Functional Characterization of the Gene Encoding ...

Genomics 69, 120 –130 (2000) doi:10.1006/geno.2000.6311, available online at http://www.idealibrary.com on

Functional Characterization of the Gene Encoding RLIM, the Corepressor of LIM Homeodomain Factors Heather P. Ostendorff,* Michael Bossenz,* Antoaneta Mincheva,† Neal G. Copeland,‡ Debra J. Gilbert,‡ Nancy A. Jenkins,‡ Peter Lichter,† and Ingolf Bach* ,1 *Center for Molecular Neurobiology, University of Hamburg, Martinistrasse 85, 20251 Hamburg, Germany; †German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; and ‡ABL-Basic Research Program, NCI–Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Received March 24, 2000; accepted June 30, 2000

RLIM is a RING H2 zinc finger protein that acts as a negative coregulator for LIM homeodomain transcription factors. We have isolated genomic ␭ clones that cover the entire mouse RLIM-encoding Rnf12 gene. The Rnf12 gene encompasses 20 kb and consists of at least five exons and four introns. Several transcriptional start sites within a 24-bp region were mapped around 300 nt upstream of the translational start site. Rnf12-specific mRNA can be detected in many tissues as revealed by Northern blot analysis. Transient cotransfections reveal that the proximal Rnf12 promoter can be activated in vitro by ubiquitously and more restrictively expressed transcription factors, some of which are known mediators of signal transduction pathways, e.g., mammalian Kru ¨ ppel-like transcription factors, Sox and ets-related proteins, and RBP-J. We isolated a cDNA encoding human RLIM, which is highly conserved with mouse and chick RLIM. By fluorescence in situ hybridization and interspecific backcross analysis, we have localized the Rnf12 gene to the central regions of mouse and human chromosome X. © 2000 Academic Press

INTRODUCTION

LIM homeodomain factors have been shown in numerous examples to specify cell lineages and regulate differentiation during the development of many species from Caenorhabditis elegans to human (Dawid et al., 1998; Jurata and Gill, 1998; Bach, 2000; Hobert and Westphal, 2000). Recently, this class of transcription factors has been demonstrated to be highly regulated. On one hand, Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. (human RLIM cDNA sequence) AJ271670; (sequence for the intron– exon junctions of the mouse Rnf12 gene) AJ272122–AJ272129, and (mouse Rnf12 promoter sequence) AJ272032. 1 To whom correspondence should be addressed. Telephone: ⫹ 49 40 42803 5668. Fax: ⫹ 49 40 42803 8023. E-mail: ingolf.bach@ zmnh.uni-hamburg.de.

0888-7543/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

the CLIM/NLI/Ldb/Chip family of cofactors, consisting of CLIM1/Ldb2 and CLIM2/NLI/Ldb1, that bind to nuclear LIM domains has been identified (Agulnick et al., 1996; Jurata et al., 1996; Bach et al., 1997; Morcillo et al., 1997; Visvader et al., 1997). It has been shown that LIM homeodomain factors require CLIM cofactors to exert their biological activity (Agulnick et al., 1996; Morcillo et al., 1997; Bach et al., 1999; Mila`n and Cohen, 1999; van Meyel et al., 1999). More recently, MRG1 has been demonstrated to bind the LIM domain of Lhx2, enhancing its transcriptional activity (Glenn and Maurer, 1999). On the other hand, functional activity of LIM homeodomain factors is inhibited by LMO proteins (Mila`n and Cohen, 1999; van Meyel et al., 1999) and the RING H2 zinc finger protein RLIM (Bach et al., 1999). Whereas it is thought that LMO proteins compete with LIM homeodomain factors for binding to CLIM cofactors, the inhibitory effect of RLIM is likely to originate from its in vivo interaction with Sin3A, a protein that is a component of the histone deacetylase corepressor complex. RLIM-encoding mRNA, like that of CLIM2, is widely expressed and can be detected as early as stage E7.5 during mouse development (Bach et al., 1999). Since RLIM can interact with the LIM domains of all tested nuclear LIM proteins, it is likely that this factor acts as a general repressor molecule for LIM homeodomain proteins, and recent studies suggest that altered RLIM protein expression may be relevant to cancer (Scanlan et al., 1999). In this paper we have localized the Rnf12 gene encoding the RLIM protein on both mouse and human chromosome X. Furthermore, we have characterized the Rnf12 gene, determined the proximal RLIM promoter, and shown that it can be activated by several classes of transcription factor families in vitro, including RBP-J, a mediator protein of the Notch signal transduction pathway. MATERIALS AND METHODS Genomic library screening and Southern hybridizations. A 2.4-kb fragment containing the entire murine RLIM coding sequences (Bach et al., 1999) and a 3⬘-specific (568 bp) NotI/HindIII fragment were labeled with [␣- 32P]dCTP (NEN) using a Random Primers DNA

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Rnf12 GENE CHARACTERIZATION Labeling System (Gibco BRL) and used to probe 7 ⫻ 10 5 plaques of a pGEM11 mouse genomic library (Promega), plated at a density of 40,000 PFU per 150-mm plate. Plating the library and taking filter replicas were performed as described (Sambrook et al., 1989). The filters were prehybridized (6⫻ SSC, 5⫻ Denhardt’s, 0.4% SDS, 0.1 mg/ml salmon sperm DNA) for 1 h at 65°C prior to the addition of at least 3 ⫻ 10 6 cpm/ml of probe. After overnight hybridization, the filters were washed for 3⫻ 15 min at 60°C in 0.5⫻ SSC, briefly dried, and exposed overnight for autoradiography. Secondary and tertiary screenings were performed as described above. ␭ phage DNA was prepared from the plaque-purified phage isolates using the Lambda Mini Kit (Qiagen). Analysis of the resulting ␭ DNA was accomplished by Southern hybridization (Sambrook et al., 1989) performed with a 5⬘-specific (442-bp fragment) and a 3⬘-specific (568-bp fragment) probe derived from the 2.4-kb mouse cDNA clone (Bach et al., 1999). The inserts of the positive clones were subcloned by SacI or XhoI into the Bluescript vector pBKS (Stratagene) and analyzed by restriction enzyme analysis and DNA sequencing. Establishment of the intron– exon structure. To find intron– exon junctions, the genomic inserts were analyzed by sequencing using primer sequences that correspond to the Rnf12 cDNA sequence. The intron– exon boundaries were determined by direct comparison of the nucleotide sequences of Rnf12 cDNA with that of the subcloned genomic fragments. Introns were indicated when the genomic Rnf12 sequence differed from that of the cDNA. Each intron was confirmed, and its borders were defined by additional sequencing from downstream of the intron boundary in the opposite direction. The size of introns was determined by PCR using specific primers and by Southern blotting. 5⬘-RACE and Northern blots. To determine the transcriptional initiation site of the Rnf12 gene, 5⬘-RACE (Gibco BRL) was performed as described by the manufacturer, using several nested antisense primers (RACE1, TSU, and ASD) located in the RLIM coding region and in the 5⬘-UTR, respectively. Two separate series of reactions were performed, using total RNA from whole mouse embryos, E9 and E13.5. The 5⬘-RACE products were subcloned into the pCR2.1-TOPO vector contained in the TOPO TA Cloning kit (Invitrogen) and sequenced using the T7 primer. The primer extension experiments (Sambrook et al., 1989) were accomplished using the Kaifu primer, which specifically hybridizes to the ⫹100 region of the RLIM-encoding mRNA employing the same sources of RNA as used previously for the 5⬘-RACE experiments. Northern blot analysis was performed using mRNA from mouse tissues and from forskolin-stimulated and unstimulated PC12 cells. The Northern blot with RNA from mouse tissues was purchased from Clontech. PC12 cells were stimulated with 50 mM forskolin, and total RNA was prepared as described (Waltereit et al., submitted for publication). The RNA was run on a 1.5% agarose gel and transferred onto nitrocellulose (Sambrook et al., 1989). The 568-bp SacI mouse RLIM cDNA fragment that was 32P-labeled by random priming (Gibco BRL) served as a probe for both blots. Transfections and isolation of human RLIM. Transient transfections were carried out on HeLa and CHO-K1 cells using the calcium phosphate method (Bach et al., 1995) and by lipofection with Qiagen SuperFect transfection reagent according to the manufacturer’s instructions. The reporter construct to test the proximal Rnf12 promoter was constructed by PCR using Kpn1-RLIM Prom and PlusProm-Sac1 as primers, thus amplifying the region from ⫺667 bp to the transcriptional start site of the Rnf12 gene, and transferred into the pGL2 Basic vector. The correct insertion of the PCR fragment was verified by DNA sequencing. Cotransfections were carried out using CMV-driven, eukaryotic expression plasmids containing the mammalian Kru¨ppel-like transcription factors Sp1, Sp3, Sp4, LKLF, and BKLF (Kuo et al., 1997; Suske and Philipson, 1999; Turner and Crossley, 1999), C/EBP (Poli, 1998), GATA-1 (Orkin, 1998), dNotch and RBP-J (Jarriault et al., 1995), Sox 4 and Sox 11 (Wegner, 1999), and an RSV-driven Spi-1/PU.1 expression plasmid (Gauthier et al., 1993).

Genomic DNA was isolated from human testis and served as template for PCR amplifications using the primers huRLIMDown and 3⬘hRLIM-Eco. The resulting PCR fragment was subcloned in the pCR2 vector using the Invitrogen TOPO TA cloning kit followed by sequencing. Oligonucleotides. For 5⬘-RACE, oligonucleotides were as follows: RACE1, 5⬘-GCAAATTGTTGTCTCTCATAA-3⬘; TSU, 5⬘-GTCTTTTAGGACATAGTATTG-3⬘; and ASD, 5⬘-CAGATGCCACAGATAAAGCAC-3⬘. For PCR of human RLIM, oligonucleotides were huRLIMDown, 5⬘-CTTCAGATGATGTGTCTAATGG-3⬘, and 3⬘hRLIM-Eco, 5⬘-AGTTCAGATCTTAATTACACAACACTTTCTCTG-3⬘. For construction of the Rnf12 promoter plasmid, oligonucleotides Kpn1-RLIM Prom, 5⬘-TTGGTACCCTAGTTAGAGCCTCGAGGAGGCCTT-3⬘, and PlusProm-Sac1, 5⬘-CCTAGGAGCTCGCCAGCTCGGAGACGTAGCTCA-3⬘, were used. For primer extension analysis, oligonucleotide Kaifu, 5⬘-CCCGGGCAAGAGACCCGCCTCTAGAACAGCGCC-3⬘, was used. Interspecific mouse backcross mapping. Interspecific backcross progeny were generated by mating (C57BL/6J ⫻ Mus spretus)F 1 females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 N 2 mice were used to map the Rnf12 locus (see below for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (Jenkins et al., 1982). All blots were prepared with Hybond-N ⫹ nylon membrane (Amersham). The probe, an ⬃800-bp fragment of mouse cDNA, was labeled with [␣- 32 P]dCTP using a random-primed labeling kit (Stratagene); washing was performed to a final stringency of 0.5⫻ SSCP, 0.1% SDS, 65°C. Fragments of 4.5, 4.1, and 2.2 kb were detected in BglII-digested C57BL/6J DNA, and fragments of 4.1, 2.6, 2.2, and 1.8 kb were detected in BglII-digested M. spretus DNA. The presence or absence of the 2.6- and 1.8-kb BglII M. spretus-specific fragments, which cosegregated, was followed in backcross mice. A description of the probes and RFLPs for the loci linked to Rnf12 including Gjb1, Pgk1, and Btk has been reported previously (Haefliger et al., 1992; Rawlings et al., 1993). Recombination distances were calculated using Map Manager, version 2.6.5. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. Fluorescence in situ hybridization. DNA encoding mouse and human RLIM was biotin-labeled by nick-translation and used as a probe for chromosomal in situ suppression hybridization as described (Lichter et al., 1990). For the hybridizations on mouse chromosomes, a 12-kb genomic Rnf12 fragment served as a probe, whereas a 1.6-kb cDNA fragment was used for the hybridizations to human chromosomes. Metaphase chromosomes were hybridized to 80 ng of the labeled probe combined with 3 ␮g of mouse and human Cot1 DNA and 7 ␮g salmon sperm DNA in a 10-␮l hybridization cocktail. The mouse chromosomes were prepared from spleen cells of a female Balb/c animal and the human chromosomes from phytohemaglutinin stimulated human lymphocytes that were cultured for 72 h, according to an established protocol (Sawyer et al., 1987). Following posthybridization washes, the hybridized probe was detected via fluoresceinated avidin (FITC). Chromosomes were counterstained with 4,6-diamino-2-phenylindone dihydrochloride (DAPI). Digitized images of emitted DAPI and FITC fluorescence were recorded separately by a CCD camera (Photometrics), electronically overlaid, and aligned.

RESULTS

Structure of the Mouse Rnf12 Gene Screening of the mouse genomic library resulted in eight ␭ clones that hybridized strongly with radiolabeled full-length RLIM cDNA or a 600-bp SacI RLIM fragment. These genomic clones were analyzed by re-

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FIG. 1. Genomic organization of the RLIM-encoding Rnf12 gene. (A) Functional domains of the Rnf12 gene, spanning approximately 20 kb on the mouse genome. Selected restriction sites are indicated. Exons are marked as boxes; solid lines indicate introns. The genomic structure of the 3⬘ untranslated region of the RNF12 gene was not investigated and is therefore displayed as a shaded box. The Rnf12 gene consists of at least five exons interrupted by four introns. (B) Three representative ␭ phage clones isolated from a mouse genomic library, collectively spanning the entire Rnf12 gene locus. (C) Lengths of introns and exon positions relative to the transcriptional start site region, with the first nucleotide numbered as ⫹1.

striction analysis and Southern hybridization. The three clones G1b, G12, and G18 containing inserts of 17, 16, and 14.5 kb in size, respectively, were found to span the entire coding part of the Rnf12 gene locus and were therefore inserted into the Bluescript plasmid for further analysis (Fig. 1). DNA sequencing revealed that the Rnf12 gene consists of at least five exons interrupted by four introns. The first two introns are 1.8 and 12 kb in length, respectively, and are located in the 5⬘ noncoding region. Introns III and IV, 1.1 and 1.2 kb in length, respectively, are located in the coding region of the Rnf12 gene. Thus, the Rnf12 gene spans at least 20 kb on the mouse genome. The first two exons at positions 1 to 208 and 209 to 270, respectively, contain the main part of the 5⬘ noncoding region. The third exon, spanning from position 271 to 460, harbors the ATG translational start site. The fifth exon contains the main portion of the sequences encoding RLIM, including the nuclear localization site, the RING H2 zinc finger, and the TAA stop codon. The intron– exon structure within the 3⬘ noncoding part of the Rnf12 gene was not investigated. Using these ␭ clones, we established a restriction map of the genomic region (Fig. 1). Sequencing of the intron– exon borders showed that all five introns displayed consensus

GT splice donor and GA splice acceptor sequences (Table 1). Determination of the Transcription Initiation Site We used 5⬘-RACE to map the transcriptional start site of the Rnf12 gene (Figs. 2A and 2B) employing the oligonucleotides TSU and ASD as specific nested primers. As template we used cDNA produced from embryonic mouse E13.5 total RNA. Sequencing of the bands that appeared in the 5⬘-RACE–PCRs for each of the nested primers showed that there are multiple transcriptional start sites centered within 24 nt in a region that contains a YYAN(T/A)YY consensus initiator sequence (Smale and Baltimore, 1989; see Fig. 2B) and another initiator-like motif that is located downstream from the first motif (Fig. 2B). This transcriptional start site was confirmed by using cDNA produced from E9 mouse embryonic RNA as template for the 5⬘-RACE procedure and by the method of primer extension analysis on mouse E13.5 embryonic RNA (data not shown). Consequently, we defined as ⫹1 position the first nucleotide of the longest RACE product.

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Rnf12 GENE CHARACTERIZATION

TABLE 1 Intron–Exon Boundary Structure of the Rnf12 Gene Splice donor site Exon Exon Exon Exon

1 2 3 4

GAAACACCGTgtaagtgaaggtacggccaccccta TAAAAGACAGgtaattatttttgtgttgtctcctg GGCACCCCAGgtaagctgcatgtaatatgtgatat GAAAATAGAGgtacattttgtttggggaggtgggt

Splice acceptor site Intron Intron Intron Intron

1 2 3 4

ataatgaatgcttttttttttctagAGCTGAGTGA tcaatttggtctttttgctttttagATAATTTTCC tttgcaacacatgttcaactttcagGTGAAAGTAC ccttccccttctttgttttaaacagCTGGAGAGTC

Exon Exon Exon Exon

2 3 4 5

Note. Exons are indicated in uppercase, and introns are indicated in lowercase. All intron sequences displayed consensus GT donor and GA acceptor sites.

Proximal Rnf12 Promoter Analysis The Rnf12 promoter sequences were elucidated by sequencing further into the 5⬘ upstream region of the Rnf12 gene using the mouse genomic ␭ clones and were searched for consensus transcription factor recognition sequences. As illustrated in Fig. 2C, the 5⬘ upstream region of the Rnf12 gene lacks both a TATA box near the ⫺30-bp region and a CCAAT element near the ⫺100 position. However, as often observed on TATAless promoters, two putative Sp1-binding sites (e.g., Letovsky and Dynan, 1989) are found at positions ⫺40 and ⫺100. Other sequences on this promoter include potential binding sites for mammalian Kru¨ppel-like transcription factors (Suske and Philipson, 1999; Turner and Crossley, 1999), as well as transcription factors RBP-J (Jarriault et al., 1995), CREB/CREM (De Cesare et al., 1999; Fimia et al., 1999), Zen1 and 2 (Rushlow et al., 1987; Hirsch et al., 1991), PU.1/Spi-1 (Gauthier et al., 1993; Scott et al., 1994), GATA (Orkin, 1998), Sox (Wegner, 1999), and C/EBP␣ (Umek et al., 1991). To verify whether members of these transcription factor families are able to activate the Rnf12 promoter, we transferred the region from ⫺667 bp to the transcriptional start site of the Rnf12 gene into the pGL2 basic vector and tested it in transient cotransfections. Transfecting this promoter alone in HeLa and CHO-K1 cells resulted in background levels of luciferase expression between 10- and 50-fold and between 20- and 120-fold, respectively, when compared to the pGL2 vector alone (data not shown). This finding is probably due to the existence of binding sites for ubiquitously expressed transcription factors present in these cell lines. These background activations were normalized to 1 (Figs. 3A and 3B). Using this promoter, we then cotransfected the mammalian Kru¨ppel-like transcription factors including Sp1, Sp4, and LKLF, which are known activators of transcription, but also Sp3 and BKLF, which are thought to be transcriptional repressors (Suske and Philipson, 1999; Turner and Crossley, 1999). Cotransfections of plasmids in which Sp1, Sp4, or LKLF cDNAs are under the control of the CMV promoter activated the Rnf12 promoter construct between 2- and 7.5-fold (Figs. 3A and 3B), whereas cotransfections of C/EBP (Poli, 1998), GATA-1 (Orkin, 1998), Sp3, and BKLF (Suske and Philipson, 1999; Turner and Crossley, 1999) did not augment the tran-

scriptional activity of this promoter. Cotransfecting dNotch, a constitutively active Notch-1 receptor or RBP-J (Jarriault et al., 1995), resulted in activation levels between 2- and 17-fold, depending upon the transfected cell line, and the known transcriptional activators Sox 4 or Sox 11 (Wegner, 1999) led to an up to 20-fold activation in CHO-K1 cells (Figs. 3A and 3B). The differences in promoter activation levels in the two cell lines HeLa and CHO-K1 are likely to be due to differences of cellular context, e.g., to the specific sets of expressed endogenous transcription factors. Consistent with previous in situ hybridizations that detected widespread Rnf12 mRNA expression with higher concentrations in certain tissues and organs (Bach et al., 1999), our results show that both tissue-specific and ubiquitously expressed transcription factor families may contribute to the regulation of expression of the Rnf12 gene in different cell types. In addition, our results suggest that the Notch signal transduction pathway may be involved in modulating Rnf12 mRNA expression levels. Northern blot analysis on RNA of different mouse tissues using a specific mouse 568-bp RLIM probe showed that RLIM-encoding mouse mRNA is widely expressed (Fig. 3C), confirming previous in situ hybridization experiments (Bach et al., 1999). In most tissues and in the PC12 cell line, a single band migrating at 6.5 kb hybridized to the RLIM probe. However, in mouse testis, in addition to the 6.5-kb band, a shorter band that migrates at 2.3 kb is detected. This may reflect differential processing of some of the RLIM-encoding mRNA or the possibility that in this tissue an mRNA that is closely related to RLIM is specifically expressed. Since the Rnf12 promoter contains a site that closely matches a consensus CREB-binding sequence (Fig. 2C), we investigated whether RLIM-encoding mRNA may be induced by cAMP by performing a Northern blot analysis using total RNA from control PC12 cells (Fig. 3C) and from PC12 cells that were treated with forskolin (data not shown), a known activator of adenyl cyclase, an enzyme that catalyzes the generation of cAMP from ATP (Kruijer et al., 1985; Fisch et al., 1989). On this blot, forskolin induction of Arg3.1/ arc mRNA, an mRNA located in dendrites, has previously been demonstrated (Waltereit et al., submit-

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FIG. 2. Rnf12 promoter. (A) The Rnf12 transcriptional start region as mapped by 5⬘-RACE. The two bands obtained in the agarose gel analysis correspond to PCRs with the two gene-specific nested primers that were used on cDNA derived from E13.5 total RNA as template. (B) Sequencing the 5⬘-RACE PCR products revealed multiple transcriptional start sites all centered within the boxed 24 bp. Each arrow indicates the 5⬘ end of at least one PCR product. (C) The 5⬘ upstream region of the mouse Rnf12 gene contains neither a TATA box near the ⫺30-bp region nor a CCAAT element near the ⫺100 position. Consensus binding sites for transcription factors are in boldface and underlined letters. The transcriptional start site is ⫹1. The beginnings of the first exon and the first intron are indicated by arrows.

ted for publication). Forskolin treatment of PC12 cells was not able to induce Rnf12 gene transcription to significant levels (data not shown).

Human RLIM Recently, a human cDNA that shows a high level of homology to mouse and chick RLIM has been isolated


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by screening antigens that are recognized by autologous antibodies in patients with renal cell carcinoma (Scanlan et al., 1999; AF155109). However, the encoded open reading frame lacks the 145 N-terminal amino acids of RLIM. To isolate human RLIM, we amplified human genomic DNA with RLIM-specific primers by PCR. Sequencing of the resulting fragment showed that the human RLIM sequence is highly conserved with mouse and chick RLIM throughout its entire length (Fig. 4). Our human sequences were very similar if not identical to the previously published sequence except for two nucleotides at positions 574 and 613 that were lacking in our RLIM clone. The absence of these two nucleotides changed the open reading frame and thereby led to a larger protein that is homologous to the mouse and chicken RLIM protein over its entire length (Fig. 4). Thus, the overall human RLIM protein sequence is 89% identical to mouse RLIM and 85% identical to chick RLIM. Chromosomal Localizations

FIG. 3. Analysis of the Rnf12 promoter. Analysis by transient cotransfections in (A) HeLa and (B) CHO-K1 cells using CMV-driven expression plasmids of the Kru¨ppel-like transcription factors Sp1, Sp3, Sp4, BKLF, and LKLF, dNotch, a constitutively active form of the Notch-1 receptor and RBP-J, both mediator proteins of the Notch signal transduction pathway. Sox 4 and Sox 11 belong to the HMG transcription factor family, and C/EPB is a leucine zipper transcription factor. The ETS-related PU.1/Spi-1 transcription factor was under the control of the RSV promoter. Results represent four individual experiments and are expressed as fold activation, mean ⫾ standard deviation of the mean, compared to activity of the Rnf12 promoter in the presence of an empty CMV expression plasmid.

The mouse chromosomal location of the Rnf12 gene that encodes RLIM was determined by both fluorescence in situ hybridization (FISH) and interspecific backcross analysis. Evaluation of the results obtained by FISH analysis revealed specific fluorescent signals of the RLIM probe on mouse chromosome XD (Fig. 5A). In 69% (22/32), hybridization signals were revealed on both chromosome XD homologs, and in 31% (10/32) the signals were found on one of the homologs. Additional specific fluorescence signals were not observed. To establish the precise genetic localization of Rnf12 on mouse chromosomes, we performed interspecific backcross analysis using progeny derived from matings of [(C57BL/6J ⫻ M. spretus)F 1 ⫻ C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 2900 loci that are well distributed among all the autosomes as well as the X chromosome (Copeland and Jenkins, 1991). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using a mouse Rnf12 cDNA probe. The 2.6- and 1.8-kb BglII M. spretus RFLPs (see Materials and Methods) were used to follow the segregation of the Rnf12 locus in backcross mice. The mapping results confirmed the results obtained by FISH analysis and indicated that Rnf12 is located in the central region of the mouse X chromosome linked to Gjb1, Pgk1, and Btk. Although 94 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 5B), up to 142 mice were typed for some pairs of markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the

(C) Northern blot analysis of total RNA from adult mouse tissues and PC12 cells. The bands specifically hybridizing with the mouse RLIM probe are indicated by arrows at 6.5 and 2.3 kb.

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FIG. 4. RLIM protein is conserved from humans to chicken. Comparison of human, mouse, and chick RLIM protein sequences. The nuclear localization signal (NLS) and the RING H2 zinc finger are indicated.

total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are as follows: centromere–Gjb1–1/122–Rnf12– 0/111–Pgk1– 6/ 142–Btk. The recombination frequencies (expressed as genetic distances in centimorgans ⫾ the standard error) are as follows: Gjb1– 0.8 ⫾ 0.8 –(Rnf12, Pgk1)– 4.2 ⫾ 1.7. No recombinants were detected between Rnf12 and Pgk1 in 111 animals typed in common, suggesting that the two loci are within 2.7 cM of each other (upper 95% confidence limit). These results match well with the results obtained by fluorescence in situ hybridizations since the D band is located in the central portion on mouse chromosome X. We have compared our interspecific map of the X chromosome with a composite mouse linkage map that reports the map location of many uncloned mouse mutations (provided from Mouse Genome Database, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Rnf12 mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). As described above, we mapped the mouse Rnf12 gene to the central region of mouse chromosome X. This region of the mouse X chromosome shares homology with the human X chromosome (summarized in Fig. 5). In particular, Pgk1 has been mapped to Xq13.3. The close linkage between Pgk1 and Rnf12 in mouse suggested that the human homolog of Rnf12 would map to the long arm of the X chromosome as well.

Indeed, microscopic evaluation of the FISH analysis on human chromosomes revealed fluorescent signals of the RLIM probe on human chromosome Xq13– q21 as well as on human chromosome 15 (Figs. 6A and 6B). In 100% (28/28) of the metaphase cells, signals were observed on chromosome Xq13– q21, and in 75% (21/28), an additional signal was observed on chromosome 15q21– q22. DISCUSSION

Structure of the Rnf12 Gene The RLIM protein, encoded by the Rnf12 gene, has been shown to be a transcriptional repressor of LIM homeodomain transcription factors. We have analyzed the gene Rnf12 that encodes the RLIM protein and determined that it consists of at least five exons and four introns. The two largest introns, introns I and II of 1.8 and 12 kb in length, respectively, are within the 5⬘ noncoding region of the Rnf12 gene, and introns III and IV are located within the N-terminal 249 bp of the coding sequence. Thus, since the 1551 bp of the Cterminal coding sequences are not interrupted by introns, the largest portion of the RLIM-encoding region of the Rnf12 gene is intron-less. Rnf12 Promoter We have determined the transcriptional start site of the Rnf12 gene using 5⬘-RACE with two independent gene-specific oligonucleotides and by primer extension


FIG. 5. Rnf12 maps in the central region of the mouse X chromosome. (A) Section of a mouse metaphase spread after in situ hybridization with the biotin-labeled probe RLIM, detected via FITC. Arrows indicate the fluorescent signals on both mouse X chromosomes in band XD. Chromosomes were counterstained with DAPI. (B) Rnf12 was placed on the mouse X chromosome by interspecific backcross analysis. The segregation patterns of Rnf12 and flanking genes in 94 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 94 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J ⫻ M. spretus)F 1 parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial X chromosome linkage map showing the location of Rnf12 in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from the Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

analysis using RNA from mouse embryonic tissues. In the 5⬘-RACE experiments, no genomic Rnf12 sequences could be amplified due to the 14 kb of mainly

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intronic sequences that lie between the gene-specific primers used for this experiment and the mapped transcriptional start site (see Fig. 1). As seen for the ldb1 gene (encoding CLIM2; Yamashita et al., 1998), the Rnf12 gene contains no TATA box. Promoters that do not have a TATA box often have multiple transcriptional start sites and have been described for many housekeeping genes, oncogenes, growth factors, and transcription factors (Novina and Roy, 1996). It has been demonstrated that initiator sequences can direct accurate transcription initiation in the absence of a TATA box by using RNA polymerase II with the first A as the transcriptional start site (Smale, 1997). Indeed, some of the 5⬘-RACE products using Rnf12 gene-specific primers started at the A position of both initiator sequences (Fig. 2B). However, the fact that several RACE products were found that extended the consensus initiator sequence toward the 5⬘ suggests that neither of these initiators is probably very strong. As often seen with TATA-less promoters, consensus binding sites for the transcription factor Sp1 are located at both ⫺40 and ⫺100 bp upstream of the transcriptional start region, and cotransfecting a CMV-driven Sp1 expression plasmid with the luciferase gene fused to the 667 bp of Rnf12 proximal promoter sequences resulted in sevenfold activation levels (Figs. 3A and 3B). Weaker but consistent transactivation levels were also obtained by cotransfecting the expression plasmids of the Kru¨ppel-like transcription factors Sp4 and LKLF, whereas cotransfection of Sp3 and BKLF did not activate the Rnf12 promoter. These results fit well with known functions of these proteins (Kuo et al., 1997; Suske and Philipson, 1999; Turner and Crossley, 1999). Furthermore, we found activation of transcription by cotransfections of the ETS-related transcription factor PU.1/Spi-1, which is involved in the development of hematopoietic and myeloid cell lineages (Gauthier et al., 1993; Scott et al., 1994; Tondravi et al., 1997), of dNotch, a constitutively active form of the Notch-1 receptor, of RBP-J (Jarriault et al., 1995), and of Sox4 and Sox11 (Wegner, 1999). The Notch-1 receptor and the transcription factor RBP-J are both mediators of the cell– cell contact-induced Notch signal transduction pathway that is involved in many cell fate determination events in vertebrates and invertebrates (Artavanis-Tsakonas et al., 1999). This pathway has been shown to be important for the development of several tissues and organs during embryogenesis that express LIM homeodomain genes (Diaz-Benjumea and Cohen, 1993; Ahlgren et al., 1997; Apelqvist et al., 1999). Our results suggest that the expression levels of Rnf12 mRNA may be modulated by influences through the Notch signal transduction pathway. It has previously been shown that the expression levels of the Fgf-8-inducible cofactor CLIM/NLI/Ldb (Tucker et al., 1999) are extremely critical for the developmental functioning of LIM homeodomain genes (FernandezFunez et al., 1998; Milań et al., 1998; Milań and Cohen, 1999; van Meyel et al., 1999), and therefore, our results

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FIG. 6. Localization of the Rnf12 gene on human chromosomes. Sections of human metaphase spreads after in situ hybridization with the biotin-labeled probe RLIM, detected via FITC (arrows). Chromosomes were counterstained with DAPI. (A) The upper field shows the localization of the RNF12 gene to Xq13– q21. On the right, an inverted image of DAPI of one chromosome X is depicted to illustrate the band assignment. (B) The lower field shows the localization of the probe to chromosome 15q21– q22.

may be of functional relevance. However, since our results were obtained by transient transfections in vitro, further experiments are necessary to confirm that these binding sites participate in the in vivo regulation of the Rnf12 gene. Because of an almost consensus recognition site for the cAMP inducible transcription factor CREB, we also tested in Northern blots whether the Rnf12 gene is inducible by forskolin. The results show that this was not the case, arguing against cAMP-mediated induction of the Rnf12 gene. However, since the existence of a CBP-independent activation by CREM in testis has been shown (Fimia et al., 1999; De Cesare et al., 1999), we cannot exclude the possibility that such a pathway could contribute to Rnf12 gene activation. Whereas the LIM homeodomain genes are expressed in a highly restricted fashion (Bach, 2000; Hobert and Westphal, 2000) and respect the boundaries of specific territories called neuromeres in the developing central nervous system (Re´taux et al., 1999), the CLIM/NLI/ Ldb and RLIM cofactors are widely expressed, and

little is known about factors controlling the expression of these genes. The fact that the Rnf12 promoter can be activated by ubiquitously (e.g., RBP-J, Sp1) and more restrictively (e.g., LKLF, PU.1, Sox) expressed transcription factors may explain the wide Rnf12 mRNA expression pattern with a higher level of expression in certain cell types and organs (Fig. 3A; Bach et al., 1999; Scanlan et al., 1999) and argues for an involvement of a complex transcription factor network that regulates the expression of the Rnf12 gene. The Rnf12 Gene Is Conserved between Species The high level of sequence homology throughout the chick, mouse, and human protein sequences suggests that RLIM serves an evolutionarily conserved function in these species. Interestingly, by hybridizing a human RLIM cDNA probe to human chromosomes, we obtained a signal on chromosomes Xq13– q21, a region that corresponds well to genomic regions of the mouse chromosome X in which we mapped the Rnf12 gene


(and neighboring genes, e.g., Pgk1 and Gjb1; see Fig. 5). The fact that we obtained a second signal on human chromosome 15q21– q22 by using a human cDNA probe that consisted of 1.6-kb RLIM-coding sequences (Fig. 6) is best explained by the assumption of a gene closely related to RNF12 in humans, thereby raising the possibility of a Rnf12-related gene on the mouse genome as well. Indeed, our results and Northern experiments using human RNA material, in which two bands migrating at positions 6.5 and 2.3 kb were detected (Fig. 3C; Scanlan et al., 1999), may also be explained by the assumption of a second Rnf12-related gene. The appearance of two representatives of a subgroup within mammalian species has been observed, as in the case of LIM homeodomain genes as well as their cofactor CLIM/NLI/Ldb (Bach, 2000; Hobert and Westphal, 2000). The fact that RLIM protein has been recognized by autologous antibodies in patients with renal cell carcinoma (Scanlan et al., 1999) suggests that RLIM protein shows altered expression in these tumors. Thus, this altered RLIM protein expression may be relevant to cancer. The isolation of human RLIM and the functional characterization and chromosomal localization of its gene will facilitate future studies on the roles of RLIM during development and in human disease. ACKNOWLEDGMENTS We thank C. Hu¨bner, N. Piwon, M. Wegner, I. Hermans-Borgmeyer, R. Waltereit, B. Bourachot, R. Peirano, M. Yaniv, F. Logeat, A. Israel, M. Crossley, G. Suske, J. Leiden, and V. Poli for providing critical plasmids and reagents and D. B. Householder for excellent technical assistance. H.P.O. is a fellow of the Graduiertenkolleg 255 of the University of Hamburg. This research was supported, in part, by the National Cancer Institute, DHHS, under contract with ABL. I.B. is supported by the Deutsche Forschungsgemeinschaft (BA1842/1-2, 1842/2-1, 1842/3-1, and SFB444). Note added in proof. Sequence comparison using the RLIM nucleotide sequence with the recently published human genome sequences revealed a second Rnf12-related gene on human chromosome 15q21– q22, confirming our mapping results obtained by FISH analysis.

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