differential cooperation between regulatory sequences required for
TRANSCRIPT
Differential cooperation between regulatory sequences required for
human CD53 gene expression
Javier Hernández-Torres, Mónica Yunta and Pedro A. Lazo *
Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del
Cáncer, Consejo Superior de Investigaciones Científicas, Universidad de Salamanca,
Campus Miguel de Unamuno, E-37007 Salamanca, Spain, and Unidad de Genética y
Medicina Molecular, Centro Nacional de Biología Fundamental, Instituto de Salud
Carlos III, E-28220 Majadahonda, Spain
Running Title: Differential Regulation of Human CD53 Promoter
Key words: gene expression; transcription factors; co-stimulatory molecules; cell-
surface molecules; enhanceosome
*Address for correspondence: Dr. Pedro A. Lazo, Centro de Investigación del Cáncer, CSIC-Universidad
de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
Tel: 34-923 294 804; Fax: 34-923 294 795; E-mail: [email protected]
This work was supported by grants from Ministerio de Ciencia y Tecnología (SAF2000/0169) and Junta
de Castilla y León (CSI-1/01). M.Y. has a fellowship from Instituto de Salud Carlos III.
1
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on July 6, 2001 as Manuscript M104723200 by guest on M
arch 4, 2018http://w
ww
.jbc.org/D
ownloaded from
SUMMARY
CD53 is a tetraspanin protein mostly expressed in to the lymphoid-myeloid lineage. We
have characterized the human CD53 gene regulatory region. Within the proximal 2
kilobases, and with opposite transcriptional orientation, is located the promoter-
enhancer of a second gene, which does not affect CD53. Twenty-four copies of a CA
dinucleotide repeat separate these two gene promoters. The proximal enhanceosome of
the human CD53 gene is comprised between residues –266 to +84, and can be
subdivided into four major subregions, two of them within exon 1. Mutational analysis
identified several cooperating sequences. An Sp1 and an ets-1 site, at positions –115
and +62 respectively, are essential for transcriptional competence in all cell lines. Other
five regulatory sequences have a dual role, activator or down-regulator, depending on
the cell line. At the end of the non-coding exon 1, +64 to +83, there is a second ets-1
regulatory element, which is required for high level of transcription, in cooperation with
the Sp1 site, in K562 and Molt 4, but not in Namalwa cells, where it functions as a
repressor. This Sp1 site also cooperates with another ets-1/PU.1 site at –172. Different
cell types use different regulatory sequences in the enhanceosome for the expression of
the same gene.
INTRODUCTION
Gene regulation in a specific cell type requires the cooperation of several cis-
acting DNA regulatory sequences, which are binding sites for proteins that transmit
molecular signals to genes (1). These sequences bind regulatory proteins and form
complexes known as enhanceosomes (2,3). The CD53 gene codes for an antigen that
belongs to the tetraspanin family of membrane proteins. These proteins are very
2
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
hydrophobic and consist of four transmembrane domains with polar residues, and have
a small and a large extra cellular loop; the latter appears to be responsible for the
functional specificity of each individual tetraspanin protein (4,5). CD53 was originally
reported to be a pan leukocyte antigen, whose expression is mostly restricted to the
lymphoid-myeloid lineage (4). Recent data suggests that tetraspanin proteins play an
important role as co-stimulatory molecules in several cell types (5). Ligation of the
CD53 antigen with monoclonal antibodies modulates several biological processes.
Among them are intracellular calcium mobilization in human B-cells and monocytes
(6) and rat macrophages (7), induction of homotypic adhesion (8,9), and in rat
macrophages also induces the expression of the inducible form of nitric oxide synthase
(7). No ligand is known for any tetraspanin antigen. Tetraspanin antigens form a protein
complex with several integrins on the cell membrane, which might require a
coordinated expression of their genes (10-13).
Loss of CD53 antigen surface level has been reported in a family with CD53
deficiency, which is characterized by the occurrence of recurrent and heterogeneous
infectious diseases caused by viruses, bacteria and fungi (14), a syndrome similar to the
clinical manifestations of defects in leukocyte adhesion properties (15). Also, down
regulation of several tetraspanin antigen gene expression, such as CD9, CD82/KAI1,
and CD63, has been correlated with poor prognosis in several types of tumors, including
breast, melanoma, lung, prostate, pancreas and esophageal carcinoma (16-26).
Reintroduction of these antigens, such as CD9 or CD82 acted as a brake reducing cell
motility (27). These effects might be a consequence of the tetraspan-integrin interaction
and their corresponding effects on cellular motility and adhesion (13). In all these
situations, the correlation was performed for a unique member of the tetraspanin family.
3
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
However, an individual cell expresses simultaneously from five to ten members of this
protein family (10,11,28), where they form a complex in the membrane that is different
depending on cell type (10,11,13,29,30). This occurs both in normal cells (10),
lymphomas (31), and carcinomas (unpublished observations). The reduction in levels of
an individual protein represents a change in the composition of such complexes (32).
Therefore, the role of low-level expression of any given antigen should be considered
in the context of the multiple antigen expression.
To understand the coordinated expression of genes coding for tetraspanin
antigens, which are located on different chromosomes, it is necessary to characterize
their gene regulatory regions. Very few promoters of this growing gene family have
been characterized. Only the CD9 promoter (33,34) and the CD63 promoter (35,36)
have been studied in some detail, and the genomic location of the CD53 transcription
initiation site has also been identified (37). In this report we have cloned and
characterized the promoter region of the human CD53 gene. By mutational analysis
several cis-acting DNA regulatory sequences were identified, which cooperate and
contribute to CD53 gene expression in different cell types. Several of these new
regulatory elements are located within the non-coding exon 1. Some of these regulatory
sequences function as positive or negative elements depending on the structure of the
enhanceosome in each cell type. The basic CD53 regulatory region has an Sp1 sequence
that cooperates with several ets-1 sequences. Also the promoter-enhancer of a very
proximal second gene does not interfere with the CD53 promoter activity.
EXPERIMENTAL PROCEDURES
4
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Genomic cloning. To isolate the CD53 gene promoter we used a human genomic library from a 3-year
old Caucasian male made by partial EcoRI digestion and cloned in phage λFIX (Stratagene, San Diego,
CA). The library was screened with a cDNA clone made by primer extension of the 5’ untranslated region
of human CD53 message kindly provided by V. Horejsi (Czech Academy of Sciences, Prague)(37). The
positive genomic clones were mapped by partial digestion of the inserts with several restriction enzymes,
followed by hybridization to end-labeled T3 or T7 primers, present in the cloning vector, as probes.
Conditions for primer hybridization and filter washes have been previously reported (38). Location of the
non-translated first exon was done by Southern blot hybridization under conditions previously reported
(39).
Deletions by Exonuclease III and mung bean nuclease and DNA sequencing. The genomic clone was
digested with selected restriction enzymes and subcloned in plasmid pBluescript SKII (-) The sub-clones
were linearized and nested deletions were made using exonuclease III and mung bean nuclease with a
commercial kit (Stratagene, San Diego, CA). The nucleotide sequence was determined by the
dideoxynucleotide termination method according to the T7 DNA polymerase Sequenase kit (Amersham-
Pharmacia Biotech). The products were analyzed in 7% polyacrylamide gels with urea. The dried gel was
exposed to Fuji-RX film (Fuji, Japan). Alternatively they were sequenced with a Dye Terminator Cycle
Sequencing kit (Perkin-Elmer Cetus, Norwalk, CT) using Thermus aquaticus FS polymerase and
universal M13-forward and reverse primers. Sequences were resolved in an ABI PRISM 377 automatic
DNA sequencer and the results were processed with the ABI Analysis software (version 2.1). Nucleotide
sequence analysis was performed using the PCGene and OMIGA packages from Oxford Molecular
(Oxford, U.K.). The nucleotide sequence with the two promoters, CD53 and the new transcriptional unit
has GenBank Accession number AJ243474. To detect specific DNA motifs specific for regulatory
elements, such as enhancers and transcription factors, the sequence was analyzed with the Transfac 4.0
program (Gesellschaft für Biotechnolosgische Forschung, Braunschweig, Germany) (40).
RNA preparation and primer extension analysis. Total RNA was extracted following the guanidinium
thiocyanate-chloroform method (41), as previously described (42). To extend the novel gene an antisense
oligonucleotide (GENA2) was used, 5’-GAGAGCTCGTGAGACAGAACTAG-3’ (positions –2175 to
5
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
–2152 in the sequence). The extension was performed with MoMuLV reverse transcriptase according to
manufactures instructions (Life Technologies, Gaithersburg, MD). The extended products were analyzed
in a 6 % polyacrylamide/7M urea sequencing gel. The radioactivity in the gel was detected by
autoradiography with Kodak XAR-5 film, or directly with a FUJIBAS1000 phosphorimager (Fuji, Fuji,
Japan).
Luciferase reporter gene constructions. To characterize the regulatory region, DNA fragments were
prepared by PCR amplification. Selected regions of the human CD53 promoter sequence were amplified
by PCR using specific oligonucleotides to defined regions upstream and downstream of the transcription
start site, which are indicated in the experiments, designed based on the CD53 genomic sequence, and
approximately 30 nucleotides long. Pairs of primers were used for the PCR reaction used for
amplification of selected genomic regions. These primers contained, at their ends, the suitable restriction
sites, MluI and SmaI, for subcloning into the appropriate luciferase reporter vector of the pGL2 family
(Promega, Madison, WI). All the constructs were cloned in the pGL2-Basic, that lacks both SV40
enhancer and promoter sequences, and in some cases in the pGL2-Promoter vector that lacks the SV40
enhancer sequence, but retains the SV40 promoter. As positive control, we used the pGL2-control
plasmid containing both SV40 promoter and enhancer sequences. The empty pGL2-Basic that lacks
promoter or enhancer sequences was also used as negative control. To introduce point mutations at
specific nucleotide locations we used the Quick Mutagenesis kit from Stratagene (San Diego, CA). To
generate the mutant two complementary primers containing the desired mutation in its center were
designed, and used to copy a target sequence with the Pfu polymerase, in such a way that the whole
plasmid was copied. The input DNA was digested with DpnI that only cuts the input plasmid of bacterial
origin because of its methylation, but does not cut the DNA made by the Pfu polymerase. The remaining
DNA was used to transfect DH5αF E. coli strain and isolate new plasmid constructs with the desired
mutation. All mutations were confirmed by nucleotide sequence. For internal control of transfection and
normalization we used plasmid pCMV-gal (Invitrogen, San Diego, CA) with the β-galactosidase gene.
Also we used the dual luciferase Renilla system for normalization (Promega, Madison, WI). The
generated light was detected with an OPTOCOM-1 luminometer (MGM Instruments, Inc., Hamden, CT).
6
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Cell lines, flow cytometry and transfections. The Molt-4 cell line from a T-cell lymphoma; the K562 cell
line from a chronic myelogenous leukemia; and the Namalwa B-cell line, derived from a Burkitt
lymphoma, was used for transfection experiments. Cells were grown in RPMI1640 supplemented with
10% fetal calf serum and antibiotics. The cell lines were transfected by electroporation with 10 µg of the
corresponding plasmid DNA using a Gene-Pulser apparatus (BioRad, Richmond, CA). The
eletroporation conditions were 250 volts and 960 µF for Molt-4 cells, 300 volts and 500 µF for Namalwa
cells, and 280 volts and 960 µF for K562 cells.
The phenotype of the cell lines was determined by flow cytometry using a FACScalibur form Becton-
Dickinson. The CD53 antigen was detected with the monoclonal antibody MEM53, and as secondary
antibody we used a FITC labeled rabbit anti-mouse IgG antibody from Sigma (St. Louis, MO).
Protein extracts and western blots. Cells were grown to a density of 5 x 106 cells as indicated. For total
protein extracts we used 6 x 107 cells that were washed in PBS and lysed in 600 µl of RIPA buffer (1%
triton X-100, 150 mM NaCl, 1 % aprotinin, 1 % leupeptin, 250 µM PMSF, 10 mM Tris-HCl pH 8.0).
The cell suspension was passed through a needle to break the DNA. The extract was centrifuged at
10.000g for 10 min. at 4ºC. The supernatant was used as the total protein extract. The protein
concentration was determined using a Bio-Rad protein assay kit.
The proteins were fractionated in a 7.5 % polyacrylamide gel loaded with 50 µg of total protein. The
proteins were transferred to a PVDF membrane (Millipore, Bedford, CT) in a Bio-Rad Trans blot cell.
The membrane was blocked with 5 % skimmed milk in TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl)-
0.1 % Tween buffer.
In the western blots, the first antibody indicated in the figure was used at a 1/1000 dilution, and for
detection we used protein-A-horse radish peroxidase (Amersham) at 1/1000 dilution or anti-mouse or
anti-goat IgG antibodies coupled to peroxidase. The blots were developed using an ECL
chemiluminiscence kit from Amersham.
Antibodies. For the transcription factor Sp1 we used a mouse monoclonal antibody, 1C6, from Santa Cruz
(Santa Cruz, CA). For Sp2 and Sp3 we used rabbit polyclonal antibodies, K-20 and D-20 respectively,
7
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
from Santa Cruz. The ets-1 transcription factor was detected with a rabbit polyclonal antibody, N-276,
from Santa Cruz. The PU.1 protein was detected with a rabbit polyclonal antibody, T-21, from Santa
Cruz. E4BP4 protein was detected with a goat polyclonal, V-19, and GATA1 transcription factor with
goat polyclonal, C-20, both from Santa Cruz. NFAT proteins were detected with a rabbit polyclonal
antibody, 06-348, from Upstate Biotechnology (Lake Placid, NY).
RESULTS
Cloning of the human 5’ CD53 gene regulatory region
To characterize the human CD53 gene promoter, located on chromosome region
1p13 (43), we cloned the upstream regulatory region by screening a normal individual
genomic library with a partial cDNA probe. The probe, clone pPET/XAG, was derived
by primer extension of the poly (A+) RNA, and contained only the 5’ non-translated
sequence (37). With this probe a genomic clone, λJHT1, containing 20 kbp was
isolated. The restriction map of the genomic clone is shown in Fig.1. The location of the
region containing the beginning of the CD53 transcriptional unit was determined by
hybridization to the same probe used for library screening (Fig.1). From the phage clone
we made several subclones in plasmid pBluescript SK-II containing the XbaI and the
EcoRI - HindIII region that comprises the start of the CD53 transcribed sequences. We
sequenced a region of 3613 nucleotides (GenBank Accession number AJ243474)
surrounding the CD53 start site (Fig. 2). The CD53 gene lacks a TATA box, but has a
sequence that plays its role, as well as a capping site (37). The sequence was analyzed
with the Transfac 4.0 program to detect sequences that are recognized by transcription
factors (40). In the sequence, shown in Fig.2, we have indicated the position of several
cis-acting consensus DNA motifs recognized by transcription factors, such as Sp1, ets-
1/PU.1, elk-1, an Ets related factor (44,45), and GATA1, which are likely to be
implicated in the regulation of CD53 gene expression. Some of these regulatory
8
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
elements are located within the non-coding exon 1 (Fig.2).
There is a second promoter-enhancer proximal to hCD53
The analysis of the nucleotide sequence upstream of the CD53 promoter also
detected a putative TATA box located in the –2000 region approximately; with opposite
orientation with respect to the CD53 gene start site (Fig.1). The presence of such
unknown gene was confirmed by the sequencing of the human genome, which is located
upstream and proximal of the humanCD53 promoter in the chromosome 1p13.3 region
(46). Before proceeding to characterize the CD53 promoter we confirmed that there is
indeed a start site for an unknown transcriptional unit, called gene A, by performing a
primer extension assay using as target RNA from two different cell lines, Molt4 and
K562 (data not shown).
To functionally demonstrate that there is a second enhancer-promoter from
another gene proximal to CD53 we subcloned it upstream of a luciferase reporter gene
in the pGL2-B (basic) vector. The constructs were of increasing length till the CD53
promoter was included in the antisense orientation. In the region from –1837 to –2127
there is a very strong enhancer-promoter that is even stronger, in the three cell lines
tested, than the positive control containing the SV40 enhancer-promoter used as
positive activation control (Fig.3). As the length of DNA towards the CD53 promoter
was increased, there was a significant drop in the activity of this novel enhancer-
promoter, the major drop in luciferase activity occurred when the region located
between –1533 to –489 was included in the construct. This region has 24 copies of a CA
dinucleotide repeat which is located between positions –1361 to –1313. The CA repeat
is thus located between the two promoters. The inclusion of the CD53 enhancer, present
9
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
in constructs extending to the +137 and +322 positions, did not prevent the drop in the
activity of Gene A promoter-enhancer (Fig.3), suggesting that the presence of the CD53
promoter-enhancer does not affect this novel transcriptional unit. The relative strength
of the Gene A promoter-enhancer (↑1837 to –2127) is stronger than the SV40 control
(pGL2-C vector) in the three cell lines.
The proximal CD53 promoter region is the major determinant of its activity
To characterize the proximal regulatory region of the human CD53 gene we used
three different cell lines from the lymphoid-myeloid lineage, such as K562 derived
from an erythroleukemia, Molt-4 derived from a T-cell lymphoma, and Namalwa cells
derived from a Burkitt lymphoma, a B-cell tumor. The three cell lines expressed CD53
antigen on their surface as determine by flow cytometry analysis (Fig.4). The expression
levels in the three cell lines were also determined at the RNA level by a northern blot
(not shown). Therefore, these different cells lines can be used to ascertain if the same
regulatory elements determine the transcriptional activity of the CD53 promoter in the
three cells lines, or alternatively if the proximal enhanceosome or transcriptional
complex (2) is different in each cell line, although located in the same genomic region.
First, to identify the location of the major regulatory elements within the human
CD53 gene regulatory region, several constructs were made from –2157 to the +1
position using vectors pGL2-B (without enhancer-promoter) and pGL2-E (lacks the
promoter, but has the SV40 enhancer). With both vectors the results obtained were
similar, therefore we continued the analysis using only the constructs made in the
pGL2-B vector.
The activation of transcription was studied in three cell lines K562, Molt-4 and
10
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Namalwa. The proximal region between nucleotides –266 and +1 (Fig.5) appeared to
contain most of the sequences required for expression of the CD53 gene, and in the
three cell lines, the values were similar to those of the positive control with the SV40
promoter-enhancer. The effect from the –266 to –998 sequence was heterogeneous and
no conclusion could be drawn from it. Although in Molt-4 cells, the region between
–509 and –667 resulted in a two fold increase in the activity of the proximal CD53
region (Fig.5). The inclusion of the Gene A enhancer-promoter and the CA repeat, up
to position –2157, did not significantly affect the CD53 promoter in K562 cells, but
resulted in a drop in activity in Molt-4 and Namalwa cells, although still with a relative
high-level transcriptional activity with respect to the empty vector (Fig.5).
We conclude that the major determinant of the transcriptional activity of the
human CD53 gene regulatory region is very proximal to the +1 position.
There are four subregions in the proximal regulatory region of CD53
To further characterize the proximal regulatory region of the human CD53 gene
we included the immediately upstream sequence, as well as the non-coding exon 1 and
intron 1, extending from nucleotides –266 to +84. This region was subdivided into four
subregions, A (↑266 to –168), B (↑168 to +1), D (+1 to +64) and C (+64 to +84), to
detect the location of putative cis-acting regulatory elements (Fig.6). All clones that
included intronic sequences beyond the splice donor at +84, such as the + 137, + 322,
and + 1040 constructs, were not active because the luciferase reporter gene was deleted
as a result of splicing into the SV40 splice acceptor signal present in the reporter vector.
Regions B + D (-163 to +64) appeared to account for the basic expression of the CD53
promoter, as originally reported (37), and deletion of region D in this construct resulted
11
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
in a very important drop in activity (↑168 to +1 in Fig.4). The region comprised from
position –266 to +84 has the highest activity, and was very similar to the activity of the
construct from –168 to +84 positions. The inclusion of region C (+64 to +84) in
constructs starting either at –266 or –168 also resulted in an increase in transcriptional
activity, suggesting the existence of another important sequence at the end of exon 1.
The fragment from +1 to +84 (D + C) was not active by itself, probably because of the
lack of TATA-like box (Fig.6). If the CD53 TATA-like box was included (↑25 to +84)
there was also no recovery of activity (Fig.6). But in the construct that extended from
–266 to the +64 position, there was a partial recovery of the activity (Fig.6). These data
indicates that within exon 1 there are at least two important regulatory sequences, one
between +1 to +64 (D), and the other between +64 and +84 (C). Because this non-
coding exon 1 has no TATA-box, we tested whether this region by itself could function
as an enhancer by activating a heterologous promoter, such as that of SV40. We cloned
this CD53 exon 1 region upstream of the SV40 promoter, in the pGL2-P vector (that
has the SV40 promoter, but not its enhancer). Neither orientation of this CD53 exon 1
was able to activate transcription directed by the SV40 promoter in Molt4 or K562 cells
(not shown). Thus, the regulatory element appears to be specific and is not active by
itself. To be functional this region, which has two regulatory subregions (D + C), needs
to act coordinated with other upstream elements located in the proximal regulatory
region of human CD53 gene, and that are not present in a heterologous promoter, such
as SV40.
Also in the proximal upstream region the deletion of sequence from –266 to
–168 (region A) resulted in a partial loss of activity, more noticeable if the construct
reached only to the +64 position (Fig.6). Thus in the proximal upstream region A there
12
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
is another regulatory sequence in addition to the previously known to be within B (–168
to +1).
We can conclude that the proximal sequences and the first exon of CD53 can be
subdivided into four subregions, each with at least one regulatory element, that
contribute to CD53 gene expression. These data obtained by deletion analysis did not
detect any significant difference among the three types of cell lines used (Fig.6).
Identification by mutagenesis of cis- acting sequences that control CD53gene
expression in different cell types
Gene expression is controlled by the assembly of the enhanceosome o
transcription complex where several cis-acting DNA sequences are bound to different
regulatory and structural proteins (2,3). We reasoned that a mutational analysis of the
potential cis-acting sequences in the region might detect the differences, if any in the
assembly of the transcription complex in each cell line. Therefore, in order to determine
if CD53 gene expression was regulated differentially depending on the cell type we
performed a mutational analysis of the proximal region. The DNA sequence was
analyzed with the Transfac 4.0 program to detect DNA elements that are candidates to
be regulatory sequences (40). Among the elements detected, we selected eight to be
studied by site-directed mutagenesis, which are distributed within the four subregions
previously identified by deletion analysis, and that did not detect differences among the
three cell lines (Fig. 6). The selected DNA target sites for mutation were modified by
the substitution of two nucleotides that form the core consensus of the site and are
required for binding of transcription factor (Table I). Two of them, A5 and C1, have a
core sequence related to the ets-1/PU.1 family of transcription factors, and another (B5)
13
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
a site for an ets related factor, elk-1. Mutations in DNA sequences that are positive
regulators will de detected by a reduction in luciferase activity, and those that play a
negative role will be detected by an increase in luciferase activity. These sequences
were mutated to identify the putative regulatory elements located in different sub-
regions of the CD53 promoter, as well as determine their relative contribution to CD53
gene expression in specific cell types.
Within region A, three sequences were mutated, but only two appeared to
modulate CD53 promoter activity, but with different roles depending on cell type. The
mutations were introduced in the construct from –266 to +64 that lacks element C. The
mutation of site A3 (PuF core) resulted in a very high increase in luciferase activity in
Molt-4 cells, and more moderately in K562, indicating that this sequence plays a
negative regulatory role in these two cell lines (Fig. 7). This A3 mutation had no effect
in Namalwa cells (Fig.7). The mutation of site A5 (ets-1 core) had no effect in K562
and Molt-4, but resulted in a very high increase of activity in Namalwa cells, where it
plays a negative regulatory role (Fig.7). Mutation of the A1 site had no effect in any cell
line (not shown).
Region B was previously identified as essential for CD53 expression and it must
have a basic component required for gene expression. In this region we mutated three
different putative target sequences. There is an SP1 consensus sequence (B1) that was
mutated in a construct from –266 to + 84 region. This mutation resulted in a complete
loss of activity despite the presence of all the other DNA binding sites as wild type, in
the three cell lines (Fig. 7). The deletion of the region with this Sp1 site, construct from
↑25 to +84, retaining element C1 as wild type, also resulted in almost complete loss of
activity (Fig. 5). These observations suggest that this SP1 site is a basic component of
14
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
the transcriptional unit in the three cell lines. Mutation of element B3 reduced by half
the expression in K562, but mainly resulted in an important increase in activity in the
two lymphoid cell lines, Molt 4 and Namalwa (Fig.7). Mutation of element B5
increased the activity in Namalwa cells, moderately reduced the activity in Molt 4, and
had no effect in K562 cells (Fig.7).
The region comprised between nucleotides +65 and +84 (region C), at the end of
the non-coding exon 1 confers a very strong transcriptional activation. This region
contains 18 nucleotides and a core recognition sequence (C1) of ets-1/PU.1 family
transcription factors. To determine if this element was implicated in transcriptional
activation, we replaced the two GG of the target sequence by two AA (nt +71 and +72)
(Table 1) in a construct containing from the –266 to +84 region, this mutation resulted
in a drop in activity to levels similar a deletion construct without that region (↑266 to
+64), suggesting that it participates in the activation of transcription, but as shown by
the construct from –168 to +64 (Fig.6), it needs to cooperate with other sequences to be
functional. Element C1 appears to play different roles depending on cell type. C1 is
required for expression in K562 and Molt-4 cells, and is a strong negative regulator in
Namalwa cells since its mutation resulted in a very high increase in transcriptional
activity (Fig.7).
Within region D, we mutated element D3 (a core consensus for GATA-1 or
NFAT). This mutation only resulted in an important activation of transcription in
Namalwa cells, with no effect on K562 or Molt4 cells (Fig.7). At the end of region D,
there is an ets-1 core element at position +62 (element D1) that overlaps the beginning
of region C, which is absolutely essential for the transcriptional competence of the
CD53 promoter, its mutation resulted in a complete loss of activity in the three cell lines
15
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
(Fig.7).
The overall picture resulting from this mutational analysis is that different
regulatory sequences account for expression of the CD53 gene in the three cell types
studied (summarized in Fig.8). Two of the elements, B1 (Sp1 core) and D1 (ets-1 core)
are essential for transcriptional competence in all cell lines. But in this context it is very
important to draw the attention to the fact that a single cis-acting DNA sequence can
have opposite roles depending on the cell type, as is the case for elements C1, B3, B5,
D3, A3 and A5.
Levels of transcription factors
The functional differences observed in the three cells lines might be a
consequence of differences in the level of transcription factors present in each of the
cell lines, or be the result of a differential assembly of the proteins in the proximal
enhanceosome. To address this issue we determined the level of several transcription
factors in the three cell lines. For this purpose whole cell extracts were prepared and
fractionated in denaturing SDS-polyacrylamide gel electrophoresis. The proteins were
transferred to a PVDF membrane, and the filters were analyzed by western blot with
different antibodies specific for transcription factors indicated in Figure 9. The level of
transcription factors that have a marked differential effect, such as Sp1, ets-1 or PU.1
appear to be similar in the three cell lines. The erythroid transcription factor GATA-1
(47) is restricted to the K562 cell line. The E4BP4 element is expressed at similar levels
in the three cell lines (Fig.9). This element is a known negative regulator of
transcription (48), and it exerts this effect in the two lymphoid cell lines, Molt-A and
Namalwa (Fig.7). The blot with antibody against NFAT is not shown because of the
16
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
very heterogeneous size of this family that contains multiple members. The pattern
observed was a smear, that was similar and within the expected size range for this
protein family in the three cell lines. The cell lines were also analyzed for other factors
of the Sp family, as well as with an antibody against β-actin as a control for gel
loading.
The similarity in the levels of transcription factors suggests that the functional
differences observed in the three cell lines are likely to be a result of a differential
protein assembly in the enhanceosome, which is specific and different for each of them.
In that way different cells achieve the expression of a common gene.
DISCUSSION
Understanding the regulation of tetraspan antigen genes expression is necessary
to know how they are coordinated among themselves and with other proteins with
which they interact, such as integrins, to form complexes on the cell membrane. For this
purpose we cloned a genomic fragment containing the human CD53 gene promoter
(Fig.1), and identified several regulatory elements within a region surrounding the start
site and including the non-coding exon 1 (Fig.2). Within the genomic clone and
approximately 2 kilobases upstream of the CD53 start, by primer extension and
luciferase reporter assays it was identified the enhancer-promoter of an unknown gene
(Fig.3), confirmed by the sequence of the human genome (46). This second enhancer-
promoter does not interfere with the activity of the CD53 promoter. However, the
activity of the novel gene regulatory region drops significantly when the region
containing a CA dinucleotide repeat is included in the construct. The twenty-four CA
repeat region can form Z-DNA and perhaps might functionally separate the two
17
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
transcriptional units (49). The activity of the CD53 promoter remains at a similar level
independent of whether the CA repeat and the gene A promoter are present in the
construct, except when the +64 to +84 element is included, which is much higher. Close
proximity of two gene promoters has been found for other genes. Thus, the murine trkA
gene is 2 kb from the IRR gene promoter, and it is not affected by its presence (50).
Also the interaction between a TATA-less promoter and a gene with conventional
TATA, close to each other and with opposite orientation has been studied
experimentally (1). In these constructs the TATA-promoter containing gene is
preferentially active. In the case of CD53 gene (TATA-less promoter), this promoter is
less active than gene A promoter (with TATA box). TATA-less promoters, as it is the
case of the CD53 gene, require the participation of Sp1 elements and members of the
Ets family of transcription factors (51).
The transcriptional analysis of the –266 to + 84 region of the CD53 gene which
included the non-coding exon 1 in the three cell lines of different lineage allowed the
identification of several cooperating regulatory sequences. The same elements
contribute to the expression of the CD53 gene, but their effect and combination of
elements is specific of the cell type (Fig.8). An Sp1 element (B1), previously postulated
to be important for expression was shown to be essential for CD53 transcription in all
cell lines. Both, its deletion (Fig. 6) or mutation (Fig. 7) resulted in a total loss of
activity in the three cell lines. This element must be a basic component of the
transcription machinery of human CD53 gene. Sp1 elements are also important for
expression of other tetraspan antigens, such as CD63 (52) and CD9 (33).
Ets/Pu.1 sequences play a major role in CD53 gene expression. The GGAA is
the core recognition sequence for the Ets family of transcription factors, consisting of
18
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
are more than forty-five different members, which are very relevant for lymphoid-
myeloid gene expression (47,53). One of them, D1, is absolutely essential for gene
expression in the three cell lines (Fig.7). Two Ets elements (A5 and C1) were identified
as well as another element (B5) with a consensus for elk-1, a factor that has an Ets
domain (45). Some of these Ets target sequences have a dual role, activator or repressor
depending on the cell type, suggesting that they modulate the activity of the basic
transcription machinery. Thus mutation of the three sites (A5, B5 and C1) resulted in a
very high activity of the promoter in Namalwa cells (Fig.7), where it must have a
repressor role, preventing activation. The small region C, +64 to +84 has a canonical Ets
core sequence (C1) that requires cooperating with another sequences within the CD53
promoter-enhancer region. This important element functions in cooperation with the
Sp1 element (B1) and by itself it has no role, and it does not activate transcription from
a heterologous SV40 promoter. This C1 element is a downregulator in Namalwa cells
and an activator in K562 and Molt4 cells. A dual role for Ets/PU.1 proteins has been
reported in the regulation of the κ locus, where it functions as a positive regulator in
pre-B cells, and as a negative regulator in mature B cell stages (54).
Previous work postulated that within region B of this report there were three
putative binding sites, and Sp1, PuF and PU.1 sites, that might account for CD53 gene
activation (37). In this report we show that they indeed contribute to the basal level of
the promoter, but that they need to cooperate with another more distal element in exon 1
(+64 to +84) to achieve a much higher level of expression.
It is interesting to note that integrin genes, proteins that complex with tetraspan
antigens on the membrane (32), are also regulated by combination of Sp1 and ets-1
DNA elements (55,56), where PU.1 may play a recruiting role (57). For example, the
19
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
integrin CD11b requires Sp1 (58) and PU.1 (59) to drive its expression. Cooperation
between Sp1 and Ets regulatory sequences have been reported in the regulation of some
integrin genes, among these genes are CD11b (58,59), the β2 (CD18) (60) and the β5
chain (61). Mutation of the Sp1 element dramatically reduces CD18 expression (60), an
effect similar to mutation of the Sp1 element (B1) in the human CD53 promoter (Fig.7).
Also some ets-1 DNA recognition sequences have a positive or negative role in these
cell lines, such as is the case for PU.1 which functions as a negative element in pre-B
cells, and as positive in mature B-cells (54). Also Sp1 and Ets cooperation has been
reported in other proteins such as the mannose receptor (62) and the btk kinase (63).
The differential assembly of the regulatory region might be regulated by methylation
(64) and the binding of other proteins, such as CTCF (65), which also have a role as
insulators of gene transcription (49).
Several regulatory elements contribute to a specific gene expression (66,67).
Depending on the type of cell, a given element might have a different role or opposite
effects as demonstrated in this report. For the same genomic region to achieve a similar
level of expression, a different composition of regulatory elements is needed in each cell
line. The different requirements for CD53 gene expression in the three cell lines are
illustrated in the diagram of Figure 8. In general, transcription regulatory protein has
been shown to function as positive or negative regulators (68). In this report by
identifying simultaneously several regulatory sequences within a promoter and
analyzing their role in combination, we have shown that there are three different
functional complexes (Fig.8). Some elements play the same role independently of the
cell type. That is the case of element B1, a consensus Sp1 site, which is a basic
component of the transcription machinery. Other elements function as positive or
20
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
negative regulators depending on the cell. Element B5 has no effect in K562 cells, but
activates transcription in Molt-4 cells, and is a negative regulator in Namalwa cells.
The B3 element is an activator in K562, and a negative regulator in both lymphoid cell
lines, this element has a consensus sequence for the E4BP4, which is a known negative
regulator in other systems (48). The C1 element, however, is a repressor of gene
expression in Namalwa cells. Element D3 is required for high-level transcription in
Namalwa cells. This element interacts with members of the ets transcription factors
(69). The positive or negative effect of a DNA sequence is a likely consequence of its
effect on the protein assembly of the enhanceosome in each specific cell type.
In this report we have shown that the regulation of the human CD53 gene,
includes exon 1 sequences and is composed of several elements. An essential SP1 and
ets-1 sites are required for transcriptional competence; and several other elements,
mainly of the Ets family, have a different role depending on the structure of the
enhanceosome in each cell type. We postulate that the combination of Sp1 and Ets
transcription factors coordinate the expression of tetraspan genes and the proteins with
which they interact on the cell membrane, such as integrins. The CD53 enhanceosome
has different protein components, reflected by different DNA sequence roles, which are
required to adjust the expression of a single gene to different cell types.
21
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
TABLE I
Mutations introduced in different potential regulatory sequences of the human CD53
proximal promoter region
Region A A1 c/EBP wt 5’-CACAGATCTG-3’ (nt ↑246 to ↑237)mt 5’-CACATTTCTG-3’
A3 PuF wt 5’-GGGTGGGCTG-3’ (nt ↑183 to ↑174)mt 5’-GGGTAAGCTG-3’
A5 Ets-1 wt 5’-GAGAGGAAGC-3’ (nt ↑172 to ↑163)mt 5’-GAGAAAAAGC-3’
Region B B1 Sp1 wt 5’-GGGCGGACTCA-3’ (nt ↑118 to ↑108)
mt 5’-GGGCTTACTCA-3’
B3 E4BP4 wt 5’-CTCCTTTTACA-3’ (nt ↑34 to ↑24)mt 5’-CTCCGGTTACA-3’
B5 elk-1 wt 5’-CCTCCTTCTTC-3’ (nt ↑98 to ↑87)mt 5’-CCTCAATCTTC-3’
Region D D3 GATA1 wt 5’-CAAGGATAATC-3’ (nt +24 to +35)mt 5’-CAAGCCTAATC-3’
D1 Ets-1 wt 5’-CTGAGGAACGGT-3’ (nt +56 to +67)
mt 5’-CTGAAAAACGGT-3’
Region C C1 Ets-1 wt 5’-GCCTTGGAAAAG-3’ (nt +66 to +76)
mt 5’-GCCTTAAAAAAG-3’
Two nucleotide changes (indicated in bold and underlined) were introduced in the
known essential nucleotides of the binding site to make the mutant sequence. Their
nucleotide position within the promoter sequence is indicated in parenthesis.
22
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
REFERENCES
1. Blackwood, E. M., and Kadonoga, J. T. (1998) Science 281, 60-63
2. Carey, M. (1998) Cell 92(1), 5-8.
3. Merika, M., and Thanos, D. (2001) Curr Opin Genet Dev 11(2), 205-208.
4. Horejsi, V., and Vlcek, C. (1991) FEBS Lett 288, 1-4
5. Maecker, H. T., Todd, S., C., and Levy, S. (1997) FASEB J. 11, 428-442
6. Olweus, J., Lund-Johansen, F., and Horejsi, V. (1993) J immunol 151, 707-716
7. Boscá, L., and Lazo, P. A. (1994) J. Exp. Med. 179, 1119-1126
8. Lazo, P. A., Cuevas, L., Gutierrez del Arroyo, A., and Orue, E. (1997)
Cell.Immunol. 178, 132-140
9. Cao, L., Yoshino, T., Kawasaki, N., Sakuma, I., Takahashi, K., and Akagi, T.
(1997) Immunobiology 197, 70-81
10. Rubinstein, E., La Naour, F., Lagaudriere-Gesbert, C., Billard, M., Conjeaud,
H., and Boucheix, C. (1996) Eur J Immunol 26, 2657-2665
11. Mannion, B. A., Berditchevski, F., Kraeft, S., Chen, L. B., and Hemler, M. E.
(1996) J immunol 157, 2039-2047
12. Hemler, M. E. (1998) Current Biol. 10, 578-585
13. Berditchevski, F., and Odintsova, E. (1999) J. Cell. Biol. 146(2), 477-492
14. Mollinedo, F., Fontán, G., Barasoaín, I., and Lazo, P. A. (1997) Clin. Diag. Lab.
Immunol. 4, 229-231
15. Anderson, D. C., Kishimoto, T. K., and Smith, C. W. (1995) in The metabolic
and molecular bases of inherited disease (Scriver, C. R., Beaudet, A. L., Sly, W.
S., and Valle, D., eds), pp. 3955-3994, McGraw-Hill, New York, N.Y.
16. Higashiyama, M., Taki, T., Ieki, Y., Adachi, M., Huang, C., Koh, T., Kodama,
23
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
K., Doi, O., and Miyake, M. (1995) Cancer Res 55, 6040-6044
17. Higashiyama, M., Doi, O., Kodama, K., Yokouchi, H., Adachi, M., Huang, C.,
Taki, T., Kasugai, T., Ishiguro, S., Nakamori, S., and Miyake, M. (1997) Int J
Cancer (74), 205-211
18. Miyake, M., Nakano, K., Ieki, Y., Adachi, M., Huang, C., Itoi, S., Koh, T., and
Taki, T. (1995) Cancer Res 55, 4127-4131
19. Huang, C. I., Kohno, N., Ogawa, E., Adachi, M., Taki, T., and Miyake, M.
(1998) Am J Pathol 153(3), 973-983
20. Uchida, S., Shimada, Y., Watanabe, G., Li, Z. G., Hong, T., Miyake, M., and
Imamura, M. (1999) Br. J. Cancer 79(7-8), 1168-1173
21. Dong, J., Suzuki, H., Pin, S. S., Bova, G. S., Schalken, J. A., Isaacs, W. B.,
Barrett, J. C., and Isaacs, J. T. (1996) Cancer Res 56, 4387-4390
22. Adachi, M., Taki, T., Ieki, Y., Huang, C., Higashiyama, M., and Miyake, M.
(1996) Cancer Res 56, 1751-1755
23. Adachi, M., Taki, T., Konishi, T., Huang, C. I., Higashiyama, M., and Miyake,
M. (1998) J Clin Oncol 16(4), 1397-406
24. Guo, X., Friess, H., Graber, H. U., Kashigawi, M., Zimmerman, A., Kore, M.,
and MW, B. c. (1996) Cancer Res 56, 4876-4880
25. Guo, X.-Z., Friess, H., Maurer, C., Berberat, P., Tang, W.-H., Zimmerman, A.,
Naef, M., Graber, H. U., Kore, M., and Buchler, M. W. (1998) Cancer Res. 58,
753-758
26. Hotta, H., Ross, A. H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M. V.,
Ricciardi, R. P., Tsujimoto, Y., Croce, C. M., and Koprowski, H. (1988) Cancer
Res. 48, 2955-2962
24
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
27. Ikeyama, S., Koyama, M., Yamaoko, M., Sasada, R., and Miyake, M. (1993) J.
Exp. Med. 177, 1231-1237
28. Angelisova, P., Hilgert, I., and Horejsi, V. (1996) Immunogenetics 39, 249-256
29. Berditchevski, F., Zutter, M. M., and Hemler, M. E. (1996) Mol. Biol. Cell. 7,
193-207
30. Rubinstein, E., Poindessous-Jazat, V., Le Naour, F., Billard, M., and Boucheix,
C. (1997) Eur.J.Immunol. 27, 1919-1927
31. Ferrer, M., Yunta, M., and Lazo, P. A. (1998) Leukemia 12, 773
32. Hemler, M. E., Mannion, B. A., and Berditchevski, F. (1996) Biochem Biophys
Acta 1287, 67-71
33. Le Naour, F., Prenan, M., Francastel, C., Rubinstein, E., Uzan, G., and
Boucheix, C. (1996) Oncogene 13, 481-486
34. Le Naour, F., Francastel, C., Prenant, M., Lantz, O., Boucheix, C., and
Rubinstein, E. (1997) Leukemia 11, 1290-1297
35. Hotta, H., Takahashi, N., and Homma, M. (1989) Jpn.J.Cancer Res. 80, 1186-
1191
36. Takahashi, N., Hotta, H., and Homma, M. (1991) Jpn J Cancer Res 82, 1239-
1244
37. Korinek, V., and Horejsi, V. (1993) Immunogenetics 38, 272-279
38. Gallego, M. I., Shoenmakers, E. P. F. M., Van de Ven, W. J. M., and Lazo, P. A.
(1997) Mol. Carcinogen. 19, 114-121
39. Gallego, M. I., and Lazo, P. A. (1995) J. Biol. Chem. 270, 24321-24326
40. Wingender, E., Chen, X., Hehl, R., Karas, H., Liebich, I., Matys, V., Meinhardt,
T., Pruss, M., Reuter, I., and Schacherer, F. (2000) Nucleic Acids Res 28(1),
25
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
316-319
41. Chomczynski, P., and Sacchi, N. (1987) Anal.Biochem. 162, 156-162
42. Feduchi, E., Gallego, M. I., and Lazo, P. A. (1994) Int. J. Cancer 58, 855-859
43. Gónzalez, M. E., Pardo-Manuel de Villena, F., Fernández-Ruiz, E., Rodriguez
de Córdoba, S., and Lazo, P. A. (1993) Genomics 18, 725-728
44. Yang, S. H., Shore, P., Willingham, N., Lakey, J. H., and Sharrocks, A. D.
(1999) Embo J 18(20), 5666-5674
45. Mo, Y., Vaessen, B., Johnston, K., and Marmorstein, R. (2000) Nat Struct Biol
7(4), 292-297
46. Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J.,
Devon, K., Dewar, K., Doyle, M., FitzHugh, W., Funke, R., Gage, D., Harris,
K., Heaford, A., Howland, J., Kann, L., Lehoczky, J., LeVine, R., McEwan, P.,
McKernan, K., Meldrim, J., Mesirov, J. P., Miranda, C., Morris, W., Naylor, J.,
Raymond, C., Rosetti, M., Santos, R., Sheridan, A., Sougnez, C., Stange-
Thomann, N., Stojanovic, N., Subramanian, A., Wyman, D., Rogers, J., Sulston,
J., Ainscough, R., Beck, S., Bentley, D., Burton, J., Clee, C., Carter, N.,
Coulson, A., Deadman, R., Deloukas, P., Dunham, A., Dunham, I., Durbin, R.,
French, L., Grafham, D., Gregory, S., Hubbard, T., Humphray, S., Hunt, A.,
Jones, M., Lloyd, C., McMurray, A., Matthews, L., Mercer, S., Milne, S.,
Mullikin, J. C., Mungall, A., Plumb, R., Ross, M., Shownkeen, R., Sims, S.,
Waterston, R. H., Wilson, R. K., Hillier, L. W., McPherson, J. D., Marra, M. A.,
Mardis, E. R., Fulton, L. A., Chinwalla, A. T., Pepin, K. H., Gish, W. R.,
Chissoe, S. L., Wendl, M. C., Delehaunty, K. D., Miner, T. L., Delehaunty, A.,
Kramer, J. B., Cook, L. L., Fulton, R. S., Johnson, D. L., Minx, P. J., Clifton, S.
26
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
W., Hawkins, T., Branscomb, E., Predki, P., Richardson, P., Wenning, S.,
Slezak, T., Doggett, N., Cheng, J. F., Olsen, A., Lucas, S., Elkin, C.,
Uberbacher, E., Frazier, M., et al. (2001) Nature 409(6822), 860-921.
47. Shivdasani, R. A., and Orkin, S. H. (1996) Blood 87, 4025-4039
48. Mitsui, S., Yamaguchi, S., Matsuo, T., Ishida, Y., and Okamura, H. (2001)
Genes Dev 15(8), 995-1006.
49. Bell, A. C., West, A. G., and Felsenfeld, G. (2001) Science 291, 447-450
50. Sacristan, M. P., de Diego, J. G., Bonilla, M., and Martin-Zanca, D. (1999)
Oncogene 18, 5836-5842
51. Ross, I. L., Yue, X., Ostrowski, M. C., and Hume, D. A. (1998) J. Biol. Chem.
273, 6662-6669
52. Hotta, H., Miyamoto, H., Hara, I., Takahashi, N., and Homma, M. (1992)
Biochem Biophys Res Commun 185, 436-442
53. Lloberas, J., Soler, C., and Celada, A. (1999) Immunol Today 20(4), 184-189
54. Liu, X., Prabhu, A., and Van Ness, B. (1998) J. Biol. Chem. 274, 3285-3293
55. Pongubala, J. M. R., and Atchison, M. L. (1995) J. Biol. Chem. 270, 10304-
10313
56. Heydemann, A., Juang, G., Hennesey, K., Parmacek, M. S., and Simon, M. C.
(1996) Mol. Cell. Biol. 16, 1676-1686
57. Pongubala, J. M. R., Nagulapalli, S., Klemsz, M. J., McKercher, S. R., Maki, R.
A., and Atchison, M. L. (1992) Mol. Cell. Biol. 12, 368-378
58. Chen, H. M., Pahl, H. L., Scheibe, R. J., Zhang, D. E., and Tenen, D. G. (1993) J
Biol Chem 268(11), 8230-8239
59. Pahl, H. L., Scheibe, R. J., Zhang, D. E., Chen, H. M., Galson, D. L., Maki, R.
27
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
A., and Tenen, D. G. (1993) J Biol Chem 268(7), 5014-5020
60. Rosmarin, A. G., Luo, M., Caprio, D. G., Shang, J., and Simkevich, C. P. (1998)
J. Biol. Chem. 273, 13097-13103
61. Feng, X., Teitelbaum, S. L., Quiroz, M. E., Cheng, S. L., Lai, C. F., Avioli, L.
V., and Ross, F. P. (2000) J Biol Chem 275(12), 8331-8340
62. Eichbaum, Q., Heney, D., Raveh, D., Chung, M., Davidson, M., Epstein, J., and
Ezekowitz, R. A. (1997) Blood 90(10), 4135-4143
63. Muller, S., Maas, A., Islam, T. C., Sideras, P., Suske, G., Philipsen, S.,
Xanthopoulos, K. G., Hendriks, R. W., and Smith, C. I. (1999) Biochem
Biophys Res Commun 259(2), 364-369
64. Bell, A. C., and Felsenfeld, G. (2000) Nature 405(6785), 482-485.
65. Bell, A. C., West, A. G., and Felsenfeld, G. (1999) Cell 98(3), 387-396.
66. Naar, A. M., Beaurang, P. A., Zhou, S., Abraham, S., Solomon, W., and Tjian,
R. (1999) Nature 398(6730), 828-832.
67. Naar, A. M., Ryu, S., and Tjian, R. (1998) Cold Spring Harb Symp Quant Biol
63, 189-199
68. Lemon, B., and Tjian, R. (2000) Genes Dev 14(20), 2551-69
69. Nerlov, C., Querfurth, E., Kulessa, H., and Graf, T. (2000) Blood 95(8), 2543-
2551.
28
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
FIGURE LEGENDS
FIGURE 1. Cloning and mapping of the promoter region of the human CD53 gene. At
the top is the restriction map of genomic clone containing 20 kb of the CD53 upstream
genomic region, and at the bottom is shown the restriction map of proximal region that
was used for transcriptional activity studies. The location of the cDNA probe
(pPET/XAG) used for library screening is indicated. The start sites of the CD53 and
Gene A are indicated by arrows. Xh: XhoI; H: HindIII; C: ClaI; R: EcoRI; B: BamHI;
Hc: HincII; Bg: BglII; S: SmaI; Nh: NheI; Xb: XbaI.
FIGURE 2. Nucleotide sequence of the proximal region surrounding the transcription
start site of CD53 and containing its proximal enhancer-promoter the location of
consensus sequences corresponding to known transcription factors is indicated. This
sequence is within the clone comprising the human CD53 gene promoter from position
↑2562 to + 1051 (EBI/GenBank Accession number AJ243474), which includes the
promoter-enhancer of an unknown gene (gene A in this report). The two gene
promoter-enhancers are separated by twenty-four copies of a CA dinucleotide repeat.
FIGURE 3. Identification of a proximal second transcriptional unit that belongs to an
unknown gene. Determination of luciferase activity of the promoter-enhancer from the
new Gene A transcriptional unit, that has a transcriptional orientation opposite with
respect to hCD53. In the diagram it is also indicated the relative position of the twenty-
four CA dinucleotide repeats.
29
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
FIGURE 4. Cell surface expression of CD53 antigen in K562, Molt.4 and Namalwa cell
lines. The three cell lines were analyzed with the monoclonal antibody MEM53, which
is specific for the human CD53 antigen. The non-specific control background is also
shown in the FACS diagram.
FIGURE 5. Characterization of the proximal genomic region, from –2157 to +1
position, upstream of the CD53 transcription start site, and lack of interference by the
second gene (Gene A) promoter-enhancer.
FIGURE 6. Characterization of the transcriptional activity in the CD53 proximal region,
from –266 to +84, detects four different cooperating elements. Identification of an
enhancer at the end of exon 1, and its cooperation with a proximal regulatory region. At
the bottom is a diagram describing the regions into which the proximal CD 53
regulatory region can be subdivided.
FIGURE 7. Mutagenesis of selected cis-acting DNA regulatory sequences in the human
CD53 gene promoter. Effect of the double mutations (Table 1) introduced in the
consensus sequences for transcription factors on the expression from the CD53
promoter in different cell lines, K562, Molt-4 and Namalwa. The empty luciferase
reporter plasmid (indicated by the position of its ends) into which the mutations were
introduced is shown to the left of the mutants.
FIGURE 8. CD53 transcriptional complex in different cell lines. Diagram illustrating
the different functional organization of the regulatory elements in the active CD53
30
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
promoter in K562, Molt4 and Namalwa cell lines. Positive elements are those that result
in a loss of activity when they are mutated (squares), negative elements are those that
activate transcription when they are mutated (circles), and elements with no effect
(triangles). The shaded elements are required for transcriptional competence in the three
cell lines.
FIGURE 9. Level of several transcription factors determined by western blot analysis.
Total cellular extracts from the different cell lines, K562, Molt-4 and Namalwa, were
used for the analysis. The proteins were fractionated in a 10 % polyacrylamide-SDS gel
and after transfer to a PVDF Immobilon-P membrane, the western blots were analyzed
with an antibody against a specific transcription factor. The detection was performed
using a chemiluminiscence commercial kit. An antibody against β-actin was included
as a control for protein loading in the gels.
31
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Javier Hernández-Torres, Mónica Yunta and Pedro A. Lazoexpression
Differential cooperation between regulatory sequences required for human CD53 gene
published online July 6, 2001J. Biol. Chem.
10.1074/jbc.M104723200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on March 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from