Sp1 Transactivation of the TCL1 Oncogene*
Samuel W.
French
,
Cindy S.
Malone§,
Rhine R.
Shen
,
Mathilde
Renard
,
Sarah E.
Henson§,
Maurine D.
Miner
,
Randolph
Wall§¶
, and
Michael A.
Teitell
¶
**
§§
From the
Department of Pathology and Laboratory
Medicine, the § Department of Microbiology, Immunology,
and Molecular Genetics, ¶ Molecular Biology Institute,
Jonsson Comprehensive Cancer Center, ** Department
of Pediatrics and 
AIDS Institute, David
Geffen School of Medicine at UCLA, Center for the Health Sciences,
Los Angeles, California 90095-1732
Received for publication, July 17, 2002, and in revised form, November 1, 2002
 |
ABSTRACT |
Cis-regions and trans-factors controlling
TCL1 oncogene expression are not known. We identified the
functional TCL1 promoter by mapping four transcriptional
start sites 24-30 bp downstream of a TATA box. A 424-bp fragment
upstream of the major start site showed robust promoter activity
comparable with SV40 in both TCL1 expressing and
non-expressing cell lines. Additional constructs spanning 10 kb
upstream and 20 kb downstream of the start site showed only modest
increases in reporter activity indicating that TCL1
expression is primarily controlled by the promoter. Ten putative Sp1-binding sites were identified within 300 bp of the start site, and
three of these specifically bound Sp1. A dose-dependent
transactivation of the TCL1 promoter with Sp1 addition in
Sp1-negative Drosophila SL2 cells was observed, and
mutation of the three identified Sp1-binding sites significantly
repressed reporter gene expression in 293T cells, confirming a key role
for Sp1 in activating the TCL1 promoter in
vivo. In TCL1 silent cell lines, CpG DNA methylation
was rarely observed at functional Sp1 sites, and methylation of a
previously reported NotI restriction site was associated
with dense CpG methylation rather than endogenous TCL1 gene
silencing. Together, these results indicate that Sp1 mediates
transactivation of the TCL1 core promoter and that
TCL1 gene silencing is not dependent on mechanisms
involving Sp1 and NotI site methylation.
 |
INTRODUCTION |
The T-cell leukemia-1 (TCL1) oncogene is expressed
mainly but not exclusively at specific stages of lymphocyte development in humans. In normal T-lineage cells, TCL1 expression is
restricted to CD3/CD4/CD8 triple-negative immature thymocytes (1).
Interestingly, mature peripheral T-cell expansions and clonal
malignancies may aberrantly express TCL1 due to
characteristic chromosomal translocations and inversions at 14q32.1
(reviewed in Ref. 2). These chromosomal rearrangements reposition
T-cell receptor
/
- or
-chain control sequences next to the
TCL1 coding region leading to T-cell-specific dysregulation.
A tumorigenic role for this aberrant expression has been confirmed from
transgenic studies in which TCL1 dysregulation targeted to
T-cells mainly in the thymus cause mice to exclusively develop mature
peripheral T-cell malignancies (3).
In normal B-lineage cells, TCL1 is expressed from early
pro-B bone marrow precursors through mature peripheral B-cell stages of
development (1, 4, 5). Terminally differentiated B-cells, such as
non-proliferating memory or plasma cells, lack TCL1
expression. TCL1 gene silencing with terminal B-cell
maturation has been shown to correlate with the conversion of
TCL1-positive B-cells to plasmacytoid cells by growth on
CD40L-expressing fibroblasts supplemented with interleukin-4 and
interleukin-10 (5). Interestingly, recent studies (4-7) have also
linked aberrant TCL1 expression to specific classes of
mature B-cell lymphoma. Approximately 30% of diffuse large B-cell
lymphomas (DLBCL)1 and about
75% of AIDS-related DLBCL abnormally express TCL1 (4, 5).
Evidence that this dysregulation promotes B-cell malignancies has been
obtained from transgenic mouse studies in which TCL1 is
aberrantly expressed only in B-cells or in both T- and B-cells (8, 13).
In this situation, dysregulation strongly favors the development of
peripheral B-cell tumors versus T-cell malignancies. The
mechanism for induction of both T- and B-cell malignancies is thought
to rely on inappropriately strong co-activation of the serine-threonine
kinase AKT by excessive TCL1 oncoprotein levels (9-14).
In contrast to known chromosomal rearrangements that cause aberrant
expression in mature T-cell tumors, little is known about the
mechanism(s) regulating TCL1 expression during development or supporting its aberrant expression in mature B-cell malignancies. The TCL1 promoter and other potential regulatory regions
have not been characterized, and there have been no reports of 14q32.1 rearrangements with associated dysregulation of TCL1 for
B-cell malignancies, including TCL1-expressing Burkitt
lymphomas (BL), B-chronic lymphocytic leukemias (
-CLL), or DLBCL
(15). Initial studies by Yuille et al. (15) reported a
correlation between the methylation status of two CpG sites within a
single NotI site in the putative TCL1 promoter
and TCL1 expression levels. Also, treatment of
TCL1 non-expressing Jurkat and CEM T-cells with the DNA
methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AC) was reported
to activate TCL1 expression, suggesting a potential role for
epigenetic modifications in TCL1 gene silencing (15). While
providing important first clues, further analyses are needed to improve
our understanding of the mechanisms regulating TCL1 in
expressing and silent cell types during development and in cancer. Here
we provide the first detailed functional characterization of the human
TCL1 promoter, and we demonstrate a key role for Sp1 in
activating TCL1 gene transcription from the core promoter.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines and Cell Culture--
EBV-immortalized fetal cord
blood lymphocyte line 75714 was created, and cell lines were obtained
and grown as described (5) except human myeloma AF10 which was provided
as a kind gift from M. Kuehl (NCI, National Institutes of Health,
Bethesda). 293T fibroblasts, Raji BL, and UC 729-6 B-lymphoblastoid
cells were purchased from ATCC. SL2 Drosophila cells (a kind
gift from L. Zipursky, UCLA, Los Angeles) were grown in Schneider's
Drosophila media (Invitrogen) supplemented with 10% fetal
bovine serum at room temperature.
S1 Nuclease Protection--
Total cellular RNA was isolated from
Ramos, 2F7, and KS-1 cells (Qiagen). A complementary oligonucleotide
was manufactured that overlapped the presumed transcriptional start
site:
5'-ACTCGGCCATGGCGTCCTCGGGCCGCCTAAGAAGCAAGAGCCAGAGCCTCTCAAGGCCGCTCGCTGGTCCCTGGGATGTG-3'. A single-stranded oligo-probe was 5' end-labeled with
[
-32P]ATP and purified by G-50 Sephadex spin column
chromatography. A G + A ladder was created using the Maxam-Gilbert
sequencing method (16). In brief, 500,000 cpm of radiolabeled probe was incubated with 5 µg of salmon sperm DNA and 1 M
piperidine/formate at 37 °C for 20 min. The reaction was frozen on
dry ice and dried to completion in a Speedvac concentrator. 20 µl of
deionized water was added, and the reaction was frozen and re-dried as
before. 100 µl of 1 M piperidine was added, and the
reaction was incubated at 90 °C for 20 min and dried. 100 µl of
deionized water was added, and the reaction was dried. The reaction was
resuspended in loading dye and boiled for 3 min before gel loading. S1
nuclease protection was performed as described (16), with some
modifications. In brief, 500,000 cpm of radiolabeled probe was
hybridized with 50 µg of total RNA at 30 °C overnight. 450 units
of S1 nuclease (Protégé) was added, and probe digestion was
carried out at 30 °C for 2 h. After ethanol precipitation, the
reaction was resuspended in loading dye, boiled for 3 min, and loaded
on an 8% acrylamide, 8 M urea denaturing gel. Gels were
run at 1400 V for 2.5 h at room temperature in 1× TBE, dried, and
exposed to film.
TCL1 Promoter-Reporter Gene Constructs--
The sequence of the
human TCL1 genomic locus at 14q32.1 (GenBankTM
HTG data base entry accession number AL139020.1) was used to engineer
PCR cloning primers. The reverse primer began at the ATG translation
start site (5'-ATGGCGTCCTCGGGCCGCCTAAGAAGCAAG-3') and was paired
with the following forward primers: (
191)
5'-ACGTAGCGCCTGCGCGGGACCCTCA-3'; (
350)
5'-AGAAAGGGCCAAGGTCACCCCGGTGCCTCT-3'; (
424)
5'-GTCGACTGTGAGTTCCCAGCAGAG-3'; (
543)
5'-AAGGCACAGGCTGGTGGAGATCCAGGGAAC-3'; (
760)
5'-GGTGGAAGGAGGGATTTCTTTTTAAG-3'; and (
943)
5'-TGATGTTTGAACCAGGCTGGAGCTGG-3'. PCR-amplified products from 200 ng of BL41 (and TC32 Ewing sarcoma) genomic DNA were isolated on a 1%
agarose gel, cloned into the pCR2.1-TOPO vector (Invitrogen), digested
with EcoRI, subcloned into the EcoRI site of the
pGL3-basic firefly luciferase expression plasmid (Promega) and cycle
sequenced. Reporter gene point mutants were generated with the
QuikChange Kit (Stratagene) and targeted CCGCCC to CCGAAC changes made
in specific Sp1 GC box-binding motifs using techniques provided by the manufacturer.
Reporter Gene Assays--
107 mammalian cells were
co-transfected with 10 µg of each test construct and 1 µg of the
Renilla transfection control plasmid pRL-SV40
(Promega) by electroporation (250 V, 1180 microfarads, low resistance,
4 °C in ice-cold supplemented RPMI 1640 plus 5% fetal calf serum;
Invitrogen Cell-Porator). Cells were harvested at 48 h, and
Renilla-normalized firefly luciferase luminescence was
measured using the Dual Luciferase Assay System (Promega). SL2
Drosophila cells were co-transfected in 6-well plates using LipofectAMINE (Invitrogen) per the manufacturer's instructions. 3 µg
of pGL3-basic, pSV40-luciferase or
p424TCL1-luciferase constructs were used along with varying
amounts of the control plasmid pPac and 0, 100, or 300 ng
of the Drosophila Sp1 expression plasmid pPacSp1
(generously provided by R. Tjian, University of California, Berkeley),
keeping the total amount of DNA constant at 3.3 µg (17). Cells were
harvested at 48 h, and protein was normalized using the Bio-Rad
Protein Assay (Bio-Rad). Firefly luciferase luminescence was measured
using the Luciferase Assay System (Promega).
EMSA Analysis--
Preparation of crude nuclear extract from
BCBL-1 and Ramos cells was as described (18). Electrophoretic mobility
shift assay (EMSA) was performed as described previously (19). Briefly, EMSA probes and cold competitors were complementary double-stranded DNA
oligonucleotides containing each of the 10 identified Sp1 consensus
sites within the TCL1 core promoter region (see Fig. 1). The probe sequences are as follows:
Sp1(consensus), 5'-ATTCGATCGGGGCGGGGCGAGC-3'; Sp1(A),
5'-CAGCAGCAGAGGGCGGCGGTCGGTG-3'; Sp1(B),
5'-CCTCGAGGAAGGCGCGGGCCAGCTG-3'; Sp1(C),
5'-CGGGACGTAGCGCCTGCGCGGGACC-3'; Sp1(D),
5'-CCCACAAACCCCCGCCCCATCCTGCC-3'; Sp1(E),
5'-GCCTTACGCCCCGCCCCAAGGTCGT-3'; Sp1(F),
5'-ACCCGGGGTCCCGCCCCAAGACCGT-3'; Sp1(G),
5'-CCGTCCTCCCGCCCCGCCGCTTGGT-3'; Sp1(H),
5'-GCCGCTTGGTGGCGCCCGCATGCTG-3'; Sp1(I),
5'-CTTGCTTCTTAGGCGGCCCGAGGAC-3'; and Sp1(J),
5'-CTTAGGCGGCCCGAGGACGCCATGG-3'. Mutant Sp1 probes were also
created by changing the GC box-binding sequence from CCGCCC to CCGAAC
to create Mut-Sp1(D), Mut-Sp1(E), Mut-Sp1(F), and Mut-Sp1(G) (20).
Probes were end-labeled with [
-32P]dATP using T4
polynucleotide kinase (New England Biolabs). 10 µg of BCBL-1 or Ramos
nuclear extract was incubated with 20 mM HEPES, pH 7.9, 0.2 mM EDTA, 50 mM KCl, 1 mM
dithiothreitol, 5% glycerol, 2 µg of poly(dI-dC), and 10 µg of
bovine serum albumin in a 20-µl reaction volume. Radiolabeled probe
(20,000 cpm per reaction) was added, and samples were incubated for 20 min at 25 °C. DNA-protein complexes were resolved by electrophoresis for 2 h at 150 V in a 4% non-denaturing polyacrylamide gel and subjected to autoradiography. Competition assays were performed with a
500-fold molar excess of unlabeled probe. Antibody blocking assays were
performed by adding 2 µg of either Sp1 antibody (clone 1C6, Santa
Cruz Biotechnology) or control normal mouse IgG (Santa Cruz
Biotechnology) to the reaction 10 min prior to incubation with
probes.

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Fig. 1.
The TCL1 promoter. The
sequence from 304 to +66 nucleotides relative to the major
transcription start site (large arrowhead) is shown. Three
minor start sites are depicted with small arrowheads. The
TATA box is underlined and enlarged whereas the
translation start site is enlarged and shown in
italics. Consensus Sp1-binding sites (CCGCCC; A, D, E,
F, and G) are boxed with a solid
line, and those shaded in gray functionally
bind Sp1 by EMSA (see Fig. 5). Potential Sp1-binding sites (B, C,
H, I, and J), based on a conserved CCGC core sequence,
are boxed with dashed lines (40). CpG
dinucleotides that were surveyed for methylation (see Fig. 8) are
indicated by boldface type and numbered
1-33.
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Western Blot Analysis--
10 µg per lane of SL2 cell extract
or 15 µg per lane of cell extracts from a variety of human
TCL1-expressing and silent lymphocyte cell lines was
resolved on a 10% denaturing polyacrylamide gel followed by transfer
to nitrocellulose membranes (Micron Separations). Western blotting was
performed as described previously (5) with the following modifications.
The membrane was incubated with Sp1 antiserum (1:2000 clone 1C6, Santa
Cruz Biotechnology), washed, and incubated with horseradish
peroxidase-conjugated mouse antiserum (Cell Signaling Technology) and
exposed to film following development with ECL+Plus (Amersham Biosciences).
Genomic Bisulfite Sequencing--
Sodium bisulfite treatment of
DNA has been described (21, 22). 2 µg of genomic DNA was
restriction-digested with HindII and NcoI to
isolate the basal TCL1 promoter, ethanol-precipitated, resuspended in 40 µl of water, heated to 97 °C for 5 min, and placed on ice. 2 µl of 6.3 M NaOH was added, and the
mixture was incubated at 39 °C for 30 min. 416 µl of fresh sodium
bisulfite solution (40.5 g of sodium bisulfite dissolved in 80 ml of
water, pH adjusted to 5.1, add 3.3 ml of 20 mM
hydroquinone; adjust final volume to 100 ml with water) was added, and
the mixture was incubated at 55 °C for 15 h, with a heat shock
to 95 °C every 3 h. Sodium bisulfite-treated DNA was desalted
with the Wizard DNA Clean-up System (Promega); 6.3 M NaOH
was added to a final concentration of 0.3 M followed by
incubation at 37 °C for 15 min. 10 M ammonium acetate,
pH 7.0, was added to a final concentration of 3 M, followed by ethanol precipitation, pellet drying, and resuspension in 100 µl
of TE. 2 µl of sodium bisulfite-treated DNA was PCR-amplified for 47 cycles using multiple sense-strand primer pair combinations for
TCL1 followed by 30 cycles of nested PCR amplification.
Primers used to amplify sodium bisulfite-treated DNA include
5'-GGGGGGGTTTTTTAGAAGAAGAAAGGG-3' and 5'-CCAAAACCTCTCAAAACCACTC-3'.
Nested primers used were either 5'-GGTGTTTTTTTAGTAGTAGTAGAGGG-3' or
5'-GTTTTAGGGAGTAAGTTAGGTTGGG-3'. PCR products were cloned into
the pCR2.1-TOPO vector and transformed into DH5
bacteria, and
individual clones were sequenced.
 |
RESULTS |
TCL1 Transcriptional Start Site Determination--
A transcription
initiation site has been suggested at 41 bp downstream of a putative
TATA box based upon the initial reverse transcriptase-PCR cloning of
the TCL1 cDNA (1, 15). However, the TCL1
start site has not been experimentally determined, and this position
would be inconsistent with known start sites from TATA
box-dependent promoters, which are generally 25-30
nucleotides downstream of the first T in the TATA sequence (reviewed in
Refs. 23 and 24). Therefore, we used S1 nuclease protection analysis to
identify the transcription start site(s) of TCL1 in order to help locate the major promoter for further investigation (Fig. 2). In TCL1 expressing 2F7 and
Ramos BL cells, the major site of transcription initiation is a
cytosine located 30 nucleotides downstream of the TATA box. There are
also three identical minor sites of transcription initiation in these
two cell lines. These sites include an adenine at 24 nucleotides, a
guanine at 25 nucleotides, and a guanine at 27 nucleotides downstream
of the TATA box (summarized in Fig. 1). These sites are consistent with
transcription initiation sites in TATA box-dependent
promoters. In addition, S1 nuclease protection products were not seen
in KS-1 primary effusion lymphoma (PEL) cells, consistent with a lack
of TCL1 transcription in this line (5), and no products were
observed with a probe incubated with S1 nuclease in the absence of
input RNA (data not shown).

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Fig. 2.
TCL1 transcription start sites
determined by S1 nuclease protection assay. A single-stranded
end-labeled probe complementary to the presumed start site(s) of
TCL1 transcription was used. The probe was incubated with 50 µg per sample of total cellular RNA from KS-1
(TCL1-silent), 2F7, and Ramos (TCL1-expressing)
B-cell lines (5). The protected products were resolved on an 8%
acrylamide, 8 M urea denaturing gel. Lane 1, G + A ladder; lane 2, KS-1 RNA; lane 3, 2F7 RNA; and
lane 4, Ramos RNA. The major transcription start site is
indicated with a heavy arrow, and the minor start sites are
indicated with smaller arrows (see also Fig. 1). The
sequence of the complementary oligonucleotide is indicated on the
left.
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TCL1 Promoter Identification--
To date, regions suspected to
contain TCL1 promoter activity have not been analyzed. We
sought to define the TCL1 promoter and began a search just
upstream of the TATA box and transcription initiation sites. A genomic
fragment was cloned beginning at the ATG translation start site and
extending to minus 424 bp from the major transcription start site using
the originally reported upstream sequence as a guide for primer design
(1). Sequencing of a BL41-derived fragment (and one derived from TC32
Ewing sarcoma cell genomic DNA, data not shown) revealed that the
originally reported 5' sequence contains an ~60-bp duplication that
is not found in clones generated here or in recent data released from GenBankTM (data base entry GI 624960) (1). This fragment
was inserted into pGL3-basic, creating p424TCL1-luciferase.
Previously it was shown that EBV infection has no effect on endogenous
TCL1 expression levels and that EBV-immortalized peripheral
blood cells, along with BL lines, express abundant TCL1 (5).
Reporter activity in three of these highly expressing B-cell lines
(75714, BL41, and 2F7) was 15-35-fold elevated over an empty vector
construct, pGL3-basic, that lacks known promoter activity (Fig.
3A). PEL lines silence
B-cell-specific gene transcription, including TCL1, and are
derived from terminal stages of B-cell differentiation that normally
lack TCL1 expression (25-27). In four silent PEL lines
(BCBL-1, BC-1, BC-3, and KS-1), the TCL1 reporter gene was expressed 15-45-fold over pGL3-basic. In addition, a 12-fold
activation was seen in Jurkat T-cells, which also do not express the
endogenous TCL1 gene. These results indicate that the
424
TCL1 gene fragment contains strong promoter activity and
lacks functional silencing elements in transient transfection assays in
lymphocytes. Furthermore, the level of activity was roughly equivalent
to the activity seen with a robust SV40 promoter
positive-control construct in all lines examined.

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Fig. 3.
TCL1 promoter activity in
TCL1-expressing and non-expressing cell types.
A, TCL1-expressing (75714, BL41, and 2F7) and
silent (Jurkat, KS-1, BC-1, BC-3, and BCBL-1) lines were transiently
transfected with either p424TCL1-luciferase or
pGL3SV40-luciferase expression and pGL3-basic (control)
constructs and assayed for luciferase activity. White bars
display SV40 reporter activity, and black bars
show TCL1 reporter activity in comparison to pGL3-basic
activity, which was set at 1. Each transfection was normalized to a
co-transfected Renilla-luciferase vector
(pRLSV40-luciferase) to control for transfection efficiency.
Error bars denote S.D. from three separate experiments using
independently isolated reporter gene DNA. B, DNA fragments
from 191 to 943 bp upstream of the major transcription start site do
not significantly affect TCL1 promoter activity in
TCL1-negative 293T fibroblast and BC-3 PEL cells.
White bars display TCL1 reporter activity in 293T
fibroblasts, and black bars display reporter activity in
BC-3 PEL cells in comparison to pGL3-basic activity, which was set at
1. Each transfection was normalized to co-transfected
pRLSV40-luciferase to control for transfection efficiency.
Error bars denote S.D. from at least four separate
experiments using independently isolated reporter gene DNA. Scales for
fold induction are shown above and below the bar
diagram for each cell line.
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We next performed an extended search for further 5' sequences affecting
TCL1 promoter activity. Overlapping fragments from 191- to
943-bp upstream of the transcription start site were cloned into
pGL3-basic and tested for promoter activity. All of the engineered constructs demonstrated robust expression in TCL1-negative
BC-3 PEL and 293T fibroblast cells (Fig. 3B). In BC-3 PEL
cells, reporter gene activity was essentially equivalent for constructs
containing 350-943 bp of 5' sequence and ranged from 17- to 25-fold
stronger than pGL3-basic. This indicates that cis-elements required for activity in transient assays are localized within 350 bp of the transcription start site and that no additional strong enhancer or
silencer motifs are within 1 kb upstream. Reporter gene expression in
293T cells was 15-35-fold higher compared with levels seen in BC-3 PEL
cells using a similar range of reporter constructs (Fig.
3B). In fact, a 191-bp 5' fragment showed reporter activity equivalent to that seen with a 424-bp fragment, suggesting that all
essential core promoter components are present within the first 200 bp
5' of the transcription start site. In sum, these findings suggest the
TCL1 core promoter region functions in a tissue-nonspecific
manner. Similar findings have been reported from the analyses of many
other core promoters from tissue-specific genes (28-35).
Sp1 Binding in the TCL1 Core Promoter--
The 350-bp core
promoter of TCL1 exhibited robust activity that was not
significantly affected by upstream (to 10 kb) or downstream (to 20 kb)
elements in expressing versus non-expressing or lymphoid versus non-lymphoid cell
types.2 This indicates that
critical regions responsible for expression in transient transfections
are present in the core promoter and that the factor(s) driving this
expression are broadly expressed in distinct cell types. MatInspector
(36) analysis of this 350-bp promoter sequence revealed 5 consensus GC
box Sp1 factor-binding motifs (CCGCCC) that are labeled A, D, E,
F, and G in Fig. 1 (1, 37-39). A previous
transcription factor-binding site study showed that the core sequence
CGCC was sufficient, in multiple sequence contexts, to facilitate Sp1
binding in EMSA (40). Therefore, sites with this core motif are labeled
B, C, H, I, and J in Fig. 1, bringing to 10 the
total number of putative Sp1-binding sites within the TCL1
core promoter region. Sp1 is a ubiquitous transcription factor
(reviewed in Refs. 41 and 42), and its known transactivating function
is consistent with a role in driving TCL1 promoter activity in all the cell types examined regardless of endogenous TCL1
expression (Fig. 3, A and B). Supporting this
postulate, Western blot analysis shows that the level of Sp1 protein in
TCL1-expressing and silent lymphocyte lines is equivalent,
which also indicates that changes in the level of Sp1 are not
responsible for the tissue-specific expression of TCL1 (Fig.
4).

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Fig. 4.
Equivalent Sp1 protein levels in
TCL1-expressing and silent cell lines.
A, Western blot using Sp1 antiserum on cell lysates from
TCL1-expressing (UC 729-6, Ramos, BL41, and Raji) and
TCL1-silent (Jurkat, BCBL-1, BC-3, and KS-1) B- and T-cell
lines. B, Coomassie Brilliant Blue-stained gel indicating
equal total protein loading for each cell lysate examined.
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Double-stranded DNA oligomers of these 10 potential Sp1 sites were
tested for binding function by EMSA with nuclear extracts from
TCL1-silent BCBL-1 PEL cells and TCL1-expressing
Ramos cells (Fig. 5 and data not shown).
Three sites, Sp1(D) Sp1(E), and Sp1(F), formed a complex resulting in a
band with decreased gel mobility. This complex appears to be specific,
as the band was competed away with unlabeled consensus cold Sp1 and
self-oligomers. It was not competed away with a nonspecific oligomer
that binds another transcription factor (LEF) (43-46). Additionally,
Sp1-specific antibody significantly blocked complex formation by
preincubation with nuclear extracts, whereas nonspecific IgG antibody
had no effect on complex formation (Fig. 5, D and
E). Furthermore, Mut-Sp1(D), Mut-Sp1(E), Mut-Sp1(F), and
Mut-Sp1(G) oligomers not only failed to generate a reduced mobility
band shift but they were also ineffective at inhibiting complex
formation with wild-type Sp1(D), Sp1(E), and SP1(F) probes (Fig. 5 and
data not shown). Taken together, the data demonstrate that Sp1
interacts with Sp1(D), Sp1(E), and Sp1(F) sites centrally located
within the first 150 bp of the TCL1 core promoter.

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Fig. 5.
Sp1 binding to 3 of 10 Sp1 sites within the
TCL1 promoter. EMSA was performed using BCBL-1
(A-E and G-I) and Ramos (F) nuclear
extracts. Radiolabeled or competitor probes Sp1(A) through Sp1(J) and
mutant probes Mut Sp1(D) through Mut Sp1(G), corresponding to the boxed
Sp1 elements depicted in Fig. 1, along with an Sp1(consensus) sequence
probe were investigated. A-C, nonspecific (LEF) and
distinct Sp1 cold competitors were used to demonstrate binding
specificity. Identical results to those presented in C were
also obtained with Sp1(D)- and Sp1(F)-labeled probes (data not shown).
D and E, EMSA was performed with radiolabeled Sp1
oligomers and pretreatment of nuclear extracts with Sp1 antiserum or
control IgG. Identical results to those presented in E were
also obtained with Sp1(E)- and Sp1(F)-labeled probes (data not shown).
F, G, and I, Mut-Sp1(D), Mut-Sp1(E), and
Mut-Sp1(F) probes cannot compete with radiolabeled Sp1(consensus) or
Sp1(E) probes for Sp1 binding. Identical results were obtained with
radiolabeled Sp1(D) and Sp1(F) probes (data not shown). H
and I, Mut-Sp1(D), Mut-Sp1(E), and Mut-Sp1(F) (data not
shown) oligomers do not bind Sp1. Overall, Sp1 consensus and Sp1(D),
Sp1(E), and Sp1(F) sequences within the TCL1 core promoter
specifically bind Sp1 from TCL1 silent (BCBL-1) and
expressing (Ramos) nuclear extracts.
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Sp1 Transactivates the TCL1 Core Promoter--
To confirm that Sp1
can functionally transactivate the TCL1 promoter,
co-transfection assays were performed in Sp1-negative Drosophila SL2 cells where the effect of exogenous Sp1
expression can be tested on Sp1-dependent promoters (17).
Promoter-less control (pGL3-basic), Sp1-dependent
pGL3SV40-luciferase, or p424TCL1-luciferase expressing constructs were transfected into Drosophila cells
with varying amounts of human Sp1 generated by a fly-specific Sp1
expression vector (pPacSp1; Fig.
6A). The level of reporter
gene expression detected for each construct in the absence of
co-expression of exogenous Sp1 was assigned the arbitrary value of 1. The SV40 promoter was induced 150-180-fold (data not
shown), whereas the TCL1 promoter was induced 9-21-fold
with exogenous Sp1 expression compared with the induction without Sp1
expression (Fig. 6B). Western analysis confirmed
dose-dependent expression of exogenous Sp1 with increasing
amounts of pPacSp1 in transfected SL2 cells. In addition,
reporter constructs containing single, double, or triple mutations
engineered into the Sp1(D), Sp1(E), and Sp1(F) sites, along with a
control mutation engineered into the non-binding Sp1(G) site, were
tested for activity in 293T fibroblast cells (Fig.
7). 293T cells are the optimal cell line
for analyzing the effects of Sp1-binding site mutations, because the
robust expression of TCL1 reporter constructs in 293T cells
(Fig. 3B) would require a strong inhibitory effect from
Sp1-binding site mutants to significantly reduce expression. In this
context, the occurrence of a statistically significant inhibitory
effect would strongly support a powerful role for Sp1 in regulating
TCL1 promoter activity. Mutations of the Sp1D, -E, and
-F-binding sites resulted in markedly decreased expression of the
TCL1 reporter construct (Fig. 7). The amount of expression
was reduced in all single site mutants relative to the unmutated
reporter construct and further decreased substantially in double and
triple mutant constructs (p
.01). Importantly, mutation of the Sp1(G)-binding site, which failed to bind Sp1 by EMSA
analysis (Fig. 5B), had no effect on TCL1
reporter construct expression (p > 0.05).
Together, the data from EMSA analysis, SL2 Drosophila cell
studies, and mutant reporter construct investigations in 293T cells
strongly indicate that Sp1 transactivating factors and cis-binding
sites Sp1(D), Sp1(E), and Sp1(F) play a dominant role in regulating
TCL1 core promoter activity.

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Fig. 6.
Sp1-induced expression of the TCL1
core promoter in Drosophila SL2 cells. SL2
cells were transfected with promoter-less pGL3-basic,
Sp1-dependent pSV40-luciferase, or
p424TCL1-luciferase expression constructs and varying
amounts of the pPacSp1 Sp1 expression vector. A,
Western analysis of Sp1 level from a representative experiment. The
amount of co-transfected pPacSp1 vector is indicated at the
top. Arrowheads at the right indicate the Sp1
specific immunoreactive band and a nonspecific (ns), lower
molecular weight band. Equal protein was loaded in each lane, followed
by confirmation with Coomassie Brilliant Blue gel staining prior to
transfer (data not shown). B, dose-dependent
induction of TCL1 expression by Sp1. Amount of
co-transfected pPacSp1 is indicated at the
bottom. The Sp1-dependent pSV40
luciferase-positive control construct exhibited a 150-180-fold
induction with Sp1 addition under identical conditions (data not
shown).
|
|

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Fig. 7.
Mutation of three Sp1-binding sites inhibits
TCL1 reporter gene expression. 293T cells were
transfected with promoter-less pGL3-basic (BASIC),
p350TCL1-luciferase ( 350), or mutant
p350TCL1-luciferase expression constructs containing
mutations in Sp1 protein-binding sites. Single mutant (Mut-D, Mut-E,
Mut-F, and Mut-G), double mutant (Mut-DE, Mut-DF, and Mut-EF), and
triple mutant (Mut-DEF) reporter constructs were tested. Black
bars show TCL1 reporter activity in comparison to
pGL3-basic activity, which was set at 1. Each transfection was
normalized to co-transfected pRLSV40-luciferase to control
for transfection efficiency. Error bars denote S.D. from at
least six separate experiments using independently isolated reporter
gene DNA. Asterisks indicate statistically significant
differences from unmutated p350TCL1-luciferase results using
a two-sided Student's t test (p .01).
Identical results were obtained using similarly mutated and unmutated
p191TCL1-luciferase and p424TCL1-luciferase
expression constructs (data not shown).
|
|
CpG Methylation Is Not Correlated with TCL1 Silencing--
The
region from +10 to
300 bp of the TCL1 core promoter
contains 33 CpG sites and corresponds to a classical "CpG island" (Fig. 1) (47). Based on differential sensitivity at a single NotI restriction enzyme site in this core promoter, Yuille
et al. (15) previously concluded that TCL1
silencing was mediated by CpG methylation. Their finding that
TCL1 expression was activated in silenced cells by 5-AC
treatment was advanced as further support for this conclusion, although
this reactivation experiment does not distinguish between direct and
indirect effects resulting from 5-AC treatment. We used genomic
bisulfite sequencing to determine definitively the methylation status
of every cytosine in the TCL1 core promoter of expressing
and non-expressing cell types (Fig. 8).
As expected, no CpG methylation was detected at any of the 33 potential
sites in core promoter clones derived from TCL1-expressing BL line BL41 (Fig. 8). The core promoter clones from five tested cell
lines in which TCL1 is silenced showed no, minimal (1 or 2 sites), moderate (5-10 sites), or dense (>50% of sites) CpG methylation. Both unmethylated and CpG-methylated promoter clones were
seen in 2 of 5 TCL1 silent cell lines (BCBL-1 and Jurkat), consistent with either one unmethylated allele in all cells or a
mixture of cells containing both methylated and unmethylated TCL1 alleles. Non-expressing BC-3 cells contained both
moderately and heavily CpG-methylated clones, with the moderately
CpG-methylated positions all clustering within 50-bp at the 5' end of
the promoter. All of the core promoter clones from the two remaining
TCL1 silent cell lines tested, BC-1 and AF10, were either
totally unmethylated or contained only minimal (1-2) methylated CpG
core promoter sites, indicating that neither allele is methylated in
these cells. Because 4 of 5 TCL1 negative cell lines
analyzed with bisulfite sequencing contained unmethylated core promoter
clones, CpG methylation is not correlated with or directly responsible
for TCL1 gene silencing. Methylation of two CpG sites
contained within the previously analyzed NotI site was only
detected in those promoter clones with the highest degree of CpG
methylation, such as in clones with
half of the 33 potential
sites methylated in BCBL-1, BC-3, and Jurkat cell lines. This indicates
that NotI methylation is only consistently associated with
the most extensive CpG methylation and is not a marker for silencing
the TCL1 core promoter in all negative cell types.
Significantly, the three confirmed functional Sp1 motifs identified in
our studies were not methylated in most TCL1 promoter
clones. CpG methylation was detected in only 1 of 3 Sp1 sites, and this
pattern was only seen in the most densely methylated promoter clones
analyzed from BCBL-1 and Jurkat cell lines. It has been shown that CpG
methylation within Sp1 sequence motifs does not affect Sp1 factor
binding (48-54). In fact, Sp1 sites may be important for maintaining
CpG-rich regions in an unmethylated state. Our finding with the
TCL1 oncogene is consistent with these combined reports in
which methylation of functional Sp1 sites does not play a role in
silencing genes. Overall, our results exclude direct TCL1
core promoter methylation as the mechanism responsible for
TCL1 gene silencing in the cell lines examined here.

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Fig. 8.
CpG DNA methylation of the TCL1
core promoter in expressing and silent cell types. Five
TCL1 silent (TCL1 ) and one TCL1-expressing
(TCL1+) cell lines were examined for CpG methylation by genomic
bisulfite sequencing. Three to four clones were determined with genomic
bisulfite sequencing from each cell line for a total of 23 clones.
Clones from each cell line were determined on distinct days to reduce
the possibility for crossover contamination, and each assessment
included a control plasmid analysis, which always showed complete
bisulfite-mediated conversion of unmethylated cytosines. The CpG
positions analyzed are numbered 1-33 and correspond to the
boldface type positions in Fig. 1. Cell lines and the number
of clones examined are listed on the left. The span of
individual sequenced clones is depicted by the extent of a
horizontal line on the right. A vertical
slash indicates a methylated CpG at that position. "X
" corresponds to a methylated CpG position contained within Sp1(D),
Sp1(E), or Sp1(F) sites within the TCL1 core promoter. A corresponds to methylated CpG positions within a previously reported
NotI restriction enzyme site (15).
|
|
 |
DISCUSSION |
In this study we have identified a 424-bp fragment that confers
robust TCL1 reporter activity in many
TCL1-expressing and silent cell types, indicating that we
have localized the TCL1 core promoter. This core promoter
directs one major and three minor sites of TCL1
transcription initiation. These mapped sites are longer by 11-17
nucleotides than the start site suggested by Yuille et al.
(15), based upon the cloning of the TCL1 cDNA (1). This
discrepancy may result from an incomplete or partially degraded 5' end
of the original library-based TCL1 cDNA. We think it
unlikely to have occurred from rare intact shorter transcripts based on
our sensitive S1 nuclease mapping procedure performed with freshly
isolated RNA. This result strongly suggests that TCL1
promoter activity depends on classic TATA-binding proteins and the TATA
box (reviewed in Refs. 55 and 56).
We searched extensively both upstream and downstream for cis-acting
elements that could affect transcription from the TCL1 promoter in TCL1 silent cell types. We were unable to
demonstrate any significant effects on core promoter activity by
sequences ~10-kb 5' and 20-kb 3' of the promoter in transient
transfections. This suggests that regulation of expression is mediated
through the core promoter rather than through upstream or downstream
cis-acting elements. The TCL1 core promoter contains 10 putative Sp1-binding sites. We confirmed that Sp1 interacts with three
of these sites by cold Sp1 oligomer competition assays and by blocking
with Sp1 antibody. Exogenously introduced Sp1 in Drosophila
SL2 cells induced significant reporter gene expression indicating that
Sp1 functions in vivo to regulate transcription from the
TCL1 core promoter. Furthermore, mutation of these three Sp1
sites, but not a site that did not bind Sp1 using EMSA, substantially
inhibited reporter gene expression in 293T cells, reiterating the
importance of these specific Sp1 interaction sites in regulating the
TCL1 core promoter.
Sp1 was previously shown to activate transcription from a spectrum of
housekeeping, tissue-specific, and cell cycle-related gene promoters
(reviewed in Ref. 57). Although Sp1 is ubiquitous and is expressed at
equivalent levels in TCL1-expressing and silent lymphoid
cell types, its function is regulated in several different ways that
could explain its involvement with modulation of a tissue-specific promoter such as TCL1. Levels of Sp1 have been shown to be
cell cycle-dependent and high in the G1 phase
of the cell cycle, whereas levels are significantly lower in other
cycle stages. Sp1 levels may be reduced and thereby contribute to
decreased TCL1 expression in terminally differentiated,
non-dividing B-lineage cells in which TCL1 expression is
extinguished (5). This is in contrast to the cell lines tested here
that are all in cycle and demonstrate abundant Sp1 protein. In these
cell lines Sp1 does not confer tissue specificity to a transiently
transfected TCL1 reporter construct.
Transcriptional control via Sp1 is also regulated by means other than
absolute protein levels. Sp1 has been shown to be glycosylated and
phosphorylated which may alter its activity in specific cell types (58,
59). Another level of regulation is through binding site competition
with other specific transcription factors, including additional members
of the Sp1 protein family. For example, promoters with multiple
Sp1-binding sites as seen in the TCL1 core promoter were
repressed by interactions with Sp3 (60, 61). However, Sp3 is also
ubiquitous so its detailed role in tissue-specific gene regulation is
not clear.
Sp1 induced SV40-mediated transcription by a significantly
larger amount than it did TCL1-mediated expression in
Drosophila SL2 cells, yet both promoters showed comparable
levels of expression in mammalian cells (Figs. 3 and 5). This implies
that, in addition to Sp1, other transcription factors are likely
involved in the regulation of the TCL1 promoter. Interaction
of tissue-specific factors with Sp1 has been shown to regulate
transcription from tissue-specific promoters through their specific
transcription factor-binding sites. Interaction of Puralpha and Sp1
results in enhanced binding of Puralpha to its binding site and
increased transcription from the myelin basic protein promoter (62).
Also MEF-2 and Sp1 synergistically activate transcription of myoglobin and muscle creatine kinase together through their individual specific binding sites (63). Further analysis for putative tissue-specific transcription factor binding sites will be necessary to characterize completely the TCL1 promoter.
In other cases, tissue-specific promoter activity was mediated by
tissue-specific factors that regulated the interaction of Sp1 with its
cognate binding site in the absence of the tissue-specific factor
binding to the promoter. For example, peroxisome proliferator-activated receptor-
blocks expression of thromboxane by binding Sp1 and preventing its binding to an Sp1 site within the thromboxane promoter (64). Also the transcription factors retinoic acid receptor and
retinoid X receptor interact with Sp1 to increase its binding to a GC
box allowing increased transcription of urokinase plasminogen activator
(65). Therefore, TCL1 promoter activity may be modulated through blocking or enhancing Sp1 interactions with the core promoter Sp1-binding sites via intervention by yet unidentified tissue-specific factors.
Of particular interest is the recent finding that the POZ domain of
BCL-6, a B-cell-specific transcriptional repressor, binds directly to
Sp1 and blocks its DNA binding activity and subsequent ability to
transactivate transcription (66). BCL-6 is expressed in germinal center
(GC) B-cells but not by post-GC B-cells (67, 68). Interestingly,
TCL1 expression begins in early B-cell development in the
bone marrow, markedly decreases in the GC, and disappears in post-GC
B-cells (4, 5). It is possible that the transient expression of BCL-6
in GCs has a role in directing TCL1 repression through an
interaction between the BCL-6 POZ domain and Sp1. This could tip the
balance toward binding of Sp3 and Sp4 transcription factors in the
TCL1 core promoter, as has been shown to mediate repression
of the ADH5/FDH promoter (69). Also, the POZ domain of BCL-6 interacts
with SMRT/N-CoR, mSin3a, B-CoR, and histone deacetylase
transcription-inhibitory factors, leading to increased gene repression
(70-72). In addition to potentially blocking Sp1 binding and
augmenting the binding of inhibitory Sp1 family member proteins, the
recruitment of these repressing co-factors may facilitate epigenetic
modifications of chromatin structure (e.g. histone deacetylation and DNA methylation) involved in gene silencing. Suggesting against a role for BCL-6 in TCL1 gene silencing,
however, is the observation of high level BCL-6 and TCL1 co-expression in multiple DLBCLs (5). If BCL-6 normally represses TCL1
expression, this co-expression argues that the repressive mechanism may
be broken in lymphoid cancers.
An association between CpG promoter methylation and TCL1
gene silencing was not found in studies presented here. Also, the association between gene silencing and methylation of two CpG sites
within a single NotI site in the core promoter as reported previously (15) was not confirmed. Instead, an association between alleles with high level CpG methylation and NotI site
methylation was demonstrated. Rather than acting as a marker for gene
silencing, NotI methylation appears to indicate dense CpG
methylation of the core TCL1 promoter. Because each of the
five TCL1-negative cell types examined here contained
unmethylated or minimally methylated DNA clones, TCL1
silencing does not appear to be due to promoter methylation. Combined
with the results presented here, the prior finding that Jurkat and CEM
T-cell lines treated with 5-AC-activated TCL1 gene
expression suggests that inhibition of DNA methyltransferase activity
likely affected DNA methylation outside of the TCL1 core promoter region (15). In fact, the lack of consistent high level CpG
methylation in the promoter does not exclude additional mechanisms of
epigenetic regulation in the control of TCL1 gene
expression. The identification of tissue- and development-specific
factors that may interfere with Sp1 binding, such as BCL-6, along with the resolution of potential epigenetic mechanisms will both be necessary to understand the regulation of TCL1 expression in
lymphocyte development and malignancy.
 |
FOOTNOTES |
*
This work was supported by the Lymphoma Research Foundation,
Amgen/U.C. BioStar Project S98-35, and National Institutes of Health
Grants T32CA009120, T32CA009056, CA74929, CA90571, CA85841, GM07185,
and GM40185.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.
§§
To whom correspondence should be addressed: David Geffen School
of Medicine at UCLA, Dept. of Pathology and Laboratory Medicine, 675 Charles Young Dr. South, 4-760 MacDonald Research Laboratories, Los
Angeles, CA 90095-1732. Tel.: 310-206-6754; Fax: 310-267-0382; E-mail:
mteitell@ucla.edu.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M207166200
2
S. W. French, C. S. Malone,
R. R. Shen, M. Renard, S. E. Henson, M. D. Miner, R. Wall, and M. A. Teitell, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
DLBCL, diffuse large
B-cell lymphoma;
BL, Burkitt lymphoma;
5-AC, 5-aza-2'-deoxycytidine;
EMSA, electrophoretic mobility shift assay;
PEL, primary effusion
lymphoma;
GC, germinal center;
EMSA, electrophoretic mobility shift
assay;
EBV, Epstein-Barr virus.
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.