From the Division of Immunology, Department of
Microbiology and Immunology, the Institute of Medical Science, the
University of Tokyo, Minato-ku, Tokyo 108-8639, Japan, the ** Department
of Immunology, University of Washington, Seattle, Washington 98195, the
§§ Cutaneous Biology Research Center,
Massachusetts General Hospital, Harvard Medical School, Charlestown,
Massachusetts 02129, and ¶¶ AMGEN, Thousand
Oaks, California 91320
Received for publication, September 13, 2000, and in revised form, February 19, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The lck gene encodes a
protein-tyrosine kinase that plays a key role in signaling
mediated through T cell receptor (TCR) and pre-TCR complexes.
Transcription of the lck gene is regulated by two
independent promoter elements: the proximal and distal promoters.
Previous studies employing transgenic mice demonstrated that the
sequence between The lck gene encodes a lymphocyte-specific
protein-tyrosine kinase, p56lck, a member of the src
kinase family (1). It has been demonstrated by co-immunoprecipitation
that p56lck associates with the cytoplasmic domains of CD4 and
CD8 co-receptors (2) and with the acidic region of the
IL-21 receptor The lck gene is transcribed from two structurally unrelated
promoters (9-13). The lck proximal promoter is positioned
immediately adjacent to the first coding exon, and is active in the
thymus, but is essentially silent in peripheral T cells. The distal
promoter is located far 5'-upstream from the proximal promoter and is
active during all developmental stages of T-lineage cells. Since the proximal promoter becomes active only at an early developmental stage
of T-lymphopoiesis (14, 15), and since the level of p56lck
greatly influences thymocyte maturation (6-8), the transcriptional regulators of this promoter play a critical role in the developmental program for T-lineage cells.
The 5'-flanking sequence of the lck proximal promoter that
is critical for the thymocyte-specific and developmental stage-specific expression has been defined by transgenic mouse models (16). Transgenic
animals bearing truncations in the mouse lck proximal promoter revealed that as little as 584 bases of the 5'-flanking sequence can confer appropriate developmentally regulated expression of
heterologous reporter genes. The 5' sequence critical for the promoter
activity contains several binding sites for nuclear proteins. Among
those nuclear proteins, "B-factor," which binds to the G-rich stretch within the In this study, we characterized the B-factor and identified an 86-kDa
Krüppel-type zinc finger protein, which had been cloned previously as a binding protein to the CD3 Cells--
EL4, LSTRA, WEHI231, and BAL17 cells were grown
in RPMI 1640 medium supplemented with 8% FCS and 50 µM
2-mercaptoethanol. FDC-P1 and Ba/F3 cells were grown in RPMI
1640 medium supplemented with 8% FCS, 50 µM
2-mercaptoethanol, and 5 units/ml mIL-3. Murine IL-3 was
prepared as a culture supernatant of X63Ag8.653 cells transfected with
mIL-3 cDNA (18). COS7 cells were grown in RPMI 1640 medium
supplemented with 8% FCS. MTH cells were grown in RPMI 1640 medium
supplemented with 8% FCS, 50 µM
2-mercaptoethanol, and 1.25 ng/ml IL-2 (Roche Molecular
Biochemicals).
Preparation of Nuclear Extracts and Electophoretic Mobility Shift
Assays (EMSAs)--
Cells were washed twice with ice-cold
phosphate-buffered saline and once with hypotonic buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 0.1 mM
EGTA, 2 mM MgCl2, 1 mM
Na2VO4, 20 mM NaF, 1 mM
dithiothreitol, 0.1 mM Pefabloc SC (Roche Molecular
Biochemicals), 10 µg/ml leupeptin). Cells were suspended in buffer I
(hypotonic buffer containing 0.2% Nonidet P-40) and incubated for 5 min on ice. Nuclei were pelleted by centrifugation at 15,000 × g for 20 min and re-suspended in buffer K (hypotonic buffer
supplemented with 420 mM NaCl and 20% glycerol). After
vigorous shaking for 30 min at 4 °C, the supernatants were collected
and used as nuclear extract (19). Oligonucleotides corresponding to the
Plasmid Construction--
The full-length mt RNA Isolation and Northern Blot Analysis--
Total RNA was
extracted from various cell lines using the acid guanidine
isothiocyanate-phenol-chloroform method. Fifteen micrograms of total
RNA were fractionated through electrophoresis on 1% agarose gel in
the presence of 0.66 M formaldehyde, transferred to nylon
membranes (GeneScreen, DuPont). Mouse multiple tissue Northern blot was
purchased from OriGene Technology (Rockville, MA). Membranes carrying
RNA were hybridized with a 2.0-kilobase EcoRI
fragment of mt Generation of Polyclonal Antibodies--
A 1135-base pair
EcoRV-ApaI fragment encoding Ser51 to
Gly428 and a 1510-base pair ScaI-NotI
fragment encoding Thr446 to Gly769 of mt Preparation of Cell Lysates and Western Blotting--
Cells were
harvested and boiled in SDS-PAGE sample buffer (50 mM Tris,
pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) for 5 min. After centrifugation at 15,000 × g for 15 min, resulting clear cell lysates were subjected to SDS-8% PAGE and then electrophoretically transferred to a
polyvinylidene difluoride membrane (Immobilon, Millipore,
Bedford, MA) in transfer buffer (25 mM Tris, 200 mM glycine, 10% methanol). After blocking with 5% bovine
serum albumin in TBS overnight at 4 °C, membranes were incubated
with appropriately diluted primary antibodies. Membranes were washed
with TBS containing 0.1% Tween 20 (TBS-T) and further incubated with
horseradish peroxidase-conjugated antibodies against rabbit IgG (Cappel
Organon Technica, Durham, NC). After washing, bound antibodies on
membranes were detected using an ECL detection system (Amersham
Pharmacia Biotech, Buckinghamshire, United Kingdom) and x-ray film
(Fuji Film). Splenic B cells were purified by negative sorting using
anti-CD43 monoclonal antibody and a magnetic cell sorting system
(Miltenyi Biotec, Bergisch Gladbach, Germany). Splenic T cells
were purified by negative sorting using anti-B220 and anti-Mac1
monoclonal antibodies and magnetic cell sorting.
RT-PCR--
Total RNA or mRNA purified using Micro-FastTrack
kit (Invitrogen) was subjected to cDNA synthesis using random
hexamers (TaKaRa, Kyoto, Japan) and Superscript II reverse
transcriptase (Life Technologies, Inc.). Serial dilution (3- or 2-fold)
of the cDNA reaction mixtures was subjected to PCR amplification
using the following primers: distal-sense, 5'-ATGTGAATAGGCCAGAAGAC-3';
proximal-sense, 5'-TCTGAGCTGACGATCTCGG-3'; lck antisense,
5'-GATCTTGTAATGTTTCACCAC-3'; Transfections and Luciferase Assays--
Cells (3 × 106) were suspended in 0.2 ml of Opti-MEM (Life
Technologies, Inc.) and transferred to a 4-mm gap cuvette and mixed with 10 µg of reporter firefly luciferase plasmids, 1 µg of renilla luciferase plasmid pRL-TK (Promega). In some experiments, 10 µg of
pcDNA3-mt Identification of a Nuclear Protein Binding to the lck Proximal
Promoter--
A nuclear factor termed "B-factor" binds to a G-rich
stretch located at mt Recombinant mt Expression of Mt
The expression level of mt Lineage-specific Transactivation by mt
To confirm the role of mt It has been reported that a close correlation exists between
transcriptional activities of the lck proximal promoter and
the presence of the B-factor complex binding to the G-rich stretch of
the promoter (16). In this work, we characterized and identified the
B-factor, a potential transcriptional regulator of the lck proximal promoter. Our results indicated mt Mt In addition to its binding to a broad range of target genes, mt mt In addition to the positive regulators directing the lck
proximal promoter activity in thymocytes, the silencers suppressing the
promoter are likely to play roles in peripheral T cells. It has been
reported that A2 complex binding to the sequence from In summary, we identified a Krüppel-type zinc finger protein,
mt584 and
240 from the transcription start site in
the mouse lck proximal promoter is required for its
tissue-specific expression in the thymus. In this study, we demonstrate
that a Krüppel-like zinc finger protein, mt
(BFCOL1, BERF-1,
ZBP-89, ZNF148), previously cloned as a protein that binds to the
CD3
gene enhancer, binds to the
365 to
328 region of the
lck proximal promoter. mt
is ubiquitously expressed in
various cell lines and mouse tissues. Overexpressed mt
is more
active in T-lineage cells than B-lineage cells for transactivating
an artificial promoter consisting of the mt
binding site and a TATA box. Activity of the lck proximal promoter was
significantly impaired by mutating the mt
binding site or by
reducing mt
protein expression level by using antisense mRNA.
Our results indicate that mt
activity is regulated in a
tissue-specific manner and that mt
is a critical transactivator for
the lck proximal promoter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain in T
cells (3). By a series of biochemical analysis, it has been shown that
p56lck plays a key role in signal transduction mediated through
the T cell receptor (TCR) complex in mature T cells (4, 5). It also
contributes to signaling through the pre-TCR complex, thereby playing
an essential role in thymocyte development. lck-deficient mice and transgenic mice overexpressing a dominant negative form of
p56lckck exhibit severe impairment in
the expansion of CD4/CD8-double negative immature thymocytes (6, 7). A
simple doubling of wild type p56lck expression levels in
immature thymocytes in transgenic mice was sufficient to block
maturation of thymocytes (8). These findings suggest that the
transcriptional control of the lck gene must be tightly
regulated to express adequate amounts of p56lck at the right
developmental stage during thymopoiesis.
365 to
328 region was reported as a candidate for the critical transcriptional regulator. B-factor is only found in
cells expressing the lck transcript derived from the
proximal promoter, namely thymocytes and thymoma cell lines such as
LSTRA and EL4 (16).
gene enhancer, as a
component of the B-factor. The NH2-terminal half of the
protein is 90% identical to ht
, a 49-kDa protein that binds to the
human TCR V
8.1 gene promoter and the TCR
gene silencer (17),
indicating that mt
is the murine homologue of ht
, and the
reported amino acid sequence of ht
is a part of its full-length
protein. mt
is ubiquitously expressed in various cell lines and
tissues. We re-evaluated distribution of the B-factor and found it is
also expressed in various cell lines and tissues. However, the
transcriptional activity of mt
measured by reporter constructs
carrying the B-factor binding site is observed only in T-lineage cells.
The transcription from the lck proximal promoter is greatly
impaired by introducing mutations in the B-factor binding site or by
expression of mt
antisense mRNAs. Our results demonstrate that
mt
is one of the critical transactivators driving the lck
proximal promoter and that its activity is regulated in a
tissue-specific manner.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
365 to
328 region of the lck proximal promoter
(
365/
328, see section below) were subcloned into the
KpnI site of pBluescript II (Stratagene). The fragment was
cut out by Asp718I, labeled with [
-32P]dATP
by Klenow large fragment (TaKaRa) and used as a probe. The
nuclear extracts and the probe were incubated for 30 min at room
temperature in the reaction buffer containing 10 mM HEPES, pH 7.9, 50 mM NaCl, 1 mM EDTA, 5% glycerol,
0.1% Nonidet P-40 (19). Each reaction contained 0.02 unit of
poly[dI-dC] (Amersham Pharmacia Biotech). In some reactions,
affinity-purified anti-mt
antibodies or normal rabbit IgG were added
prior to incubation with the probe. Reactions were then subjected to
4% polyacrylamide gel electrophoresis in 0.25 × TBE buffer (Tris
borate/EDTA buffer) and analyzed by autoradiography.
Oligonucleotides with the following sequences (binding sequences
for nuclear proteins are boldface) were used in EMSAs:
365/
328, 5'-TGTGGTTGAGTGGTGGGGGTAGGGGTGCTGGGGTAC-3' and
3'-CATGACACCAACTCACCACCCCCATCCCCACGACCC-5';
365/328mut, 5'-GTACTGTGGTTGAGTGGTGCTGGTAGGGGTGCTGGG-3' and
3'-ACACCAACTCACCACGACCATCCCCACGACCCCATG-5';
A,
5'-AGAAGTTTCCATGACATCATGAATGGGGGTGGCAGA-3' and 3'-TTCAAAGGTACTGTAGTACTTACCCCCACCGTCTCT-5';
A-CRE, 5'-AGAAGTTTCCATAAGATGATGAATGGGGGTGGCAGA-3' and
3'-TTCAAAGGTATTCTACTACTTACCCCCACCGTCTCT-5';
A-G,
5'-AGAAGTTTCCATGACATCATGAATGGGGTGGCAGAG-3' and
3'-TCTTCAAAGGTACTGTAGTACTTACCCCACCGTCTCT-5'; IkarosBS, 5'-TCAGCTTTTGGGAATGTATTCCCTGTCA-3' and
3'-AGTCGAAAACCCTTACATAAGGGACAGT-5'.
cDNA2 was subcloned into
the EcoRI site of pcDNA3 (Stratagene), a eukaryotic
expression vector driven by the human cytomegalovirus enhancer and
promoter, resulting in pcDNA3-mt
. For the mt
antisense
plasmid (pcDNA3-ASmt
), the full-length mt
cDNA was
subcloned in the opposite direction into the EcoRI site of
pcDNA3. Various truncated fragments from the mouse lck
proximal promoter were subcloned into the pGL2-Basic plasmid (Promega),
which has a firefly luciferase gene without promoter or enhancer. For
3200/pGL2, the NotI-BamHI (positions
3200 to
+37) fragment of the p1017 plasmid (20) containing the entire
lck proximal promoter region was blunt-ended and ligated to
the XhoI, HindIII-digested, blunt-ended
pGL2-Basic plasmid. For
433GL2, two SmaI fragments
(position
3200 to
1675 and
1675 to
433) were removed
from
3200/pGL2 and self-ligated. For
240/pGL2, two KpnI
fragments (position
3200 to
584 and
584 to
240) were removed from
3200/pGL2. For
584/pGL2, the KpnI fragment
(position
584 to
240) from
3200/pGL2 was inserted into the
KpnI site of
240/pGL2. To construct reporter plasmids
carrying the B-factor binding site and TATA box,
365/
328 and
365/
328mut oligonucleotides (see section above) were inserted
upstream of the TATA box of pLuc-S (gift from Drs. P. Doerfler and M. Busslinger (21)), resulting in pLuc-wild and pLuc-mut, respectively.
Point mutations were introduced into the mt
binding sites of
3200/pGL2 and
433/pGL2 by PCR-based directed mutagenesis using
365/
328mut oligonucleotides to generate
3200-mut/pGL2 and
433-mut/pGL2, respectively.
cDNA labeled with [
-32P]dCTP by
the random priming method. After hybridization, membranes were analyzed
using a BAS1000 Bio-Image Analyzer (Fuji Film, Tokyo, Japan).
After removing the mt
probe, the membranes were re-hybridized with a
human
-actin probe to normalize the amount of RNA loaded per lane.
protein were subcloned into the bacterial expression vectors pGEX-4T-1
and pGEX-4T-2 (Amersham Pharmacia Biotech), respectively. The resulting
plasmids were used to transform BL21(DE3)/pLysS (Novagen, Madison, WI).
Recombinant glutathione S-transferase-mt
fusion proteins
were induced with 1 mM
isopropyl-
-D-thiogalactopyranoside and affinity-purified
by binding to glutathione-linked Sepharose beads (Amersham Pharmacia
Biotech). The fusion proteins were further purified by gel filtration
and used to immunize rabbits. Rabbit polyclonal anti-mt
antibodies
were immunopurified on Sepharose-4B beads covalently coupled with the
respective glutathione S-transferase-mt
fusion proteins
used as immunogens.
-actin-sense,
5'-ACACTGTGCCCATCTACCAG-3';
-actin-antisense,
5'-CTAGAAGCACTTGCGGTGCA-3'; G3PDH (glyceraldehyde-3-phophate dehydrogenase)-sense, 5'-ACCACAGTCCATGCCATCAC-3'; G3PDH-antisense, 5'-TCCACCACCCTGTTGCTGTA-3'; HGPRT (hypoxantine guanine
phophoribosyltransferase)-sense, 5'-CGTCGTGATTAGCGATGATGAACC-3'; HGPRT-antisense,
5'-ACTGCTTAACCAGGGAAAGCAAAG-3'. The conditions for PCR
amplification were 30 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min for the lck or HGPRT transcripts
and 27 cycles of the above for
-actin or G3PDH transcripts using a
PCR Thermal Cycler MP (TaKaRa). The resulting PCR products were
separated by electrophoresis on 1% agarose gels and visualized by
ethidium bromide staining.
or a control pcDNA3 was added in addition to the reporter plasmids. Cells were transfected at 960 microfarads and 250 V using a Gene Pulser electroporation apparatus (Bio-Rad). Cells were harvested 12 h after transfection, and luciferase
activity in cell lysates was measured using the Dual luciferase assay
system (Promega) according to the manufacturer's recommendation. The firefly luciferase activity was normalized by the renilla luciferase activity to normalize for the transfection efficiency of each sample.
In experiments using pcDNA3-ASmt
, EL4 cells were transfected with 15 µg of pcDNA3-ASmt
or control pcDNA3, together with
3 µg of reporter firefly luciferase plasmids and 1 µg of pRL-TK. Cells were harvested 30 h after transfection, and luciferase
activity was measured. To analyze the amounts of mt
protein and the
endogenous lck transcripts, cells were transfected
with 10 µg of pcDNA3-ASmt
or pcDNA3, together with 1 µg
of pEGFP-N1 (CLONTECH). Cells expressing GFP were
sorted using a FACS Vantage cell sorter (Becton-Dickinson) at 30 or 36 h after transfection. Sorted cells were lysed and subjected
to immunoblot analysis using anti-mt
or anti-tubulin antibodies or
were subjected to RT-PCR analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
365 to
328 from the transcriptional initiation
site of the lck proximal promoter, and its expression
correlates well with the activity of the promoter (16). Because
lck expression driven by the proximal promoter occurs early
during lymphopoiesis (14, 15), and the level of p56lck greatly
influences thymocyte maturation (6-8), we hypothesized that the
B-factor would be one of the transcription factors playing critical
roles in early lymphopoiesis. Ikaros regulates the early lymphopoiesis or the commitment for lymphocytes as demonstrated in the
Ikaros-deficient mice (22). Ikaros has been shown
to bind to a G-rich sequence of the CD3
gene enhancer (23), although the high affinity binding sites for Ikaros are not G-rich
(24). We examined whether the B-factor contains Ikaros or
Ikaros-related proteins by EMSAs. Nuclear extract of a
thymoma cell line, LSTRA, contains the B-factor binding to the
365 to
328 G-rich sequence (
365/
328) of the lck proximal
promoter (Fig. 1A) as shown
previously (16). The binding was specific, since it was competed by
unlabeled
365/
328 oligonucleotides, but not by the
365/
328
oligonucleotides carrying mutations in the G-rich sequence
(
365/
328mut). As shown in Fig. 1B, the binding of
B-factor to the
365/
328 probe was competed by
A sequences, a
functional element of the CD3
gene enhancer. The
A consists of a
CRE (cyclic AMP response element)-like region and a G-rich site similar
to the B-factor binding site (25). The binding of B-factor to
A was
mediated by the G-rich site, because mutation at the G-rich site
(
A-G) but not at the CRE-like site (
A-CRE) abrogated the
competition with the
365/
328 probe. This binding characteristic of
B-factor to
A sequence was similar to that of Ikaros
(23). However, the high affinity Ikaros binding
oligonucleotides (IkarosBS) (24) did not show any competition with
365/
328. During the cloning of Ikaros, a cDNA clone
encoding a zinc finger protein (mt
, see below) was simultaneously
cloned by its ability to bind to the CD3
enhancer.2 We
examined whether antibodies against Ikaros or mt
could
react with B-factor. Anti-Ikaros antibodies did not affect
B-factor complex formation (Fig. 1C), confirming that
Ikaros is not a component of B-factor. Interestingly,
anti-mt
antibodies efficiently supershifted the B-factor complex
(Fig. 1D). A similar result was obtained using nuclear
extracts of EL4, a lymphoma cell line in which the lck
proximal promoter is active (Fig. 1E). These results
demonstrate that mt
is a component of the B-factor that binds to the
365 to
328 region of the mouse lck proximal
promoter.
View larger version (49K):
[in a new window]
Fig. 1.
Characterization of the nuclear protein,
"B-factor," that binds to the sequence from 365 to
328 of the
lck proximal promoter. A, the B-factor
(arrow) in nuclear extracts from LSTRA. EMSA was performed
using a fragment containing the sequence from
365 to
328 of the
lck proximal promoter as a probe. The
365 to
328
(
365/
328) oligonucleotides or the oligonucleotides carrying
mutations in the G-rich region (
365/
328mut) were used as
competitors (10- and 50-fold molar excess over the labeled
probe) to determine the binding specificity of the B-factor.
B, the B-factor binds to
A, the core enhancer sequences
of the CD3
gene enhancer. Unlabeled oligonucleotides with various
G-rich sequences were used as competitors.
A-CRE,
A carrying the
mutation in the CRE binding site;
A-G,
A carrying the mutation in
the G-rich sequence; and Ikaros BS, a high affinity Ikaros binding
sequence. C, Ikaros is not a component of the B-factor.
Anti-Ikaros antiserum did not react with the B-factor. D,
zinc finger protein; mt
that binds to
A sequence is a component
of the B-factor. The B-factor complex was supershifted by anti-mt
antibodies. E, the B-factor present in nuclear extracts from
EL4.
Is a Krüppel-type Zinc Finger Protein--
The deduced
amino acid sequence of mt
contains an amino-terminal acidic domain,
four tandem C2H2 Krüppel-type zinc finger motifs (26), and two
basic domains, located upstream and downstream of the zinc finger
cluster (data not shown). A data base search by BLAST identified a
human homologue, ht
, a 49-kDa protein that binds to the V
8.1
promoter and the V
silencer of the T cell receptor genes (17). The
cDNA sequence encoding the NH2-terminal half of the
mt
is 90% identical to ht
, and their deduced amino acid
sequences are 95% identical. The 3'-half of the mt
coding region
has 91% identity with the 3'-untranslated region of the reported ht
cDNA. These indicate that mt
is the murine homologue of ht
and that the reported ht
cDNA sequence has a one-base deletion
that causes a frameshift and a premature stop codon. During this study,
several cDNAs that have identical sequences with mt
have been
reported: BFCOL1 that binds to the proximal promoters of the type I
collagen genes (27) and BERF-1, a 89-kDa protein that binds to a
muscle-specific enhancer of the
-enolase gene (28). In addition, the
rat and human homologue of the protein, ZBP-89, has been shown to bind
to promoter regions of the gastrin gene (29) and the ornithine
decarboxylase promoter (30). It has subsequently been reported that the
same zinc finger protein also binds to the p21WAF1 gene (31), the
matrix metalloproteinase-3 gene (32), the pT
gene (33), and the
vimentin gene (34).
Binds to the lck Proximal Promoter--
We then
asked whether recombinant mt
forms the B-factor complex. Recombinant
mt
expressed in COS7 cells was detected as a band around 105 kDa in
immunoblots (Fig. 2A). An
endogenous simian homologue of mt
in COS7 cells was detected at the
same position as the recombinant mt
when the blot was overexposed (data not shown). mt
protein appeared to migrate more slowly in
SDS-PAGE than its estimated molecular size, as is consistent with the
observation for BFCOL1 by Hasegawa et al. (27). In EMSA, a
residual amount of the B-factor complex was detected in COS7 cells that
derived from the endogenous simian mt
-homologue protein.
Overexpression of mt
resulted in a significant increase of the
amount of the B-factor complex (Fig. 2B). The entire complex was supershifted by the addition of anti-mt
antibodies. These results strongly indicate that mtB by itself, or in combination with
proteins present in COS7 cells, forms the B-factor complex that binds
to the
365 to
328 region of the lck promoter.
View larger version (39K):
[in a new window]
Fig. 2.
The B-factor complex formation by recombinant
mt . A, Western blot analysis.
COS7 cells were transfected with 10 µg of mt
expression vector or
control plasmid. Cells were harvested at 48 h after transfection,
and nuclear extracts were prepared. The resulting extracts (1 × 106 cells/lane) were separated, transferred to membrane,
and were probed with anti-mt
antibodies. Molecular size markers are
indicated on the left. B, EMSA. The amount of
B-factor complex (arrow) was increased by expressing the
recombinant mt
protein. Anti-mt
antibodies supershifted the
B-factor complex.
in Various Cell Lines and Tissues--
In a
previous study, the strong correlation between the lck
proximal promoter activity and amounts of B-factor has been reported (16). We therefore examined the expression of mt
mRNA in various cell lines and tissues. As shown in Fig.
3A, two mRNA species, with
estimated sizes of 9.0 and 4.2 kilobase, were detected. Mt
mRNA expression was observed in all cell lines tested and was independent of the lck proximal promoter activity. In mice,
the mt
transcripts were ubiquitously expressed in various tissues. The mRNA was abundant in thymus where the proximal promoter is active; however, significant amounts of mRNA were also detected in
all tissues, especially in the heart, kidney, and liver (Fig. 3B). We conclude that there is no correlation between
lck proximal promoter activity and the expression levels of
mt
mRNA.
View larger version (61K):
[in a new window]
Fig. 3.
Northern blot analysis. Expression of
mt transcripts in various cell lines (A) and mouse
tissues (B). RNA was prepared from LSTRA (thymoma), MTH
(mature T), Ba/F3 (pro-B), WEHI231 (Immature B), BAL17 (mature B), and
FDC-P1 (myeloid). Total RNA (15 µg) was separated and transferred
onto a nylon membrane. A prepared multi-tissue membrane was purchased
from OriGene Technologies. The blots were hybridized with mt
cDNA. The blots were stripped and subsequently hybridized with a
-actin probe to normalize for the amount of loaded RNA.
protein might be controlled by
post-transcriptional mechanisms. To test this possibility, we measured mt
protein levels by immunoblots of whole cell extracts isolated from various cell lines and primary mouse lymphoid cells. The mt
protein was detected in all tested cell lines, LSTRA (thymoma), BAL17
(mature B), Ba/F3 (proB), MTH (mature T), and EL4 (lymphoma) cell lines
(Fig. 4A, left
panel). Significant amounts of the mt
protein were also
detected not only in thymocytes but also in splenic B cells and T cells
(Fig. 4A, right panel). The expression of mt
protein was further confirmed by EMSAs. The B-factor was detected in
the nuclear extract prepared from thymocytes as reported previously
(16). We initially failed to detect either mt
protein or B-factor
complex in extract from total splenocytes. However, the B-factor was
present in the nuclear extract prepared from purified splenic B cells
as well as mature T cells (Fig. 4B). High proteinase
activity in total splenocytes may have caused degradation and prevented
detection of mt
protein and the B-factor complex. Careful
preparation of nuclear extracts revealed the presence of the B-factor,
even in BAL17, Ba/F3, and MTH cell lines and mature splenic T cells
(Fig. 4B).
View larger version (34K):
[in a new window]
Fig. 4.
Expression of mt
protein and the B-factor is not restricted in T lineage
cells. A, Western blot analysis of mt
in LSTRA
(thymoma), BAL17 (mature B), Ba/F3 (pro-B), MTH (mature T), EL4
(lymphoma), thymocytes, purified splenic T cells (95% was
CD3+), and purified splenic B cells (95% was
B220+). Cells (2 × 105 cells) were boiled
in 1 × SDS-PAGE sample buffer, and the insoluble materials were
removed by centrifugation. Resulting cell lysates were separated,
transferred to membrane, and were probed with anti-mt
antibodies.
Molecular size markers are indicated on the left.
B, EMSA. The B-factor (arrow) was detected in
nuclear extracts prepared from thymocytes, purified splenic B cells
(95% was B220+), BAL17, Ba/F3, MTH, and purified splenic T
cells (95% was CD3+). Anti-mt
antibodies supershifted
the B-factor complexes in all of tested extracts.
and Its Critical Function
for the lck Proximal Promoter Activity--
To clarify the role of
mt
in transactivation of the lck proximal promoter,
various reporter plasmids were constructed and introduced into EL4 or
mature BAL17. EL4 expressed mainly type I transcripts (9) transcribed
from the proximal promoter, while BAL17 expressed only type II
transcripts (9) from the distal promoter (Fig.
5A). First, we studied
transactivation of a reporter construct that contains only the B-factor
binding site of the proximal promoter and a TATA box (pLuc-wild) (Fig.
5B). The pLuc-wild construct showed significant promoter
activity in parental EL4 cells, and the activity was augmented
~3-fold by overexpression of mt
. The promoter activity was not
observed with a reporter (pLuc-mut) carrying mutations at the B-factor
binding site on which mt
does not bind. Interestingly, pLuc-wild did
not show significant promoter activity in BAL17, and the activity was
not increased when mt
was overexpressed. We next studied the
activity of reporter constructs with various deletions and mutations in the lck proximal promoter sequences (Fig. 5C).
The fragment from
3200 to 0 of the promoter was active in EL4.
Deletion of the fragment up to
584 did not affect promoter activity,
whereas an additional deletion up to
433 resulted in a 3-fold
increase of the activity. Further deletion of the fragment, including
the B-factor binding site up to
240, did not impair the activity. However, the introduction of mutations into the B-factor binding site
that abolishes mt
binding resulted in a significant reduction of the
promoter activity of the
3200 and
433 fragments (compare
3200
versus
3200-mut and
433 versus
433-mut).
All reporter constructs with the lck proximal promoter
sequence did not show significant promoter activity in BAL17.
View larger version (35K):
[in a new window]
Fig. 5.
Transcriptional activity of
mt on the lck proximal
promoter. A, schematic representation of the
lck gene promoter regions, and position of primers used for
RT-PCR analysis are shown (left). Type I and II
lck mRNAs are transcribed from the proximal and distal
promoters, respectively. The relative ratio of type I and type II
mRNAs in EL4 and BAL17 cells were measured by semiquantitative
RT-PCR analysis. Serial dilutions (3-fold) of cDNA prepared from
each cell line were subjected to PCR using sets of primers for type I
(primers A and C) and type II (primers
B and C) lck transcripts.
-Actin
cDNA was amplified (right lower panel) to calibrate the
amounts of cDNA templates in each sample. The proximal
lck promoter is mainly active in EL4, while the distal
promoter is active in BAL17. B, mt
activates
transcription from an artificial promoter consisting of the mt
binding site of the lck proximal promoter and a TATA-box
(pLuc-wild) in EL4 but not in BAL17. Cells were transfected with 10 µg of reporter plasmid and 10 µg of mt
expression (+) or vector
plasmid (
). Twelve hours after transfection, cells were harvested,
lysed, and subjected to luciferase activity measurement. The luciferase
activities are represented as percent activity of that produced by pGL2
control vector driven by the SV40 promoter. In pLuc-mut, mutations were
introduced into the mt
binding site. The activity produced by a
promoterless plasmid (0) is also shown. The results
represent mean ± S.D. of multiple independent transfections.
C, the binding of mt
is critical for the lck
proximal promoter activity. Cells were transfected with 10 µg of
luciferase reporter constructs carrying the various lengths of the
lck proximal promoter region (
3200,
584,
433,
240,
and 0) or the mutated promoter sequences (
3200-mut and
433-mut).
The mt
binding site was destroyed by point mutations in the
3200-mut and
433-mut reporter constructs. The results are
represented as percent luciferase activity observed with pGL2 control
vector driven by SV40 promoter. The results represent mean ± S.D.
of multiple independent transfections. In each experiment, the
luciferase activities were normalized for transfection efficiency by
measuring renilla luciferase activities encoded by a co-transfected
pRL-TK plasmid. ND, not determined.
in transactivation of the lck
proximal promoter, we reduced the protein expression level of mt
in
EL4 by expressing mt
antisense mRNA. As shown in Fig.
6A, the amount of mt
protein was reduced to about 50% of control by transfection of the
antisense plasmid. The activity of
3200 lck promoter
fragment was significantly diminished, whereas that of the control SV40
promoter activity was not affected (Fig. 6B). Moreover,
expression levels of the endogenous lck transcripts were also reduced (Fig. 6C). These results indicate that
there is lineage-specific control for the mt
activity and that mt
plays a critical role in transactivation of the lck proximal
promoter.
View larger version (28K):
[in a new window]
Fig. 6.
Expression of mt
antisense reduces the lck proximal promoter
activity. A, reduction of mt
protein level by
expression of antisense mt
. EL4 cells were transfected with 10 µg
of pcDNA3-ASmt
or pcDNA3, together with 1 µg of pEGFP-N1.
Cells expressing GFP were sorted at 36 h after transfection.
GFP-positive cells (5 × 104 cells) were lysed, and
mt
protein levels were analyzed by immunoblot. Molecular size
markers are indicated on the left. The blots were stripped
and subsequently probed with anti-tubulin antibodies to normalize the
amount of loaded proteins. The relative expression levels of mt
are
indicated by mt
/tubulin ratio, which is set to 1 in cells
transfected with vector control plasmid. B, relative
luciferase activities of cells expressing mt
antisense plasmid. EL4
cells were transfected with 15 µg of pcDNA3-ASmt
or control
pcDNA3, together with 3 µg of reporter plasmids (
3200/pGL2 or
pGL2) and 1 µg of pRL-TK. Cells were harvested at 30 h after
transfection, and luciferase activity in cell lysates was measured. The
results represent mean ± S.D. of three independent transfections.
C, reduced expression of the endogenous
lck transcripts by expression of antisense mt
.
EL4 cells were co-transfected with pcDNA3-ASmt
or control
pcDNA3, together with pEGFP-N1 and GFP-positive cells were sorted
at 30 h after transfection. cDNAs were synthesized, and serial
dilutions (2-fold) of cDNA templates were subjected to PCR
amplifications for lck, G3PDH, and HGPRT.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which has been
previously cloned by its binding to the CD3
enhancer in
vitro, is a component of the B-factor. Anti-mt
antibodies
supershifted the B-factor complex, and the recombinant mt
expressed
in cell lines formed the B-factor complex.
is an 89-kDa zinc finger protein belonging to the
Krüppel-type subfamily, whose members recognize the GC-rich or
GT-rich sequences with their conserved DNA-binding zinc finger domains (26, 35). Mt
(identical to BFCOL1, BERF-1) and its human and rat
homologues (ht
, ZBP-89, ZFP148) are reported to bind regulatory
regions of various genes, such as the V
8.1 promoter and the V
silencer of the T-cell receptor genes (17), the gastrin promoter (29),
the type I collagen promoter (27), the
-enolase enhancer (28), the
ornithine decarboxylase promoter (30), the p21WAF1 promoter (31), the
matrix metalloproteinase-3 promoter (32), the pT
enhancer (33), and
the silencer element of the vimentin gene (34). Our data showing that
mt
is ubiquitously expressed at both mRNA and protein levels are
consistent with previously published reports and the fact that mt
functions in the various promoters and enhancers in various tissues.
However, it should be noted that mt
regulates genes critical for
maturation of T cells, such as lck, pT
gene, as well as
TCR
and
genes. Thus, mt
is one of the key transcriptional
regulators controlling the T cell development.
appears to act as both transcriptional activator and repressor. As
shown in this study, mt
is required for transactivating the proximal
lck promoter. It also activates transcription from the V
8.1 promoter of the TCR gene and counteracts the silencing effect of the TCR
gene silencer (17), and increased promoter activity of
the p21WAF1 gene (31) and the matrix metalloproteinase-3 gene (32).
Moreover, the binding site of mt
appears to be important for the
pT
enhancer element (33). In contrast, mt
represses transcription
from the gastrin gene (29), the
-enolase gene (28), the ornithine
decarboxylase gene (30), and the vimentin gene (34). It is currently
unknown how mt
/BFCOL1/BERF-1 (ht
/ZBP-89/ZFP148 in humans)
manifests opposite activities on transcription of different genes. It
has been shown that ZBP-89 competes with Sp1 for binding to the same
element in the gastrin promoter (29) and inhibits the activation of the
ornithine decarboxylase promoter by Sp1 (30). This might be one of
the potential mechanisms by which mt
/BFCOL1/BERF-1 suppresses
transcription from several promoter elements. A fascinating possibility
is that mt
has different isoforms derived from alternative splicing,
and each isoform has distinct transcriptional activities. To test this
hypothesis, we performed RT-PCR to amplify various fragments of mt
cDNA using several combinations of primers. However, we could not
detect any splicing variants of mt
cDNA either in thymocytes or
splenocytes (data not shown). Another possibility is that interacting
proteins exist that determine the DNA binding specificity and the
transactivating activities of the mt
complex and whose expression is
regulated in a tissue-specific manner. It is also possible that mt
receives post-transcriptional modifications in a tissue-specific
manner. Endogenous as well as overexpressed mt
transactivated
transcription from an artificial promoter consisting of B-factor
binding sites and TATA-box in EL4 but not in BAL17. Moreover, mt
generally transactivates genes expressed in T cells such as
lck and TCR
and
genes, but represses gastrin,
collagen, and
-enolase genes expressed in non-T cells. These
observations support the idea that there are tissue-specific mechanisms
regulating activity of mt
. Basic Krüppel-like factor (BKLF),
which is widely expressed in various tissues and binds to the CACCC
motifs, is also a member of the Krüppel-like zinc finger protein
subfamily (36). Although BKLF positively regulates the transcription
from a promoter containing a single BKLF binding site, it represses
activity of glucocorticoid receptor-mediated activation of a promoter
containing three copies of CACCC motifs and glucocorticoid-responsive
elements (36, 37). The NH2-terminal domain of BKLF is
responsible for its repressive activity and interacts with a
co-repressor protein, murine COOH-terminal-binding protein 2 (mCtBP2)
(37). mCtBP2 interacts with BKLF and a number of mammalian
transcription factors, such as Evi-1, AREB6, ZEB, and FOG, via the
Pro-X-Asp-Leu-Ser (PXDLS) motif on the
transcription factors (37). The mt
also carries several
PXDLS-like motifs (PVDLQ (amino acids 112-116), PKDNS
(amino acids 282-286)). mt
may associate with mCtBP2 or related
molecules and exhibits suppressing activity in cells that fail to
support proximal lck promoter activity. Our initial
attempts, however, to detect associating molecules using glutathione
S-transferase-mt
fusion proteins or modification of mt
such as phosphorylation have not been successful.
is critical for the full activation of the lck
proximal promoter activity, since the mutation of the mt
binding
site of the promoter or the reduction of the mt
protein level
significantly impaired the promoter activity. However, the
overexpression of mt
in EL4 did not augment the lck
proximal activity (data not shown). This may indicate that the
coordinated interaction of mt
with T cell-specific transcription
factors (whose expression level is limiting) is involved in the full
activation of the lck proximal promoter in thymocytes.
Binding sites for the T cell-specific factors TCF-1, LEF, and TCF-1
are present in a region highly homologous between the murine and human
proximal promoters (16). TCF-1 expressed ectotopically in BAL17 cells,
however, failed to drive the lck proximal promoter activity
with endogenous mt
(data not shown), suggesting a complex
cooperation of multiple transcription factors in transactivating the
lck proximal promoter. A
240 lck promoter
fragment lacking a mt
binding site is still active in EL4. It should
be noted, however, that the
240 fragment (but not the
584 promoter
fragment carrying a mt
binding site) failed to support
thymocyte-specific transcription of the lacZ-hGH transgene
construct in mice (16). The EL4 cell line might lack a negative
regulator expressed in primary cells that suppresses the
240 promoter
activity in the absence of mt
. Alternatively, EL4 might abundantly
express positive transactivators binding to the
240 fragment whose
activity is repressed by proteins bound to the
584 to
240 region.
The mechanism that accounts for the discrepancy between the
240
promoter activity in EL4 and that in thymocytes remains unknown.
477 to
460 in
the murine proximal promoter was detected in extracts from cells
negative for the lck proximal promoter activity (16). It has
also been reported that the
474 to
466 region in the human
lck proximal promoter acts as a strong repressor in human tumor cell lines that do not express lck and binds proteins
with molecular masses of 35 and 75 kDa (38). Deletion of the
584 to
433 region from our luciferase reporter constructs resulted in the
enhancement of the promoter activity. These suppressive elements and
binding factors are also critical in achieving tissue-specific expression of the lck proximal promoter in concert with the
positive regulators, including mt
.
, as a transactivator of the lck proximal promoter.
mt
is ubiquitously expressed and manifests a broad range of
activities on various genes. However, mt
is presumably a critical
transcription factor for the T cell development, since it positively
regulates lck and pT
genes as well as TCR
and
genes. Overexpressed mt
is active only in T-lineage cells,
suggesting that there exists tissue-specific regulatory mechanisms to
control mt
activity. Understanding the function of mt
should
provide important insight into how T cell development and the
thymocyte-specific expression of the lck proximal promoter
are controlled and how one DNA-binding protein regulates different
promoters positively and negatively.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Drs. P. Doerfler and M. Busslinger for providing pLuc-S reporter constructs, Dr. J. Allen for providing oligonucleotides and probes, and all our colleagues for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.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.
§ These authors contributed equally to this work.
¶ Supported in part by a Center of Excellence Fellowship from the Institute of Medical Science Program.
To whom correspondence should be addressed. Tel.:
81-3-5449-5264; Fax: 81-3-5449-5407; E-mail:
takakis@ims.u-tokyo.ac.jp.
Supported by the Howard Hughes Medical Institute Predoctoral
Fellowship in Biological Science.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M008387200
2 K. Georgopoulos, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IL, interleukin; TCR, T cell receptor; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; FCS, fetal calf serum; RT-PCR, reverse transcriptase polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HGPRT, hypoxantine guanine phophoribosyltransferase; CRE, cyclic AMP response element; BKLF, basic Krüppel-like factor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Marth, J. D., Peet, R., Krebs, E. G., and Perlmutter, R. M. (1985) Cell 43, 393-404[Medline] [Order article via Infotrieve] |
2. | Barber, E. K., Dasgupta, J. D., Schlossman, S. F., Trevillyan, J. M., and Rudd, C. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3277-3281[Abstract] |
3. | Hatakeyama, M., Kono, T., Kobayashi, N., Kawahara, A., Levin, S. D., Perlmutter, R. M., and Taniguchi, T. (1991) Science 252, 1523-1528[Medline] [Order article via Infotrieve] |
4. | Alberola-Ila, J., Takaki, S., Kerner, J. D., and Perlmutter, R. M. (1997) Annu. Rev. Immunol. 15, 125-154[CrossRef][Medline] [Order article via Infotrieve] |
5. | Kane, L. P., Lin, J., and Weiss, A. (2000) Curr. Opin. Immunol. 12, 242-249[CrossRef][Medline] [Order article via Infotrieve] |
6. | Molina, T. J., Kishihara, K., Siderovski, D. P., van Ewijk, W., Narendran, A., Timms, E., Wakeham, A., Paige, C. J., Hartmann, K. U., Veillette, A., and Mak, T. W. (1992) Nature 357, 161-164[CrossRef][Medline] [Order article via Infotrieve] |
7. | Levin, S. D., Anderson, S. J., Forbush, K. A., and Perlmutter, R. M. (1993) EMBO J. 12, 1671-1680[Abstract] |
8. | Abraham, K. M., Levin, S. D., Marth, J. D., Forbush, K. A., and Perlmutter, R. M. (1991) J. Exp. Med. 173, 1421-1432[Abstract] |
9. | Voronova, A. F., Adler, H. T., and Sefton, B. M. (1987) Mol. Cell. Biol. 7, 4407-4413[Medline] [Order article via Infotrieve] |
10. | Garvin, A. M., Pawar, S., Marth, J. D., and Perlmutter, R. M. (1988) Mol. Cell. Biol. 8, 3058-3064[Medline] [Order article via Infotrieve] |
11. | Adler, H. T., Reynolds, P. J., Kelley, C. M., and Sefton, B. M. (1988) J. Virol. 62, 4113-4122[Medline] [Order article via Infotrieve] |
12. | Takadera, T., Leung, S., Gernone, A., Koga, Y., Takihara, Y., Miyamoto, N. G., and Mak, T. W. (1989) Mol. Cell. Biol. 9, 2173-2180[Medline] [Order article via Infotrieve] |
13. | Wildin, R. S., Wang, H. U., Forbush, K. A., and Perlmutter, R. M. (1995) J. Immunol. 155, 1286-1295[Abstract] |
14. | Reynolds, P. J., Lesley, J., Trotter, J., Schulte, R., Hyman, R., and Sefton, B. M. (1990) Mol. Cell. Biol. 10, 4266-4270[Medline] [Order article via Infotrieve] |
15. | Wildin, R. S., Garvin, A. M., Pawar, S., Lewis, D. B., Abraham, K. M., Forbush, K. A., Ziegler, S. F., Allen, J. M., and Perlmutter, R. M. (1991) J. Exp. Med. 173, 383-393[Abstract] |
16. | Allen, J. M., Forbush, K. A., and Perlmutter, R. M. (1992) Mol. Cell. Biol. 12, 2758-2768[Abstract] |
17. | Wang, Y., Kobori, J. A., and Hood, L. (1993) Mol. Cell. Biol. 13, 5691-5701[Abstract] |
18. | Karasuyama, H., Rolink, A., and Melchers, F. (1988) J. Exp. Med. 167, 1377-1390[Abstract] |
19. | Wakao, H., Gouilleux, F., and Groner, B. (1994) EMBO J. 13, 2182-2191[Abstract] |
20. | Chaffin, K. E., Beals, C. R., Wilkie, T. M., Forbush, K. A., Simon, M. I., and Perlmutter, R. M. (1990) EMBO J. 9, 3821-3829[Abstract] |
21. |
Kozmik, Z.,
Czerny, T.,
and Busslinger, M.
(1997)
EMBO J.
16,
6793-6803 |
22. | Georgopoulos, K., Bigby, M., Wang, J. H., Molnar, A., Wu, P., Winandy, S., and Sharpe, A. (1994) Cell 79, 143-156[Medline] [Order article via Infotrieve] |
23. | Georgopoulos, K., Moore, D. D., and Derfler, B. (1992) Science 258, 808-812[Medline] [Order article via Infotrieve] |
24. | Molnar, A., and Georgopoulos, K. (1994) Mol. Cell. Biol. 14, 8292-8303[Abstract] |
25. | Georgopoulos, K., Morgan, B. A., and Moore, D. D. (1992) Mol. Cell. Biol. 12, 747-757[Abstract] |
26. | Bray, P., Lichter, P., Thiesen, H. J., Ward, D. C., and Dawid, I. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9563-9567[Abstract] |
27. |
Hasegawa, T.,
Takeuchi, A.,
Miyaishi, O.,
Isobe, K.,
and de Crombrugghe, B.
(1997)
J. Biol. Chem.
272,
4915-4923 |
28. |
Passantino, R.,
Antona, V.,
Barbieri, G.,
Rubino, P.,
Melchionna, R.,
Cossu, G.,
Feo, S.,
and Giallongo, A.
(1998)
J. Biol. Chem.
273,
484-494 |
29. | Merchant, J. L., Iyer, G. R., Taylor, B. R., Kitchen, J. R., Mortensen, E. R., Wang, Z., Flintoft, R. J., Michel, J. B., and Bassel-Duby, R. (1996) Mol. Cell. Biol. 16, 6644-6653[Abstract] |
30. |
Law, G. L.,
Itoh, H.,
Law, D. J.,
Mize, G. J.,
Merchant, J. L.,
and Morris, D. R.
(1998)
J. Biol. Chem.
273,
19955-19964 |
31. | Hasegawa, T., Xiao, H., and Isobe, K. (1999) Biochem. Biophys. Res. Commun. 256, 249-254[CrossRef][Medline] [Order article via Infotrieve] |
32. | Ye, S., Whatling, C., Watkins, H., and Henney, A. (1999) FEBS Lett. 450, 268-272[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Reizis, B.,
and Leder, P.
(1999)
J. Exp. Med.
189,
1669-1678 |
34. |
Wieczorek, E.,
Lin, Z.,
Perkins, E. B.,
Law, D. J.,
Merchant, J. L.,
and Zehner, Z. E.
(2000)
J. Biol. Chem.
275,
12879-12888 |
35. | Turner, J., and Crossley, M. (1999) Trends Biochem. Sci. 24, 236-240[CrossRef][Medline] [Order article via Infotrieve] |
36. | Crossley, M., Whitelaw, E., Perkins, A., Williams, G., Fujiwara, Y., and Orkin, S. H. (1996) Mol. Cell. Biol. 16, 1695-1705[Abstract] |
37. |
Turner, J.,
and Crossley, M.
(1998)
EMBO J.
17,
5129-5140 |
38. |
Muise-Helmericks, R. C.,
and Rosen, N.
(1995)
J. Biol. Chem.
270,
27538-27543 |