(Received for publication, September 27, 1996, and in revised form, November 22, 1996)
From the Department of Biochemistry and Molecular Biology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030
PAX6, a member of the highly
conserved paired-type homeobox gene family, is expressed in a spatially
and temporally restricted pattern during early embryogenesis, and its
mutation is responsible for human aniridia. Here we examined the
transcriptional regulation of the PAX6 gene by transient
transfection assays and identified multiple cis-regulatory elements
that function differently in different cell lines. The transcriptional
initiation site was identified by RNase protection and primer extension
assay. Examination of the genomic DNA sequence indicated that the
PAX6 promoter has a TATA like-box (ATATTTT) at 26 base
pairs (bp), and two CCAAT boxes are positioned at
70 and
100 bp. A
38-bp poly(CA) sequence was located 992 bp upstream from the initiation
site. Transient transfection assays in glioblastoma cells and leukemia
cells indicate that a 92-bp region was required for basal level
PAX6 promoter activity. A negative transcriptional element,
silencer (bases
1518 to
1268), functioned differently in different
cell lines. The activation of the promoter is positively correlated
with the expression of PAX6 transcripts in all cells
tested. These results indicate that a cis-regulatory element or
elements is responsible for selective activation of the
PAX6 promoter in cells that can express PAX6
mRNA.
The paired box (pax) gene family was originally
identified in Drosophila as segmentation genes such as
paired (prd), gooseberry-distal (gsb-d), and
gooseberry-proximal (gsb-p) (Bopp et al., 1986)
and was subsequently found in many other species (Burri et
al., 1989
; Ton et al., 1991
; Chisholm et
al., 1995). Based on their sequence homology to the
Drosophila gsb-d paired box, nine pax genes have been found in mice (pax1-9) (Walther and Gruss, 1991
) and
humans (PAX1-9) (Gruss and Walther, 1992
). All the
pax genes contain a 128-amino acid N-terminal paired box
that can be present alone (as in Pax1 and Pax9) or with a full-length
(as in Pax3, Pax4, Pax6, and Pax7) or truncated (as in Pax2, Pax5, and
Pax8) paired-type homeodomain. The Pax proteins make up a family of
transcription factors involved in the regulation of cell morphogenesis
and differentiation. The paired domain alone or in combination with the
paired-type homeodomain, confers a novel DNA binding specificity
(Czerny et al., 1993
; Wilson et al., 1995
).
Pax6 plays a key role in eye morphogenesis and has been implicated in
the secondary inductive interaction of lens formation (Quiring et
al., 1994; Li et al., 1994
; Grindley et al.,
1995
). Ectopic expression studies indicate that pax6 is
probably a master control gene for eye development (Halder et
al., 1995
). The expression pattern for PAX6 (Pax6) has
been extensively characterized by northern and in situ
hybridization analysis (Ton et al., 1991
, 1992
). Pax6
expression is restricted to defined regions of the forebrain, optic
cup, hindbrain, and spinal cord, as well as the lens placode and nasal
epithelium (Walther and Gruss, 1991
; Puschel et al., 1992
;
Li et al., 1994
). Heterozygous mutations in PAX6 result in eye abnormalities in human (aniridia) (Glaser et
al., 1992
; Jordan et al., 1992
; Martha et
al., 1994
, 1995
), mouse (small eye) (Hill et al., 1991
;
Hogan et al., 1988
), and Drosophila melanogaster (eyeless) (Quiring et al., 1994
). We know relatively little
about the transcriptional regulation of PAX genes compared
with those of the homeobox family (Sham et al., 1993
) and
Drosophila paired box genes (Gutjahr et al.,
1993
). Examining transcriptional regulation of PAX6 may
reveal how it functions in different tissues during early
embryogenesis.
In this paper, we describe analyses of the human PAX6 promoter and upstream regulatory region and report the presence of multiple repressors and activators within the regulatory region. One of the repressive elements was a strong silencer (SX250) that could specifically repress PAX6 promoter activity in glioblastoma and HeLa cells.
HeLa (a human cervical carcinoma cell line), U87 (a human glioblastoma cell line), and 293 (a human embryonic kidney cell line) were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum, 50 mM glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin. NIH 3T3 fibroblasts, K562 (a human chronic myelogenous leukemia cell line), and a normal human lymphoblastoid cell line were grown in RPMI 1640 medium containing 10% fetal calf serum, 50 mM glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin. HBL100 (a human breast cell line) was grown in McCoy's 5a medium containing 10% fetal calf serum, 50 mM glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin.
RNA Preparation and Northern AnalysisTotal RNA was
prepared from cultured cells by the guanidinium thiocyanate method
(Chomczynski and Sacchi, 1987), and mRNA was isolated with the
poly(A) tract system (Promega) and analyzed by Northern blotting. The
probe used for hybridization of the Northern blots was a random primed
1.6-kilobase (kb)1 human PAX6
cDNA clone (ph-12) (Ton et al., 1991
); the membranes were then stripped and rehybridized with a human
-actin cDNA probe.
The PAX6 genomic clone cH1-7 (Ton
et al., 1991) was isolated from a pWE15 cosmid library by
screening with a 1.6-kb PAX6 cDNA (ph-12). Restriction
endonuclease mapping revealed that cH1-7 contained a 36-kb insert. A
300-base pair (bp) probe derived from the 5
end (from the
HindIII site to the StuI site) of a human PAX6 cDNA clone (ph-12) was used to localize the most 5
genomic sequence.
A 6.5-kb EcoRI-XbaI
genomic DNA fragment, which hybridized to the 300-bp ph-12 probe was
cloned into pBluescript II KS (Stratagene). The insert was then further
dissected to generate small overlapping subclones. The sequence of the
2.9 kb between the 5-HindIII site and the 3
end of exon 2 was determined by the dideoxy chain termination method. The sequence
was analyzed for candidate transcription factor binding sites by using
the Genetics Computer Group (Madison, WI) program.
Total cellular RNA (30 µg) was
hybridized with gel-purified labeled antisense transcripts
(105 cpm). The probe was transcribed with T7 polymerase
using [-32P]CTP from a genomic fragment corresponding
to the 5
region of the PAX6 cDNA (
180 to +166 bp)
cloned into the vector pBluescript SK+. The hybridization was carried
out at 42 °C overnight. Hybrids were digested for 30 min at 30 °C
with RNase A and T1 (ribonuclease protection assay kit, Ambion). The
sizes of the protected fragments were analyzed by electrophoresis on a
6% denaturing polyacrylamide gel using a DNA sequencing ladder as size
marker.
A 33-mer antisense primer corresponding to
bases +133 to +101 of the human PAX6 cDNA sequence was
end-labeled with T4 polynucleotide kinase using
[-32P]ATP. Total cellular RNA (15 µg) was hybridized
with 105 cpm of the 32P-labeled oligonucleotide
by heating at 90 °C for 5 min in 20 µl of hybridization buffer (50 mM Tris-Cl, pH 8.3, 150 mM KCl, 1 mM EDTA) and then immediately incubated at 42 °C
overnight. The DNA-RNA hybrid was then collected by ethanol
precipitation and dissolved in 20 µl of reverse transcription buffer
(50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTP). The primer was extended by SuperScriptTM II
RNaseH
Reverse Transcriptase (Life Technologies, Inc.) at
42 °C for 1 h. After completion of the reaction, samples were
extracted with phenol-chloroform, precipitated with ethanol, and
analyzed on 6% polyacrylamide gel. The sequencing ladder was done with the same primer used for primer extension, and the DNA template is a
genomic fragment corresponding to the 5
region of the human PAX6 gene.
DNA fragment (6.5-kb
EcoRI-XbaI) derived from the 5-flanking region
clone described above was inserted immediately upstream from the
chloramphenicol acetyltransferase (CAT) reporter gene in the
promoterless and enhancerless CAT expression vector pCAT-basic (Promega). We then made a series of CAT expression constructs that
contained various lengths of the 5
-upstream sequence of the
PAX6 gene and had the same 3
end, the NaeI site
in exon 1. DNA fragments for minimal promoter analyses were prepared by
PCR amplification with the following oligonucleotides:
5
-CCCAAGCTTCGGCTGGCGCGAGGCC-3
(bases
66 to
50) for the 5
ends of the PAX6 inserts and
5
-GCTCTAGAGTCATCATCCTCCAGCA-3
(bases +93 to +109),
5
-GCTCTAGACCCACTAATCACTCCG-3
(bases +13 to +29) for the 3
end of the
PAX6 inserts. The amplified products were digested with
HindIII and XbaI and inserted immediately
upstream of the CAT reporter gene in the vector's multiple cloning
site.
A 1147-bp
HindIII-PstI (bases 2404 to
1256) fragment
was amplified by PCR and inserted downstream of the CAT gene, which was
driven by a 346-bp SmaI-NaeI PAX6
promoter fragment. A series of deletions from the 5
and 3
ends were
generated by PCR with the 5
primers and 3
primers. The PCR fragments
were digested with BamHI and BglII and inserted
into the BamHI site of the PAX6 promoter
construct pCSMNA. 5
primers: 5
-CGGGATCCTCGGAAAGAAGCAGCC-3
, 5
-CGGGATCCCTCTGCCAAGTACCG-3
, 5
-CGGGATCCCATCAGGCCTTCGG-3
, and 5
-CGGGATCCAAACTCTCTCTTTC-3
; and 3
primers:
5
-CGGGATCCCAGGGTCTGCAAA-3
and 5
-GAAGATCTGCTTGACCGGGGAA-3
.
CAT expression constructs were
transfected into cells growing in monolayers or suspension by
electroporation. 20 µg of plasmid DNA and 5 µg of internal control
(pSV--gal) were electroporated normally into 5 × 106 cells in 0.3 ml of culture medium with a Bio-Rad gene
pulser at 260 V and 960 microfarads. The transfected cells were
harvested after 48 h, and cell lysates were prepared by three
cycles of freezing and thawing. The
-galactosidase activity was
determined by the method of Rosenthal (1987)
, and the protein
concentration was determined with a Bio-Rad protein assay kit. Aliquots
of cell extracts containing 30-50 µg of protein were used for the
CAT assay. The acetylated chloramphenicol was separated from the
unacetylated form by thin-layer chromatography in chloroform:methanol
(97:3, v/v). The acetylated [14C]chloramphenicol was
quantitated with a PhosphorImager (Molecular Dynamics). The CAT
activity of each construct was determined with at least three
independent transfection assays and normalized to the
-galactosidase
activity.
Nuclear
extracts were prepared following the method described by Schreiber
et al. (1989). The nuclei were isolated following the
disruption of cells by the addition of 0.4% Nonidet P-40 after being
swollen on ice in the lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 2.0 µg/ml leupeptin, 2.0 µ/ml aprotinin, and 0.5 mg/ml
benzamidine). The nuclear pellet was resuspended in nuclear extraction
buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml leupeptin, 2.0 µg/ml aprotinin, and 0.5 mg/ml benzamidine)
and centrifuged for 5 min at 4 °C, and the supernatant (nuclear
extracts) was stored at
70 °C.
The in vitro translated protein and nuclear extracts were resolved on 10% SDS-polyacrylamide gels, electrotransferred to nitrocellulose, probed with a rabbit polyclonal antibody against the full-length PAX6 protein, and detected by chemiluminescence (ECL, Amersham Corp.).
Nucleotide Sequence Accession NumberThe nucleotide sequence of the human PAX6 promoter has been deposited in GenBankTM (accession number U63833[GenBank]).
A
human genomic library constructed in the cosmid vector pWE15 was
screened with a 1.6-kb human PAX6 cDNA probe. Cosmid
cH1-7 was selected and found to contain the first seven exons of the PAX6 gene. cH1-7 spans 36 kb, including 24 kb of the
untranscribed 5 sequence.
cH1-7 was subcloned into pBluescript II SK+ and further analyzed by
Southern blotting and restriction mapping (Fig. 1). The genomic structure obtained is similar to that reported by Glaser et al. (1992). Sequence analysis of the 5
end of the clone
(Fig. 2) revealed two untranslated exons (exons 1 and 2)
separated by a 99-bp first intron. The translational start site was
located in exon 4.
In order to identify the PAX6 promoter region, it was
necessary to first identify the transcriptional start site. For this purpose we used both primer extension and RNase protection analysis. A
5-labeled 33-mer antisense primer (corresponding to position +133 to
+101) was used for primer extension and hybridized to total RNA
isolated from PAX6-expressing cells and
non-PAX6-expressing cells. A single 134-bp primer extension
product was generated in three PAX6-expressing cells (U87,
K562, and HeLa cells) but not in the Y79 cells that do not contain
endogenous PAX6 mRNA (Fig.
3A). By comparison with a DNA ladder
generated on a genomic plasmid by the same oligonucleotide primer, the
transcriptional initiation site was located 108 bp upstream from the
beginning of the reported longest cDNA sequence (Glaser et
al., 1992
). This result was confirmed by the RNase protection
assay (Fig. 3B). A 32P-labeled antisense RNA
probe corresponding to a 346-bp SmaI-NaeI fragment containing the 66-bp published 5
-terminal sequence was used
to hybridize with 15 µg of total RNA isolated from
PAX6-expressing cells and nonexpressing cells. A single
protected fragment of approximately 166 bp was observed in
PAX6-expressing cells but not in
non-PAX6-expressing cells. In addition to primer extension and RNase protection analyses, we also carried out a 5
RACE assay of
the isolated total RNA from PAX6-expressing cell lines (data not shown). Using this experiment, the primer extended cDNA
sequence exactly matched with the published 5
-terminal sequence and
the adjacent 5
genomic sequence. RNase protection and 5
RACE assays both indicate that no intron sequence is located between the initiation site and the identified 5
cDNA sequence. Therefore, the
transcriptional start site was positioned 100 bp upstream of the
beginning of the identified longest cDNA and numbered base
+1.
Sequence analyses of the 2.9-kb upstream promoter region (Fig. 2) from
the HindIII site to the end of exon 2 revealed a TATA-like sequence (ATATTTT) located 26 bp from the initiation site, and two
CCAAT boxes located 70 and
100 bp, respectively. The position of
TATA-like box and CCAAT boxes are consistent with those found in other
eukaryotes. A third consensus sequence (PyAPyPyPy), a pyrimidine-rich
initiator element, is also found at the PAX6 transcriptional start site (Breathnach et al. 1981; Burley et al.
1996).
Several putative transcriptional regulatory consensus sequences (Faisst
and Meyer, 1992) were identified within the
NotI-NaeI proximal promoter region (at bases
452 to +166), including single binding sites for Sp1 protein (at
bases
395 to
387), Myc protein (at bases
379 to
373), PuF
protein (at bases
410 to
405), CCCT binding factor (at bases
334
to
329), and TCF-2 (at bases +81 to +86). In addition, we found two
consensus TCF-1 binding sites (at bases +33 to +37 and
348 to
344),
three potential binding sites for activator protein-2 (AP2) (at bases
51 to
44,
145 to
136, and
281 to
274), and four potential
candidate binding sites for GCF (at bases
450 to
444,
392 to
386,
236 to
230, and
49 to
43). A 38-bp poly(CA) sequence was
located at bases
992 to
955.
To
examine the PAX6 promoter activity and identify important
regulatory domains within the 5 region, a series of fragments with 5
and 3
deletions in the 5
region of the PAX6 gene were ligated upstream of the bacterial CAT gene in the promoterless and
enhancerless CAT expression vector pCAT-basic (Fig.
4A). These CAT constructs were tested for
promoter activity by transfection into U87, K562, and NIH 3T3 cells.
For comparison, the cells were also transfected with two control CAT
constructs, one with a simian virus 40 promoter and enhancer
(pCAT-control) and one without promoter and enhancer (pCAT-basic). The
-galactosidase-expressing vector pSV-
-gal was used as an internal
control for transfection efficiency, and CAT activity was normalized to
-galactosidase activity. Fig. 4 shows the structures of the deletion
constructs and their promoter activity determined by transient
expression assays. The longest construct, pCENA-b (bases
4900 to
+166), had high promoter activity in both U87 and K562 cells (Fig.
4B). The 1.4-kb PstI-NaeI construct
(pCPNA, bases
1254 to +166) had a very high level of CAT activity in
K562 and U87 cells. It is interesting that the 346-bp
SmaI-NaeI fragment (pCSMNA, bases
180 to +166)
had the highest CAT activity in U87 and K562 cells. Further deletion to
the BstXI site, which contained two CCAAT motifs, still
exhibited a very high promoter activity in U87 cells. The deletion data
indicate that these two CCAAT motifs are not critical to the
PAX6 promoter activity, because deleting them from pCSMNA
did not substantially affect its promoter activity. However, the
deletion of the 3
228-bp BstXI-NaeI fragment
(construct pCPBS) from pCPNA dramatically decreased the promoter
activity to the basal level in both U87 and K562 cells. This indicates that the 228-bp BstXI-NaeI fragment, which
contained the initiation sites, was required for normal PAX6
promoter activity.
Deletion of a 2.4-kb EcoRI-HindIII fragment from pCENA to form pCHNA caused a 9-fold decrease in CAT activity in U87 cells but not in K562 cells. Deletion of a 1.1-kb HindIII-PstI fragment from pCHNA to form pCPNA caused a 16-fold increase in CAT activity in U87 cells but only a 2-fold increase in K562 cells. This suggested that there was a strong repressor between the HindIII and PstI sites that could regulate cell type-specific expression of the PAX6 gene. Further deletion of the 0.7-kb PstI-SacII fragment from pCPNA to form pCSANA caused 3-fold and 2.5-fold decreases in CAT activity in K562 and U87 cells, respectively. The deletion of a 363-bp SacII-SmaI fragment from pCSANA to form pCSMNA produced 3.5-fold increases in CAT activity in K562 and U87 cells. These results indicated that there is an activator sequence between the PstI and SacII sites and a repressor between the SacII and SmaI sites, and these regulatory elements were active in both K562 and U87 cells. However, the activator between the EcoRI and HindIII sites was only active in U87 cells, and the strong repressor between the HindIII and PstI sites functioned differently in U87 and K562 cells. The multiple activators and repressors in the 4.9-kb upstream promoter region may function differently in combination and in different cells.
To determine the tissue specificity of the PAX6 promoter,
murine and human cell lines of different origins were tested for promoter activity by using the constructs pCHNA and pCPNA.
PAX6 promoter activity was detected in cell lines that
expressed PAX6 mRNA (as revealed by Northern blotting,
Fig. 5), including U87 glioblastoma cells, K562
erythroleukemia cells, 293 kidney cells, and HeLa cells (data not
shown). No CAT activity was observed in cell lines that did not express
pax6 mRNA, including NIH 3T3 murine fibroblast cells and
HBL100 cells (data not shown). To confirm the expression of PAX6
protein in cell lines, we did Western analysis (Fig. 5B).
The in vitro translated PAX6 protein was made from a human
PAX6 expression construct (RC-CMV-PAX6) in rabbit reticulocyte lysate
in the presence of [35S]methionine. The result revealed a
major 46-kDa band in U87 cells, K562 cells, and HeLa cells but not in
NIH 3T3 cells. The rabbit anti-PAX6 serum (Tang et al., in
press) can specifically recognize the PAX6 protein, because no
cross-interaction was observed using an in vitro translated
PAX8 protein. The size of the in vitro translated PAX6
protein is consistent with the predicted molecular weight of 46,500 for
the 422-amino acid human PAX6 protein. The human PAX6 transcripts are
well correlated with the protein level, indicating that PAX6 expression
is mainly controlled at the transcriptional level, at least in the cell
lines tested.
Identification of the PAX6 Minimal Promoter
Functional
analysis of the PAX6 promoter region showed that the 228-bp
BstXI-NaeI fragment (bases 62 to +166, pCBSNA)
had very high CAT activity (Fig. 4B), especially in U87
cells. Four potential transcriptional factor binding sites in this
region were identified in the nucleotide sequence, including AP2, GCF, TCF-1, and TCF-2 (Fig. 2). The relative activity of the 3
deletion constructs created by PCR amplification was tested in U87 and K562
cells (Fig. 6). The 3
deletion of bases +109 to +166
(pCHX-176) resulted in a nearly 50% decrease in promoter activity in
both U87 and K562 cells, indicating that the 57-bp sequence (bases +109
to +166) contained a positive regulatory element or elements for
PAX6 promoter function. Further deletion of the 79-bp 3
fragment (bases +30 to +109, pCHX-95) still retained appreciable
promoter activity. These observations demonstrated that sequences both upstream and downstream of the transcriptional start sites are required
for PAX6 promoter activity. From the deletion analysis, we
can conclude that the 92-bp sequence flanking the TATA-like box and the
initiation start site is important for promoter activity. The TATA-like
sequence (ATATTTT) identified in human PAX6 promoter is
similar to that found in the quail pax6 promoter (ATATTAA) (Plaza et al., 1993
).
Functional Analysis of PAX6 5
Analysis of the 5
deletion constructs shown in Fig. 4 suggested the presence of a
repressor between the HindIII and PstI sites
(
2404 to
1256). This repressor repressed the PAX6
promoter in U87 cells but not in K562 cells. We then tested the
repressive activity of this 1.1-kb fragment when it was inserted
downstream of the PAX6 promoter. The fragment acted on the
PAX6 promoter and a SV40 promoter as a strong repressor in a
position- and orientation-independent manner in U87 cells but not in
K562 cells (data not shown).
Next, we dissected the 1.1-kb repressor to more precisely locate the
sequence responsible for its repressive activity. After deletion of the
5 406-bp sequence (bases
2404 to
1998) from the 1.1-kb fragment,
the remaining 730 bp (SX730) showed repressor activity in both U87 and
HeLa cells but not in K562 cells (Fig. 7, A
and B). Further dissection analysis of the 730-bp fragment showed that the 5
481-bp region (bases
1998 to
1519, SX480) had no
repressor activity in all the cell lines tested, but the 3
250 bp
(bases
1518 to
1268, SX250) caused a 2.5-fold decrease of CAT
activity in both U87 and HeLa cells. The repressor activity was absent
when the 250-bp sequence was split into two overlapping 5
and 3
fragments. Because the 5
SX45 and 3
SX54 (Fig. 7A) have a
31-bp overlap sequence, it is likely that cis-elements upstream and
downstream of the 31-bp region are required for the repressor activity.
The 250-bp repressor sequence functions equally well in either
orientation (Fig. 7B). These results demonstrate that more
than 31 bp overlap sequence is required for its repressor activity, and
it functions as a cell type-specific silencer. Sequence analysis of the
250-bp silencer showed several transcription factor binding sites, but
no potential binding site within 31 bp overlapped sequence (Fig.
7C).
In this study, we report the isolation and characterization of the
human PAX6 promoter. A 92-bp basal promoter (bases 62 to
+30) was able to drive transcription of a CAT reporter gene in
PAX6-expressing glioblastoma and erythroleukemia cell lines. The promoter activity was well correlated with the expression level of
endogenous PAX6, and high CAT activity was found in cell lines with high levels of PAX6 transcripts. Deletion
analysis of the 346-bp fragment (
180 to +166, pCSMNA) indicates that
multiple cis-elements located upstream and downstream of the initiation start site are responsible for high level basal promoter activity and
cell specificity.
The 346-bp PAX6 minimal promoter region contains a single
consensus binding site for the transcription factors TCF-1, TCF-2, and
GCF and two binding sites for the AP2 protein. The PAX6
promoter has a typical initiator element, a TATA-like sequence and two CCAAT boxes at positions 70 and
100. This structure is similar to
that of many tissue-specific genes that have a typical TATA box and
initiator sequences (Arcioni et al., 1992
; Hu et
al., 1993
). Because the quail pax6 promoter has been
well characterized, we did sequence comparison of the 2.9-kb human
PAX6 promoter and the quail 1.5-kb pax6 promoter
(Plaza et al., 1993
). Like many TATA box containing gene,
they both have a TATA-like box and CCAAT box(es) in the corresponding
position.
One of the most intriguing results of the functional analysis of the
PAX6 upstream region was the discovery of multiple
repressors and activators. A series of 5 deletions of these
cis-regulatory elements altered promoter activity (Fig. 4). The various
cis-regulatory elements had various levels of activity in different
cell lines. No single cis-element was shown to be essential for
PAX6 transcriptional activation. The tissue-specific and
temporal expression of the PAX6 gene may be controlled by a
combination of different cis-regulatory elements upstream and
downstream of the initiation site. The cis-regulatory regions of the
Drosophila paired box genes prd, gsb,
and gsbn (Li and Noll, 1994
) control the temporal and
spatial expression of functionally equivalent genes. The three paired
box genes play different roles during early embryogenesis and are
controlled by cis-regulatory elements. All pax genes contain
a very conserved paired domain and bind to a consensus recognition
sequence (Czerny et al., 1993
; Epstein et al.,
1994
). It is possible that several pax genes also have some
redundant functions as do the homeobox genes. Therefore, some
cis-regulatory elements may be responsible for the functional diversity
of the pax genes. This hypothesis can be confirmed by
mutation analysis of candidate cis-regulatory elements in aniridia
patients. These elements can be positive or negative, because either
haploinsufficiency or overdosage of PAX6 protein can cause
eye defects (Schedl et al., 1996
). So far, less than half of
the aniridia patients examined have been found to have mutations in the
coding region of the PAX6 gene.2
It is likely that some of the unidentified mutations are present in
regulatory elements. This is the case for the Drosophila
pax6 (eyeless) gene, in which two eyeless phenotypes were found to be caused by mutation in a 200-bp cis-regulatory element (Quiring et al., 1994
).
Transient transfection analysis of the PAX6 promoter
constructs showed that there is a transcriptional (silencer) in the
upstream region. This silencer (bases 1518 to
1268) had different
activities in different cell lines (U87, HeLa, and K562 cells).
Sequence analysis showed multiple potential transcription factor
binding sites in the silencer region (Fig. 7C). One binding
site for GCF, a negative regulator of the epidermal growth factor
receptor gene (Kageyama and Pastan, 1989
), was identified in the
silencer region. Notochord transplantation experiments indicated that
signals produced by the notochord can repress the region-specific
expression of pax6 in the developing spinal cord (Goulding
et al., 1993
). It was suggested that one of the signal
molecules, activin A, is involved in the dorsal and ventral
down-regulation of pax6 gene expression (Pituello et
al., 1995
). Another signal molecule, sonic hedgehog, appears to
either directly or indirectly inhibit the expression of PAX6 (MacDonald
et al., 1995
). The cell-specific silencer in combination
with other positive and negative elements may contribute to
tissue-specific and temporal expression of the PAX6
gene.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U63833[GenBank].
We thank Gail Fraizer, Hank Tang, Lian Chao, Kathy Tucker, and Ruby Desiderio for assistance. We also thank Swayampakula Ramakanth for critical review of this paper.