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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 214-221
Transcriptional Regulatory Elements Within the First Intron of
Bruton's Tyrosine Kinase
By
Jurg Rohrer and
Mary Ellen Conley
From the Department of Immunology, St Jude Children's Research
Hospital; and the Department of Pediatrics, University of Tennessee
College of Medicine, Memphis.
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ABSTRACT |
Defects in the gene for Bruton's tyrosine kinase (Btk) result in
the disorder X-linked agammaglobulinemia (XLA). Whereas XLA is
characterized by a profound defect in B-cell development, Btk is
expressed in both the B lymphocyte and myeloid cell lineages. We
evaluated a patient with XLA who had reduced amounts of Btk transcript
but no abnormalities in his coding sequence. A single base-pair
substitution in the first intron of Btk was identified in this patient,
suggesting that this region may contain regulatory elements. Using
reporter constructs we identified two transcriptional control elements
in the first 500 bp of intron 1. A strong positive regulator, active in
both pre-B cells and B cells, was identified within the first 43 bp of
the intron. Gel-shift assays identified two Sp1 binding sites within
this element. The patient's mutation results in an altered binding
specificity of the proximal Sp1 binding site. A negative regulator,
active in pre-B cells only, was located between base pairs 281 and 491
of the intron. These findings indicate that regulation of Btk
transcription is complex and may involve several transcriptional
regulatory factors at the different stages of B-cell differentiation.
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INTRODUCTION |
BRUTON'S TYROSINE kinase (Btk) has been
identified as a key regulator of B-cell development.1-3
Mutations in the Btk gene result in the human immunodeficiency X-linked
agammaglobulinemia (XLA),1,2 which is characterized by low
levels of circulating immunoglobulins and a severe decrease in B-cell
numbers.4 In mice, a defective Btk gives rise to the
xid phenotype,5 a milder defect in B-cell
development in which affected mice fail to make antibody to some
T-independent antigens and have about half the normal number of B
cells.6,7
Btk is a cytoplasmic tyrosine kinase that is expressed throughout
B-cell and myeloid development but it is not expressed in T cells or
other nonhematopoietic cell lineages.8-10 Amino acid
sequence identity places Btk in an emerging family of Src-related
tyrosine kinases including Itk,11 Tec,12
DrSrc28C,13 and Bmx.14 A unique feature of this
family is the long amino terminal end, which includes a proline rich
region and a pleckstrin homology domain.15 The proline rich
region has been shown to bind the SH3 domains of the protein tyrosine
kinases; Fyn, Lyn, and Hck.16 For Itk, in vitro studies
have shown that the proline rich region is able to bind its SH3 domain
in an intramolecular reaction that regulates the binding of these two
domains to their respective targets.17 Current data suggest
that the pleckstrin homology (PH) domain is likely to be involved in
the intracellular translocation of Btk15,18-20 and perhaps
in binding to critical substrates.21 Although Btk can be
activated by cross-linking of surface immunoglobulin,22,23
Fc R,24 interleukin-5 receptor (IL-5R),25 and
IL-6,26 the mechanisms by which mutations in Btk result in
a failure of B-cell development are not known.
Btk consists of 19 exons in a 37-kb stretch of DNA at
Xq22.27-30 The first exon and 30 bp of the second exon
constitute the 5 untranslated region of the Btk mRNA. Expression of
Btk has been shown in the CD34+ progenitor cell line KG-1
and begins before immunoglobulin heavy and light chain gene
rearrangements.10 Multiple transcriptional start sites
between base pairs  5 to 30 upstream of exon 1 (numbering
according to the original sequence published by Vetrie et al) have been
identified,30,31 which is consistent with the lack of a
clear TATAA box in the promoter region. Studies on the promoter region
have identified binding sites critical for lineage-specific expression
within the first 200-bp upstream of exon 1. Transcription factor
binding sites included a PU.1 site at 61 bp, an Sp1 site at 169
bp, and an Sp3 site at 38 bp.31,32 When a more upstream
sequence was included in promoter reporter constructs activity dropped
approximately twofold in B cells, suggesting that regulation of Btk
expression was likely to be more complex and involve more than just the
Sp1 and PU.1 family members binding at the minimal
promoter.31 Both phorbol ester and retinoic acid were able
to increase Btk expression in myeloid cells, whereas addition of
phorbol ester and TGF- 1 to B cells decreased Btk mRNA
levels.9
Over 200 different mutations in Btk have been
identified.33-39 These varied mutations include single base
pair substitutions (70%), small insertions or deletions (27%), and
gross deletions (3%). However, no mutations affecting the regulation
of the gene have been reported. In this report we describe a mutation
in Btk that results in decreased amounts of Btk transcript but does not
affect the integrity of the coding sequence. Analysis of this mutation
led to the identification of two regulatory elements within the first
500 bp of intron 1.
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MATERIALS AND METHODS |
RNA and DNA analysis.
Northern blots, polymerase chain reaction (PCR), single-strand
conformation polymorphism (SSCP) analyses of Btk, and cloning and
sequencing of PCR fragments were performed as previously
described.33 RNA was isolated from Epstein-Barr virus
(EBV)-transformed cell lines or freshly isolated peripheral blood
lymphocytes (PBLs) using the Qiagen RNeasy spin column (Qiagen, Santa
Clarita, CA). cDNA was prepared by reverse transcription
of the RNA using reverse transcriptase (Superscript; GIBCO-BRL,
Gaithersburg, MD) and then PCR amplified using the
appropriate primers. Forward primers used for amplifying the exon
1/exon 2 border sequence of Btk were: 5-CAGACTGTCCTTCCTCTC-3 and
5-AATGCATCTGGGAAGCTA-3, and the reverse primer was
5-CAAGAGAAACAGGCGCTT-3. Amplification of genomic DNA at the Xq22
polymorphic loci DXS101 and DXS178 was performed as previously
described.40
Luciferase reporter constructs.
A 3.4-kb EcoRI-HindIII fragment, beginning 2.3 kb
upstream of the first exon of Btk and ending 1 kb downstream of the
exon, was subcloned from cosmid 237D10 into pBluescript (pBSK,
Stratagene, La Jolla, CA).27 From this construct a 2.9-kb
Sma I to Xho I fragment was subcloned into the
luciferase reporter backbone pGL2 (Promega, Madison, WI). The
Sma I site is 1.8 kb upstream of exon 1, and the Xho I
site is 1.0 kb downstream of the exon. Reporter pBtkpro was constructed
by deleting the sequence between the Ppu10I site
(within exon 1) and the HindIII site (within the pGL2 cloning
cassette). All other constructs that contained a splice donor site and
varying lengths of intron 1 also included a splice acceptor sequence.
The intron 1 acceptor site was derived from a PCR product, which was
amplified using the forward primer 5-AAACCTGACAGATCTGGG-3 and
the reverse primer 5-TGCGGCCAAAGCTTCTTC-3. This product was cloned
into the pGL2 vector using the BglII and HindIII sites
included in the above primer sequences. The pGL2 construct, containing
the 2.9-kb Sma I-Xho I fragment and the acceptor
sequence was called pBtkpro+1029 because it contained 1,029 bp of 5
intron 1 sequence. Constructs pBtkpro+491, pBtkpro+281, and
pBtkpro+43 were deletion constructs of pBtkpro +1029 from
the Stu I, Kpn I, and Rsa I sites,
respectively.
The pBtkpro+6 reporter was also derived from a PCR-amplified product.
Sense primer 5-CAGACTGTCCTTCCTCTC-3 and antisense primer
5-AGATCTACCCACCTCAGTCCTGA-3 were used to amplify exon 1 and the first
6 bp of the intron. This fragment was cloned into the pBtkpro construct
to give pBtkpro+6.
Introduction of the patient mutation into the pBtkpro+ constructs was
done using a PCR-based strategy. The same sense primer as used in the
construction of pBtkpro+6 was used with the antisense primer
5-AGCCAGCTCTGACCCTGG-3 to amplify patient genomic DNA. The PCR
product was cloned into pBtkpro+1029 at the Ppu10I and
Kpn I sites. Again, deletion constructs as described above were
made using the Stu I, Kpn I, and Rsa I sites.
All constructs were sequenced to check for PCR errors and correct
ligation. Plasmid DNA for transient transfections was prepared by
CsCl/EtBr density gradient centrifugation as described by Maniatis et
al.41
Cell culture.
Human pre-B-cell line REH, B-cell lines Daudi and Raji, and T-cell
line Jurkat were maintained as suspension cultures in RPMI supplemented
with heat inactivated fetal bovine serum (FBS; 15%), 2-ME
(2.5 × 10 5 mol/L), and gentamicin (250 ng/mL).
Transient transfections.
Transient transfection assays were done by electroporation using the
BioRad Gene Pulsar (BioRad Laboratories, Richmond,
CA).42 Cells were grown to early log phase,
harvested, resuspended in serum-free Iscove's modified Dulbecco's
media (IMDM) at 2 × 107 cells per 200 µL and placed in
a cuvette (0.4 cm electrode gap) containing 20 µg of the test plasmid
and 8 µg of an internal control reporter construct, secreted alkaline
phosphatase driven by a PGK promoter (PGKSEAP; kindly
provided by Dr L.H. Shapiro; St Jude Children's Research Hospital,
Memphis, TN). After a 10-minute room temperature (RT) incubation the
cells were subjected to a predetermined electrical field (REH and
Jurkat 250V, Daudi 190V, and Raji 220V), with the capacitance extender
set to 960 µF. After another 10 minutes at RT the cells were plated
into 100-mm tissue culture plates containing 10 mL of IMDM supplemented
with 15% FBS and gentamicin. The cells were left to recover for 24
hours, at which stage 300 µL was drawn off to be assayed for secreted
alkaline phosphatase, and the remaining cells were harvested, lysed,
and resuspended in 200 µL of assay buffer, and 100 µL was used in
the assay as described elsewhere.42 Once the
luciferase units had been normalized using the PGKSEAP to control
for transfection efficiency, all results were expressed as a fold
increase or decrease over the basic pBtkpro construct. All experiments
were repeated at least three times using two different preparations for
each construct tested.
Gel-shift assays.
Nuclear extracts were prepared as described elsewhere.43
Two double-stranded oligonucleotide probes that covered the 5 to +32
bp region at the exon 1/intron 1 border were made. Probe sequences were
as follows: 5+16 5-ACTGAGGTGGGTCTGGGGTATG-3 and +12+32
5-GTATGGCAGGGGCTGGGCAGC-3. An additional 5+16 probe including the
patient's T to G mutation at position +6 was also made (5+16M). The
Sp1 consensus oligonucleotide, 5-ATTCGATCGGGGCGGGGCGAGC-3, was
purchased from Promega (Madison, WI). One hundred micrograms of each
complementary strand was annealed in the presence of 100 mmol/L NaCl by
heating the mixture to 95°C for 5 minutes and then allowing the
samples to cool to RT for 1 hour followed by a 1 hour incubation at
37°C. Double-stranded probes were radiolabeled with
 32P-adenosine triphosphate (ATP) using polynucleotide
kinase (Pharmacia Biotech, Piscataway, NJ) and purified over a G50
Sephadex spin column (Boehringer Mannheim, Indianapolis, IN). For the
gel-shift, 0.1 ng of probe was incubated with 10 to 15 µg of nuclear
extract, 2 µg of poly(dI-dC; Boehringer Mannheim), and 20 µg bovine
serum albumin (BSA; New England Biolabs, Beverly, MA) in a 20 µL
volume with 10% glycerol, 10 mmol/L HEPES pH 7.9, 2 mmol/L Tris pH
7.9, 100 mmol/L KCl, 1 mmol/L DTT, 1 mmol/L EDTA, 1 mmol/L 2-ME, and
0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF) at RT for 25 minutes.
Samples were electrophoresed at 4°C on 6% polyacrylamide gels in
0.4× Tris borate EDTA (TBE) at 10 V/cm for 3 hours. Gels were dried
under vacuum and exposed to x-ray film (Eastman Kodak, Rochester, NY)
for 3 to 6 hours.
| |
RESULTS |
Patient 0030 has reduced amounts of normal Btk mRNA.
We have previously shown that most mutations in Btk that cause
premature stop codons, frameshifts, or splice defects are associated
with markedly reduced amounts of Btk transcript, whereas mutations
resulting in amino acid substitutions are associated with normal
amounts of Btk mRNA as detected by Northern blot
analysis.33 A single EBV-transformed cell line from a
patient with late onset XLA (UPN 0030) consistently showed decreased
but easily detectable Btk transcripts. Because EBV transformation and
prolonged in vitro culture can alter the phenotype of a cell line, mRNA
from freshly isolated patient neutrophils was also analyzed by Northern
blot. Figure 1 shows that the amount of Btk
transcript was equivalently decreased in the cell line and in the
freshly isolated cells.
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| Fig 1.
Northern blot analysis. The top panel shows RNA from
EBV-transformed cell lines (B) and neutrophils (N) from an XLA patient
with a 4-bp deletion (1); the patient described in this study (2); an
XLA patient with an amino acid substitution (3); and a normal control
(4). To control for sample loading, the blot was stripped and
rehybridized with a -actin specific probe, shown in the lower
panel.
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To exclude the possibility of a mutation that altered the stability of
the Btk message in the patient, the coding region and 5 and 3
untranslated regions of the cDNA were sequenced, but no abnormalities
were identified. The absence of mutations in Btk mRNA and the
preservation of significant amounts of transcript raised questions
about the reliability of the XLA diagnosis. However, this patient was
part of a four-generation pedigree that included at least five other
affected males (Fig 2). Linkage studies in
this family had mapped the defect to the site of the Btk gene at Xq22
with a lod score greater than 3.0.44
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| Fig 2.
Pedigree of the patient described in this study. Squares
represent males and circles denote females. The shaded symbols
represent patients with XLA. A slash through a symbol indicates that
the individual is deceased. Patient numbers are written below their
respective symbols.
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Mutation detection in patient 0030.
Screening for mutations in Btk outside the coding region was performed
using SSCP analysis.33 Primer pairs that flanked each of
the 19 exons were used to examine the genomic DNA of the patient.
Analysis of exon 1, the noncoding exon, showed an abnormal migration
pattern (Fig 3A); all other exons were
normal. The same alteration was seen in DNA from a second XLA patient
who had no known family history of immunodeficiency. Similar to Patient
0030, this patient had slightly higher concentrations of serum
immunoglobulins than patients with typical XLA, and he had no other
alterations in the remaining 18 exons as tested by SSCP. The altered
SSCP pattern for exon 1 was not seen in 111 unrelated X chromosomes,
making it unlikely that this alteration represented a polymorphism.
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| Fig 3.
Mutation detection in the genomic DNA from Patient 0300.
(A) SSCP analysis of exon 1 and flanking sequences from a control (lane
labeled c) and patients with XLA. Patients 0030 and 0025 are shown in
lanes 1 and 10, respectively. (B) DNA sequence of the 5 part of intron
1 from a control and from Patient 0030. The 3 end of exon 1 is
indicated in the control sequence. The T to G transversion in the
patient is denoted by an asterisk.
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Exon 1 and its flanking regions were sequenced, and a T to G
transversion at position +6 of the intron 1 splice donor site was
identified in both patients (Fig 3B). To determine whether this
alteration occurred independently in the two patients or as a result of
common descent, highly polymorphic markers, DXS178 and
DXS101,40 near the Btk gene at Xq22 were used to establish
an XLA haplotype. Both boys had inherited the same alleles at DXS178
and DXS101, suggesting common descent. Further inquiries established
that the patients were descendants of a common ancestor (Fig 2).
The proximity of the base pair substitution to a splice donor site
suggested that the alteration might affect splicing, although defects
at the +6 position in an intron are rarely responsible for splice
defects.45 To determine if any cryptic splice sites were
activated because of the +6 alteration, PCR primers were designed to
amplify the cDNA region surrounding the exon 1/exon 2 cDNA border. Two
5 primers, one beginning at the transcriptional start site position
28 (numbering according to the original cDNA sequence published by
Vetrie et al) and the other closer to the 3 end of exon 1 (position
+62) were used with a 3 primer from exon 2 (position +210). When cDNA
from freshly isolated peripheral blood mononuclear cells from the
patient was used as the template, both PCR reactions showed the
expected products. No products using cryptic splice sites were
identified. In the absence of evidence that the base-pair substitution
was influencing splicing, we explored the possibility that the
alteration affected transcription regulatory elements within intron 1
of Btk.
Identification of transcriptional regulators within the first 1,029
bp of intron 1.
A Btk promoter reporter construct (pBtkpro, Fig
4) was designed by subcloning 1.8 kb of Btk
promoter DNA upstream of exon 1 plus 66 bp of this exon into the
promoterless pGL2 luciferase expression vector. The activity of pBtkpro
was compared with that of the empty pGL2 vector by transient
transfection in the pre-B-cell line REH, the B-cell lines Raji and
Daudi, and the T-cell line Jurkat (Table
1). Activities were consistently above
background and were in agreement with previous observations showing
that the Btk promoter was more active in B cells than in T
cells.31,32 To identify any cis acting elements within
intron 1 that could affect Btk expression, a construct containing the
same 1.8-kb upstream region plus the entire 102 bp of exon 1 and 1,029
bp of intron 1, pBtkpro+1029 (Fig 4A) was designed. Because this
construct contained a splice donor site, an acceptor site was included
to facilitate splicing. The acceptor site was derived from a 182-bp PCR
fragment that included the last 152 bp of intron 1 and the first 30 bp
of exon 2, ending 1 bp upstream of the Btk start codon. When comparing
pBtkpro+1029 with pGL2, marked increases above background were observed
in the two B-cell lines, a less dramatic increase was seen for the
pre-B-cell line, and no change was observed for the T-cell line (Table
1). The 20- to 50-fold increase in luciferase activity seen in the two
B-cell lines for the intron 1 containing construct, when compared with
pBtkpro, suggested that intron 1 contained a strong positive regulatory
element active in B cells. In the pre-B-cell line, the approximate
threefold increase suggested that this element was either nonfunctional
or was balanced by negative regulatory elements.
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| Fig 4.
Functional characterization of the 5 portion of Btk
intron 1. (A) The diagram shows the extent of the Btk genomic sequence
flanking exon 1 that was subcloned to produce the largest reporter
construct pBtkpro+1029. The Sma I site (S) 1.8 kb upstream of
exon 1 and the HindIII site (H) 1.0 kb downstream indicate the
5 and 3 extents of the subclone, respectively. Also shown are the Sp1
and PU.1 transcription factor binding sites.31,32
Restriction sites used to make the deletions included: P,
Ppu10I; R, Rsa I; K, Kpn I; and St, Stu
I. The intron 1 acceptor sequence is shown 5 to the start of the
luciferase gene (LUC). (B) The deletion constructs with the extent of
the remaining intron given in base pairs. The final construct
represents the fusion between 5 exon 1 and 3 exon 15 and the first
119 bp of intron 15. The relative luciferase activity for each
construct in the three cell lines tested is given in the table on the
right. Reporter construct pBtkpro was assigned the relative activity
level of 1 and all other values indicate the fold increase from this
basal level. Experiments were performed in duplicate and repeated three
times. Values within experiments and between experiments consistently
differed by less than 10%.
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Serial 3 end deletions were made in pBtkpro+1029 (Fig 4B) to localize
potential regulatory elements active in the pre-B and B cells. No
differences were seen when pBtkpro+491 was compared with pBtkpro+1029
in either the B-cell or pre-B-cell lines; however, deletion of an
additional 210 bp (pBtkpro+281) resulted in a fivefold increase in
activity in the pre-B-cell line REH. This indicated the presence of a
pre-B cell-specific negative regulatory element between base-pairs 281
and 491 of intron 1. By specifically deleting the 210 bp between the
Kpn I and Stu I sites in pBtkpro+1029, no change in
activity was seen in the B cells, but a twofold to threefold increase
was observed in the pre-B cells confirming the presence of a negative
regulator within this sequence. No significant differences in activity
were observed for the next serial deletion, pBtkpro+43, when compared
with pBtkpro+281. This suggested that the positive regulatory element,
first identified in the pBtkpro+1029 construct, was active in both B
cells and pre-B cells and was localized within the first 43 bp of the
intron. To confirm the presence of the positive regulatory element
within the first 43 bp of intron 1, a luciferase construct containing
only the first 6 bp of the intron, pBtkpro+6, was compared with pBtkpro
and pBtkpro+43. pBtkpro+6 showed a fivefold to sevenfold reduction in
luciferase activity in the B-cell lines Daudi and Raji and in the
pre-B-cell line REH when compared with pBtkpro+43; however, the
activity remained higher than that of the pBtkpro construct.
A 79-bp fragment containing the first 43 bp of intron 1 and spanning
the exon 1/intron 1 border sequence was analyzed for enhancer activity
by reversing the orientation and/or moving the fragment
upstream of the transcriptional start site. However, this Btk sequence
was only active in its original position and orientation, suggesting
that it was not a "classical" enhancer element.
It has been shown that the presence of an intron in a reporter
construct can increase expression levels by stabilizing the
mRNA.46 To determine if the residual increase in activity
of pBtkpro+6 over pBtkpro could be attributed to splicing, the last 36
bp of exon 1 and the intron 1 sequence were replaced with the last 44
bp of exon 15 and the first 119 bp of intron 15. The 182-bp acceptor
site remained the same in the new construct, pBtkpro-int15. The
luciferase activity seen in cells transfected with pBtkpro-int15 was
marginally higher than that of pBtkpro but lower than all of the intron
1-containing constructs, indicating that splicing was making only a
minor contribution to the luciferase activity of the intron 1
containing constructs.
The effect of the base pair substitution found in the patient, a T to G
transversion at position +6 in intron 1, was evaluated in all of the
constructs containing intron 1 sequence. Luciferase activities elicited
by these constructs were consistently lower when compared with their
normal counterparts (Fig 5). The sixfold to
eightfold reduction seen in the Daudi and REH cells when transfected
with the altered pBtkpro+1029 construct, containing the most intron 1
sequence, approximated the reduction in Btk mRNA as originally seen on
the Northern blots.
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| Fig 5.
Relative luciferase activities of the normal constructs
( ) compared with the mutant constructs ( ). The fold increase is
relative to pBtkpro, the construct with no intron 1 sequence. Construct
numbers are given below their respective bars: 6,
pBtkpro+6; 43, pBtkpro+43; 281, pBtkpro+281; 491,
pBtkpro+491; and 1029, pBtkpro+1029. Cell lines are indicated in
the top left-hand corner of each series.
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Nuclear extracts specifically bind sequences within the first 43 bp
of intron 1.
DNA sequence analysis of the first 43 bp of intron 1 identified a
putative Sp1 binding site47 between base pairs +20 and +28.
The T to G transversion at position +6 identified in the patient's DNA
created an identical Sp1 binding site at the mutant locus between base
pairs +3 and +11. To determine whether these sites or additional sites
within this region could bind nuclear extracts, we performed gel-shift
assays.
Using a sequence spanning nucleotides 5 to +16 at the exon/intron
border (probe 5+16) to identify nuclear DNA binding factors, a
single gel-shift was observed (Fig 6A, band
1). This band could be competed with cold
wild type 5+16 probe, a probe bearing the +6 T to G transversion
(5+16M), and by an Sp1 consensus oligonucleotide. When the mutant
probe, 5+16M, was used as a target for DNA binding proteins, the
single band observed for the normal probe was again seen but an
additional doublet (Fig 6A, bands 2 and 3) was also seen, suggesting
that the mutation created additional binding sites in this region. In
the competition assays, all three shifts were inhibited by the addition
of cold 5+16M, 5+16, or Sp1 oligonucleotides, indicating the
involvement of Sp1 transcription factors. Finally the +12+32 probe was
used to establish if the putative Sp1 binding site between base pairs
20 to 28 could bind Sp1 family members. In these gel-shifts, a doublet
corresponding to bands 2 and 3 seen for the mutant probe was observed
(Fig 5B); however, no clear band corresponding to band 1 was seen. The
doublet was competed by both the cold +12+32 and Sp1 probes.
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| Fig 6.
Gel-shift analyses of the sequence at the exon 1/intron 1
border. Raji nuclear extracts were prepared and tested for their
ability to shift three 32P-labeled oligonucleotides by
EMSA. (A) The normal 5+16 probe and the mutant 5+16M probe,
indicated above their respective lanes, were incubated in the absence
or presence of 200× excess unlabeled oligonucleotide, shown below
each lane. (B) The +12+32 labeled probe, indicated above the lanes,
was incubated in the absence or presence of 200× excess cold probe as
shown below. The three arrows indicate the observed gel-shifts.
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As expected, because of the ubiquitous expression of Sp1, similar band
shifts were identified when REH or Jurkat nuclear extracts were used,
and in all cases a nonspecific unlabeled oligonucleotide was unable to
compete for the specific band shifts 1, 2, and 3 (data not shown).
| |
DISCUSSION |
In this report we document the location of two transcriptional control
elements within the first 1,029 bp of intron 1 of Btk. These two
regulatory elements were identified through a series of 3 end
deletions of the intron 1 sequence. The dramatic increase in activity
of pBtkpro+1029 compared with pBtkpro in the B-cell lines indicated the
presence of a positive regulator within intron 1. The substantial loss
in activity of pBtkpro+6 compared with pBtkpro+43 placed this element
within the first 43 bp of the intron. This drop in activity was not
caused by an unacceptably short intron as the construct still contained
162 nucleotides between the splice donor and acceptor sites, a sequence
long enough for efficient splicing. Replacing the exon 1/intron 1
border sequence with that of exon/intron 15 (pBtkpro-int15, intron
length 287 bp) gave similar results to that of pBtkpro+6 and not of the
other constructs containing more of the intron 1 sequence. This
confirmed that splicing was only playing a minor role in the activity
of the reporter constructs and that intron 1 contained a positive
regulator of transcription.
The positive regulator also increased luciferase activity in pre-B
cells by fivefold, but its activity was reduced by the presence of a
negative regulator located between base pairs 281 and 491. The
potential importance of the two regulatory elements in intron 1 of Btk
is underscored by the high DNA sequence conservation in this region
between the murine and human genes.48
DNA sequence analysis of the positive regulator identified a putative
Sp1 binding site between +20 and +28 bp. Using a +12 to +32 bp probe we
were able to show specific binding of one or more proteins belonging to
the Sp1 family of transcription factors to this site. When a 5 to
+16 bp probe spanning the patient mutation region at +6 bp was used in
an EMSA, a different gel-shift was seen; however, it was also competed
by the Sp1-specific probe, suggesting that another combination of Sp1
family members was able to bind this region. The T to G alteration at
position +6 in the patient created an Sp1 binding site identical to the
one identified between base pairs +20 to +28. When the patient 5+16M
probe was used in the EMSA, the doublet seen for the +12 to +32 bp
probe was seen in addition to the single band detected for the 5 to
+16 bp normal probe. These data taken together suggest that the first
32 bp of intron 1 can bind two different proteins or sets of proteins
that belong to the Sp1 family of transcription factors. The patient
mutation, by altering the specificity of the 5 binding site, probably
disrupts the orderly binding of these factors leading to inefficient
initiation of transcription.
Transcription control elements have been identified within the introns
of several lymphoid associated genes, including those found within the
immunoglobulin49 and T-cell receptor50 loci.
Intron 1 of the CD4,51 c-Fes,52 and adenosine
deaminase genes53 and intron 2 of the IL-4
gene54 have all been shown to contain regulatory elements.
Some of these elements, like the T-cell receptor 
enhancer,55 the c-Fes negative regulator, and the adenosine
deaminase enhancer, differ from the classical enhancer in that they are
both site- and orientation-dependent for correct functioning. Other
orientation-dependent transcription control elements include the
interferon- virus inducible enhancer56 and the c-fos
negative regulator.57 These elements have a common mode of
action that involves modifying the architecture of the surrounding
chromatin to facilitate the functioning of the enhancer or repressor.
Of note therefore is the fact that DNA-bound Sp1 can self-associate to
bring together distant DNA segments that facilitate transcriptional
enhancer function.58 It is possible therefore that the
elements identified in Btk function in an analogous manner.
Studies done in both the human and the mouse suggest that expression of
Btk is likely to be tightly regulated and complex. We have previously
shown that intron 10 of Btk has a methylated site in pre-B cells that
becomes specifically demethylated in B cells. This and the presence of
several transcription regulator binding sites near the methylation
site, including two E box motifs and a silencer element, imply that the
intron is involved in gene regulation.59
An indication that timely expression of Btk is crucial for B-cell
development comes from transgenic experiments done in the xid
mouse. The xid defect was not corrected by a Btk transgene that
was under the transcriptional control of an immunoglobulin
promoter/enhancer.60 Although the possibility remains that
this could have been caused by a dominant negative effect of the mutant
Btk, the fact that the normal enzyme is expressed before immunoglobulin
heavy and light chain gene rearrangement suggests that Btk may be
required at the earliest stages of B-cell development to promote B-cell
maturation. The presence of disease in our patient also indicates that
the amount of Btk may be crucial.
The patient's mutation is unique with respect to previously reported
mutations in Btk; although his cDNA sequence was normal he had lower
amounts of mRNA and protein. Identification of the mutation at position
+6 of the intron 1 donor site suggested that the observed phenotype
could have resulted from a regulatory or splicing defect. The normal
splice donor site of intron 1 closely resembles the splice donor
consensus sequence (Shapiro Senapathy61 score of 85.9) and
the alteration at position +6 did not significantly alter the score
(81.4). In a study of 63 different donor site mutations only two were
at the +6 position. In both cases, the wild type sequence corresponded
poorly with the donor consensus sequence and the alterations compared
even less favorably,45 making it unlikely that the T to G
transversion in the patient was affecting the intron deletion reaction.
A consistent drop in luciferase activity was noted when the patient
mutation was substituted for the normal T at position +6 in the
reporter constructs. However, the most striking difference,
approximating the decrease in mRNA seen on the Northern blots, was
noticed for the mutant pBtkpro+1029 containing the most intron 1
sequence. The much smaller drop in luciferase activity for mutant
pBtkpro+6 suggested that the mutation was only having a negligible
effect on the splicing reaction.
This study emphasizes that B-cell development will not progress
appropriately unless sufficient Btk is provided at the earliest stages
of B-cell development. We have identified regulatory elements within
intron 1 that may account for the fine tuning of Btk expression.
Examination of the intron 1 mutation shows the importance of patient
mutation analyses not only with respect to structure/function studies
of a protein, but also for the identification of sequences involved in
the regulation of transcription.
| |
FOOTNOTES |
Submitted July 24, 1997;
accepted September 5, 1997.
Supported in part by grants from the National Institutes of Health
(Grant No. AI 25129) and NCI CORE (Grant Nos. PO1 CA20180 and P30
CA21765); by funds from the Federal Express Chair of Excellence; by the
American Lebanese and Syrian Associated Charities; and by the Assisi
foundation.
Address reprint requests to Jurg Rohrer, PhD, Department
of Immunology, St Jude Children's Research Hospital, 332 N Lauderdale,
Memphis, TN 38101.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
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Abstract
Introduction
Abstract