|
|
|
|||||||
|
|||||||||
(Received for publication, July 6, 1995) From the
Genomic DNA containing the first exon and 5`-flanking region of
the human protein tyrosine kinase, blk, was isolated. Sequence
analysis identified a TG repeat element in this region with enhancer
activity, but no TATA or CCAAT sequences were found. Two blk transcripts of 2.2 and 2.5 kilobases were identified in various
B-cell lines by Northern blot analyses, and primer extension
experiments demonstrated two clusters of multiple transcription start
sites. Subsequent promoter analyses by transient transfection assays
with a reporter gene identified two promoter elements in the human blk gene. Promoter P1 contains sequences that have been shown
to regulate the expression of immunoglobulin genes and promoter P2
contains elements that are highly conserved in the promoter of major
histocompatibility complex class II genes, as well as a B-cell-specific
activator protein- (BSAP) binding site. Electrophoretic mobility shift
assays demonstrated that the binding of a protein to the BSAP-binding
site was correlated with the presence of the 2.5-kilobase blk transcript. These data suggest that the two human blk RNAs arise from the transcription of the blk gene by two
distinct promoters and that these promoters may be subject to
regulation by different trans-acting factors.
The blk gene is a member of the src family of
protein tyrosine kinases(1, 2) . The product of the blk gene, as well as other src-family members
including fyn, lyn, and lck, has been shown
to associate with the immunoglobulin receptor
complex(3, 4) . Since signal transduction via the
B-cell antigen receptor is mediated by protein tyrosine
phosphorylation(5, 6) , blk may play a role
in B-cell activation and the initial steps of the intracellular signal
pathway. Little is known about the regulatory mechanisms controlling
the restricted expression of blk. In the mouse, blk expression is restricted to B-lymphoid cells and is
developmentally regulated(7) . blk transcripts are
first detected in pro-B-cells and persist through differentiation to
mature-B-cells, but are absent in plasma cells. This expression pattern
is similar to that of two other B-cell-specific genes, mb-1
and CD19(8, 9) . The control of CD19 gene expression
has been shown to involve a B-cell-specific activating protein
(BSAP)( In contrast to the murine blk gene, the human blk gene, although predominantly
expressed in B-cells, is also found in some T-cells (14, and this
study). In order to understand the mechanisms regulating human blk expression, we have isolated and characterized the first exon and
the 5`-flanking region of the human blk gene. Northern blot
analyses identified the presence of two blk RNAs in various
B-cell lines, and the transcription start sites of these RNAs were
mapped to two clusters. Electrophoretic mobility shift assays
demonstrated that expression of one of the blk RNAs was
correlated with the presence of BSAP. Luciferase reporter gene assays
and deletion analyses identified two promoter elements in the
5`-flanking region of the blk gene. An enhancer-like element,
containing a TG repeat sequence, was also identified upstream of these
promoters.
All B- and T-lymphoid cell lines were
grown in RPMI 1640 medium (Hyclone Laboratories, Inc., Logan, UT)
supplemented with 10% fetal bovine serum (Hyclone Laboratories), 2
mML-glutamine, 100 units/ml penicillin, and 100
µg/ml streptomycin. DU 145 and T-47D cells were maintained in
minimum essential medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum, 25 mM HEPES, 26 mM sodium
bicarbonate, 0.006 µg/ml bovine insulin (Sigma), 100 units/ml
penicillin G (Life Technologies, Inc.), and 100 µg/ml streptomycin
(Life Technologies, Inc.).
To amplify blk RNAs, two sense
primers, blkpro26 (5`-TGAAAACTGATTGAGATGAG-3`, +51 to
+70) and blkpro28 (5`-GAAGGGCATTGTGACCCACG-3`, -192
to -173), derived from the first exon, were used for the
detection of 2.2- and 2.5-kb RNAs, respectively. To exclude the
possibility of contamination with genomic DNA template, the antisense
primer, race 1 (5`-GCGGTGTAGTCATACAGAGCCACCACG-3`, +391 to
+417), was derived from the third exon of blk. One µg
of total RNA was reverse transcribed and then followed by 40 cycles of
PCR using a GeneAmp PCR system 9600 (Perkin-Elmer). Each cycle
consisted of a denaturing temperature of 95 °C for 30 s, an
annealing temperature of 55 °C for 30 s, and an extension
temperature of 72 °C for 1 min. The PCR products were analyzed by
gel electrophoresis and by Southern blot analysis(17) .
A series of 5` deletions were
constructed from pL74. Plasmids pL76, pL78, pL82, and pL90 were
generated by cleavage of pL74 with SmaI, KpnI, StuI combined with SmaI and PstI,
respectively, and religation to delete the intervening sequences.
Plasmids pL86 and pL88 were produced by cleavage with HindIII
and either BglII or StuI, respectively, filling in
with Klenow and religation. For internal promoter deletion, plasmid
pL84 was generated by cleavage of pL76 with PstI and then
religation. Plasmid pL27 was constructed in several steps. The 1.8-kb HindIII to BamHI fragment of blk The RSSV-CAT plasmid, in which the
RSSV promoter was placed upstream of the chloramphenicol
acetyltransferase (CAT) gene, was a gift from Dr. R. Weigel (Stanford
University, CA).
Luciferase activity was
measured from 20 µl of the cell extract reacting with the
luciferase reagents as described by the supplier (Analytical
Luminescence Laboratory, San Diego, CA). The light emission was
measured with a Monolight 2010 instrument (Analytical Luminescence
Laboratory), reading relative light for 10 s. Luciferase activities
were normalized for CAT-specific activity. For CAT assays, cell
extracts were heated to 65 °C for 15 min to inactivate endogenous
acetylases. Subsequently, CAT activity was measured by incubating 50
µl of the cell extract with
[
The 5`BSAP and 3`BSAP
oligonucleotides correspond to nucleotides -404 to -391 of
the blk 5`-flanking region and span a putative BSAP-binding
site. The 5`mBSAP and 3`mBSAP oligonucleotides correspond to the same
region but contain two mutations, C to A and G to A, as indicated by
lower case letters. Each pair of oligonucleotides was annealed and
fill-in labeled with Klenow DNA Polymerase (Pharmacia Biotech Inc.) and
[
Figure 1:
Restriction enzyme map of the human blk gene. All clones were isolated from a human genomic DNA
library in the bacteriophage vector lambda-DASH
A 2.3-kb SmaI-EcoRI fragment containing the 5`-flanking region
and exon 1 of the human blk gene was subcloned and sequenced (Fig. 2). Nucleotide position -386 corresponds to the 5`
end of the cDNA(24) . Computer analysis demonstrated an absence
of TATA or CCAAT motifs located at the optimal distance (25, 26, 27) from the -386 site or the
+1 major transcription initiation site (later identified in this
study). However, several potential motifs known to be involved in the
expression of other B-cell-specific genes were found in two clusters
separated by approximately 400 bp. One cluster contains E(28) ,
PU.1(29, 30) , X- and Y-like boxes(31) , and
c-MYB (32) motifs. Another cluster located upstream of the
+1 position contains E, PU.1, PEA3(33, 34) , and
SP1 (35) motifs. In between, a potential BSAP-binding site (10, 12) that has been shown to be important for the
expression of the B-cell-specific gene, CD19, was identified at
position -404 to -391. The other notable feature of the
nucleotide sequence in the 5`-flanking region of the blk gene
was the presence of a 36-bp TG repeat (TG element) at position
-2071 to -2106.
Figure 2:
Nucleotide sequence of the 5`-flanking
region of the human blk gene. The major transcription
initiation site of the Daudi, ARH-77, and RPMI 7666 cell lines,
determined by primer extension experiments, is shown by the solid
triangle and is designated nucleotide +1 of the gene. TG
repeats are labeled and underlined. Sequences similar
to those of the binding sites for controlling the B-cell specificity of
immunoglobulin genes (E box, PU.1, and PEA3) and MHC class II genes
(X-box and Y-box) are labeled and underlined. The boxed sequence is a potential BSAP-binding site. The putative
motifs of
Figure 3:
Differential expression of two blk transcripts in human B-lymphoid cell lines. Upper panel,
total RNA from human B-cell lines were analyzed by Northern blotting
methods for hybridization to a probe specific for blk. Lower panel, the same filter was hybridized with a human
glyceraldehyde-3-phosphate dehydrogenase probe. The two major blk transcripts, with approximate lengths of 2.2 and 2.5 kb, are shown
by the arrows.
Figure 4:
Primer extension mapping of the
transcription initiation sites of the 2.2- and 2.5-kb blk transcripts. Total RNA was prepared from Daudi, ARH-77, RPMI 7666,
and CEM cell lines. Oligonucleotides complementary to nucleotides
+43 to +70 (A) and -256 to -287 (B) of the blk sequence were hybridized to 20 µg
of RNA. The hybridized primers were extended with reverse
transcriptase. Extension products as well as a sequence ladder were
separated on a 6% polyacrylamide gel. The arrow indicates the
most abundant product.
A pattern of heterogeneous
start sites was also found using the blkEXT primer; however,
we failed to detect extension products in the RPMI 7666 cell line using
this primer (Fig. 4B). To confirm the lack of
expression of the 2.5-kb blk message in RPMI 7666 cells and
other cell lines, reverse transcribed-PCR was performed. Total RNAs
from these cell lines were reverse transcribed and amplified with an
upstream primer, either blkpro28 (-173 to -192),
for the detection of the 2.5-kb RNA, or blkpro26 (+51 to
+70), for the detection of the 2.2-kb RNA, and a downstream
primer, race 1 (from exon 3). The results demonstrated that SUP-B12 and
Jurkat cell-line cDNA can be amplified by blkpro26, but not by blkpro28, whereas RPMI 6666 and RPMI 7666 cell line cDNA can
be amplified by both primers (Table 1). This suggests that the
2.5-kb transcript, although undetectable by Northern or primer
extension analyses in RPMI 6666 and RPMI 7666 cells, can be detected by
the more sensitive reverse transcribed-PCR method. In contrast, the
SUP-B12 and Jurkat cell lines were found to lack the 2.5-kb transcript
by all these assays. The presence of only the 2.2-kb blk RNA
in some cell lines and both transcripts in other cell lines suggested
that the expression of these two RNAs may be controlled by different
regulatory elements.
To identify the regulatory elements
responsible for the tissue-specific expression and the promoter
activity of the human blk gene, we have constructed 5`
deletion mutations of the pL76 luciferase reporter gene construct (Fig. 5). The different deletion constructs were transfected
into Daudi cells, and luciferase activities were measured. The results
show that removal of sequences from -2258 to -1628 (pL82,
-1628 to +75) significantly reduced luciferase expression (Fig. 5), indicating the presence of an enhancer element in this
region. Further deletion to -338 (pL90, -338 to +75)
resulted in an increase in luciferase activity, suggesting the presence
of a negative regulatory element upstream of -338 and the
presence of an element with promoter activity between -338 and
+75 (designated P1). Interestingly, combining the P1 promoter
fragment with the enhancer containing fragment (-2258 to
-1628), which yields plasmid pL84, dramatically increased
activity to nearly the same level as the pL76 construct.
Figure 5:
Deletion analysis of the human blk 5` regulatory region. The schematic diagram represents the human blk promoter from -2261 to +75. The two clusters of
transcription initiation sites are indicated by arrows.
Putative nuclear factor-binding sites and restriction sites used to
construct the deletion plasmids are also shown. The region of the blk promoter contained in each construct is indicated by the bars at the left, and the open box represents the luciferase coding sequence. Daudi cells were
cotransfected with the indicated luciferase plasmids and pRSSV-CAT.
Activity is presented relative to the pGL2-basic vector after
normalization for transfection efficiency. Data represent the mean + S.E. from four independent
experiments.
Deletion of
the pL76 construct from the 3` end (pL86, -2258 to -338)
resulted in a construct with significant promoter activity, albeit
weaker than the P1-containing pL90 construct. To map this element
further, it was subdivided into two fragments, one containing the
enhancer element (pL88, -2258 to -1628) and the other
containing a cluster of potential regulatory motifs (pL97, -801
to -338). Neither of these constructs had significant activity,
suggesting the presence of a weak promoter that requires an enhancer
element for activity.
Figure 6:
Interaction of BSAP with the human blk promoter. Electrophoretic mobility shift analyses with nuclear
extracts prepared from several different cell lines are shown. A, a labeled probe containing the BSAP-binding site from the blk promoter (nucleotide positions -426 to -377)
was prepared and used for gel shift assays as described (see
``Materials and Methods''). The specific BSAP complexes as
well as the position of free probe are indicated. B, two point
mutations in the BSAP recognition sequence prevent the formation of
protein
To confirm that the bound protein was BSAP, we mutated the
50-bp BSAP probe at positions -406 (C to A) and -402 (G to
A). Mutations at the corresponding positions in the murine blk promoter have been shown to impair the binding of
BSAP(13) . Data presented in Fig. 6B show that
the mutated probe, m-BSAP, was unable to interact with the protein
identified by the wild-type (wt-BSAP) probe. In previous studies, it has been shown that the expression of
the murine blk gene is B-lineage restricted and
developmentally regulated(2, 7) . The murine blk gene is expressed in pre-B through mature B-cell stages of
differentiation but not in plasma cells. Our studies and others (14) have demonstrated the expression of the human blk gene in B-cell lines representing all stages of differentiation
(pre-B through plasma cells), and in at least one T-cell line (Jurkat),
but not in any of the non-lymphoid cell lines examined. The expression
of human blk in the early stages of T-cell development has
been reported previously(14) . The function of blk in
these T-cells is not understood. It is possible that human blk may play a role in signal transduction early in T-cell
development. In most of the human B-cell lines examined in this
study, we found two blk transcripts (2.2 and 2.5 kb).
Interestingly, these two transcripts appeared to be differentially
expressed in the various cell lines. Some cell lines expressed higher
levels of the 2.2-kb message as compared to the 2.5-kb transcript,
others expressed relatively equal levels of both transcripts, while
still others expressed only the 2.2-kb message. To begin to understand
the mechanism(s) by which these two blk transcripts are
differentially regulated, we examined their structural differences
using Northern and PCR analyses. We have previously described the
isolation of a human blk cDNA clone(24) . This cDNA
corresponds in length to the 2.5-kb blk transcript. Northern
analysis with a cDNA probe containing protein-coding sequences detected
both the 2.2- and the 2.5-kb transcripts. However, a probe containing
the first 210 bp (from -386 to -177) of the 5`-untranslated
region of the cDNA clone specifically hybridized to the 2.5-kb RNA, but
not to the 2.2-kb RNA. These observations on the structural differences between the two blk transcripts and the differential expression of these
transcripts in different cell lines suggest that the human blk gene may contain two promoters that are regulated by different trans-acting factors. To investigate this possibility, the
5`-flanking region of the human blk gene was sequenced and
examined for potential transcriptional regulatory elements surrounding
the two clusters of transcription start sites. As with other src-family member genes(1) , the 5`-flanking sequence
of the human blk gene lacks TATA box elements near the
transcriptional start sites. However, two clusters of possible binding
sites for B-cell-specific transcription factors were found. One
cluster, located near the upstream group of transcription start sites,
contains X-box, CRE, and Y-box motifs. Interestingly, the spacing
between these elements is similar to that in the MHC class II promoter (31, 43) . The molecular mechanisms controlling
transcription of MHC class II genes have been studied extensively, and
the nuclear factors binding to these sequences have been
identified(44, 45, 46, 47) . These
boxes all contribute to the B-cell-specific expression of MHC class II
genes. Another DNA-binding sequence that may contribute to the
B-cell-specific expression of blk is the BSAP site at position
-404 to -391 (see Fig. 2). Previous studies on the
CD19 (10) and murine blk promoters (7, 13) have shown the presence of a site recognized
by the B-lymphoid transcription factor BSAP. This factor, like Sp1 and
GCN4(48) , may play a role in activating transcription from
TATA-less promoters. Another cluster of sequences, located just
upstream of the major transcription start site at +1, contains E,
PU.1, and PEA3 boxes. These sequences are present in the promoter and
enhancer regions of immunoglobulin genes and are important for the
B-cell-specific expression of these
genes(49, 50, 51, 52) . A binding
site for the general transcription factor Sp1 (35) is also
located in this region. Sp1 sites are often found in TATA-less
promoters. Whether cooperation of Sp1 with the other binding factors
(E, PU.1, and PEA3) is necessary for the activity of the blk promoter requires further investigation. To test the functional
activity of the 5`-flanking region of the human blk gene, a
series of luciferase reporter gene plasmids were constructed and
transfected into various cell lines. Results from these assays
demonstrated that a 2.3-kb DNA fragment from the 5`-flanking region of
the human blk gene contains tissue-specific promoter function (Table 2). This fragment drives transcription in Daudi and SUP-B8
B-cell lines and Jurkat T-cells but not in T-47D breast cancer cells or
DU 145 prostate cancer cells. Deletion analysis of this fragment in
Daudi cells demonstrated three functionally important regions. One
region (-2258 to -1628) does not stimulate transcription
independently but does act as an enhancer element. The combination of
this region with promoter P1 or P2 (described below) increased
transcription activity. However, this region also enhanced expression
from the SV40 promoter approximately 3-fold in lymphoid and
non-lymphoid cell lines, indicating that this is not a tissue-specific
enhancer. We have identified a TG repeat element within this region as
being responsible for the enhancer function. Poly(dT-dG) is capable of
forming right-handed Z-DNA and has been reported to modulate promoter
activity from a distance(41, 42) . Recently, the study
of Z-DNA-binding proteins has suggested that the TG element is bound by
the high mobility group proteins(53) . These proteins contain
an high mobility group protein domain that appears to be able to bend
or loop DNA to achieve the correct conformation for transcription and
various classes of DNA
rearrangement(54, 55, 56) . The finding of a
TG element within the 5`-flanking region of the blk gene
suggests that the expression of blk may be under such
influences. The other two regions with functional activity,
-1628 to -338 and -338 to +75, are able to
promote transcription independently. Deletion of either region only
partially reduces transcription of the reporter gene. Removal of both
regions completely abolished the transcriptional activity. These
regions each contain one of the clusters of transcription initiation
sites as well as a cluster of potential transcription factor-binding
sites as described above. These results are consistent with the
hypothesis that the blk gene contains two promoters. We have
designated the putative promoter between -338 and +75 as
``P1'' and the promoter between -1628 to -338 as
``P2''. Two promoters controlling single gene expression are
also present in another member of the src family, lck(39) . Similarly, the mouse Thy-1.2 glycoprotein gene(57) , The
presence of two promoters in the blk gene was further
supported by the data from electrophoretic mobility shift assays. The
P2 promoter, which drives the expression of the 2.5-kb blk transcript, contains a possible BSAP-binding site. We have
demonstrated that the expression of the 2.5-kb transcript is correlated
with the presence of a protein that binds to a probe containing this
BSAP site. In contrast, cell lines that express the 2.2-kb blk transcript but lack the 2.5-kb transcript, lack this binding
protein. These results suggest that the expression of the 2.5-kb
transcript is regulated by BSAP or a related protein whereas the
expression of the 2.2-kb transcript is not under regulation by this
factor. In summary, we have reported studies to support the
conclusion that the human blk gene is expressed from two
distinct promoters. The presence of different clusters of known
B-cell-specific regulatory motifs within these two promoter regions and
the different expression patterns of the two blk transcripts
in various B-cell lines suggest that these promoters may be subject to
regulation by different trans-acting factors. Consistent with
this hypothesis is our identification of a protein that binds to the
BSAP site in promoter P2 and the demonstration that the presence of
this protein is correlated with the expression of the 2.5- but not the
2.2-kb blk transcript. The human blk gene is
expressed throughout B-cell development, from very immature B-cells (as
well as immature T-cells) to plasma cells. Two distinct blk promoters may be required to allow efficient transcription of the blk gene throughout B-cell development, since different trans-acting factors may be present at different stages of
differentiation. Investigations are underway to identify these factors
and to further elucidate the regulatory mechanisms controlling blk expression in human B lymphocytes.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
U34859[GenBank].
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25968-25975
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)(10) . BSAP is a member of the paired domain
family of transcription factors and is encoded by the paired box gene Pax-5(11, 12) . Recently, a BSAP-binding site
was identified in the murine blk promoter region(13) .
The correlation of the expression of BSAP and blk suggested
that BSAP may, at least partially, account for the B-cell-specific
expression of murine blk.
Cell Lines and Cell Culture
The following cell
lines were obtained from the American Type Culture Collection: Reh
(acute B-cell lymphocytic leukemia), ARH-77 (plasma cell leukemia),
Raji (Burkitt's lymphoma), RPMI 6666 (B lymphoblastoid cell
line), RPMI 7666 (B lymphoblastoid cell line), UC 729 (B lymphoblastoid
cell line), CEM (T-cell acute lymphoblastic leukemia), U-937
(histiocytic lymphoma, macrophage/monocyte like), LNCaP (prostate
adenocarcinoma), DU 145 (prostate carcinoma), T-47D (ductal carcinoma
of the breast), and 293 (transformed primary embryonal kidney cells).
SUP-B8 (Burkitt's lymphoma), SUP-B12 (Burkitt's lymphoma),
SUP-B17 (Burkitt's lymphoma), SU-DHL4 (diffuse histiocytic
lymphoma), Daudi (Burkitt's lymphoma), Nalm6 (precursor-B acute
lymphoblastic leukemia), OCI-Ly8 (B cell immunoblastic lymphoma), and
Jurkat (acute T cell leukemia) cell lines were kindly provided by Dr.
Ron Levy, Stanford University.Isolation of the blk Promoter
Genomic DNA clones
were isolated from a human lymphocyte genomic library in the
bacteriophage Lambda Dash (Stratagene, La Jolla, CA), using a P-labeled blk cDNA as the probe. Six positive
hybridizing clones were isolated after screening approximately 1
10
plaques. Two clones containing blk 5`-flanking sequences were identified by restriction mapping and
Southern blot analyses with a P-labeled first exon region
probe of the blk cDNA (-386 to +75). A 6.0-kb EcoRI fragment of the
blk29 clone was inserted
into the EcoRI site of Bluescript II SK (Stratagene) to yield
plasmid pL62. The nucleotide sequence of the blk promoter was
determined by the dideoxy-chain termination method following the
manufacturer's protocol (Sequenase; United State Biochemical
Corp.).
Northern Blotting and Reverse Transcribed-PCR
Analysis
Total cellular RNA was prepared according to
Gough(15) . The RNA samples (20 µg/lane) were separated by
electrophoresis on a 1% agarose-formaldehyde gel and transferred to a
Nytran membrane (Schleicher & Schuell) as described
previously(16) . cDNA fragments were labeled with
[P]dCTP by random oligonucleotide
priming(17) . The membranes with transferred RNA were probed
with either a 450-bp EcoRI fragment containing the
5`-untranslated region of the human blk cDNA or a 900-bp blk cDNA fragment that encodes the amino-terminal region of
the blk protein.
Primer Extension
Primer extension was performed as
described previously(18) . Two synthetic oligonucleotide
primers were used in the extension reaction. Primer blkEXT
(5`-CAAGTCCGGCAGAGTTGCAAACCTCCATGC-3`, -256 to -287) was
used to map upstream start sites, while primer blk-pro29
(5`-CTCATCTCAATCAGTTTTCATGGCTTGT-3`, +43 to +70) was used to
map the downstream start sites. Each P-end-labeled primer
was annealed to 40 µg of total RNA in a hybridization buffer (5
µl) containing 0.4 M NaCl, 40 mM PIPES (pH 6.4),
and 1 mM EDTA at 80 °C for 2 min and then at 60 °C for
16 h. The annealed products were extended with 200 units of superscript
reverse transcriptase (Life Technologies, Inc.) in an extension mixture
(50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl
, 0.8 mM deoxynucleotide triphosphates,
10 mM dithiothreitol) at 42 °C for 1 h. The extension
products and a sequencing ladder generated with the same primers were
analyzed on a 7 M urea, 6% polyacrylamide sequencing gel.Plasmid Constructs
To generate luciferase reporter
plasmids, the 6.0-kb SacI-XhoI fragment from pL62 was
cloned into the SacI and XhoI sites of pGL2-basic
(Promega Corp., Madison, WI). The plasmid was digested with HindIII and religated to remove extra multiple cloning sites.
The resulting construct, pL74, contained 6.0 kb of the blk 5`-flanking region upstream of the luciferase coding sequence and
the simian virus 40 splice and polyadenylation sites present in the
original pGL2-basic vector.29 was
first cloned into the HindIII and BamHI sites of
Bluescript. The resulting plasmid was digested with EcoRI and HindIII, filled in, and religated to create plasmid pL23.
Then, the XhoI to BamHI fragment of pL23 was cloned
into the BglII site of pGL2-basic vector. Plasmid pL97 was
made by polymerase chain reaction (PCR) amplification of a fragment
(from -801 to -256) using a mutant primer, blkpro5
(5`-CAGgaGCTCACTGTGTCTGGCT-3`, -801 to -780), and the blkEXT primer. Blkpro5 introduced a SacI
site at the 5` end of the PCR product. The fragment was cleaved with SacI and BglII and inserted into the corresponding
sites of the pGL2-basic vector.
Transfection, Luciferase, and CAT Assays
Daudi,
SUP-B8, SUP-B12, ARH-77, and Jurkat cells were transfected by
electroporation as described previously(19) . Cells (6
10) were washed once and resuspended in 1 ml of RPMI
(without serum or antibiotics) containing 10 µg/ml DEAE-dextran, 5
µg of the reporter plasmid, and 1 µg of RSSV-CAT plasmid and
electroporated using 0.4-cm cuvettes in a Bio-Rad gene pulser (Bio-Rad)
at 300 V, 960 microfarads for B-cells and 250 V, 960 microfarads for
T-cells. After incubation in RPMI complete medium for 24-36 h,
the cells were harvested and lysed in 150 µl of 250 mM Tris (pH 7.7) by three freeze-thaw cycles. The cell extracts were
then analyzed for luciferase and CAT activities. DU 145, T-47D, and 293
cell lines were transfected using the calcium phosphate precipitation
method as described previously(20) .
C]chloramphenicol and butyryl-coenzyme A as
described previously(21, 22) .Electrophoretic Mobility Shift
Assays
Electrophoretic mobility shift assays were carried out as
described previously(23) . Two pairs of complementary
oligonucleotides were designed with 3` complementarity as follows:
5`BSAP, 5`-ATTTAAAGACAAAGCAAAACCAGTGAGGCTG-3`; 3`BSAP,
5`-ACCAGGGCAGCCGTTCTTTCAGCCTCACTG-3`; 5`mBSAP,
5`-ATTTAAAGACAAAGCAAAACaAGTaAGGCTG-3`; 3`mBSAP,
5`-ACCAGGGCAGCCGTTCTTTCAGCCTtACTtG-3`.
-P]dCTP. Ten fmol of each labeled DNA
probe was incubated with 10 µg of whole cell extract in 1
binding buffer (40 mM KCl, 20 mM HEPES (pH 7.7), 1
mM MgCl, 0.1 mM EDTA, 0.4 mM dithiothreitol) containing 4% Ficoll and 1 µg of salmon sperm
DNA in a volume of 25 µl at room temperature for 30 min.
Protein
DNA complexes were analyzed on a native 4% polyacrylamide
gel in 0.25 Tris borate-EDTA. Gels were dried and
autoradiographed.
Cloning and Sequence Analysis of Human blk 5`-Flanking
Region
To isolate the genetic elements involved in the
regulation of blk gene expression, we initially screened a
human lymphocyte genomic library with the full-length blk cDNA (24) . Five phage clones identified by Southern blot analysis
are diagrammed in Fig. 1. Restriction enzyme mapping showed that
the human blk gene spans greater than 60 kb of genomic DNA.
Like other members of the src gene family, the first exon of
human blk contains only 5`-untranslated sequence.
. Clones
blk26A and
blk29A contain the first exon
(indicated by the filled box) and the 5`-flanking region. The
second exon is located in the
blk21A clone and contains
the translational start site (indicated by the arrow).
Restriction enzymes are indicated as follows: B, BamHI; E, EcoRI; H, HindIII; S, SalI; Xb, XbaI; Xh, XhoI.
-interferon response elements are doubly
underlined. c-myb and cyclic-AMP response element are
also labeled and underlined.
Two Sizes of blk Transcripts Differentially Expressed in
B-cell Lines
The presence of a cluster of B-cell-specific motifs
downstream of position -386 suggests that the expression of blk may have two positive regulatory regions. To test this
hypothesis, we first investigated the expression pattern of the blk gene by Northern blot analysis. An RNA blot containing total RNA
isolated from various lymphoid cell lines was hybridized with a labeled blk cDNA probe (Fig. 3, upper panel). The same
blot was reprobed with a labeled human glyceraldehyde-3-phosphate
dehydrogenase DNA probe as a control (Fig. 3, lower
panel). Two major blk transcripts with approximate sizes
of 2.2 and 2.5 kb were detected (Fig. 3). The relative abundance
of these two RNAs were different in the tested cell lines. As shown in Table 1, four patterns of blk expression were observed.
The 2.2-kb message was expressed at a higher level than the 2.5-kb RNA
in ARH-77, SU-DHL4, Daudi, Raji, SUP-B8, and Reh. Both transcripts were
detected with equal intensity in SUP-B17, OCI-Ly8, Nalm 6, and normal
spleen. SUP-B12, RPMI 6666, RPMI 7666, and Jurkat expressed only the
2.2-kb RNA; the 2.5-kb transcript was not detected. Finally, in two
B-lymphoblastoid cell lines (PW and UC 729) and several non-B-cell
lines (CEM, U-937, LNCaP, T-47D, and 293), no blk transcripts
were detected.
Mapping of the blk Transcription Initiation
Sites
To determine whether the 2.2- and 2.5-kb transcripts
initiate from the same or different start sites, we carried out primer
extension experiments using two different DNA primers. The primers blkpro29 (from +43 to +70) and blkEXT (from
-256 to -287) were designed for the extension of the 2.2-
and 2.5-kb RNA species, respectively. Total RNA isolated from Daudi,
RPMI 7666, ARH-77, and CEM was hybridized with each 5`-end-labeled
primer, extended with reverse transcriptase, and analyzed on
polyacrylamide gels. As shown in Fig. 4, a pattern of
heterogeneous start sites was observed using the blkpro29
primer. Multiple initiation sites are commonly observed with promoters
lacking a TATA
box(7, 33, 36, 37, 38, 39, 40) .
A major transcription start site was found at position +1 in
Daudi, ARH-77, and RPMI 7666 cell lines but was absent in the control
cell line CEM (Fig. 4A). This suggests that the 2.2-kb
RNA that is highly expressed in Daudi, ARH-77, and RPMI 7666 cells
initiates mainly from this start site.
Functional Analysis of the Human blk Promoters
To
determine whether the 5`-flanking region contains regulatory elements
that control the tissue-specific expression of the blk gene,
transient transfection assays were performed in a variety of cell
lines. A SmaI-EcoRI (-2258 to +75) and KpnI-EcoRI (about -3000 to +75) fragment
containing 5`-flanking sequences and part of the first exon region of
the human blk gene were inserted upstream of the luciferase
gene in the plasmid pGL2-basic. The resulting plasmids, pL76 and pL78,
were transfected into B-cell lines (Daudi and SUP-B8) and non-B-cell
lines (DU 145, T-47D, and Jurkat), and luciferase activity was
determined. Two plasmids, an SV40 promoter-driven luciferase construct
(pGL2-control) and a promoter-less luciferase plasmid (pGL2-basic),
were used for controls. In addition, each construct was cotransfected
with the RSSV-CAT plasmid to normalize for transfection efficiency. As
shown in Table 2, both pL76 and pL78 constructs gave much higher
levels of luciferase activity in B cell lines (Daudi or SUP-B8) than in
non-B-cell lines (DU 145 or T-47D). These constructs were also active
in the T-cell line, Jurkat which we have shown expresses blk (Table 1).
A TG element within the blk 5`-Flanking Region Shows
Non-tissue-specific Enhancer Activity
To test whether the
fragment from -2258 to -1628 could enhance transcription in
a B-cell-specific manner, the fragment was inserted downstream of an
SV40 promoter-driven luciferase gene. The resulting construct pL104 was
transfected into Daudi, DU145, T-47D, ARH-77, Jurkat, and 293 cells. As
shown in Table 3, in all of the cell lines tested, the
-2258 to -1628 fragment in pL104-enhanced SV40 promoter
activity by about 3-fold compared to the pGL2-promoter plasmid. Thus
this region contains an enhancer, but it is not B-cell-specific.
Deletion analyses localized this enhancer to a TG repeat element (see Fig. 1). ()These results are in agreement with other
observations that TG elements can act as enhancers (41, 42) .
The Binding of a Protein at the BSAP Site Is Correlated
with the Expression of the 2.5-kb blk RNA
Recent studies (13) have shown that the expression of murine blk is
correlated with the presence of BSAP and have suggested that BSAP is a
positive regulator of murine blk transcription. To determine
whether BSAP may regulate the expression of one of the human blk RNAs, we examined the putative BSAP site in the human blk gene (position -404 to -391) by electrophoretic
mobility shift assay. A 50-bp probe (from -426 to -375)
spanning the BSAP-binding site was prepared from the 5`-flanking region
of blk. This probe was mixed with cell extracts prepared from
various lymphocytic cell lines. As shown in Fig. 6A, a
DNAprotein complex was found in cell extracts of Daudi, ARH-77,
SU-DHL4, OCI-Ly8, SUP-B17, Raji, SUP-B8, RPMI 6666, and RPMI 7666 cell
lines, whereas it was not present in extract of SUP-B12, Jurkat, CEM,
or UC 729 cells (Fig. 6, A and B). As
summarized in Table 1, the presence of this DNA-bound protein
correlated with the presence of the 2.5-kb transcript, indicating that
this protein may be a positive regulator of the 2.5-kb blk transcript. Interestingly, the expression of the 2.2-kb blk transcript in SUP-B12 and Jurkat apparently does not require this
protein.
DNA complexes. Mutations were introduced at position
-406 (C A) and at position -410 (G
A). The
wild-type (wt-BSAP) and mutant (m-BSAP)
oligonucleotides were used for the mobility shift assays with whole
cell extracts of human B- and T-cell lines. For a description of the
oligonucleotides, see ``Materials and
Methods.''
PCR analyses, using an upstream
primer that hybridizes within this 210-bp 5`-untranslated region and a
downstream primer within exon 3, demonstrated amplification products
only in cell lines that expressed the 2.5-kb transcript. In addition,
primer extension experiments identified one major transcription
initiation site, designated +1, as well as two clusters of
multiple transcription initiation sites, one located around the major
start site at +1 and another located 300-400 bp upstream of
this site. These data suggest that the 2.2- and 2.5-kb blk transcripts initiate at the +1 and -300 transcription
start site clusters, respectively, and that the two transcripts differ
in the length of their 5`-untranslated regions by approximately 300 bp. -amylase gene (58) rat 
(59) gene, and B-50/GAP-43
gene (60) are also transcribed from two promoters.
))
We thank Dr. Susan Dymecki and Dr. Ronald J. Weigel
for helpful advice and critical review of this manuscript. We thank Dr.
Allison C. Chin for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Krejsgaard, C. S. Vetter-Kauczok, A. Woetmann, H. Kneitz, K. W. Eriksen, P. Lovato, Q. Zhang, M. A. Wasik, C. Geisler, E. Ralfkiaer, et al. Ectopic expression of B-lymphoid kinase in cutaneous T-cell lymphoma Blood, June 4, 2009; 113(23): 5896 - 5904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kawamata, S. Ogawa, M. Zimmermann, B. Niebuhr, C. Stocking, M. Sanada, K. Hemminki, G. Yamatomo, Y. Nannya, R. Koehler, et al. Cloning of genes involved in chromosomal translocations by high-resolution single nucleotide polymorphism genomic microarray PNAS, August 19, 2008; 105(33): 11921 - 11926. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Merluzzi, M. Moretti, S. Altamura, P. Zwollo, M. Sigvardsson, G. Vitale, and C. Pucillo CD40 Stimulation Induces Pax5/BSAP and EBF Activation through a APE/Ref-1-dependent Redox Mechanism J. Biol. Chem., January 16, 2004; 279(3): 1777 - 1786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rothenburg, F. Koch-Nolte, A. Rich, and F. Haag A polymorphic dinucleotide repeat in the rat nucleolin gene forms Z-DNA and inhibits promoter activity PNAS, July 5, 2001; (2001) 121176998. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Jiang, M. Troyanovskaya, G. Jayaraman, and D. P. Healy Aminopeptidase-A. II. Genomic cloning and characterization of the rat promoter Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2000; 278(2): R425 - R434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Akerblad and M. Sigvardsson Early B Cell Factor Is an Activator of the B Lymphoid Kinase Promoter in Early B Cell Development J. Immunol., November 15, 1999; 163(10): 5453 - 5461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ying, J. I. Healy, C. C. Goodnow, and J. R. Parnes Regulation of Mouse CD72 Gene Expression During B Lymphocyte Development J. Immunol., November 1, 1998; 161(9): 4760 - 4767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Qiu and J. Stavnezer Overexpression of BSAP/Pax-5 Inhibits Switching to IgA and Enhances Switching to IgE in the I.29{micro} B Cell Line J. Immunol., September 15, 1998; 161(6): 2906 - 2918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-H. Lee, M. T. Minowa, and M. M. Mouradian Two Distinct Promoters Drive Transcription of the Human D1A Dopamine Receptor Gene J. Biol. Chem., October 11, 1996; 271(41): 25292 - 25299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rothenburg, F. Koch-Nolte, A. Rich, and F. Haag A polymorphic dinucleotide repeat in the rat nucleolin gene forms Z-DNA and inhibits promoter activity PNAS, July 31, 2001; 98(16): 8985 - 8990. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |