 lacked specific interaction with Myc GST, very likely due to the predominance of the 12 zinc finger domains (Fig. 4a).
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To show that Krim-1 proteins could also be purified from c-Myc complexes in
mammalian cells, COS-7 cells were transiently transfected with expression
vectors for HA-tagged Krim-1A or Krim-1B, along with c-Myc or the
G1 phase-specific kinase subunit Cdk4 as a control. Lysates from
these cells were incubated with peptide-specific polyclonal antibodies
directed against c-Myc or Cdk4. The resulting immune complexes were collected,
extensively washed, and separated by SDS-PAGE. Immunoblot analyses with
-HA antibodies demonstrated that Krim-1A and Krim-1B are able to form a
complex with c-Myc but not with Cdk4 (Fig.
4b). Next, we wished to more closely dissect the region
in the N terminus of c-Myc that could be responsible for the interaction with
Krim-1 proteins. We used the yeast RRS and generated a series of constructs in
the pADNS bait vector for expression of fusion proteins between various Myc
mutants (40) and the Ha-Ras
sequence already used for the original bait
(Fig. 4c). The Myc
mutants were made on the basis of the original first 262 N-terminal amino
acids and contained small deletions covering either Myc box I (aa
4563), Myc box II (aa 128143), the region responsible for the
interaction with the adenoviral protein E1A (aa 109126), or both Myc
box II and the E1A-interacting region aa 104136
(Fig. 4c). In
addition, we have also used the mutants Ala58/Ala62 that
delete putative phosphorylation sites at aa 58 and 62 and
Asp58/Asp62, mimicking constitutively phosphorylated
residues (Fig. 4c).
These constructs were cotransfected with the Krim-1-containing, originally
isolated Yes library plasmid (clone Y5a) into the yeast strain
cdc25-2, and the cells were grown at the restrictive
temperature (36 °C). Deletion of the Myc box I (aa 4563) and the
E1A-binding region (aa 109126) did not have any effect on Krim-1
binding. However, mutants lacking sequences covering Myc box II and the
N-terminal adjacent region did not interact with the Krim-1 protein
(Fig. 4c), suggesting
that the interaction of Krim-1 with Myc requires sequences of Myc box II.
The Transcriptional Corepressor Tif1 Stabilizes Krim-1
ExpressionTo search for proteins that interact with the KRAB box
domain of Krim-1, we used the KRAB domain as a bait in a screen with the
GC-library. Several bait-specific clones were obtained that contained the same
insert and encoded the rat Tif-1
/Kap-1 protein (data not shown). This
protein contains multiple subdomains and has already been described as a
cofactor with a role in transcriptional regulation that binds to Krab domains
(32,
45,
46). We have obtained the
FLAG-tagged full-length cDNA in a CMV-dependent expression vector (kind gift
of W. Schaffner, University of Zürich, Switzerland) and used it to
transfect HeLa cells along with a c-Myc expression vector and vectors for
HA-tagged Krim-1A and Krim-1B in order to confirm the interaction between
Krim-1 proteins and Tif-1
/Kap-1 by co-immunoprecipitation. We observed
that the HA-Krim-1 proteins were expressed at significantly higher levels when
Tif-1
was co-expressed (Fig.
5a). In addition, immunoprecipitations with
c-Myc antibodies (9E10) delivered more c-Myc-Krim-1 complexes in
the presence of Tif-1
than in the absence of this protein
(Fig. 5a). Both
findings suggest a stabilizing function of Tif-1
for both Krim-1
proteins.
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The Second N-terminal Zinc Finger of Krim-1A Is Essential for the
Interaction with c-MycTo quantify the binding of Krim-1A or Krim-1
mutants and to identify the region in Krim-1A that binds to the Myc N
terminus, we performed a mammalian two-hybrid assay. For this assay, we
generated expression constructs that allowed the production of a fusion
protein between Myc and the transactivating domain of VP16. The cDNAs for
Krim-1A and several Krim-1A mutants were expressed as fusions with a GAL4 DNA
binding domain (GAL4-DBD). The interaction between Krim-1A or Krim-1A mutants
and Myc was measured through the transcriptional activation of a luciferase
reporter construct regulated by four GAL4-DBD-binding sites in the promoter
(Fig. 5b). The correct
expression of the VP16-Myc and the Gal4-Krim-1 fusion constructs was
determined by Western blotting (data not shown). Clearly, the Krim-1A wild
type protein and the mutant 65360 are still able to interact with Myc
(Fig. 5b), whereas all
other mutants, including a mutant comprising aa 65360 where
specifically zinc finger 2 was destroyed (Znf265360) by
replacing the cysteines in positions 219 and 222 against arginines, failed to
show such an interaction (Fig.
5b). This suggests that the zinc finger region and in
particular an intact zinc finger 2 in Krim-1 is needed to bind the Myc TAD.
Next, we transfected HeLa cells with expression vectors for FLAG-Tif-1
and HA-Krim-1A or the full-length HA-Krim-1A protein with the mutations in the
zinc finger 2 (1AZn2) or with an expression vector for the
irrelevant HA-tagged Akt kinase as a control. Co-immune precipitation analyses
with an
c-Myc antibody were developed with an
-HA
antibody and showed that indeed the Krim-1A mutant with the destroyed zinc
finger 2 as well as the irrelevant Akt kinase did not interact with the
endogenous c-Myc protein (Fig.
5c, lanes 5 and 6). However, both
Krim-1A and Krim-1B could be precipitated with antibodies against the
endogenous c-Myc protein, without a previous transfection of a c-Myc
expression vector (Fig.
5c, lanes 3 and 4). In addition, immune
precipitations with an
HA antibody and development with an
-FLAG antibody confirmed that both Krim-1 proteins can interact with
Tif-1
(Fig.
5c).
Krim-1 Proteins Are Able to Negatively Regulate Myc FunctionsIt has been demonstrated before that KRAB box-containing proteins are transcriptional transrepressors (42). We wanted to test whether the KRAB box domain of Krim-1 has transrepressing activity as compared with, for example, the KRAB domain of the Kox-1 zinc finger protein. Therefore, the Krim-1 KRAB domain was expressed as a fusion with the DNA-binding domain of the doxycyclin-dependent Tet repressor protein (TetR-DBD). As expected, a TetR-DBD-Kox fusion protein was able to repress the transcriptional activity of a Tet operator-dependent CMV promoter element in the absence of antibiotic (Fig. 6a). To show that this effect was dependent on DNA binding, repression could be relieved by the addition of doxycyclin, which renders the TetR domain unable to bind to DNA (Fig. 6a). The TetR-Krim fusion showed a very similar activity, confirming that the Krim-1A or Krim-1B proteins contain bona fide KRAB domains with strong repressor activity, which is dependent on DNA binding (Fig. 6a). The repressor activity of both Kox-1 or Krim-1 KRAB domains was not affected by the presence of c-Myc (data not shown). Similarly, none of the Krim-1 proteins showed any effect on the transcription of the adenovirus major late promoter (17), the ferritin promoter (43), or the p15ink4b promoter (21) that are all thought to be repressed by Myc through interaction with the INR element (data not shown). Next we tested whether Krim-1 could alter the transactivating function of c-Myc, and we transfected a Gal4 binding site-dependent luciferase reporter (H17-MX-Luc) construct together with a plasmid expressing a fusion of the Gal4 DNA binding domain and the c-Myc N terminus (TAD). The Gal4-Myc fusion was able to activate the reporter gene about 45-fold, and coexpression of either Krim-1A or Krim-1B could repress this activation (Fig. 6b). Similar results were obtained when a luciferase reporter construct driven by the E-box-containing promoter of the prothymosin gene (39, 44), which represents a bona fide Myc target gene promoter, was cotransfected together with plasmids expressing Myc or the Krim-1 proteins. (Fig. 6, c and d). Both Krim-1A and Krim-1B were able to repress E-box-dependent transcriptional activation of the prothymosin promoter in HeLa cells and in HepG2 cells (Fig. 6, c and d). Since HeLa cells contain very high endogenous levels of c-Myc, the E-box-dependent activation is dependent on limiting levels of Max protein (Fig. 6c). In HepG2 cells, this effect was also seen with Myc alone and without transfection of a Max expression construct. Coexpression of Krim-1A led to a repression of E-box-dependent transactivation by Myc to about 50% and of Krim-1B to even 10% in HeLa cells (Fig. 6c). In HepG2 cells, both proteins led to a repression of reporter gene activity to about 60% (Fig. 6d). The activity of SV40 promoter-driven reporter gene constructs was unaffected by Krim-1 proteins, indicating that the observed effects on the prothymosin and Gal-binding site reporter construct are not due to a nonspecific inhibition of transcription (data not shown).
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Another well established function of c-Myc is its ability to cooperate with activated Ha-Ras in the malignant transformation of rat embryo fibroblasts (REFs) (5). The cotransfection of Myc and Ha-Ras expression plasmids leads to the formation of foci of transformed cells. When constructs that drive the expression of HA-tagged Krim-1A or Krim-1B were cotransfected along with c-Myc and the oncogenically activated Ha-Ras mutant, we found that both Krim-1A and Krim-1B were able to repress focus formation (Fig. 7, a and b). Although both Krim-1 expression constructs were able to direct the production of almost identical levels of both proteins in primary REFs, the effect of Krim-1B on focus formation was more pronounced (Fig. 7, a and b). None of the foci that arose from Myc/Ras/Krim transfection and that were established as cell lines contained measurable Krim-1 protein, suggesting that foci do not form in the presence of Krim-1 proteins (not shown). In focus formation experiments using SV40 large T-antigen (SV40LT) and Ha-Ras, the coexpression of Krim-1 proteins did not show any effect (Fig. 7, a and b). Since the SV40 large T-antigen uses different pathways to transform primary cells than Myc, this finding suggested that the inhibitory effect of Krim-1B on focus formation is specific for the Myc function and does not affect the transformation of primary fibroblasts in general (e.g. due to toxic effects). In addition, when the Krim-1AZnf265360 mutant that was no longer able to interact with Myc was used in cotransfections with Ha-Ras, a suppression of focus formation was markedly relieved in four of five independent experiments (Table I).
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DISCUSSION |
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To investigate in more detail the mechanisms that are involved in c-Myc-mediated transactivation or transrepression, we have used a novel yeast interaction cloning system, the RRS, to identify proteins that bind to the TAD of c-Myc. We were able to isolate a novel 527-aa widely expressed nuclear protein, Krim-1, that contains a KRAB domain at the N terminus and 12 C2H2 zinc finger domains at its carboxyl-terminal end. The protein exists in two isoforms: a longer Krim-1A and a shorter Krim-1B protein that only contains 9 instead of 12 zinc finger domains. Both Krim-1A and Krim-1B require sequences of the Myc box II homology region to be able to bind to c-Myc. The interaction between Krim-1 and c-Myc proteins was also supported by biochemical evidence. Krim-1-c-Myc complexes could be precipitated from mixtures of in vitro translated Krim-1 and GST-Myc fusion proteins but also from COS-7 or HeLa cells that overexpress both proteins or only Krim-1 proteins after transient transfection. In addition, we could demonstrate that both proteins interact in vivo in a mammalian two-hybrid assay. Our mutational analysis demonstrated that the Krim-1 protein requires the second zinc finger domain for this interaction; the KRAB box itself was found to be dispensable for c-Myc binding.
The KRAB domain is a conserved motif at the N terminus of a large number of zinc finger proteins and comprises a 75-amino acid stretch that can be divided into the KRAB A domain, a 45-aa-long minimal repression module, and the KRAB B domain (42). The Krim-1 proteins contain the KRAB A domain only. KRAB box-containing zinc finger proteins have been shown to act as very potent transcriptional repressors that are dependent on DNA binding (32, 4652). Our experiment with TetR-Krim fusion proteins clearly demonstrated that Krim-1 is no exception to this rule and also bears this repressor activity. Since the zinc finger domains of KRAB box proteins can interact with a cognate DNA recognition sequence (51) and thus at least have the potential to act as upstream transcriptional regulators, the interaction between Krim-1 and c-Myc could suggest that c-Myc regulates the repressor activity of Krim-1. Whereas our experiments with TetR-Krim fusion proteins support a function of Krim-1 as a transcriptional repressor, we were unable to detect any effect of c-Myc on this activity of Krim-1. Therefore, a regulatory role of c-Myc on DNA-bound Krim-1 seems unlikely. A second possible model would implicate a negative regulatory function of Krim-1 on c-Myc activity given the potential of KRAB box proteins as transcriptional repressors. Our data point to the possibility that Krim-1 proteins could be involved in the regulation of E-box-dependent transactivation of c-Myc. This is supported by assays with the E-box-containing and c-Myc-dependent prothymosin reporter. We observed that Krim-1A and Krim-1B were able to repress c-Myc-mediated activation of both types of reporter gene in three different cell lines. Additional evidence for a role of Krim-1 proteins as repressors of c-Myc activity is provided by transfection experiments with primary REFs. The formation of malignantly transformed foci after cotransfection of constructs directing the expression of c-Myc and the activated human Ha-ras gene was found to be repressed severalfold in the presence of Krim-1A and Krim-1B. The loss of interaction mutant Krim-1AZnf265360 was unable to exert this repression in four of five assays, providing additional support for the specificity of the Krim-1/Myc interaction. It has been shown that binding to Max is required for malignant transformation by c-Myc (53) and that target genes that are transcriptionally activated by c-Myc are themselves active, dominant oncogenes in the REF transformation assay, for instance genes encoding specific regulators of cell cycle progression (28) (reviewed in Refs. 1 and 2). Both findings argue that the E-box-dependent activation of target genes is critical for c-Myc in malignant transformation of fibroblasts. Therefore, our observation that Krim-1 proteins repress malignant transformation by c-Myc supports our view that Krim-1 proteins interfere with E-box-mediated transcriptional transactivation of c-Myc.
The E-box dependent transactivation of target genes by c-Myc requires the recruitment of enzymatic activities to the vicinity of the c-Myc binding site that allow chromatin modification, which in eukaryotic cells is executed by ATP-dependent nucleosome remodeling and by histone acetylation. It is known that c-Myc TAD binds to TRRAP, which is part of multiprotein complexes that also contain histone acetyltransferase activity (2528), suggesting that one mechanism of c-Myc-mediated activation of PolII-dependent transcription is the recruitment of histone acetyltransferases and the induction of histone acetylation. In this respect, it is interesting to note that, very similar to the c-Myc/Krim-1 interaction, the recruitment of TRRAP by c-Myc depends on the integrity of Myc box II sequences. This finding underscores the critical role of this homology domain in the process of transcriptional transregulation by c-Myc and may suggest that TRRAP-histone acetyltransferase complexes may compete with other multiprotein complexes for binding to the c-Myc TAD, possibly with Krim-1 or associated proteins.
We present evidence that the KRAB box of Krim-1 is dispensable for c-Myc
binding and that a region in the Krim-1 protein that contains this domain can
interact with the transcriptional co-repressor Tif-1/Kap-1. The
interaction of KRAB box repressor domains with Tif-1
is supported by
many experiments with other, similar KRAB box-containing zinc finger proteins
reported in the literature
(45,
46,
49,
50,
52). Tif-1
is a 97-kDa
multisubunit phosphoprotein that interacts with the 45-amino acid minimal
repression KRAB A box domain as a trimer through the N-terminal Ring-B
box-coiled-coil domains, which have been suggested to function as a
protein-protein interface
(50). Several studies suggest
that Tif-1
is able to function as a universal transcriptional
co-repressor for KRAB box proteins
(32,
47,
48,
50,
52). The carboxyl terminus of
Tif-1
contains a plant homeo domain finger and Bromo domains, both of
which can also function as transcriptional repressors when linked to
heterologous DNA binding domains
(50,
54,
55). Whereas it is widely
accepted that Tif-1
is required for KRAB domain-mediated repression, the
exact mechanisms of how this is achieved remain to be clarified. Experimental
evidence suggests that Tif-1
recruits the HP-1 proteins, which represent
chromatin constituents associated with silencing of euchromatic genes
(52,
56,
57). Recently, the methylation
of lysine in histone H3 has emerged as one critical enzymatic activity in
heterochromatin formation and gene silencing, which provides a signal to
enhance the recruitment and binding of HP-1 proteins
(58). In addition, Tif-1
can use its plant homeo domain and Bromo domains to link KRAB box zinc finger
proteins to Mi-2
and other components of the nucleosome remodeling and
deacetylase complex (55). By
this interaction, Tif-1
is able to recruit not only HP-1 proteins but
also histone deacetylases to KRAB box zinc finger proteins
(55). This suggests that
Tif-1
-mediated repression relies on multiple mechanisms involving
histone modification. Given that these enzymatic activities can be activated
or recruited by Tif-1
, we hypothesize that pathways exists that link the
TAD of c-Myc via the Krim-1 protein to the Tif-1
co-repressor complex by
which both histone methyltransferases and histone deacetylases can be
recruited to c-Myc-occupied sites on the chromatin. Although we can clearly
demonstrate that c-Myc binds to Krim-1 and that Krim-1 can interact with
Tif-1
, further experiments are needed to show whether indeed a trimeric
complex between c-Myc-Krim-1 and Tif-1
is formed in cells. This would
support the possibility that Krim-1 can serve as a bridging molecule to
recruit Tif-1
and the appended chromatin remodeling machinery consisting
of histone deacetylases and HP-1 to c-Myc occupied sites to silence gene
transcription.
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FOOTNOTES |
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Present address: caesar, center of advanced european studies and research,
Friedensplatz 16, D-53111 Bonn, Germany.
¶ The first three authors contributed equally to this work.
|| Present address: The Burnham Institute, Signal Transduction Program, 10901
N. Torrey Pines Rd., La Jolla, CA 92037.
To whom correspondence should be addressed. Tel.: 49-201-723-3380; Fax:
49-201-723-5904; E-mail:
moeroey{at}uni-essen.de.
1 The abbreviations used are: TAD, transactivation domain; RRS, Ras
recruitment system; CMV, cytomegalovirus; RACE, rapid amplification of cDNA
ends; PBS, phosphate-buffered saline; HA, hemagglutinin; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST,
glutathione S-transferase; aa, amino acid(s); REF, rat embryo
fibroblast; b-HLH-LZip, basic helix loop helix-leucine zipper motif.
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ACKNOWLEDGMENTS |
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REFERENCES |
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