Identification in the Human Candidate Tumor Suppressor Gene HIC-1 of a New Major Alternative TATA-less Promoter Positively Regulated by p53*

Cateline Guerardel, Sophie Deltour§, Sébastien Pinte, Didier Monte, Agnès Begue, Andrew K. Godwin, and Dominique Leprince||

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma 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).

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).



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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 gamma FBP is a single-copy gene subject to alternative splicing mechanisms because three transcripts with alternate 5' ends (gamma FBP-A, gamma FBP-B and gamma 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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 [gamma -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-beta -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 lambda 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-beta 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 beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   Sequence comparison of the two 5' RACE products with the human and murine HIC-1 genomic sequences. A, partial nucleotide sequence of the 5' RACE products highlighting their divergent 5' ends. Sequences derived from the common BTB/POZ domain are underlined. B, nucleotide sequences of the human and murine exon 1a. These sequences of human (Hu) and murine (Mu) HIC-1 start at the conserved TATA box and are derived from GenBankTM and from our type A 5' RACE products. The noncoding and intronic sequences are shown as lowercase letters. A splice donor site validated by RT-PCR analyses of human and mouse ovaries mRNAs (this study) is underlined. The newly identified coding sequences in frame with exon 2 (see below) are indicated as capital letters, using the one-letter code for the amino acids. An upstream termination stop codon is indicated by *** . C, partial nucleotide sequences of the human and murine exon 1b. These sequences are derived from our type B 5' RACE products, from the sequencing of human (Hu) and murine (Mu) HIC-1 genomic clones. The noncoding and intronic sequences are shown as lowercase letters. A splice donor site validated by RT-PCR and cDNA cloning is underlined. The putative coding sequences (see "Results") are shown as capital letters. Dashes represent gaps introduced to maximize the alignments. D, 5' boundary of the large human and murine coding exon, exon 2. The previously identified initiation codons are indicated in gray (15, 28, 29). The new amino acids encoded by 5' sequences, which are in frame with the ATG codon in exon 1a, are indicated as capital letters, using the one-letter code for the amino acids. A splice acceptor site validated by RT-PCR analyses of human and mouse mRNAs and cDNA cloning is underlined.

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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Fig. 3.   Characterization of the human HIC-1 exon 1b and comparison with its murine counterpart. A, schematic drawing and comparison of the human and murine HIC-1 genomic sequences encompassing the alternative exon 1b. A 1631-bp genomic fragment containing the new exon 1b sequences identified by the 5' RACE experiments (dashed open box) displayed a global 61% homology with the murine HIC-1 sequence (positions 1-1462) using the LALIGN program. The region of strong homology (91%) identified using the BLAST program is shown as a black box. The published TATA box and ATG codon in the murine sequence are printed in bold type (28). A putative downstream TATA box conserved between the two sequences and a downstream in-frame ATG codon in the murine sequence are also indicated. This exon would encode for 172 upstream residues in the putative murine isoform (28). s.d., splice donor site. B, sequence comparison of the human and murine HIC-1 exon 1b. The sequences are shown as capital letters, whereas the 3'-flanking genomic sequences including the splice donor site (s.d.) are shown as lowercase letters. Identical nucleotide positions are indicated by double dots. In the human sequence, numbers on the left refer to the position relative to the major transcription initiation start site (+1), which is indicated by a bent arrow (see below). The murine sequence is numbered as the HIC-1 genomic sequence deposited in GenBankTM (AF036582). The previously published TATA box and ATG codon are double underlined (28). A downstream putative TATA box conserved in both species as well as a putative in frame ATG codon in the murine sequence are underlined. In the human HIC-1 sequence an in-frame termination stop codon is boxed. The conserved and functional GC boxes identified in this study are underlined in bold. The arrow indicates the boundary of the highly homologous region detected by the BLAST algorithm. The filled arrowheads refer to the 5' ends of the partial human (1.0 kb) and full-size murine (3.0 kb) HIC-1 cDNAs that we have isolated. The opened arrowhead corresponds to the partial murine HIC-1 cDNA previously described (28). The small squares and circles correspond to the ends of the various 5' RACE products we have obtained with various sources of Marathon ready human cDNAs (CLONTECH). R, RV, and B refer, respectively, to a RsaI, an EcoRV, and a conserved BamHI restriction site used for the synthesis of the RNase protection probes. C, identification of a major transcription start site of the human HIC-1 exon 1b by RNase protection assay. Left panel, to define the size of the human 1b exon (hatched box), we used an uniformly labeled antisense probe of a HIC-1 genomic fragment RsaI-BamHI (white box) as schematically drawn. Right panel, products protected from RNase digestion after hybridization of the probe (lane 2) to 20 µg of total RNAs were separated on acrylamide gels. The size of the protected fragments was precisely defined by comparison with a labeled 100-bp ladder (lane 1) and a sequencing ladder (data not shown). Protected fragments are observed in two "mortal" ovarian epithelial cell lines, HIO 121 and HIO 106 (lanes 3 and 4); in normal fibroblast, MRC-5 (lane 5) and in RNAs obtained from total human prostate (CLONTECH) (lane 6). By contrast, no protected fragments are observed when RNAs from the breast cancer cell line MCF-7 (lane 7) or from yeast (lane 8) were assayed in the same conditions. Similar results were obtained in several independent experiments. D, the major transcription start site of the murine HIC-1 exon 1b is not the previously proposed TATA box. Left panel, the structure of the murine HIC-1 exon 1b deduced from sequencing analyses (28) of a genomic clone is schematically drawn. To define the size of the murine 1b exon, we used an uniformly labeled antisense probe of a genomic fragment (EcoRV-BamHI) as shown below. Right panel, products protected from RNase digestion after hybridization of the probe (lane 10) to 20 µg of total RNAs extracted from various adult tissues (lanes 2-7) or to 2.5 µg of mouse ovaries poly(A)+ RNAs (lane 8) were separated on acrylamide gels. The size of the protected fragments was precisely defined by comparison with a labeled 100-bp ladder (lane 1) and a sequencing ladder (data not shown). Protected fragments are observed in most tissues tested (lanes 2-8). By contrast, no protected fragments are observed when RNAs from yeast were assayed in the same experimental conditions (lane 9). When 2.5 µg of poly(A)+ RNAs prepared from mouse ovaries were analyzed (lane 8), we noticed, in addition to the major protected fragment, the presence of a slightly larger fragment (~140 bp) that could represent an upstream minor initiation site.

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).



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Fig. 4.   Deletion analysis of the human and murine HIC-1 exon 1b promoters. From top to bottom, the localization of the HIC-1 1b exon in the 1.58-kbp BamHI fragment is indicated as well as the relevant restriction sites used to construct the various deletions. * refers to restriction sites, which are not unique in the sequence. ° refers to exogenous restriction sites added to allow the construction of the required deletion. The bent arrow indicates the transcription initiation site. Left panel shows a schematic drawing of various fragments of the human HIC-1 exon 1b 5' sequences subcloned upstream of the luciferase reporter plasmid pGL3-basic. Reporter constructs were transfected in triplicate in RK13 cells, harvested and assayed for luciferase activity. Luciferase activity is shown in the right panel (black boxes). Data shown represent the averages of three independent experiments with standard deviations. Two murine HIC-1 constructs lacking both the previously published and a downstream conserved TATA box sequence and containing or not the conserved GC box, as drawn below, have been analyzed in the same conditions. A shaded box represents their respective luciferase activity.

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).



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Fig. 5.   Tissue-specific expression pattern of the HIC-1 alternative transcripts. Blots containing 2 µg of poly(A)+ selected mRNAs from the indicated human tissues (CLONTECH) were hybridized with the indicated probes. S, spleen; Th, thymus; P, prostate; Te, testis; O, ovary; SI, small intestine; C, colon; PBL, peripheral blood leukocyte. Note that the tissues are heterogeneous and that the percentage of epithelial cells in the ovary can be variable in the two blots used. Size standards are in kb. A, a blot was probed with a fragment specific for exon 1b and exposed for 3 days. Note a predominant 3.0-kb transcript (HIC-1) seen in mRNAs from most tissues. B, another blot was probed with a fragment specific for exon 1a. The HIC-1 transcript was detected after a very long exposure (3 weeks). C, the blot used in B was hybridized with a BTB/POZ-specific probe. The HIC-1 transcripts were observed in most tissues after a classical 3-day exposure. The open arrow indicates a ~7.5-kb band, which has not been fully characterized but could correspond to another alternative splice product. The two blots used in these studies were reprobed with beta -actin as internal control (data not shown).

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).



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Fig. 6.   RNase protection analyses of HIC-1 expression and 5' alternative exons utilization in various normal and neoplastic human cell lines and tissue. A, a schematic representation of the probes used for RNase protection analyses. Antisense RNA probes were transcribed from 5' RACE cDNAs templates comprising of 135 bp from the 3' end of exon 1a and 312 bp from the 5' end of the common exon 2 containing the BTB/POZ domain (Probe a, left panel) or 119 bp from the 3' end of exon 1b fused to 312 bp from exon 2 (Probe b, right panel). B, Representative autoradiographs of RNase protection analyses carried out on total RNAs (20 µg) isolated from the above indicated human cells or tissue with Probe a (lanes 2-8). The integrity of the probe is shown in lane 1. Yeast denotes the negative control containing yeast tRNA. The sizes of the fully protected and common (BTB/POZ) probe fragments are indicated by arrows and correspond to those predicted in A. A similar experiment was conducted with Probe b (lane 10) and various RNAs including HIO 121 (lane 9) and other controls (data not shown).

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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Fig. 7.   HIC-1 expression is induced in the human SaOs-2 cancer cell line upon infection by an adenovirus vector containing the wild-type human p53 gene. Total RNAs (20 µg) obtained from SaOs-2 cells infected either with an adenovirus expressing wild-type TP53 (lane 1) or an adenovirus expressing the GFP gene as a control (lane 2) were analyzed by RNase protection analyses. In both cases, the RNA was cohybridized with the HIC-1 Probe a (see Fig. 6 for details) and with an antisense probe from the ubiquitously expressed human beta  actin gene (Ambion) as a control for RNA loading. A remnant of undigested actin probe (160 bp) is marked by an asterisk, and the protected fragment (127 bp) is labeled Actin. The fully protected (1a+BTB/POZ) and the common (BTB/POZ) HIC-1 protected fragments are indicated.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



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Fig. 8.   Schematic structure of the human HIC-1 locus. The structure of the human HIC-1 locus as derived from this study is schematically drawn. The two alternative 5' exons, exon 1b and exon 1a, as well as the large coding exon, exon 2, are shown. Symbols and keys to this figure are as in Fig. 1 with minor additions. The untranslated exon 1b and the putative open reading frame identified in exon 1a are shown, respectively, as a dashed box and a black box. B, BamHI restriction site. An upstream p53 binding site shown to be functional (Ref. 15 and this study) and the microsatellite marker YNZ22/D17S30/D17S5 are also shown.

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 gamma FBP locus (18). Indeed, because of alternative splicing mechanisms, this locus encodes an isoform called gamma FBP-B, which is highly related to the isoform encoded by the murine and human exon 1b-containing RNAs and another isoform called gamma FBP-A containing 24 upstream residues. Interestingly enough, whereas gamma FBP-B is able to repress transcription through binding to the gamma F1 motif in the promoter of the gamma F-crystallin gene, the gamma FBP-A isoform is unable to do so (18). The HIC-1 and gamma 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 gamma 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 beta -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.


    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.


    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


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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