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Analysis of the human LHX3 neuroendocrine transcription factor gene and mapping to the subtelomeric region of chromosome 9
Gene 245 (2000) 237–243 www.elsevier.com/locate/gene
Analysis of the human LHX3 neuroendocrine transcription factor gene and mapping to the subtelomeric region of chromosome 9 k Kyle W. Sloop a, Aaron D. Showalter a, Christopher Von Kap-Herr b, Mark J. Pettenati b, Simon J. Rhodes a, * a Department of Biology, Indiana University–Purdue University Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202-5132, USA b Department of Pediatrics, Section on Medical Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157-0001, USA Received 6 December 1999; received in revised form 5 January 2000; accepted 13 January 2000 Received by A.J. van Wijnen
Abstract The Lhx3 LIM homeodomain transcription factor is critical to pituitary organogenesis and motor neuron development. We determined the genomic structure and chromosomal localization of human LHX3. The gene contains seven coding exons and six introns that span 8.7 kilobases in length. The LHX3 gene codes for two functionally distinct isoforms that differ in their amino termini but share common LIM domains and a homeodomain. The functional domains of the LHX3 proteins are encoded by distinct exons. The alternate amino termini and LIM domains lie within individual exons, and the homeodomain is coded by two exons interrupted by a small intron. Human LHX3 maps to the subtelomeric region of chromosome 9 at band 9q34.3, within a region noted for chromosomal translocation and insertion events. Characterization of the genomic organization and chromosomal localization of LHX3 will enable molecular evaluation and genetic diagnoses of pituitary diseases and central nervous system developmental disorders in humans. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Development; Homeodomain; LIM; Pituitary; Spinal cord
1. Introduction The LIM homeodomain (HD) proteins constitute a subfamily of HD transcription factors that regulate many aspects of mammalian organogenesis and development. These factors contain two cysteine-rich zinc finger-like LIM motifs that mediate protein–protein interactions with other transcription factors and nuclear coactivator/corepressor proteins (reviewed in Dawid et al., 1998; Jurata and Gill, 1998; Bach et al., 1999). In addition, regulatory factors in this class possess a characteristic DNA-binding HD. These functional protein motifs are highly conserved throughout evolution. LIM HD transcription factors play critical roles in cell lineage specification and organ development. For examk
The human LHX3 gene intron sequences described in this paper are submitted under the following GenBank entries: AF188738, AF188739, AF188740, AF188741, AF188742, and AF188743. Abbreviations: FISH, fluorescence in-situ hybridization; LXH3, LIM homeobox gene 3; PCR, polymerase chain reaction. * Corresponding author. Tel.: +1-317-278-1797; fax: +1-317-274-2846. E-mail address: [email protected] (S.J. Rhodes)
ple, mice lacking a functional Isl-1 gene exhibit defects in the development of motor neurons and the pancreas (Pfaff et al., 1996; Ahlgren et al., 1997). Lim-1/Lhx1 is essential for the formation of anterior structures, kidneys, and gonads (Shawlot and Behringer, 1995). Lhx2/LH-2 is required for eye, forebrain, and hematopoietic development (Porter et al., 1997). Lhx4/Gsh4 is important for motor neuron pathfinding and pituitary development, including pituitary cell-type proliferation and differentiation (Li et al., 1994; Sheng et al., 1997; Sharma et al., 1998). Recently, Lhx5 has been shown to be critical for hippocampal morphogenesis and neuronal differentiation ( Zhao et al., 1999). Lhx3/Lim3/P-LIM is a LIM HD transcription factor important for motor neuron specification and the development of the anterior and intermediate lobes of the pituitary gland (Seidah et al., 1994; Bach et al., 1995; Zhadanov et al., 1995a; Sheng et al., 1996, 1997; Sharma et al., 1998). Cross-species comparison of Lhx3 protein sequences reveals conservation of the LIM and HD domains and of a motif in the carboxyl-termini of these orthologs, known as the Lhx3/LIM3-specific domain (Glasgow et al., 1997; Meier et al., 1999; Sloop et al.,
0378-1119/00/$ - see front matter © 2000 Published by Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 02 5 - 1
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1999; Thor et al., 1999). A specific function of the latter motif has not been determined. Lhx3 is transiently expressed in the developing neural cord and brainstem and is then detected during pituitary organogenesis, first appearing in the primordial structure known as Rathke’s pouch and persisting in the pituitary throughout adulthood (Seidah et al., 1994; Bach et al., 1995; Zhadanov et al., 1995a; Meier et al., 1999; Sloop et al., 1999). Lhx3 null mutant mice are stillborn or die soon after birth and lack the anterior and intermediate lobes of the pituitary gland (Sheng et al., 1996). These and other studies suggest that Lhx3 and the related Lhx4 factor are required to transform Rathke’s pouch into the mature pituitary gland, and that Lhx3 is critical for the specification and proliferation of the pituitary lactotrope, somatotrope, gonadotrope, and thyrotrope cell lineages (Sheng et al., 1996, 1997). These cell types secrete hormones that regulate many physiological functions, including lactation, growth, reproduction, and metabolic homeostasis. Two isoform products, Lhx3a and Lhx3b, identified in mice and humans, possess unique amino-terminal domains but are otherwise identical (Bach et al., 1995; Zhadanov et al., 1995a; Sloop et al., 1999). Recently, we described distinct gene activation properties of human LHX3a and LHX3b that are based on their abilities to bind specific DNA target sequences within pituitary trophic hormone genes (Sloop et al., 1999). These factors differentially activate the alpha-glycoprotein and thyroid-stimulating hormone beta genes. The LHX3a isoform binds to DNA elements within the regulatory regions of these genes and can readily activate gene expression, acting alone or in synergy with other pituitary transcription factors. By contrast, LHX3b binds with a lower affinity to these DNA-binding sites and is either inactive or only weakly capable of gene activation. In order to further characterize the LHX3 factors, we analyzed the exon–intron organization of human LHX3 and determined that the isoforms are generated from a single gene. Human LHX3 contains seven exons and six introns and is located on chromosome 9 in the subtelomeric region at 9q34.3. The functional domains of LHX3a and LHX3b are encoded by individual exons. Identification and chromosomal localization of LHX3 will facilitate future genetic evaluation of humans with pituitary and neuronal developmental disorders that may be caused by a loss of function of this gene.
2. Materials and methods 2.1. Characterization of the structure of the human LHX3 gene Human genomic DNA was extracted from the peripheral blood of two normal adults using a QIAmp
Blood Maxi Kit (Qiagen). Human LHX3 introns were amplified by the polymerase chain reaction (PCR). PCR reactions contained 2.5 U of Expand High Fidelity DNA polymerase mixture (Roche Biochemical ), 10 mM dATP, dCTP, dGTP, and dTTP, 200 ng of human genomic DNA, and 10 pmol of each forward and reverse primer. Introns 1a and 1b were amplified using 5∞-tgacctcggaggagcgcgtct-3∞ and 5∞-tcgtccttgcagtaaacgct3∞. Intron 2 was amplified using 5∞-agcgtttactgcaaggacga-3∞ and 5∞-cgcacttggtcccgaagcgc-3∞. Introns 3 and 4 were amplified using 5∞-gcgcttcgggaccaagtgcg-3∞ and 5∞-cggggaaggagacctcagcgt-3∞. Intron 5 was amplified using 5∞-ggacaaggacagcgttcag-3∞ and 5∞-ctcccgtagaggccattg-3∞. The cycling parameters were as follows: 94°C for 10 s, 58°C for 10 s, 72°C for 1–5 min for 25 cycles. Reaction products were analyzed on 1% agarose, Tris– borate gels. PCR products were ligated into pCRIITOPO ( Invitrogen) and sequenced on both strands by automated DNA sequencing using a Perkin Elmer DNA Sequencer (Biochemistry Biotechnology Facility, Indiana University School of Medicine). Gene sequences were assembled using the Wisconsin Genetics GCG computer package and DNASIS software (Hitachi). To confirm the LHX3 gene structure, exonic regions were also amplified from human genomic DNA and sequenced as described above for intronic sequences. 2.2. Molecular cytogenetics Fluorescence in-situ hybridization ( FISH ) was performed as described previously (Pettenati et al., 1988) with modifications. A 4.5 kb genomic DNA fragment containing exons Ia and Ib of the human LHX3 gene was labeled with biotin-14-dATP using the BioNick labeling system (Life Technologies). Metaphase chromosome spreads were prepared from chromosomally normal peripheral blood lymphocytes obtained by standard clinical laboratory techniques. Slides were pretreated with 0.005% pepsin in 0.01 N HCl prior to denaturation. The LHX3 probe (200 ng) was combined with Cot-1 DNA (1 mg/ml ), herring testes DNA (1 mg/ml ), and either with a probe specific for the centromere of chromosome 9 (CEP 9. VYSIS Inc.) or with a probe specific for the subtelomeric region of chromosome 9 ( TelVysion 9q, VYSIS Inc.). Probes were precipitated with ethanol and resuspended in 10 ml of Hybrisol VII (Oncor). The probes were denatured at 70°C for 5 min and pre-annealed at 37°C for at least 20 min. The hybridization mix was applied to the metaphase chromosome spreads for 24 h at 37°C. Postwashing was done in 50% formamide/2× SSC in phosphate-buffered detergent at 45°C (Pettenati et al., 1988). Probe signal detection was by indirect staining with Avidin-Texas Red, and the chromosomes were counterstained with DAPI ( VYSIS Inc.) diluted 1:1 with antifade ( Vector Laboratories). Hybridized metaphase chro-
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mosome spread images and hybridized G-band metaphase cells were obtained and viewed using the M-FISH version 3.012 software program in the VYSIS QUIPS system. Images were captured using a CCD photometric camera, digitized to an 8-bit gray scale and then merged automatically and pseudo-colored to resemble the original dyes by the software. The brightness and contrast were sharpened using a linear filter, and the signal intensity was adjusted to help remove background noise. A final image was produced using an Olympus digital color printer.
3. Results and discussion The Lhx3 neuroendocrine transcription factor protein has been conserved throughout evolution. From Drosophila to humans, these proteins contain two LIM domains, a HD, and a carboxyl Lhx3/LIM3-specific domain (Glasgow et al., 1997; Sloop et al., 1999; Thor et al., 1999). In mammals, two Lhx3 isoforms exist that share these domains but also possess distinct aminoterminal protein sequences (Bach et al., 1995; Zhadanov et al., 1995a; Sloop et al., 1999). Here, we analyze the human LHX3 gene structure and map its location in the subtelomeric region of chromosome 9. 3.1. Characterization of the human LHX3 gene In order to test the hypothesis that the human LHX3 gene structure is conserved in mammals, potential exon– intron boundaries of human LHX3 were predicted by alignment of human LHX3 cDNA sequences (Sloop et al., 1999) with the mouse Lhx3 gene (Zhadanov et al., 1995b). PCR primers were designed near potential exon– intron boundaries and used to amplify each human
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LHX3 intron. Intron DNA fragments were subsequently sequenced on both strands. Alignment of LHX3 cDNAs with isolated LHX3 intronic genomic DNAs enabled the analysis of splice site junctions. Each exon–intron boundary conforms to the GT/AG splice donor/splice acceptor consensus sequence ( Table 1). To confirm that no additional introns were located within the predicted exonic regions of the gene, LHX3 exons also were amplified from genomic DNA and sequenced. The human LHX3 gene contains seven coding exons and six introns, and the entire locus is 66% GC-rich and CpG dinucleotide-rich in composition (Fig. 1A and B; and data not shown). Similar to other proteins in this class of transcription factors (Singh et al., 1991; Zhadanov et al., 1995b; Bertuzzi et al., 1996; Bozzi et al., 1996), the coding regions for the functional protein domains of LHX3 are each contained within individual exons ( Fig. 1C ). Exons Ia and Ib code for the amino termini of alternate LHX3 isoforms ( Fig. 1C ) that possess different abilities to activate pituitary hormone genes (Sloop et al., 1999). Exon II and exon III code for the LIM1 and LIM2 domains, respectively ( Fig. 1C ). These motifs mediate interactions with coregulatory factors and other transcription factors to regulate transcriptional activity (Bach et al., 1997, 1999; Dawid et al., 1998; Jurata and Gill, 1998; Meier et al., 1999). The DNA-binding HD is encoded by exons IV and V ( Fig. 1C ). These exons are separated by an 87 bp intron (intron 4). Finally, although a function has not yet been ascribed to the conserved Lhx3/LIM-specific domain, it is entirely contained within exon VI (Fig. 1C ). By comparison to the mouse gene (Zhadanov et al., 1995b), the genomic organization of human LHX3 appears to be conserved in mammals. A similar overall gene structure is also present in the Drosophila and zebrafish Lhx3/LIM3 genes (Glasgow et al., 1997; Thor et al.,
Table 1 Characteristics of human LHX3 gene exons and introns and sequences of exon–intron boundaries
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Fig. 1. Genomic organization of the human LHX3 gene. (A) Structure of the LHX3 gene. Exons are represented by boxes and labeled in Roman numerals. White boxes indicate untranslated regions; black boxes denote protein coding exons. Introns are labeled using Arabic numerals. The asterisk denotes the location of a conserved ATTTA motif. (B) Location of CpG dinucleotide sequences (vertical lines) within the LHX3 locus. (C ) Structure of LHX3 protein isoforms and correlation of protein domains with the gene exon structure.
1999), with the important difference that these genes appear to produce only one form of the protein. The gene structure of LHX3 is similar to other factors in its class. Human LHX1 and the mouse Lhx4 and Lhx5 genes also contain separate exons that code for two LIM domains and a HD sequence broken up by a small intron (Singh et al., 1991; Zhadanov et al., 1995b; Bertuzzi et al., 1996; Bozzi et al., 1996). By contrast, the first LIM domain of mouse Lhx8 is coded by two exons, and the HD coding region for this factor contains two introns, one in the same position as the LHX3 gene and an additional intron separating the coding region of the HD helix 1 ( Kitanaka et al., 1998). Interestingly, the homeobox intron is not found in the human and mouse Isl-1 LIM HD factor genes but is found in genes encoding the LIM HD factor Xlim1 and the non-LIM HD factors evx1 and evx2 (Dawid et al., 1995). It is likely that the location of protein functional domains within individual exons has allowed exon duplication and shuffling processes and higher order gene duplication events to occur in the generation of multiple LIM and HD encoding genes. Our results provide further evidence that the assignment of coding sequences for functional domains to specific exons has enabled the expansion of this class of proteins throughout evolution. The human LHX3 gene also shares regions of similarity with the mouse Lhx3 gene outside protein coding regions, suggesting critical functional roles for these DNA elements. For example, the 3∞ end of intron 1a (Fig. 1) contains the presumed LHX3b promoter. This sequence is a highly conserved CpG-rich region ( Fig. 1B) that contains three putative Sp-1 transcription factor
binding sites located −195, −175, −159 bp relative to the start of the LHX3b coding sequence. These sites are conserved in the mouse Lhx3 gene (Zhadanov et al., 1995b), supporting the suggestion that the two LHX3 isoforms are generated from separate promoters. In addition, further comparison of this region fails to locate TATA-box or initiator elements in either species. Many ubiquitously expressed genes contain TATA-less promoters that are CpG dinucleotide-rich in their promoter regions. By contrast, Lhx3 expression is highly restricted to the developing and adult pituitary gland after being transiently expressed in specific structures of the developing nervous system (Seidah et al., 1994; Bach et al., 1995; Zhadanov et al., 1995a; Meier et al., 1999; Sloop et al., 1999). Assessment of the level of Lhx3a and Lhx3b isoform expression in established rodent pituitary cell lines suggests differential promoter activity in specific pituitary cell types (Sloop et al., 1999), consistent with the hypothesis that Lhx3a and Lhx3b promoters are subject to cell-specific regulation. Another element found to be conserved between the human and mouse Lhx3 genes is an ATTTA motif located in the 3∞ untranslated region of both genes ( located 1016 bp downstream of the LHX3 stop site, Fig. 1A). Although the 3∞ untranslated region sequences of human and mouse Lhx3 share little overall sequence similarity, this site appears in the same location in both genes. This element has been previously shown to be present in genes that produce mRNAs with short half-lives (Shaw and Kamen, 1986; Sachs, 1993). Indeed, recent experiments assaying the function of human LHX3 isoforms with and without this 3∞ element have demonstrated an
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increased capacity to activate target genes when this region is removed (Sloop et al., 1999). 3.2. Chromosomal localization of human LHX3 Using FISH, we mapped the human LHX3 gene to band 9q34.3 ( Fig. 2A and B). A signal was clearly observed on both homologous Chr 9s in 15 metaphase spreads examined. The identity of Chr 9 was confirmed by co-hybridization with a probe specific for the Chr 9 centromere (Fig. 2A). This region also includes the Notch homologue, TAN-1, and the retinoid X receptor alpha genes. The mouse Lhx3 gene has been mapped to Chr 2 near the Notch1 locus (Mbikay et al., 1995; Zhadanov et al., 1995b). To date, a number of genes have been localized to chromosomal band 9q34.3. Other genes mapped to this region include carboxyl ester lipase, dopamine-b-hydroxylase, fucosyltransferase, guanine nucleotide-releasing factor 2, lysophosphatidic acid acyltransferase, N-methyl--aspartate receptor 1, orosomucoid 1 and 2, prostaglandin D2 synthase, and retinoid X receptor alpha. The 9q34.3 band is the most distal region of chromosome 9 and includes the telomeric/subtelomeric region. Hybridization experiments were also performed using LHX3 probes in tandem with a 9q specific subtelomeric region probe that maps within 300 kb of the telomere. The signals for the two probes could not be clearly separated, as
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indicated by the overlapping probe colors (Fig. 2C ). This indicates that the LHX3 gene is located close to the telomeric region. Several reports have described chromosomal rearrangements in this region of chromosome 9, including translocation and insertion events ( Ellisen, et al., 1991; Vieira et al., 1999). In addition, it has been demonstrated that the subtelomeric regions of chromosomes are often high in GC and CpG dinucleotide content ( Flint et al., 1997). This is in accord with our observations of the LHX3 gene composition ( Fig. 1B, and above). Mutations in genes encoding many transcription factors critical during development have recently been shown to cause human disease. Mutations in the POU HD factor, PIT-1 (POU1F1), and the paired-like HD factor, prophet of Pit-1 (PROP1), lead to combined pituitary hormone deficiency (reviewed by Procter et al., 1998). The thyroid transcription factor 2 (TTF-2) is a forkhead domain protein that has been shown to cause thyroid agenesis when mutated (Clifton-Bligh et al., 1998). Reiger syndrome is caused by mutations in the bicoid-like homeobox transcription factor, PITX2 (Semina et al., 1996; Amendt et al., 1998). Also, mutations in the LIM homeobox gene, LMX1B, cause Nail– Patella syndrome (Dreyer et al., 1998). Characterization and determination of the chromosomal location of human LHX3 provide a new candidate gene potentially responsible for genetic disorders associated with this
Fig. 2. Chromosomal localization of the human LHX3 gene by FISH. (A) Metaphase chromosome spread showing the presence of the LHX3 gene (red, large arrow) on both chromosome 9s (identified by the green centromeric chromosome 9 probe, small arrow) at terminal region of band 9q34.3. (B) Ideogram of chromosome 9 showing location of LHX3. (C ) Colocalization of LHX3 gene probe (red, large arrow) with the chromosome subtelomeric probe (green). Note the yellow color resulting from the close proximity of the red and green labeled probes. The centromere is marked using a green fluorescent probe as in (A) (small arrow).
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chromosome region. Although we are unaware of any human disease linked to this specific chromosomal location, we speculate that pituitary or central nervous system developmental disorders may result from mutations in LHX3. Although targeted ablation of the entire Lhx3 gene is lethal in mice (Sheng et al., 1996), less severe or unanticipated phenotypes may result from mutations in the functional domains of LHX3, particularly lesions in the alternate LHX3 ‘a’ or ‘b’ domains. Indeed, loss of function experiments targeting different isoforms of the alternatively spliced Drosophila Prickle LIM domain protein result in phenotypes dissimilar to that of the complete null mutant (Gubb et al., 1999). Identification of the LHX3 intron locations and sequences will permit protocols to directly test for gene mutations in prospective patients. Associating human disease with mutations in LHX3 will provide a further insight into the biological functions of this transcription factor.
4. Conclusions $
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The human LHX3 gene contains seven exons and six introns and encodes functionally distinct neuroendocrine transcription factors. The structure of the LHX3 gene is conserved between humans and mice. The LHX3 gene locus maps to human chromosome 9q34.3 and lies close to the telomeric region. Characterization of the human LHX3 gene will enable examination of LHX3 as a candidate gene for human pituitary and neurological disorders.
Acknowledgements We are grateful to Matthew Kennedy, Dr O. Pescovitz, and Dr E. Walvoord for materials and useful advice. This work was supported by grants to S.J.R. from the National Science Foundation and the NRICGP/USDA.
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Analysis of the human LHX3 neuroendocrine transcription factor gene and mapping to the subtelomeric region of chromosome 9 k Kyle W. Sloop a, Aaron D. Showalter a, Christopher Von Kap-Herr b, Mark J. Pettenati b, Simon J. Rhodes a, * a Department of Biology, Indiana University–Purdue University Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202-5132, USA b Department of Pediatrics, Section on Medical Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157-0001, USA Received 6 December 1999; received in revised form 5 January 2000; accepted 13 January 2000 Received by A.J. van Wijnen
Abstract The Lhx3 LIM homeodomain transcription factor is critical to pituitary organogenesis and motor neuron development. We determined the genomic structure and chromosomal localization of human LHX3. The gene contains seven coding exons and six introns that span 8.7 kilobases in length. The LHX3 gene codes for two functionally distinct isoforms that differ in their amino termini but share common LIM domains and a homeodomain. The functional domains of the LHX3 proteins are encoded by distinct exons. The alternate amino termini and LIM domains lie within individual exons, and the homeodomain is coded by two exons interrupted by a small intron. Human LHX3 maps to the subtelomeric region of chromosome 9 at band 9q34.3, within a region noted for chromosomal translocation and insertion events. Characterization of the genomic organization and chromosomal localization of LHX3 will enable molecular evaluation and genetic diagnoses of pituitary diseases and central nervous system developmental disorders in humans. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Development; Homeodomain; LIM; Pituitary; Spinal cord
1. Introduction The LIM homeodomain (HD) proteins constitute a subfamily of HD transcription factors that regulate many aspects of mammalian organogenesis and development. These factors contain two cysteine-rich zinc finger-like LIM motifs that mediate protein–protein interactions with other transcription factors and nuclear coactivator/corepressor proteins (reviewed in Dawid et al., 1998; Jurata and Gill, 1998; Bach et al., 1999). In addition, regulatory factors in this class possess a characteristic DNA-binding HD. These functional protein motifs are highly conserved throughout evolution. LIM HD transcription factors play critical roles in cell lineage specification and organ development. For examk
The human LHX3 gene intron sequences described in this paper are submitted under the following GenBank entries: AF188738, AF188739, AF188740, AF188741, AF188742, and AF188743. Abbreviations: FISH, fluorescence in-situ hybridization; LXH3, LIM homeobox gene 3; PCR, polymerase chain reaction. * Corresponding author. Tel.: +1-317-278-1797; fax: +1-317-274-2846. E-mail address: [email protected] (S.J. Rhodes)
ple, mice lacking a functional Isl-1 gene exhibit defects in the development of motor neurons and the pancreas (Pfaff et al., 1996; Ahlgren et al., 1997). Lim-1/Lhx1 is essential for the formation of anterior structures, kidneys, and gonads (Shawlot and Behringer, 1995). Lhx2/LH-2 is required for eye, forebrain, and hematopoietic development (Porter et al., 1997). Lhx4/Gsh4 is important for motor neuron pathfinding and pituitary development, including pituitary cell-type proliferation and differentiation (Li et al., 1994; Sheng et al., 1997; Sharma et al., 1998). Recently, Lhx5 has been shown to be critical for hippocampal morphogenesis and neuronal differentiation ( Zhao et al., 1999). Lhx3/Lim3/P-LIM is a LIM HD transcription factor important for motor neuron specification and the development of the anterior and intermediate lobes of the pituitary gland (Seidah et al., 1994; Bach et al., 1995; Zhadanov et al., 1995a; Sheng et al., 1996, 1997; Sharma et al., 1998). Cross-species comparison of Lhx3 protein sequences reveals conservation of the LIM and HD domains and of a motif in the carboxyl-termini of these orthologs, known as the Lhx3/LIM3-specific domain (Glasgow et al., 1997; Meier et al., 1999; Sloop et al.,
0378-1119/00/$ - see front matter © 2000 Published by Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 02 5 - 1
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1999; Thor et al., 1999). A specific function of the latter motif has not been determined. Lhx3 is transiently expressed in the developing neural cord and brainstem and is then detected during pituitary organogenesis, first appearing in the primordial structure known as Rathke’s pouch and persisting in the pituitary throughout adulthood (Seidah et al., 1994; Bach et al., 1995; Zhadanov et al., 1995a; Meier et al., 1999; Sloop et al., 1999). Lhx3 null mutant mice are stillborn or die soon after birth and lack the anterior and intermediate lobes of the pituitary gland (Sheng et al., 1996). These and other studies suggest that Lhx3 and the related Lhx4 factor are required to transform Rathke’s pouch into the mature pituitary gland, and that Lhx3 is critical for the specification and proliferation of the pituitary lactotrope, somatotrope, gonadotrope, and thyrotrope cell lineages (Sheng et al., 1996, 1997). These cell types secrete hormones that regulate many physiological functions, including lactation, growth, reproduction, and metabolic homeostasis. Two isoform products, Lhx3a and Lhx3b, identified in mice and humans, possess unique amino-terminal domains but are otherwise identical (Bach et al., 1995; Zhadanov et al., 1995a; Sloop et al., 1999). Recently, we described distinct gene activation properties of human LHX3a and LHX3b that are based on their abilities to bind specific DNA target sequences within pituitary trophic hormone genes (Sloop et al., 1999). These factors differentially activate the alpha-glycoprotein and thyroid-stimulating hormone beta genes. The LHX3a isoform binds to DNA elements within the regulatory regions of these genes and can readily activate gene expression, acting alone or in synergy with other pituitary transcription factors. By contrast, LHX3b binds with a lower affinity to these DNA-binding sites and is either inactive or only weakly capable of gene activation. In order to further characterize the LHX3 factors, we analyzed the exon–intron organization of human LHX3 and determined that the isoforms are generated from a single gene. Human LHX3 contains seven exons and six introns and is located on chromosome 9 in the subtelomeric region at 9q34.3. The functional domains of LHX3a and LHX3b are encoded by individual exons. Identification and chromosomal localization of LHX3 will facilitate future genetic evaluation of humans with pituitary and neuronal developmental disorders that may be caused by a loss of function of this gene.
2. Materials and methods 2.1. Characterization of the structure of the human LHX3 gene Human genomic DNA was extracted from the peripheral blood of two normal adults using a QIAmp
Blood Maxi Kit (Qiagen). Human LHX3 introns were amplified by the polymerase chain reaction (PCR). PCR reactions contained 2.5 U of Expand High Fidelity DNA polymerase mixture (Roche Biochemical ), 10 mM dATP, dCTP, dGTP, and dTTP, 200 ng of human genomic DNA, and 10 pmol of each forward and reverse primer. Introns 1a and 1b were amplified using 5∞-tgacctcggaggagcgcgtct-3∞ and 5∞-tcgtccttgcagtaaacgct3∞. Intron 2 was amplified using 5∞-agcgtttactgcaaggacga-3∞ and 5∞-cgcacttggtcccgaagcgc-3∞. Introns 3 and 4 were amplified using 5∞-gcgcttcgggaccaagtgcg-3∞ and 5∞-cggggaaggagacctcagcgt-3∞. Intron 5 was amplified using 5∞-ggacaaggacagcgttcag-3∞ and 5∞-ctcccgtagaggccattg-3∞. The cycling parameters were as follows: 94°C for 10 s, 58°C for 10 s, 72°C for 1–5 min for 25 cycles. Reaction products were analyzed on 1% agarose, Tris– borate gels. PCR products were ligated into pCRIITOPO ( Invitrogen) and sequenced on both strands by automated DNA sequencing using a Perkin Elmer DNA Sequencer (Biochemistry Biotechnology Facility, Indiana University School of Medicine). Gene sequences were assembled using the Wisconsin Genetics GCG computer package and DNASIS software (Hitachi). To confirm the LHX3 gene structure, exonic regions were also amplified from human genomic DNA and sequenced as described above for intronic sequences. 2.2. Molecular cytogenetics Fluorescence in-situ hybridization ( FISH ) was performed as described previously (Pettenati et al., 1988) with modifications. A 4.5 kb genomic DNA fragment containing exons Ia and Ib of the human LHX3 gene was labeled with biotin-14-dATP using the BioNick labeling system (Life Technologies). Metaphase chromosome spreads were prepared from chromosomally normal peripheral blood lymphocytes obtained by standard clinical laboratory techniques. Slides were pretreated with 0.005% pepsin in 0.01 N HCl prior to denaturation. The LHX3 probe (200 ng) was combined with Cot-1 DNA (1 mg/ml ), herring testes DNA (1 mg/ml ), and either with a probe specific for the centromere of chromosome 9 (CEP 9. VYSIS Inc.) or with a probe specific for the subtelomeric region of chromosome 9 ( TelVysion 9q, VYSIS Inc.). Probes were precipitated with ethanol and resuspended in 10 ml of Hybrisol VII (Oncor). The probes were denatured at 70°C for 5 min and pre-annealed at 37°C for at least 20 min. The hybridization mix was applied to the metaphase chromosome spreads for 24 h at 37°C. Postwashing was done in 50% formamide/2× SSC in phosphate-buffered detergent at 45°C (Pettenati et al., 1988). Probe signal detection was by indirect staining with Avidin-Texas Red, and the chromosomes were counterstained with DAPI ( VYSIS Inc.) diluted 1:1 with antifade ( Vector Laboratories). Hybridized metaphase chro-
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mosome spread images and hybridized G-band metaphase cells were obtained and viewed using the M-FISH version 3.012 software program in the VYSIS QUIPS system. Images were captured using a CCD photometric camera, digitized to an 8-bit gray scale and then merged automatically and pseudo-colored to resemble the original dyes by the software. The brightness and contrast were sharpened using a linear filter, and the signal intensity was adjusted to help remove background noise. A final image was produced using an Olympus digital color printer.
3. Results and discussion The Lhx3 neuroendocrine transcription factor protein has been conserved throughout evolution. From Drosophila to humans, these proteins contain two LIM domains, a HD, and a carboxyl Lhx3/LIM3-specific domain (Glasgow et al., 1997; Sloop et al., 1999; Thor et al., 1999). In mammals, two Lhx3 isoforms exist that share these domains but also possess distinct aminoterminal protein sequences (Bach et al., 1995; Zhadanov et al., 1995a; Sloop et al., 1999). Here, we analyze the human LHX3 gene structure and map its location in the subtelomeric region of chromosome 9. 3.1. Characterization of the human LHX3 gene In order to test the hypothesis that the human LHX3 gene structure is conserved in mammals, potential exon– intron boundaries of human LHX3 were predicted by alignment of human LHX3 cDNA sequences (Sloop et al., 1999) with the mouse Lhx3 gene (Zhadanov et al., 1995b). PCR primers were designed near potential exon– intron boundaries and used to amplify each human
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LHX3 intron. Intron DNA fragments were subsequently sequenced on both strands. Alignment of LHX3 cDNAs with isolated LHX3 intronic genomic DNAs enabled the analysis of splice site junctions. Each exon–intron boundary conforms to the GT/AG splice donor/splice acceptor consensus sequence ( Table 1). To confirm that no additional introns were located within the predicted exonic regions of the gene, LHX3 exons also were amplified from genomic DNA and sequenced. The human LHX3 gene contains seven coding exons and six introns, and the entire locus is 66% GC-rich and CpG dinucleotide-rich in composition (Fig. 1A and B; and data not shown). Similar to other proteins in this class of transcription factors (Singh et al., 1991; Zhadanov et al., 1995b; Bertuzzi et al., 1996; Bozzi et al., 1996), the coding regions for the functional protein domains of LHX3 are each contained within individual exons ( Fig. 1C ). Exons Ia and Ib code for the amino termini of alternate LHX3 isoforms ( Fig. 1C ) that possess different abilities to activate pituitary hormone genes (Sloop et al., 1999). Exon II and exon III code for the LIM1 and LIM2 domains, respectively ( Fig. 1C ). These motifs mediate interactions with coregulatory factors and other transcription factors to regulate transcriptional activity (Bach et al., 1997, 1999; Dawid et al., 1998; Jurata and Gill, 1998; Meier et al., 1999). The DNA-binding HD is encoded by exons IV and V ( Fig. 1C ). These exons are separated by an 87 bp intron (intron 4). Finally, although a function has not yet been ascribed to the conserved Lhx3/LIM-specific domain, it is entirely contained within exon VI (Fig. 1C ). By comparison to the mouse gene (Zhadanov et al., 1995b), the genomic organization of human LHX3 appears to be conserved in mammals. A similar overall gene structure is also present in the Drosophila and zebrafish Lhx3/LIM3 genes (Glasgow et al., 1997; Thor et al.,
Table 1 Characteristics of human LHX3 gene exons and introns and sequences of exon–intron boundaries
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Fig. 1. Genomic organization of the human LHX3 gene. (A) Structure of the LHX3 gene. Exons are represented by boxes and labeled in Roman numerals. White boxes indicate untranslated regions; black boxes denote protein coding exons. Introns are labeled using Arabic numerals. The asterisk denotes the location of a conserved ATTTA motif. (B) Location of CpG dinucleotide sequences (vertical lines) within the LHX3 locus. (C ) Structure of LHX3 protein isoforms and correlation of protein domains with the gene exon structure.
1999), with the important difference that these genes appear to produce only one form of the protein. The gene structure of LHX3 is similar to other factors in its class. Human LHX1 and the mouse Lhx4 and Lhx5 genes also contain separate exons that code for two LIM domains and a HD sequence broken up by a small intron (Singh et al., 1991; Zhadanov et al., 1995b; Bertuzzi et al., 1996; Bozzi et al., 1996). By contrast, the first LIM domain of mouse Lhx8 is coded by two exons, and the HD coding region for this factor contains two introns, one in the same position as the LHX3 gene and an additional intron separating the coding region of the HD helix 1 ( Kitanaka et al., 1998). Interestingly, the homeobox intron is not found in the human and mouse Isl-1 LIM HD factor genes but is found in genes encoding the LIM HD factor Xlim1 and the non-LIM HD factors evx1 and evx2 (Dawid et al., 1995). It is likely that the location of protein functional domains within individual exons has allowed exon duplication and shuffling processes and higher order gene duplication events to occur in the generation of multiple LIM and HD encoding genes. Our results provide further evidence that the assignment of coding sequences for functional domains to specific exons has enabled the expansion of this class of proteins throughout evolution. The human LHX3 gene also shares regions of similarity with the mouse Lhx3 gene outside protein coding regions, suggesting critical functional roles for these DNA elements. For example, the 3∞ end of intron 1a (Fig. 1) contains the presumed LHX3b promoter. This sequence is a highly conserved CpG-rich region ( Fig. 1B) that contains three putative Sp-1 transcription factor
binding sites located −195, −175, −159 bp relative to the start of the LHX3b coding sequence. These sites are conserved in the mouse Lhx3 gene (Zhadanov et al., 1995b), supporting the suggestion that the two LHX3 isoforms are generated from separate promoters. In addition, further comparison of this region fails to locate TATA-box or initiator elements in either species. Many ubiquitously expressed genes contain TATA-less promoters that are CpG dinucleotide-rich in their promoter regions. By contrast, Lhx3 expression is highly restricted to the developing and adult pituitary gland after being transiently expressed in specific structures of the developing nervous system (Seidah et al., 1994; Bach et al., 1995; Zhadanov et al., 1995a; Meier et al., 1999; Sloop et al., 1999). Assessment of the level of Lhx3a and Lhx3b isoform expression in established rodent pituitary cell lines suggests differential promoter activity in specific pituitary cell types (Sloop et al., 1999), consistent with the hypothesis that Lhx3a and Lhx3b promoters are subject to cell-specific regulation. Another element found to be conserved between the human and mouse Lhx3 genes is an ATTTA motif located in the 3∞ untranslated region of both genes ( located 1016 bp downstream of the LHX3 stop site, Fig. 1A). Although the 3∞ untranslated region sequences of human and mouse Lhx3 share little overall sequence similarity, this site appears in the same location in both genes. This element has been previously shown to be present in genes that produce mRNAs with short half-lives (Shaw and Kamen, 1986; Sachs, 1993). Indeed, recent experiments assaying the function of human LHX3 isoforms with and without this 3∞ element have demonstrated an
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increased capacity to activate target genes when this region is removed (Sloop et al., 1999). 3.2. Chromosomal localization of human LHX3 Using FISH, we mapped the human LHX3 gene to band 9q34.3 ( Fig. 2A and B). A signal was clearly observed on both homologous Chr 9s in 15 metaphase spreads examined. The identity of Chr 9 was confirmed by co-hybridization with a probe specific for the Chr 9 centromere (Fig. 2A). This region also includes the Notch homologue, TAN-1, and the retinoid X receptor alpha genes. The mouse Lhx3 gene has been mapped to Chr 2 near the Notch1 locus (Mbikay et al., 1995; Zhadanov et al., 1995b). To date, a number of genes have been localized to chromosomal band 9q34.3. Other genes mapped to this region include carboxyl ester lipase, dopamine-b-hydroxylase, fucosyltransferase, guanine nucleotide-releasing factor 2, lysophosphatidic acid acyltransferase, N-methyl--aspartate receptor 1, orosomucoid 1 and 2, prostaglandin D2 synthase, and retinoid X receptor alpha. The 9q34.3 band is the most distal region of chromosome 9 and includes the telomeric/subtelomeric region. Hybridization experiments were also performed using LHX3 probes in tandem with a 9q specific subtelomeric region probe that maps within 300 kb of the telomere. The signals for the two probes could not be clearly separated, as
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indicated by the overlapping probe colors (Fig. 2C ). This indicates that the LHX3 gene is located close to the telomeric region. Several reports have described chromosomal rearrangements in this region of chromosome 9, including translocation and insertion events ( Ellisen, et al., 1991; Vieira et al., 1999). In addition, it has been demonstrated that the subtelomeric regions of chromosomes are often high in GC and CpG dinucleotide content ( Flint et al., 1997). This is in accord with our observations of the LHX3 gene composition ( Fig. 1B, and above). Mutations in genes encoding many transcription factors critical during development have recently been shown to cause human disease. Mutations in the POU HD factor, PIT-1 (POU1F1), and the paired-like HD factor, prophet of Pit-1 (PROP1), lead to combined pituitary hormone deficiency (reviewed by Procter et al., 1998). The thyroid transcription factor 2 (TTF-2) is a forkhead domain protein that has been shown to cause thyroid agenesis when mutated (Clifton-Bligh et al., 1998). Reiger syndrome is caused by mutations in the bicoid-like homeobox transcription factor, PITX2 (Semina et al., 1996; Amendt et al., 1998). Also, mutations in the LIM homeobox gene, LMX1B, cause Nail– Patella syndrome (Dreyer et al., 1998). Characterization and determination of the chromosomal location of human LHX3 provide a new candidate gene potentially responsible for genetic disorders associated with this
Fig. 2. Chromosomal localization of the human LHX3 gene by FISH. (A) Metaphase chromosome spread showing the presence of the LHX3 gene (red, large arrow) on both chromosome 9s (identified by the green centromeric chromosome 9 probe, small arrow) at terminal region of band 9q34.3. (B) Ideogram of chromosome 9 showing location of LHX3. (C ) Colocalization of LHX3 gene probe (red, large arrow) with the chromosome subtelomeric probe (green). Note the yellow color resulting from the close proximity of the red and green labeled probes. The centromere is marked using a green fluorescent probe as in (A) (small arrow).
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chromosome region. Although we are unaware of any human disease linked to this specific chromosomal location, we speculate that pituitary or central nervous system developmental disorders may result from mutations in LHX3. Although targeted ablation of the entire Lhx3 gene is lethal in mice (Sheng et al., 1996), less severe or unanticipated phenotypes may result from mutations in the functional domains of LHX3, particularly lesions in the alternate LHX3 ‘a’ or ‘b’ domains. Indeed, loss of function experiments targeting different isoforms of the alternatively spliced Drosophila Prickle LIM domain protein result in phenotypes dissimilar to that of the complete null mutant (Gubb et al., 1999). Identification of the LHX3 intron locations and sequences will permit protocols to directly test for gene mutations in prospective patients. Associating human disease with mutations in LHX3 will provide a further insight into the biological functions of this transcription factor.
4. Conclusions $
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The human LHX3 gene contains seven exons and six introns and encodes functionally distinct neuroendocrine transcription factors. The structure of the LHX3 gene is conserved between humans and mice. The LHX3 gene locus maps to human chromosome 9q34.3 and lies close to the telomeric region. Characterization of the human LHX3 gene will enable examination of LHX3 as a candidate gene for human pituitary and neurological disorders.
Acknowledgements We are grateful to Matthew Kennedy, Dr O. Pescovitz, and Dr E. Walvoord for materials and useful advice. This work was supported by grants to S.J.R. from the National Science Foundation and the NRICGP/USDA.
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