From the CNRS UMR 8526, Institut de Biologie de Lille, Institut Pasteur de Lille, 1 Rue Calmette, 59017 Lille, Cedex, France and the ¶ Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
Received for publication, September 22, 2000
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ABSTRACT |
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HIC-1 (hypermethylated
in cancer 1), a BTB/POZ
transcriptional repressor, was isolated as a candidate tumor suppressor
gene located at 17p13.3, a region hypermethylated or subject to
allelic loss in many human cancers and in the Miller-Dieker syndrome. The human HIC-1 gene is composed of two exons, a short
5'-untranslated exon and a large second coding exon. Recently, two
murine HIC-1 isoforms generated by alternative splicing
have been described. To determine whether such isoforms also exist in
human, we have further analyzed the human HIC-1 locus.
Here, we describe and extensively characterize a novel alternative
noncoding upstream exon, exon 1b, associated with a major GC-rich
promoter. We demonstrate using functional assays that the murine exon
1b previously described as coding from computer analyses of genomic
sequences is in fact a noncoding exon highly homologous to its human
counterpart. In addition, we report that the human untranslated exon is
presumably a coding exon, renamed exon 1a, both in mice and humans.
Both types of transcripts are detected in various normal human tissues with a predominance for exon 1b containing transcripts and are up-regulated by TP53, confirming that HIC-1 is
a TP53 target gene. Thus, HIC-1 function in the
cell is controlled by a complex interplay of transcriptional and
translational regulation, which could be differently affected in many
human cancers.
Chromosome 17p is frequently altered in human cancers and allelic
losses often coincide with mutations in the TP53 gene at 17p13.1 (1). However, in some tumor types, 17p allelic losses occur at
high frequency in regions distal to TP53 and in absence of
TP53 mutations in breast (2) and ovarian tumors (3) for instance. Consequently, many studies led to the proposal that one or
more tumor suppressor genes whose loss of function is required for
early tumorigenesis may reside in a region telomeric to TP53 in 17p. A good candidate is region 17p13.3 and especially the region
around the variable number of tandem repeats
(VNTR)1 marker
YNZ22/D17S5/D17S30 that has shown frequent loss of heterozygosity or
alteration in DNA methylation (4, 5) in various common types of solid
tumors (6-12) and in some leukemia (13, 14).
DNA hypermethylation changes of the NotI restriction sites
at the D17S5 locus in many cancers allowed the positional cloning of a
candidate tumor suppressor gene, HIC-1
(hypermethylated in cancer
1) (15). HIC-1 encodes a protein with five
Krüppel-like C2H2 zinc finger motifs and
a N-terminal BTB/POZ domain characteristic of a subset of zinc finger
transcription factors that includes two proteins involved in
translocations in human neoplasia, BCL-6 and PLZF (16, 17). HIC-1 and
its avian homologue This same 17p13.3 region has also shown frequent loss of heterozygosity
in breast (20) and ovarian cancers as well as in many other cancer
types (Refs. 21-24 and references therein). In particular, extensive
analyses of different cohorts of ovarian tumors have identified an
approximately 15-kbp minimum region of allelic loss extending from
markers D17S28 to D17S30/YNZ22/D17S5, which is deleted in 80%
of all ovarian epithelial malignancies (21-23). Two candidate tumor
suppressor genes, OVCA1 and OVCA2 have been
identified that map to this commonly deleted region (Refs. 21-24 and
Fig. 1A). No known functional
motif can be identified in the amino acid sequence of OVCA1,
which displays sequence similarity (20% identity) to one of the yeast
enzymes in the diphtamide pathway (21-24) and was recently shown to
interact with RBM8A, a new RNA-binding motif protein (25). When the
ovarian cell line A2780 is transfected with plasmids expressing
OVCA1, a significant 50-60% decrease in colony number is
observed, suggesting that overexpression of OVCA1 either
blocks growth or is toxic to these cells (24). Even though the
HIC-1 coding sequence is not included in and lies a few kbp
centromeric to this minimal region of deletion, HIC-1 still
remains a strong candidate because critical 5' regulatory elements may
reside within the D17S28-D17S30 interval (Fig. 1A).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FBP-B (18) are transcriptional repressors, but
in sharp contrast to the related BCL-6 and PLZF repressors they are
unable to recruit silencing mediator of retinoid and thyroid
receptor/nuclear receptor corepressor histone deacetylase
complexes (19). In addition, HIC-1 expression can be
up-regulated, at least in a colon cancer cell line, by p53 through a
functional p53 binding site found in its 5'-flanking region (15).
Finally, constitutive expression of HIC-1 by stable transfection in various cancer cell lines results in a significant decrease in their clonogenic survival, suggesting that HIC-1
might suppress cell growth (15).
View larger version (15K):
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Fig. 1.
Schematic structure of the human and murine
HIC-1 locus. A, schematic drawing of
the D17S28-YNZ22/D17S30/D17S5 region on chromosome 17p13.3 showing the
localization and orientation of the HIC-1 and
OVCA1 genes (21-23). B, the previously published
structures of the human and murine HIC-1 locus are
respectively derived from a correction in GenBankTM (Carter
and Baylin, direct submission) of the original publication (15) and
from Refs. 28 and 29. The first human noncoding exon (UTR) and the 3'-
and 5'-untranslated sequences appear as narrow boxes,
whereas large boxes represent the coding sequences. The
introns are drawn as thin lines. The dashed line
represents unknown sequences (~2.3 kbp, not drawn to scale) localized
between HIC-1 and the microsatellite marker
YNZ22/D17S30/D17S5. Potential TATA boxes, the putative start and stop
codons and the polyadenylation signals identified by nucleotide
sequencing of the genomic clones are indicated. The region encoding the
BTB/POZ domain is shown as a gray box, and the region
encoding the five C2H2 zinc fingers is
indicated by ZF.
Finally, HIC-1 is located within the 350-kbp critical region
deleted in most patients with Miller-Dieker syndrome (MDS). Although the lysencephaly and mental retardation seen in MDS have been clearly
attributed to haploinsufficiency in the LIS1 gene, MDS patients have other developmental anomalies including craniofacial dysmorphology, defects of the limbs and digits, and omphalocele (26).
Interestingly enough, most of these defects are also observed in
HIC-1/
mouse embryos together with perinatal
death and a reduction in overall size (27). In addition, parts of the
HIC-1 expression territories as defined by in
situ hybridization studies of mouse embryos overlap with regions
that exhibit abnormalities in MDS patients (28). These observations
thus strengthen the candidacy of HIC-1 as a gene involved in
MDS.
Thus, the genomic region including the YNZ22/D17S5/D17S30 marker in the
close vicinity of the HIC-1 gene is a putative target for
genetic alterations (loss of heterozygosity, hypermethylation, or
deletion) frequently associated with many common human diseases. From
sequencing analyses of human HIC-1 genomic clones, the
exon-intron structure was predicted to have two exons, a short
5'-untranslated region (UTR) preceded by a TATA box and of a large
second coding exon also containing the 3'-untranslated region (Refs. 15
and 29; GenBankTM accession number L41919; Fig.
1B). However, the HIC-1 avian homologue
FBP is a single-copy gene subject to
alternative splicing mechanisms because three transcripts with
alternate 5' ends (
FBP-A,
FBP-B and
FBP-C) have
been described by cDNA cloning (18). Furthermore, two recent
studies (27, 28) have provided evidence in mouse genomic DNA of an
alternative exon distinct from the unique 5'-UTR previously described
in the human HIC-1 gene (Ref. 15 and Fig. 1B).
All these observations led us to reinvestigate in detail the structure
of the human HIC-1 locus.
In this study, we report that the previously described UTR is
presumably a coding exon both in humans and in mice. In addition, we
describe and characterize by a combination of 5' RACE, cDNA cloning, RNase protection analyses, exon-specific Northern blot hybridization, and functional assays, a novel alternative noncoding upstream exon transcribed from a major TATA-less promoter.
Significantly, we demonstrate using the same functional assays that the
alternative murine exon described as coding from computer analyses of
genomic sequences is in fact a noncoding exon highly homologous to its human counterpart. Finally, we show that p53 is able to up-regulate both types of HIC-1 transcripts. Thus, the two mammalian
HIC-1 genes display a very similar structural organization
but exhibit a higher complexity in their regulation at both the levels
of transcription and translation than previously suspected.
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EXPERIMENTAL PROCEDURES |
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5' RACE and Nucleotide Sequencing-- The 5' end of our alternative HIC-1 transcript was cloned by 5' RACE using the Marathon Ready cDNA amplification kit according to the manufacturer's instructions (CLONTECH, Palo Alto, CA). The double-stranded cDNAs prepared from normal human ovary poly(A)+ RNAs and ligated to the Marathon Adaptor were amplified in a primary PCR with adaptor primer 1 (AP1) and the HIC-1 BTB/POZ gene-specific antisense primer M5 (5'-TGGCGCTTGAGGCGTTTCTTGC-3', positions 1013-1035 in the corrected HIC-1 sequence) followed by a secondary PCR using as nested oligonucleotides, adaptor primer 2 (AP2), and an antisense HIC-1 specific primer (M6) (5'-GAGGTCGGGGATCTGCAGGTA-3', positions 982-1003). After a first cycle of denaturation (95 °C for 6 min), PCR was carried out with AmpliTaq Gold (PERKIN ELMER) for 35 cycles (95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min) with a final extension at 72 °C for 8 min. Because of the very high GC content of the HIC-1 gene, the PCR reactions were conducted in the presence of Me2SO (10% v/v final). PCR products were analyzed and purified by agarose gel electrophoresis, cloned using the TOPO TA Cloning Kit (Invitrogen, Leek, The Netherlands) and sequenced using an ABI PRISM 377 automated DNA sequencer (PerkinElmer Life Sciences) with reverse/forward universal and internal primers. To obtain further 5' sequences, a second round of 5' RACE was performed on various Marathon Ready cDNAs using in the primary PCR the AP1 primer with a HIC-1 antisense primer M19 located close to the 5' boundary of the BTB/POZ domain (5'-GCCGGGCGCCTCCATCGTGTCCAGCATCG-3', positions 635-663 in the HIC-1 sequence) followed by a secondary PCR using the AP2 primer and a primer, M20 specific for the new 5' end (5'-CACTCTCCTGGGGGGCATGTCG-3').
In parallel, the 1.5-kbp BamHI genomic fragment derived from our previously described human HIC-1 recombinant phage (30) was cloned into pBluescript SK (Stratagene) and sequenced. The sequencing was repeated at least three times on both strands. Sequence analyses were performed using the LALIGN (31) and the BLAST (32) network services.
Cell Culture and RNA Preparation-- MRC-5 is a normal human fibroblast line. IGR-OV-2 and MCF-7 are, respectively, human ovary and breast cancer cell lines. RK13 is a rabbit kidney cell line, and CHO K1 is a subclone of the original CHO cells. These cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. HIO 106 and HIO 121 are two mortal human ovary epithelial lines selected from cultures of human ovary surface epithelial cells transfected by the SV40 T antigen (24). They were cultured in a mixture of M199:MCDB105 (1:1) culture medium supplemented with 15% fetal bovine serum, antibiotics, and insulin (0.29 unit/ml).
Total RNAs were extracted from subconfluent cultures or from tissues dissected from 2-week-old mice using the guanidinium/CsCl gradient protocol (33). Total RNAs from normal human prostate were purchased from CLONTECH. Poly(A)+ RNAs from mouse ovaries were prepared using a mRNA purification kit (Amersham Pharmacia Biotech).
RNase Protection Assays--
Preparation of the
-P32-labeled RNA probes from the various plasmids,
liquid hybridization to RNA samples, and post-hybridization to RNase A
and T1 were performed with the Promega Riboprobe combination system and
the Ambion RPAIII kits according to the manufacturer's specifications.
In general, 150,000 cpm of probe were hybridized to 20 µg of total
RNAs for 16 h at 55 °C. Products of RNase digestion were
analyzed on a 6% acrylamide/8 M urea gel. Lengths of the protected fragments were determined by comparison with a 100-bp ladder
(Amersham Pharmacia Biotech) that was labeled with 10 µCi of
[
-32P]ATP or with a sequencing reaction run on
parallel on the same gel. The RNase protection probes used to map the
5' boundary of human and murine exons 1b were, respectively,
a 302-bp RsaI-BamHI human genomic fragment and a
306-bp EcoRV-BamHI murine genomic fragment cloned
into the SmaI-BamHI-digested pBluescript II SK
vector (Stratagene). After linearization by EcoRI, antisense RNAs were transcribed with T3 polymerase (Promega). Because of their
high GC content, the hybrization of these particular probes to total
RNAs was performed at 67 °C. The RNase protection probes that
spanned the exon 1a-2 or exon 1b-2 boundaries were constructed by cloning NotI restriction fragments from
the 5' RACE products into the pBluescript II SK or KS vectors. The 5'
NotI restriction site is located at the 3' end of the nested adaptor primer AP2, whereas the 3' NotI site is located in
the HIC-1 BTB/POZ domain (positions 919-926). These clones
were sequenced to determine the orientation of the insert. The
1a-BTB/POZ probe (Probe a) was obtained after
linearization by EcoRI and transcription with T7 polymerase
of a KS clone. Similarly, the 1b-BTB/POZ probe (Probe
b) was obtained after linearization by EcoRI and
transcription with T3 polymerase of a SK clone. For the actin control,
the pTRI-
-actin human antisense control template linearized by
XbaI (Ambion) was transcribed with T3 polymerase.
Construction of Truncated Promoter Fusion
Plasmids--
pGL3-Basic (Promega) contains a firefly luciferase gene
as a reporter and lacks eukaryotic promoter and enhancer sequences. The
human 1.58-kbp BamHI fragment containing most of exon
1b as well as 5'-flanking sequences was cloned in both
orientation into the pGL3 basic vector to yield pGL3-BB and
pGL3-BB-rev, respectively. To subclone various portions of this genomic
clone for promoter analyses, restriction fragments of 1.38 kbp
(KpnI-BamHI), 1.05 kbp
(ScaI-BamHI), 0.47 kbp
(SacI-BamHI), 0.37 kbp
(RsaI*-BamHI), 0.29 kbp
(RsaI*-BamHI), and 0.13 kbp
(SmaI*-BamHI) were cloned into the appropriate
sites in the pGL3 polylinker (BglII for BamHI, KpnI, SacI, or SmaI for the blunt
ends) to yield, respectively, pGL3-KB, pGL3-ScB, pGL3-SaB, pGL3-R1B,
pGL3-R2B, and pGL3-SmB. In the last three cases, the clones have been
obtained after partial digestion with RsaI or
SmaI. The smallest construct (pGL3-Sa°B) was obtained by
PCR with a proofreading DNA polymerase to ensure fidelity using the
following HIC-1 primers
5'-ATGCTCGAGCTCACCAGGACGCGGGGAGGACG-3' (sense) and
5'-GACGGCGGATCCAGGGGGGACGTGGC-3' (antisense). The restriction sites used for the subsequent cloning, a SacI
site incorporated in the sense oligonucleotide and the natural
BamHI site found in the HIC-1 sequence, are
underlined. Two pGL3 clones containing HIC-1 murine genomic
sequences were also constructed. The EcoRV-BamHI
fragment (position 1091-1410 in AF035682) was obtained from our murine
HIC-1 genomic clone (29) and inserted into
SmaI-BglII digested pGL3 to yield pGL3-EVB-(Mu).
The pGL3-BB-(Mu) clone was obtained by cloning into
BglII-digested pGL3 vector a BamHI fragment
obtained from a partial murine HIC-1 ovary cDNA clone.
This fragment contains HIC-1 genomic sequences (positions 1323-1420 in AF036582) flanked by a partial polylinker from the
Dash vector. All these clones have been verified by restriction mapping and sequencing using oligonucleotides flanking the pGL3 polylinker.
Transient Transfection and Luciferase Assays--
RK13 or CHO K1
cells were plated at 50-60% confluence in 6-well plates the day
before transfection. For transfection, cells were incubated with 950 ng
of pGL3-Basic or its derivative and 4 µl of polyethyleneimine
(Euromedex) for 6 h in 1 ml of OptiMEM and then in fresh complete
medium (19). The pSG5-Gal vector (50 ng) was cotransfected in each
assay to correct for variations in transfection efficiency. Cells were
rinsed in phosphate-buffered saline 48 h after transfection and
lysed in universal lysis buffer (Promega). Luciferase and
-galactosidase activities were measured using respectively beetle
luciferin (Promega) and the Galacto-light Kit (Tropix) with a Berthold
chimioluminometer as previously described (19). Results presented are
the means of at least three transfections.
Northern Blot Analyses-- The pre-made multi-tissue Northern blots (CLONTECH) were hybridized to the indicated 32P-probes labeled by random priming (Rediprime kit; Amersham Pharmacia Biotech) according to the manufacturer's specifications. The fragments used in the labeling reactions were obtained by PCR amplification with primers specific for each HIC-1 exon as follows. The exon 1a specific probe is a 137-bp fragment corresponding to positions 155-292 in the HIC-1 genomic sequence (L41919) and has been generated by PCR using as template our HIC-1 genomic clone with the human 1a sense primer 5'-CTCCGTATCACTTCCCCCAA-3' and the human 1a antisense primer 5'-CCGATTTAAGTAAAATGTCCGC-3'. The exon 1b specific probe is a 119-bp fragment corresponding to positions +21 to +140 of exon 1b and has been generated by PCR using the 5.0-kbp XhoI fragment containing the complete exon 1b as template with the human 1b sense primer 5'-GCGGGGAGGACGGACCAGC-3' and the human 1b antisense primer 5' CACTCTCCTGGGGGGCATGTCG-3'. The BTB/POZ (exon 2) specific probe is a 403-bp fragment corresponding to positions 631-1034 in the HIC-1 genomic sequence (L41919) and has been generated by PCR using as template our HIC-1 genomic clone with the HIC-1 BTB/POZ gene-specific sense primer P4 5'-CAGACGATGCTGGACACGATGGAG-3' and antisense primer M5 5'-TGGCGCTTGAGGCGTTTCTTGC-3'. All these fragments were cloned into the PCR-TOPO vector and verified by sequencing before use.
Infection of the SaOs-2 Cancer Cell Line with an Adenovirus
Vector Expressing the Wild-type Human TP53 or the GFP
Gene--
Efficient generation of recombinant adenovirus vectors
(Ad-GFP and Ad-p53) was obtained by homologous recombination in
Escherichia coli (34) after insertion of the cDNAs into
the pAdCMV2 vector (details of construction are available upon
request). Viral stocks were then created as previously described (35).
Viral titers were determined by a plaque assay on 293 cells and defined
as plaque-forming units/ml. Cells were infected at an input
multiplicity of 100 virus particles/cells by adding virus stocks
directly to the culture medium. Cells were lysed, and RNA was prepared
24 h after infection.
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RESULTS |
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Characterization of an Alternate Human HIC-1 Exon by 5'
RACE--
To search for such alternate transcripts in human
HIC-1, we performed 5' RACE using normal human ovary
cDNAs tailed with an adaptor sequence (Marathon Ready cDNAs,
CLONTECH). A primary PCR was performed with Adaptor
primer AP1 and the HIC-1 antisense-specific primer M5
(positions 1013-1035 in HIC-1). Electrophoretic analysis of
the RACE products obtained with the AP2 and the HIC-1 M6
(positions 982-1003) nested primers showed an unique DNA fragment of
~550 bp. However, after cloning in the PCR-TOPO vector and nucleotide sequencing, individual clones can be resolved into two classes (Fig.
2A).
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The type A RACE products correspond to the splicing of the previously described HIC-1 UTR to the unique coding exon using canonical splice donor and splice acceptor sites (15) (Fig. 2, A and B). Strikingly, careful analyses of the RT-PCR products and of the human HIC-1 genomic sequences reveal in this previously reported untranslated exon an upstream ATG codon in frame with the BTB/POZ and zinc fingers containing exon. This UTR exon is in fact a potentially new coding exon that we have renamed exon 1a because it directs the synthesis of 12 amino acids in frame with the previously described large HIC-1 coding exon that was renamed exon 2. Thus, transcripts containing this human exon 1a could encode an alternative protein containing 19 amino acids (12 derived from exon 1a and 7 derived from 5' sequences in exon 2 that are converted into coding sequences) in addition to the previously described 714 amino acids reading frame encoded by exon 2. Interestingly, we noticed that an exon highly homologous (85%) to the human HIC-1 exon 1a could be identified in the mouse genomic sequence at positions 2261-2553 (28). In particular, the TATA box, the consensus splice donor site and the 19 amino acids upstream open reading frame are perfectly conserved (Fig. 2B). To validate the existence of this putative new transcript, we performed RT-PCR experiments using RNAs prepared from adult mouse ovaries. Two rounds of PCR with a sense primer in the putative mouse exon 1a (positions 2435-2456 in the published genomic sequence) (28) and an antisense primer in the BTB/POZ domain (positions 3063-3082) yielded the predicted 300-bp cDNA fragment. Its nucleotide sequence unambiguously demonstrated that this band corresponded to the splicing of the mouse exon 1a to the second mouse coding exon, exon 2 (Fig. 2, B and D).
The second type of 5' RACE products we have obtained, type B, diverges precisely from the type A products at the exon 2 splice junction and is similar to the HIC-1 transcript recently described in the mouse (28) (Fig. 2, A and C). Searches in the nonredundant data base identified the 3' portion of our new sequence flanked by a canonical splice donor site in three previously described human genomic clones (VNTRA2, B2, and C2; accessions numbers M21146, M21147, and M21148) (36), corresponding to 650-bp BamHI-PstI genomic fragments located ~1.8 kbp distal to the YNZ22 VNTR on its 3' flank. Finally this new human HIC-1 alternative 5' end is perfectly conserved in a partial expressed sequence tag of the kidney (GenBankTM accession number AA910862). Although these alternative human and murine exons that we have named exons 1b utilize similar splice sites, they are not totally conserved at the nucleotide level. Indeed, the partial human exon 1b sequence significantly diverges from its murine homologue by a 6-nucleotide gap, in sharp contrast with the human and murine exon 1a, which are well conserved and are both coding exons (Fig. 2C; see below). All of these results thus implicated the existence of additional and similar HIC-1 transcripts in mouse and human, albeit possibly endowed with different coding capacities.
Sequencing of the Human Genomic Sequences Homologous to the Murine
HIC-1 Exon 1b--
The putative human HIC-1 exon
1b was localized in our HIC-1 genomic clone (30)
to a 5.0-kbp XhoI fragment. This fragment contains three
BamHI restriction sites that yield a 1578-bp fragment possessing the major portion of exon 1b and an ~1.3-kbp fragment containing the YNZ22/D17S30/D17S5 VNTR. A composite 1631-bp genomic sequence derived from the 1578-bp BamHI fragment and
from a short BamHI-XhoI fragment containing the
3' end of exon 1b (EMBL accession number AJ404688) was
compared with the murine HIC-1 genomic sequence (EMBL
accession number AF036582) and analyzed with several software tools
(Fig. 3A). Using the LALIGN program, a global 61% identity in a 1542-nucleotide overlap was observed between the two sequences (data not shown). With the BLAST
network facility (32), the main region of statistically significant
homology (91% identities, expected value 7e-92)
underscored between the human and murine sequences corresponded only to
the 3' part of exon 1b (Fig. 3A) and included the splice donor site as expected from the type B RT-PCR products (Fig. 2)
and cDNA sequences (28). The murine exon 1b contains a
predicted 172-amino acid open reading frame in frame with the murine
exon 2 (Fig. 3A and Ref. 28). However, the 5'
part of the murine exon 1b, including the putative initiation codon, is poorly conserved in the human sequence. In the
human sequence there is no other ATG codon in frame with the large open
reading frame in exon 2 that encodes the BTB/POZ and zinc
finger domains (Fig. 3B) but instead an in-frame stop codon,
strongly suggesting that the human exon 1b is not translated. Finally, we noticed that a TATA box proposed as the putative transcription start site of the murine HIC-1 gene
from sequencing data but not validated by functional assays (28) is not
conserved in the human genomic sequence (Fig. 3B).
Nevertheless, analyses of the human HIC-1 genomic sequences
with several software tools identified a downstream putative TATA box,
which is conserved in the murine sequence (Fig. 3, A and
B). The prediction scores (37) for these alternative TATA
boxes are very similar to the score of the previously published TATA
box for the murine HIC-1 gene (28) and to the scores
obtained with the murine and human TATA boxes initiating the exon
1a-containing transcripts (15) (Figs. 1 and 2), suggesting
that they could be functional as well. Noteworthy, the most 3' TATA box
in the murine sequence is also followed by an in-frame ATG codon,
indicating that the murine HIC-1 1b exon could be
a coding exon regardless of the TATA box used for the transcriptional
initiation.
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Besides these alternative TATA boxes, the same computer analyses have also identified other putative consensus sequences for transcription initiation signals in the two genomic sequences. In particular, a GC box with a high significance score (37) is conserved in the murine and human sequences and found ~150 bp upstream of the exon 1b splice donor site (Fig. 3B). These GC boxes are located respectively 20 bp upstream of the 5' end of our type B 5' RACE product obtained with human ovary RNAs and 90 bp upstream of the 5' end of the partial murine cDNA clone previously described (Ref. 28 and Fig. 3B). These putative transcription initiation sites are not followed by an ATG codon in frame with the open reading frame that initiates from exon 2. Hence, if these GC boxes were functional, both the murine and human exon 1b would be untranslated.
In conclusion, these analyses clearly demonstrate that in sharp contrast with the murine exon 1b, which has been described as coding, based on computer analyses of its genomic sequence (28), its human homologue is an untranslated exon. In addition, they strongly question the localization of the bona fide transcriptional initiation sites for the human and murine HIC-1 transcripts containing the alternative exon 1b and hence the true coding capacity of this exon 1b in mice.
Identification of Human and Murine HIC-1 Exon 1b Transcriptional Initiation Sites-- To determine the transcriptional initiation sites of the alternative human HIC-1 transcripts containing exon 1b, we used several complementary strategies, including 5' RACE experiments, cDNA cloning, and RNase protection assays.
We performed a new set of primer extension analyses on tailed human ovary cDNAs with nested primers located just at the 5' boundary of the common exon, exon 2. After two rounds of PCR, a major 220-bp band was observed. Nucleotide sequence analyses demonstrated that these new 5' RACE products ended at similar or very close positions (Fig. 3B). Similar results were obtained with distinct sources of human cDNAs obtained from normal mammary gland or peripheral blood lymphocytes (Fig. 3B and data not shown).
In another attempt to obtain further 5' sequences, extensive screenings of several commercial human cDNAs libraries with an exon 1b or a BTB/POZ probe were carried out. However, in these experiments, we only obtained from a human mammary gland cDNA library a partial 1.0-kb cDNA clone whose 5' end roughly coincided with that of the 5' RACE products. In parallel, screening of a newly constructed mouse adult ovary cDNA library with the same probes yielded an ~3.0-kb cDNA clone containing 126 bp from the murine exon 1b fused to the complete exon 2 till the polyadenylation site (28). Strikingly, the 5' end of this murine cDNA colocalized with the human 5' RACE products, a few nucleotides downstream of the conserved GC boxes (Fig. 3B). Thus, the primer extension results matched the size of the longest cDNAs isolated in both species.
To determine whether this region represented a true transcription initiation site or a strong premature retrotranscription arrest during the synthesis of the cDNAs, we performed RNase protection experiments. A 32P-labeled RNA probe complementary to the coding strand of a 302-bp RsaI-BamHI human genomic DNA fragment encompassing the 3' part of the 1.5-kbp BamHI fragment, and hence the identified exon 1b sequences was synthesized (Fig. 3C). A RsaI digestion was chosen because this RsaI restriction site is located downstream of a putative TATA box conserved in mice and human (Fig. 3, A and B). Thus, the size of the protected fragments could allow discrimination between the two putative transcription initiation signals identified in the human HIC-1 nucleotide sequence, namely the GC or the TATA boxes (Fig. 3B). The major protected fragment (101 bp) corresponds well to the size predicted by the 5' RACE experiments and strongly suggests that the conserved GC box is indeed functional, at least in humans (Fig. 3C). These protected fragments were detected only after hybridization of the probe at 67 °C (because of its high GC content) with RNAs extracted from two mortal human epithelial ovarian cell lines transfected by the SV40 T antigen, HIO106 and HIO121 (24), from normal human prostate (CLONTECH), and from an established culture line of normal fibroblast (MRC-5) (Fig. 3C). As a control no protected fragments were observed with yeast tRNA or with RNAs from the breast cancer cell line MCF-7 (Fig. 3C), in close agreement with the absence of HIC-1 expression in neoplastic cells (9, 15).
The results of these functional analyses together with the strong conservation of this GC box in the murine sequence (Fig. 3B) prompted us to investigate the functional relevance of the proposed murine HIC-1 transcription start site, a putative TATA box identified during the sequencing of the murine HIC-1 gene (28). To that end, similar RNase protection experiments were conducted with a 32P-labeled RNA probe complementary to the coding strand of a 319-bp EcoRV-BamHI genomic fragment. This fragment (positions 1091-1410 in the murine HIC-1 sequence; AF036582) is completely included in the putative coding region of the murine 1b exon (28) (Fig. 3, B and D). Thus, if the putative TATA box deduced from genomic sequencing is really functional, this probe should be fully protected. However, in sharp contrast with this prediction, the size of the major protected fragments detected after hybridization with RNAs prepared from various adult mouse tissues, and especially with poly(A)+ RNAs from ovaries, was around 100 bp (Fig. 3D) and strongly suggested that the conserved GC box, rather than the proposed TATA box, is also functional in the murine HIC-1 gene. In conclusion, the human and murine HIC-1 1b exons appear as very homologous exons initiating mainly at a conserved GC box, and more importantly both are noncoding exons.
Localization of Human and Murine HIC-1 Exon 1b Promoter--
To
determine whether bona fide start sites of transcription
have been detected, fragments surrounding this genomic region were
fused to the luciferase gene into a promoter-less reporter plasmid
(pGL3-basic, Promega) and transiently transfected into rabbit kidney
cells (RK13) (Fig. 4). A construct
(pGL3-BB) that contains approximately 1.57-kbp sequences upstream from
the transcription start site exhibited an approximately 57-fold
increase in luciferase expression above the promoter-less vector. In
contrast, a construct containing the same fragment inserted in the
opposite orientation (pGL3-BBrev) produced only a 4-fold increase in
luciferase activity. These data strongly suggest that a region within
this BamHI fragment is capable of directing transcription in
the proper direction. To define the 5' border of this fragment and to
further delineate cis-acting sequences in the HIC-1
1b promoter that might be important for its transcriptional
regulation, we created seven additional nested deletion mutants. As
shown in Fig. 4, conspicuous luciferase activity is obtained with the
different 5' deletion mutants from 1480 to
202. However, a dramatic
decrease in luciferase activity was observed when sequences between
202 and
41 were deleted (Fig. 4, compare pGL3-R2B with pGL3-SmB),
indicating the presence of key regulatory element(s) in this fragment.
Nevertheless, this shortest promoter construct (pGL3-SmB;
41/+95)
still possesses a 12-fold higher luciferase activity as compared with
pGL3-basic. Removing the region containing the identified start site of
transcription (pGL3-Sa°B; +13/+95) drops the activity to background
levels. Similar results were obtained in the ovary cell line, CHO K1
(data not shown).
|
To further substantiate the functional homologies found between the murine and human HIC-1 gene, the murine genomic sequence was assayed using the same experimental approach. The EcoRV-BamHI murine fragment lacking both the published and the downstream TATA box sequences but containing the functional GC box identified by RNase protection experiments in the murine exon 1b (Fig. 3, A, B, and D) exhibited a significant increase in luciferase activity (70-fold) when compared with the pGL3-basic vector (Fig. 4, bottom). Thus, this independent and complementary functional assay demonstrates that the two TATA boxes previously identified in the murine genomic sequence are not required for efficient promoter activity. Likewise, deletion of the conserved GC box drops the luciferase activity to background levels (3-fold), strongly suggesting a functional role in the transcription of both murine and human HIC-1 1b transcripts, in close agreement with the data obtained by primer extension, cDNA cloning, and RNase protection experiments.
Expression Patterns of the Alternate HIC-1 Transcripts--
In a
previous study, HIC-1 was found to be ubiquitously expressed
in normal human tissues as a major approximately 3.0-kb mRNA as
well as a 1.1-kb transcript, which was proposed to represent an
alternatively spliced product because it was only detected with a probe
from the BTB/POZ domain (15). A similar commercially available
multi-tissue Northern blot (CLONTECH) was
hybridized with a probe specific of the human exon 1b. This
probe detected only the predominant 3.0-kb transcript, which was found in all adult tissues tested with high levels in ovary, spleen, and
prostate after a classical 3-day exposure (Fig.
5A). Another similar blot was
hybridized with a probe specific for the exon 1a sequence.
As shown in Fig. 5B, this probe revealed a weak transcript
of similar size detected mainly in the ovary, but only after a very
long 3-week exposure. Thus, the two human alternative 5' exons are
contained within similarly sized 3.0-kb transcripts, which are
expressed at different levels but failed to hybridize to the 1.1-kb
mRNA described earlier (15). To address the nature of this smaller
transcript, the blot hybridized with the exon 1a probe was
then rehybridized with a BTB/POZ-specific probe. This BTB/POZ probe
(positions 630-1034 in the HIC-1 genomic sequence) was
different from the restriction fragment used previously (15). In
particular, our probe has been obtained by PCR with oligonucleotides
designed to exclude the 8 GGC codons located at the 3' end of the
BTB/POZ domain (29) because this sequence is reminiscent of the RRY
(26+) cryptic repeats that are enriched in encoding regions of
DNA-binding/transcription factors (38). As shown in Fig. 5C,
our HIC-1 BTB/POZ probe failed to hybridize to the small
1.1-kb mRNA species but readily recognized the 3.0-kb mRNAs
species. In addition, we noticed the appearance of an ~7.5-kb ubiquitous mRNAs species that could correspond to another
alternative splicing product of the HIC-1 gene, to a
partially spliced product, or to a related BTB/POZ gene (39).
|
Relative quantification of the two human HIC-1 transcripts
was more accurately carried out by RNase protection analyses using probes spanning either the exon 1a/2 boundary or the exon 1b/2 boundary. For all samples where HIC-1 was
expressed, both exon 1a- and exon 1b-containing
transcripts can be detected (Fig. 6 and
data not shown). Thus, using this sensitive assay, exon
1a-containing transcripts are more widely expressed than
suggested by the Northern blot analysis, as clearly shown by normal
prostate RNAs (compare Fig. 5, lane P with Fig. 6,
lane 7), but appeared consistently as a minor
HIC-1 mRNA species. Likewise, when HIC-1
expression was analyzed with the two RNase protection probes in the HIO
121 cells, we observed a coordinate and reciprocal expression of exon
1a- and exon 1b-containing transcripts, strongly
suggesting that they represent the two major HIC-1
transcripts (Fig. 6B, compare lanes 4 and
9).
|
Wild-type TP53 Induces Expression of Both HIC-1 Transcripts in
SaOs-2 Cells--
A p53-binding site located ~4.0 kbp upstream of
the exon 1a TATA box has been shown to be functional, at
least in the colon cancer cell line, SW480, because the low basal
HIC-1 expression was up-regulated upon infection with an
adenovirus expressing an exogenous wild-type TP53 gene (15).
This p53-binding site is thus located upstream and closer of the exon
1b promoter. Noteworthy, the zinc finger domain probe used
in the RNase protection experiment (15) could not discriminate between
the two HIC-1 mRNAs species that we have identified. To
do so, we infected the p53/
osteosarcoma cell line
SaOs-2 with an adenovirus expressing an exogenous wild-type
TP53 gene or a GFP gene as control. Using the exon
1a/2 probe in our sensitive RNase protection assay, we were
unable to detect any basal HIC-1 expression in SaOs-2 cells
infected by the control Adenovirus (Fig.
7, lane 2). By contrast,
exogenous wild-type TP53 was able to induce the expression of the two HIC-1 transcripts with again a predominant effect
on the exon 1b-containing transcripts (Fig. 7, lane
1). To confirm that the protected BTB/POZ fragments originated
from exon 1b-containing transcripts, we used the 302-bp
RsaI-BamHI genomic fragment (Fig. 3C)
as a probe in a similar RNase protection assay. Again, the 101-bp
protected fragments typical of the exon 1b transcripts (Fig.
3C) were observed (data not shown).
|
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DISCUSSION |
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Chromosomal band 17p13.3 and in particular, the region encompassing the YNZ22/D17S5/D17S30 marker has been implicated in major human diseases including common solid tissues cancers (6-12), leukemia (13, 14) and very recently in the Miller-Dieker syndrome (26-28). Two candidate tumor suppressor genes, OVCA1 (21-24) and BTB/POZ, and zinc finger transcriptional repressor HIC-1 (15) have been localized on each side of this microsatellite marker. Clearly, a detailed structural and functional analysis of these genes is a prerequisite to unravel their putative respective role in these malignancies. In this paper, by a combination of 5'RACE, cDNA cloning, RNase protection assays, and functional analyses, we describe a new major alternative upstream noncoding exon, exon 1b, of the human HIC-1 gene. This exon and its associated promoter are also strongly conserved in mice in contrast to a previous report based only on in silico analyses (28). In addition, our results further suggest the existence of two conserved HIC-1 isoforms both in mice and human.
Transcripts containing the new alternative exon 1b appear as
the major products of the HIC-1 locus and are expressed in
many normal tissues, in particular in prostate and ovary, tissues that
are subject to hypermethylation and/or loss of heterozygosity at the
YNZ22/D17S5/D17S30 marker in case of cancer (6, 21-24). The synthesis
of these transcripts is driven by a major TATA-less, GC-rich promoter.
However, our results also suggest the existence of further upstream,
albeit weaker, transcription initiation sites at least in mice ovaries
(Fig. 3D), a situation frequently observed with TATA-less
promoters. Preliminary data also support the existence of such upstream
minor initiation sites in some human tissues (data not shown). We have
also confirmed using adenovirus-mediated gene transfer that
HIC-1 is a TP53 target gene in the
TP53/
SaOS-2 osteosarcoma cell line, as
previously shown in the SW480 colon carcinoma cell line (15). Both exon
1b- and 1a-containing RNAs appear to be
up-regulated by TP53. Strikingly, the relative ratio between
these two types of transcripts observed upon TP53 induction
is very similar to their ratio in different normal tissues, suggesting
a general enhancer-like effect of TP53 on the
HIC-1 locus rather than an effect dedicated to a specific
promoter. The new GC-rich promoter is located upstream of the
previously described exon 1a and is thus closer to the
D17S5/YNZ22/D17S30 marker. Deletion in many ovarian tumors of the
approximately 15.0-kbp interval between the D17S30 and D17S28 markers
has been identified because of their lack of amplification in PCR
analyses of tumor DNAs using couples of specific primers for each
marker (21-23). It must be emphasized, however, that the exact 3'
boundary of the deletion is not known. It could vary from tumors to
tumors and could be located either in the D17S5 marker, thus precluding its amplification by PCR, or even extend in the adjacent
BamHI fragment containing 5' regulatory sequences and the
promoter associated with the newly identified exon 1b (Fig.
8). Nevertheless, in all cases, and at
the very least, the upstream p53-binding site, which can up-regulate
HIC-1 transcripts and part of the VNTR D17S30 marker, would be deleted.
In some instances, VNTR have been shown to influence the
transcriptional status of a downstream gene (Refs. 40 and 41 and
references therein). Taken together, the D17S30-D17S28 deletion would
thus affect not only the expression of the OVCA1 gene, which
is contained within this interval, but could also decrease the
expression level of the HIC-1 transcripts, thus mimicking
the effects of the hypermethylation of this region associated with many
tumors.
|
Another important aspect of our work is the identification of two
conserved isoforms in the HIC-1 locus. The major
HIC-1 1b transcripts that we have characterized
by several independent and complementary assays would encode both in
mice and human, a "typical" BTB/POZ and zinc finger protein
initiating at an ATG codon located in the large coding exon, exon
2, a few amino acids upstream of the BTB/POZ domain. We
cannot totally rule out the existence in mice of a protein initiated
from the upstream ATG codon (28). However, with respect to our RNase
protection analyses of various adult mice tissues (Fig. 3D),
the expression of the transcript encoding this isoform should be very
low and/or restricted to particular tissues or cell types at particular
developmental stages. Furthermore, such a large N-terminal extension
(172 amino acids) in front of the BTB/POZ domain is quite unusual in
this protein family. In addition, we have shown that the previously described UTR in human is in fact a putative coding exon also conserved
in mice. Strikingly, these exon 1a-containing transcripts
seem rather ubiquitous but are weakly expressed because they can be
only detected using long exposure of Northern blots or sensitive RNase
protection assays and RT-PCR analyses of total organs or of individual
cell types. Thus, in addition to the "classical" 714-amino acid
BTB/POZ protein, the HIC-1 locus could also encode a protein
with 19 additional residues located upstream of the BTB/POZ domain.
Although there is no clear sequence conservation between these upstream
coding exons, this situation is highly reminiscent of the chicken
FBP locus (18). Indeed, because of alternative
splicing mechanisms, this locus encodes an isoform called
FBP-B,
which is highly related to the isoform encoded by the murine and human
exon 1b-containing RNAs and another isoform called
FBP-A
containing 24 upstream residues. Interestingly enough, whereas
FBP-B
is able to repress transcription through binding to the
F1 motif in
the promoter of the
F-crystallin gene, the
FBP-A isoform is
unable to do so (18). The HIC-1 and
FBP-B BTB/POZ domains are
autonomous transcriptional repression domains, but their mechanisms of
repression remain still elusive (19). However, we can speculate that
these N-terminal extensions in mammalian type 1a and in the
chicken
FBP-A isoforms could preclude the recruitment of
corepressors or engage them in nonproductive interactions, similarly to
the anti-repression mechanism demonstrated for a thyroid hormone
receptor isoform, TR
-2 (42). Because both human HIC-1
isoforms share the same DNA-binding domains and because the two
transcripts can be coexpressed in the same cell-type (e.g.
MRC-5 and HIO 121, Fig. 3C), the expression from differently
regulated promoters of two isoforms possibly endowed with distinct
functional properties could provide a broad range of mechanisms to
regulate the HIC-1 target-genes. Answers to these questions
would shed light onto the roles of HIC-1 in pathological and
normal differentiation processes.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. Dominique Stehelin for constant interest and support and J. Coll for critical reading of the manuscript. We thank P. Dumont for dissection of mouse tissues, C. Lagrou for expert technical help with cell culture, and L. Coutte and Fabrice Soncin for patient help with some computer graphics.
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FOOTNOTES |
---|
* This work was supported by funds from CNRS, the Pasteur Institute, and the Association pour la Recherche contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ404688.
§ Recipient of a Fellowship from the Ligue Nationale contre le Cancer.
To whom correspondence should be addressed. Tel.:
33-3-20-87-1119; Fax: 33-3-20-87-1111; E-mail:
dominique.leprince@ibl.fr.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M008690200
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ABBREVIATIONS |
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The abbreviations used are: VNTR, variable number of tandem repeats; UTR, untranslated region; RACE, rapid amplification of cDNA ends; bp, base pair(s); kb, kilobase(s); kbp, kilobase pair(s); MDS, Miller-Dieker syndrome; PCR, polymerase chain reaction; RT, reverse transcriptase; AP, adaptor primer; CHO, Chinese hamster ovary; GFP, green fluorescent protein.
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