(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)()(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.
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 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.
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
[C]chloramphenicol and butyryl-coenzyme A as
described previously(21, 22) .
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
[-
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.
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. 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.
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 -interferon response elements are doubly
underlined. c-myb and cyclic-AMP response element are
also labeled and underlined.
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
proteinDNA 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.''
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. 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.
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) , -amylase gene (58) rat
(59) gene, and B-50/GAP-43
gene (60) are also transcribed from two promoters.
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].