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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 March 4, 2018 http://www.jbc.org/ Downloaded from
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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.

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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

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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

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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.

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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

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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

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–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).

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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,

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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

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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

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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

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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

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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

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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)

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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

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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

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(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

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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

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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

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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

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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

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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.

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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.

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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.

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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

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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.

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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:

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