Organization, Sequence, Chromosomal Localization, and Promoter Identification of the Mouse Orphan Nuclear Receptor Nurr1 Gene

GENOMICS

41, 250–257 (1997) GE974677

ARTICLE NO.

Organization, Sequence, Chromosomal Localization, and Promoter Identification of the Mouse Orphan Nuclear Receptor Nurr1 Gene SUSAN O. CASTILLO,* QIANXUN XIAO,* MYUNG S. LYU,† CHRISTINE A. KOZAK,† AND VERA M. NIKODEM*,1 *National Institute of Diabetes and Digestive and Kidney Diseases and †National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892-1766 Received November 27, 1996; accepted February 7, 1997

We have cloned and characterized the organization of the mouse orphan nuclear receptor Nurr1 gene. The Nurr1 gene is approximately 7 kb long, contains eight exons and seven introns, and mapped to mouse chromosome 2. Although the exon/intron structure of Nurr1 is nearly identical to that of Nur77, Nurr1 possesses an additional untranslated exon. Primer extension was used to identify two major transcription initiation sites mapped 37 nucleotides apart in the first untranslated exon. Functional studies of chimeric Nurr1–luciferase reporter genes delineated the promoter region and underscored the importance of the /1 transcription start site. Sequence analysis of the 5* flanking region surrounding /1 revealed several possible response elements such as a hexanucleotide glucocorticoid binding site, a cAMP-response element, a CArG box, and two c-Jun-binding sites. These data help to explain the different response characteristics of two closely related early response genes, Nurr1 and Nur77. q 1997 Academic Press

INTRODUCTION

Differentiation, development, and oncogenesis are just some of the important processes in which members of the steroid/thyroid hormone nuclear receptor superfamily play an substantial role (Kastner et al., 1995; Mangelsdorf et al., 1995; Schuchard et al., 1993; Thummel, 1995). Within this superfamily, orphan nuclear receptors are transcription factors that have no known ligand (Mangelsdorf and Evans, 1995; O’Malley and Conneely, 1992). The orphan nuclear receptor, Nurr1 (Law et al., 1992), also known as RNR-1 (Scearce et al., 1993), HZF3 (Pena de Ortiz et al., 1994), and human homolog NOT (Mages et al., 1994), is highly homologous with Nur77, Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. U86783. 1 To whom correspondence should be addressed at NIH, NIDDK, GBB, 10 Center Drive MSC 1766, Bethesda, MD 20892-1766. Telephone: (301) 496-0944. Fax: (301) 402-0387.

0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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also known as NGFI-B (Milbrandt, 1988), N10 (Ryseck et al., 1989), or TR3 (Chang et al., 1989), and with NOR1 (Ohkura et al., 1994), also known as MINOR (Hedvat and Irving, 1995). This unique subclass of orphan nuclear receptors binds DNA sequences (hormone-reseponse elements) as a monomer and can heterodimerize with the retinoid X receptor to activate transcription. These three family members have been shown to have greater than 90% amino acid conservation in the DNAbinding domain, a Cys2-Cys2 zinc-finger structure which allows for DNA–protein interaction (Hedvat and Irving, 1995; Okabe et al., 1995). Although the sequences indicate similarity among receptor family members, differences are well documented. Immediate-early response genes such as Nurr1, Nur77, and NOR-1 differ in their spatial and temporal tissue distribution and in their responses to a variety of stimuli, such as membrane depolarization or growth factor stimulation (Bartel et al., 1989; Davis and Lau, 1994; Law et al., 1992; Mages et al., 1994; Maruyama et al., 1996; Milbrandt, 1988; Ryseck et al., 1989; Scearce et al., 1993; Xiao et al., 1996; Yoon and Lau, 1993). Although several reports implicate Nur77 in T cell receptor-mediated apoptosis in T cell hybridomas (Liu et al., 1994) or in thymocytes undergoing apoptosis (Woronicz et al., 1994), mice with a targeted disruption of this gene are viable and show no requirement for Nur77 in thymic or peripheral T cell death (Lee et al., 1995). Thus, it has been suggested that Nurr1 may play a role through functional redundancy. To address this possibility, information about the Nurr1 gene is necessary for in vivo inactivation studies. In this paper we report the initial characterization of the Nurr1 genomic locus and its promoter region. A structural comparison with a closely related orphan nuclear receptor Nur77 gene reveals similar genomic organization of encoded exons but significant differences in both promoter sequence and untranslated exons. MATERIALS AND METHODS Genomic cloning. A mouse genomic 129/SvJ l phage library was screened for phage containing the Nurr1 locus. Briefly, 7 1 105 PFU

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FIG. 1. Structure and comparison of the mouse Nurr1 gene with Nur77. (A) Overlapping Nurr1 l genomic and PCR clones are shown with a partial restriction enzyme map in which E, B, X, and S represent EcoRI, BamHI, XhoI, and SalI restriction sites. At the Nurr1 locus, exon regions are numbered and designated by boxes. Open boxes represent untranslated regions and shaded boxes are regions of encoded sequence. Intron lengths in nucleotides are shown in parentheses. (B) An alternative splice site in exon 7 generated two Nurr1 cDNAs. The Nurr1 cDNA consists of exons 1–8 spliced together (598 aa) while Nurr1a has exon 7a (solid black box) substituted for exon 7, resulting in an insertion of a stop codon (*) at aa 455. (C) The genomic organization of the Nur77 gene (Accession No. X16995). The Nur77 gene is schematically represented; open and filled boxes indicate untranslated and translated exons, respectively, and sizes of introns in nucleotides are in parentheses. of phage were plated, duplicated on filters, and probed with a random-primed 32P-labeled fragment from the 5* end of Nurr1 cDNA (nucleotides Ç700 nt). After four rounds of phage purification, three positive clones were identified and isolated. The l phage DNA was prepared according to standard methods and was used for Southern blot analysis. The l clones were mapped with restriction enzymes, and exon-containing fragments were subsequently subcloned into plasmid Bluescript (Stratagene, LaJolla, CA). In addition, some small regions were obtained through amplification of 129/SvJ mouse genomic DNA by PCR as previously described (submitted for publication). PCR products were gel purified using the GeneClean kit (Bio 101, Vista, CA) and subcloned according to PCRscript manufacturer’s recommendations (Stratagene). Sequencing of the Nurr1 gene. Primers for sequencing were designed such that overlapping sequence data could be used for comparison and alignment. ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin–Elmer, Foster City, CA) and the Sequenase Version 2.0 kit from United States Biochemicals (USB, Cleveland, OH) were used for sequence analysis on the ABI PRISM 373A DNA sequencer or conventional sequencing gel apparatus, respectively. Sequence analysis software from the Genetics Computer Group (Madison, WI) was used to align and compile sequence data. Nur77 genomic sequence (Accession No. X16995, referred to as N10) was used for structural alignment to Nurr1 genomic sequence.

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Primer extension. Putative Nurr1 transcription start sites were identified in Postnatal Day 2 mouse brain RNA by primer extension. Two oligonucleotide primers specific for Nurr1 exon 1 (oligonucleotide 1, 5*-tgctgctgccaacatgcacctaaagtctgcgcgcgt-3*; and 2, 5*-ccgaaagaagtgtgacttctgcaacccaggtccgtg-3*) were end labeled with [g-32P]ATP and T4 polynucleotide kinase. Unincorportated [g-32P]ATP was separated from labeled oligonucleotides with the Sephadex G-25 spin column (5*–3*, Inc., Boulder, CO). Total RNA (2 mg) isolated from frozen brain tissue samples by extraction with STAT-60 (Tel-Test, Friendswood, TX) was hybridized to labeled oligonucleotides. Primer extension reactions were carried out according to recommended conditions for the Superscript preamplification system for first-strand cDNA synthesis (Gibco BRL, Gaithersburg, MD) with brain RNA and tRNA for 30 min at 507C. The cDNA products were size separated on a 6% denaturing polyacrylamide gel, and a 32P-end-labeled MspI digest of pBR322 was used as a size standard. Construction and transfection of Nurr1 LUC plasmids. A series of 5* flanking Nurr1 gene luciferase (LUC) reporter constructs was created using both fortuitous restriction enzyme sites and PCR amplification. Nurr1 genomic fragments were inserted into the pGL3 luciferase reporter vector at the 5* end utilizing SfiI and NheI restriction sites and an XhoI or a HindIII site at the 3 * end. The 5* ends of SfiI and NheI restriction sites are located 1395 and 597 nt from the exon 1/intron splice junction, respectively, and the 5* ends of

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TABLE 1 Exon/Intron Boundaries of the Nurr1 Gene Exon (donor site)

Intron

Exon (acceptor site)

1 AAGAGAGCGGACAA . . . . . . . . . . gtgagtagct . . . . . tgtatttcag . . . . . . . 2 GGAGATCTGACGGG 2 TAACTCTGCTGAAG . . . . . . . . . . gtcagtgagc . . . . . tcgtttccag. . . . . . . . 3 CCATGCCTTGTGTT MetProCysVal 3 AAGGTTTCTTTAAG . . . . . . . . . . gtgagcaaga . . . . . ccctctacag. . . . . . . . 4 CGCACGGTGCAAAA ysGlyPhePheLys ArgThrValGlnLy 4 GATGGTTAAAGAAG . . . . . . . . . . gtaggtcgag . . . . . tcctttacag. . . . . . . . 5 TGGTTCGCACGGAC yMetValLysGluV alValArgThrAsp 5 TGGACTATTCCAGG . . . . . . . . . . gtaagaagcc . . . . . gaaattccag. . . . . . . . 6 TTCCAGGCAAACCC euAspTyrSerArg PheGlnAlaAsnPr 6 CGCTTAGCATACAG . . . . . . . . . . gtaatgaatg . . . . . ttaattgcag. . . . . . . . 7 GTCCAACCCAGTGG ArgLeuAlaTyrAr gSerAsnProVa1G . . . . . . . . . caacttgcag. . . . . . . . 7a AATATGAACATCGA gIle*** 7 GGCTATGGTCACAG . . . . . . . . . . gtcagtactg . . . . . ttttctgcag. . . . . . . . 8 AGAGACACGGGCTC uAlaMetValThrG luArgHisGlyLeu . . . CCTTACCTTTCTAA hrLeuProPhe*** Note. Exon and intron nucleotide sequences are shown in upper- and lowercase letters, respectively. Amino acid sequence is shown below the coding nucleotide sequence in italics. Stop codons are designated by three asterisks. XhoI and HindIII sites are located 237 and 559 nt from the exon 1/ intron splice junction, respectively. These insertions generate constructs SX-1164, NX-366, and NH-44. The numbers following the restriction site abbreviation refer to the size of fragments utilized with the addition of 6 nt to account for the inclusion of the 3* restriction site. Reporter clones P27-93 and P28-142 were produced by PCR in which an XhoI site was created for subsequent ligation and contain sequence 072 to 21 and 072 to 70, respectively (Fig. 3). NIH 3T3 cells (4.4 1 106 cells) were cotransfected by electroporation with 10 mg of Nurr1-LUC reporter plasmid and 2 mg CMV-bGal (b-galactosidase) control plasmid. Cells were cultured for 35 h in medium containing 10% resin-stripped serum. Following incubation, cells were washed twice in cold PBS and lysed with cell culture lysis reagent (400 ml) from the Promega Luciferase Assay System (Promega). Lysates were collected and centrifuged for 5 min in 1.5ml microcentrifuge tubes. The supernatant, 10 and 5–10 ml, was used to assay for b-Gal (Norton and Coffin, 1985) and luciferase activity, respectively. Three independent experiments were performed in duplicate. Values of activation were normalized against bGal activity and are expressed as a percentage of SX-LUC construct. Expression of endogenous Nurr1 and Nurr1a transcripts in NIH 3T3 cells was detected by RT-PCR (data not shown). Genetic mapping. A 700-nt fragment of amino-terminal coding sequence was used as a probe for determining the location of the Nurr1 locus. Nurr1 was mapped by analysis of the progeny of two sets of miltilocus crosses: (NFS/N or C58/J 1 Mus mus musculus) 1 M. m. musculus (Kozak et al., 1990) and (NFS/N 1 Mus spretus) 1 M. spretus or C58/J (Adamson et al., 1991). Progeny of these crosses have been typed for over 1000 markers distributed over the 19 autosomes and the X chromosome including the Chr 2 markers Abl (Abelson oncogene), Cchb4 (calcium channel, beta 4), Scn9a and Scn3a (sodium channels 9a and 3a), and Gad1 (glutamic acid decarboxylase 1) as described previously (Kozak and Sangameswaran, 1996). Recombination was determined according to Green (1981), and loci were ordered by minimizing the number of recombinants.

RESULTS

Structural organization of the Nurr1 gene. To determine the sequence and structure of the mouse Nurr1 gene, three l clones were isolated from a mouse 129/ SvJ genomic l phage library. The longest of these was

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found by restriction enzyme mapping to hybridize to oligonucleotides derived from both the Nurr1 cDNA sequence previously reported by Law et al. (1992) and the Nur77 genomic organization (Ryseck et al., 1989; Watson and Milbrandt, 1989). A total of Ç8 kb of contiguous genomic DNA was sequenced and compared with the Nurr1 cDNA. Sequence alignment was used to determine the structural organization of the mouse Nurr1 gene. The arrangement of the eight Nurr1 exons and flanking introns spans Ç7 kb of genomic sequence and is shown in Fig. 1A (Accession No. U86783). The mouse Nurr1 transcript initially described by Law et al. (1992) consists of exons 1–8 spliced in sequential order, encoding a 598-aa protein (Fig. 1B). We have recently shown that alternative splicing of Nurr1 mRNA generates two transcripts that are both expressed in developing brain tissue (submitted for publication). The second transcript, Nurr1a, has exons 1–6 spliced to an internal splice site found within exon 7, which results in a frame shift generating a stop codon. Thus, Nurr1a encodes a 455-aa protein with a truncated COOH-terminal region. All of the exon/intron splice junctions including exon 7a agree with the GT/ AG rule (Mount, 1982), and the sequences of these junctions are outlined in Table 1. A comparison of Nurr1 and Nur77 shows a similar genomic structural organization. Nurr1 and Nur77 (Figs. 1A and 1C, respectively) have the same number of exons that contain coding sequence for each protein (shaded boxes), and the sizes of introns (in parentheses) are comparable. More interestingly, the locations of the exon/intron junctions within the coding regions are nearly identical (Ryseck et al., 1989; Watson and Milbrandt, 1989). However, structural differences, especially in the 5* untranslated and promoter regions of Nurr1 and Nur77, are striking. Unlike coding exon

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FIG. 2. Identification of transcription initiation sites of the Nurr1 gene. A schematic representation shows the 5* flanking region, the first untranslated exon (open box) of the Nurr1 gene, and location of nested primers 1 and 2 complementary to the 3* end of the first exon used for primer extension analysis (arrows). Two major extension products from 2-day-old mouse brain RNA were detected with both primers 1 (lane 3) and 2 (lane 4) and are shown by arrows. The size of each product was determined by a labeled MspI-digested marker of pBR322 run in parallel. Control reactions for primers 1 and 2 with yeast tRNA are shown in lanes 1 and 2, respectively. The major starts of transcription are depicted as /1 and /38.

regions, the sequences of two Nurr1 untranslated exons located upstream of the first coding exon (3) are not homologous to either the first and only untranslated Nur77 exon or the intronic sequence between Nur77 exons 1 and 2 (Ç3 kb). Transcription initiation start site. Primer extension analysis was used to determine Nurr1 transcription initiation sites (Fig. 2). Two major initiation sites were identified using two individual oligonucleotides derived from untranslated exon 1. Nurr1 extension products (300 and 263 nt, lane 3) elongated from primer 1 correlate with the products synthesized from primer 2 (100 and 63 nt, lane 4). Additional bands were seen with primer 1 but were significantly weaker than the two major bands. The most 5* site of transcription initiation (/1) mapped to the 5* region of exon 1 at an adenine and is flanked by pyrimidines. This appears to be the

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sequence most frequently found in the transcriptional start sites in eukaryotic genes (Kozak, 1987). The second band mapped to a location downstream from /1 at nt /38 and is also an adenine flanked with a pyrimidine. Initiation sites /1 and /38 correspond to nt 537 and 500 from the exon 1/intron junction. Thus, the entire Nurr1 exon 1 is 537 nt in length, including an additional 152 nt upstream of the previously reported 5* cDNA. Nucleotide sequence of Nurr1 5* flanking region. The sequence shown in Fig. 3 extends from an SfiI restriction enzyme site through the transcription start sites to include 537 nt of the first untranslated exon and a portion of the first intron. No recognizable TATA or CCAAT boxes were found near the two major transcription start sites. Only a single SP1 element was found between these two sites. Figure 3 also highlights several other putative regulatory elements, such as a hexanucleotide glucocorticoid response element (GRE; TGTTCTT), SP1 site (GGCGGG), cAMP-response element (CRE; TGACGTCA), CArG box [CC(A/T)7GG], and two c-Jun (TAGC) sites flanked and spaced by GCrich sequence (Greene et al., 1987; Mitchell and Tjian, 1989; Renkawitz et al., 1984; Sassone-Corsi, 1988). Although a serum-response element known to be associated with other immediate-early response gene promoters was searched for, none were found (Chavrier et al., 1989; Lemaire et al., 1988; Treisman, 1985). Functional analysis of the 5* flanking sequence of the Nurr1 gene. To localize sequences necessary for promoter activity, we utilized deletion analysis at the 5* and 3* ends of the sequence shown in Fig. 3. Since the SfiI restriction site is located 958 nt upstream of the two putative starts of transcription, we expected that this sequence would include the Nurr1 promoter region. LUC–reporter clones containing various fragments of this putative promoter were tested in transient transfection assays for their ability to express luciferase activity in NIH 3T3 cells (Fig. 4). The constructs SX-1164 and NX-366 contain 958 and 60 nt, respectively, upstream of the /1 start site, while the construct NH-44 contains neither /1 nor /38. P28142 includes both putative starts of transcription (/1, /38), whereas P27-93 contains only the /1 start. The highest transcriptional activity was obtained with the NX-366 reporter, which contains both starts of transcription and an SP1 site; this was about 40% greater than the activity of SX-1164, indicating the likelihood of a repressor element upstream of the NheI restriction site. Activity was nearly abolished when both starts of transcription were deleted (NH-44), further confirming the importance of /1 and/or /38 starts of transcription. Two additional reporter genes were tested to investigate the role of these two start sites. Interestingly, the construct P27-93, which contains only the transcriptional start site at /1, had greater activity than P28-142, in which both start sites are present, but it was somehow lower than the activity of NX-366. Thus,

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FIG. 3. Partial nucleotide sequence of the mouse Nurr1 5* flanking region and first untranslated exon. Nucleotide sequence of the first untranslated exon of the mouse Nurr1 is shown in uppercase letters, and 5* flanking and intronic sequences are shown in lowercase letters. The sequence is numbered beginning with the transcription initiation start site at /1. Both transcription initiation start sites (/1 and /38) are underscored. Putative regulatory elements, such as a hexanucleotide GRE, SP1 site (GGCGGG), CRE, CArG box [CC(A/T)6GG], and two c-Jun (TAGC) sites flanked and spaced by GC-rich sequences, are in boldface. Restriction enzyme sites are in italics, and an asterisk indicates the 5* end of the Nurr1 cDNA reported by Law et al. (1992).

it appears that the /1 transcriptional start site and the sequence located between NheI and /1 are necessary for transcription of the Nurr1 gene. Genetic mapping of the Nurr1 gene. A 700-nt fragment homologous to the Nurr1 amino-terminal coding sequence was used as a hybridization probe for genetic mapping. Southern blot analysis identified ScaI fragments of 22 kb in M. spretus and 21 kb in NFS/N and C58/J. HindIII produced fragments of 3.0 kb in M. m. musculus and 3.8 kb in NFS/N and C58/J. Inheritance of the variant fragments was followed in two sets of multilocus crosses, and linkage was observed with markers on Chr 2 (Fig. 5). Nurr1 was mapped just distal to Cchb4 in the proximal region of this chromosome (Accession No. MGD-JNUM-37733). This region is homologous to human 9q and 2q, consistent with the location of the human homolog on 2q22–q23 (Mages et al., 1994). DISCUSSION

We have cloned and sequenced the genomic DNA of the mouse Nurr1 locus and compared it with the

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sequence and structure of the Nur77 gene. These two orphan nuclear receptors are similar in exon/intron structure. The first exon of both genes is composed of 5* untranslated sequence, although Nurr1 contains an additional untranslated exon. The same number of coding exons denoting the ligand-binding domain, zincfinger DNA-binding domain, hinge region, and COOHterminal region was found in both genes. Intron sizes are also similar except for the region including the first two Nurr1 5* untranslated exons. Sequence and structural comparison of Nurr1 and Nur77 with other hormone nuclear receptors also shows structural similarity, supporting the idea of a common ancestral gene evolving into a number of family members. Nurr1, Nur77, retinoic acid receptors, thyroid hormone receptors, and steroid hormone receptors all have an exon/intron junction following the first zinc finger of the DNA-binding domain and a separate exon to encode the second zinc finger (Green and Chambon, 1988; Ryseck et al., 1989). The intron splice site for many family members is located three amino acids downstream of the conserved amino acid sequence PhePhe-Lys-Arg. Interestingly, for both Nurr1 and Nur77,

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FIG. 4. Deletion mutants of the Nurr1–LUC reporter gene and their activation activity. Organization of deletion mutants is shown on the left. The constructs were made and designated as described under Materials and Methods utilizing restriction sites and synthetic double-stranded oligonucleotides. Relative LUC activities of the Nurr1–LUC reporter constructs are shown on the right. LUC activities were normalized for transfection efficiency to b-Gal after subtracting the activity of pGL3 basic reporter (Ç3000 light units). SX-1164 activity of Ç35,000 light units was arbitrarily set to 100%. Values shown are the averages of three independent transfection experiments performed in duplicate and expressed as means { SD.

the exon splice site occurs at a unique location between the Lys and the Arg, supporting the close relationship of these two genes. The genomic organization of Nurr1 and Nur77, in which the intron positions are identical to each other and different from other members of the steroid/thyroid family of genes, indicates that they constitute a distinct subfamily. If so, presumably one of them is derived from the other by gene duplication. This same duplication preserved many of the important promoter/enhancer elements in these two genes. Although the genomic organization and sequence of NOR1 have not been reported, comparative analysis would predict this orphan receptor to be an additional member of this subfamily. Nurr1 and Nur77 are immediate-early response genes that respond very rapidly to a variety of stimuli. Dramatic and rapid increases in mRNA levels of both genes have been seen when PC12 cells are treated with membrane depolarization factors such as KCl (Davis et al., 1991; Hazel et al., 1991). However, stimulation with nerve growth factor (NGF) is selective for Nur77 (Law et al., 1992). Therefore, the functional analysis of Nurr1 5* flanking sequence and its comparison with that of Nur77 is necessary to uncover a molecular basis underlying these differences in activation. Two major Nurr1 transcription start sites were identified and, like Nur77, no TATA consensus sequences were found in these locations. Extensive mutation analysis of the Nur77 promoter region shows multiple genetic elements involved in the transcriptional activation of Nur77 (Williams and Lau, 1993; Yoon and Lau, 1993). Two distinct Nur77 transcriptional responses have been described: an immediate-early response and a delayed-early response, the former is independent of

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new protein synthesis while the latter requires new protein synthesis (Williams and Lau, 1993). Sequence analysis of the elements required for each activation type points out both similarities and differences between Nurr1 and Nur77. For immediate-early activation, the Nur77 promoter region requires a CArG-like element [CC(A/T)6GG] and an Ets site. The Nur77 CArG element has an interrupted A/T core with a G unlike that found in the promoter of another immediate-early response gene, c-fos. The Nurr1 promoter region also has a CArG-like element that is similar to cfos, but contains seven A/Ts rather than six. No Ets site was found in Nurr1. Interestingly, elements associated with the delayedearly response appear to be conserved. Like Nur77, Nurr1 has two AP1/c-Jun-binding sites spaced by a GCrich/SP1-like sequence. However, these sequences have been shown to be necessary and sufficient to mediate Nur77 induction by both NGF and KCl (Yoon and Lau, 1993), although Nurr1 has not been shown to be inducible by NGF. Furthermore, the CRE site present in the Nurr1 promoter, but not in Nur77, is also found in the c-fos promoter. KCl and cAMP have been shown to activate cfos gene expression through the interaction of CREB at a CRE binding site. Thus, CREB may also mediate the effects of membrane depolarization on the Nurr1 promoter. The presence of a Nurr1 promoter hexanucleotide 5*-TGTTCT-3* site (GRE) previously shown to bind glucocorticoid receptor in the lysozyme gene promoter suggests a putative role for hormonal induction of Nurr1 gene expression. Neither Nur77 nor c-fos contains this GRE site. In conclusion, we have shown that Nurr1 and Nur77

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FIG. 5. Genetic map location of Nurr1 on mouse Chr 2. To the right of the map are recombination fractions for adjacent loci; the first fraction is from the M. m. musculus crosses, and the second is from the M. spretus crosses. In parentheses are the recombinational distances { standard error. To the left of the map are the map locations of the human homologs of the underlined genes (MGD Accession No. MGD-JNUM-37733).

are similar in both coding sequence and gene structure, but sequence dissimilarities in the 5* flanking and untranslated regions most likely account for differences in tissue expression and immediate-early responses. ACKNOWLEDGMENTS We thank and recognize Marcia Phyillaier for assistance with transfections and George Poy for his assistance in automated sequencing and oligonucleotide primer synthesis. We also express appreciation to J. E. Rall and R. Proia for advice and comments during the preparation of the manuscript.

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