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Complex organization and structure of sense and antisense transcripts expressed from the DIO3 gene imprinted locus
Genomics 83 (2004) 413 – 424 www.elsevier.com/locate/ygeno
Complex organization and structure of sense and antisense transcripts $ expressed from the DIO3 gene imprinted locus Arturo Hernandez, * Maria E. Martinez, Walburga Croteau, and Donald L. St. Germain Department of Medicine, Dartmouth Medical School, Lebanon, NH 03756, USA Received 25 April 2003; accepted 22 August 2003
Abstract The human DIO3 gene and its mouse homolog, Dio3, map to chromosomes 14q32 and 12F1, respectively, and code for the type 3 deiodinase, an enzyme that inactivates thyroid hormones and is highly expressed during pregnancy and development. Mouse Dio3 is imprinted and preferentially expressed from the paternal allele in the fetus. We analyzed the human DIO3 genomic region and identified a gene (DIO3OS) that is transcribed in the antisense orientation. Multiple DIO3OS transcripts are expressed in most tissues. The structure of several DIO3OS cDNAs obtained by RT-PCR-based techniques reveals the occurrence of numerous splice variants. The exon – intron structures of DIO3OS are similar in mouse and human, but the homology of the exonic sequence is very low, except for the first exon, and no conserved open reading frame is present. We also detected DIO3 transcripts containing additional 5Vuntranslated sequence and a potential alternative upstream promoter for mouse Dio3. Exonic sequence of a Dio3os cDNA overlaps with the Dio3 promoter and strong promoter activity in the antisense orientation is detected in a genomic fragment located 3Vof mouse and human DIO3 but not in the DIO3 promoter region. These results suggest that the DIO3 gene may lie within the structure of the antisense gene, a complex arrangement often observed in imprinted loci. D 2003 Published by Elsevier Inc. Keywords: DIO3 promoter; Type 3 deiodinase; Antisense gene; Genomic imprinting
The Dio3 gene codes for the type 3 deiodinase (D3), a selenoprotein that plays a central role in thyroid hormone metabolism [1]. D3 enzymatic activity results in the inactivation of thyroid hormones, since it transforms both the prohormone T4 and the active hormone T3 into metabolites that are biologically inactive [1]. D3 displays a marked developmental pattern of expression: it is highly expressed in pregnant uterus [2], placenta [3,4], and the fetus [5], while in the adult, expression is low and limited to a few selected tissues [6,7]. D3 also is strongly induced by serum, growth factors, and phorbol esters in cell culture systems [8,9]. D3 cDNAs have been isolated from several species [10 – 13] and the mouse and human D3 genes (Dio3 and DIO3, $ Sequence data from this article have been deposited with the EMBL/ GenBank Data Libraries under Accession Nos. AF469199 – AF469208, AY077457 – AY077459, AY283181, AY283182, and W97869. * Corresponding author. Department of Medicine, Dartmouth Medical School, Borwell Building, Room 720 West, One Medical Center Drive, Lebanon, NH 03756. Fax: +1-603-650-6130. E-mail address: [email protected] (A. Hernandez).
0888-7543/$ - see front matter D 2003 Published by Elsevier Inc. doi:10.1016/j.ygeno.2003.08.024
respectively) have been localized to mouse chromosome 12F1 and human 14q32 [14]. Characterization of the mouse Dio3 gene has shown that the coding and 3V-untranslated regions are contained in a single exon, approximately 1.9 kb in length (GenBank Accession No. AF426023), which is transcribed using a promoter located immediately upstream [15]. The expression of the Dio3 gene results in a transcript of 2.1 kb in size that is most abundant in those rat and mouse tissues showing the highest D3 activity, such as decidual tissue [2] and placenta [11], and in growth factorstimulated cell culture systems [8,9]. However, larger transcripts have also been detected in the brain of hyperthyroid adult rats, when a rat D3 cDNA is used as a probe [16]. Interestingly, the mouse Dio3 gene is imprinted and preferentially expressed from the paternal allele in the developing fetus [17,18]. To characterize the DIO3 and Dio3 loci further, we have identified and partially characterized both human and mouse genes that overlap with the DIO3 and Dio3 promoter regions and are transcribed in an antisense orientation. We have designated these
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human and mouse genes DIO3OS and Dio3os, respectively, and speculate that they may play a role in the maintenance of Dio3 preferential monoallelic expression.
Results Identification and structure of human DIO3OS About 10 kb of the mouse and human DIO3 5V flanking regions was subcloned from previously isolated P1 genomic clones [14] and sequenced, and their sequences were compared. The first 1.5 kb of 5V flanking region was found to be extremely G+C rich (80% of the sequence) and was highly conserved between mouse and human (83% homology, Fig. 1). Further 5V the homology is low (less than 60%), except for a 180-bp region that is 80% homologous, contains a conserved BamHI site, and is located approximately at positions
Fig. 1. Structure of the human DIO3OS gene. The exon – intron structures, compared to the genomic sequence, of 13 cDNAs for the DIO3OS gene are shown. The conserved short region containing the proximal polyadenylation site is indicated in black and circled in the genomic diagram. Exons are depicted as thick bars and introns as thin bars. The first 3 cDNAs were found in GenBank (accession numbers shown), while the other 10 (numbered from 1 to 10) were generated in these studies from RNA of different human tissues by RT-PCR and RACE methods. The specific method and human RNA samples used to generate cDNAs 1 – 10, along with their GenBank accession numbers, are listed for each cDNA under Materials and methods. The six exons most consistently observed in the DIO3OS gene are designated at the bottom as 1 through 6.
4200 and 4000 from the human and mouse D3 transcription start sites, respectively (circled in Fig. 1). Mouse and human genomic sequences containing this short, conserved region were found to match the sequences of several mouse and human expressed sequence tags deposited in GenBank (Fig. 1). All these human and mouse ESTs contained a consensus polyadenylation site (referred to as the proximal site) and a poly(A)+ tail at their 3V end. The orientation of their 3V ends indicated that they were transcribed in an orientation antisense to that of DIO3. The 180 bp of conserved sequence was found to contain and flank the consensus polyadenylation site. Further analysis identified another group of human ESTs in GenBank that matched a human genomic region located 9.25 kb upstream of the DIO3 start site (Fig. 1). This second group of ESTs also contained putative polyadenylation sites (referred to as the distal site) and their 3V ends were oriented in the same direction, that is, antisense with respect to DIO3. In the mouse, no additional ESTs were found to match a similar, distal genomic region. These data showed that at least one other gene, which we have designated in the human and the mouse DIO3OS and Dio3os, respectively (OS for opposite strand), is transcribed from this locus in an orientation that is antisense to that of DIO3 and Dio3. Three human ESTs for DIO3OS deposited with GenBank (GenBank Accession Nos. AI290455, R73460, and AF305836) that contain the proximal polyadenylation site were sequenced. Comparison of these data with the genomic sequence revealed the occurrence of alternative splicing (Fig. 1, top three cDNAs). The most 5V sequence of these cDNAs corresponds to an exon (designated as 1) that shows 96% conservation between mouse and human and is located 1 kb upstream of the DIO3 transcription start, within the G+C-enriched region (Fig. 1). To characterize the exon – intron structure of DIO3OS further and to clarify whether both human proximal and distal polyadenylation sites belong to the same gene, we performed 3V and 5V rapid amplification of cDNA ends (RACE) and RT-PCR using RNA from several human tissues. Several DIO3OS cDNAs were obtained, subcloned, sequenced, and assigned a number for use in this article. The structures of these cDNAs are shown in Fig. 1, in which, at the bottom, we have numbered the exons most consistently observed from 1 to 6. The structures of these cDNAs reveal a complex splicing pattern. The sequence of the five cDNAs at the top of Fig. 1 shows alternative splicing of introns between exons 2, 3, and 4 and within exon 4. The structures of DIO3OS cDNAs 6 to 10 demonstrate that the distal polyadenylation site is also used for transcription from the DIO3OS gene. A very short variant of exon 4 is seen in cDNA 6, while up to three different splicing variants of exon 5 are observed in cDNAs 5, 8, and 9. This variable pattern of splicing leads to a complex mix of transcripts encoded by the DIO3OS gene.
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Expression pattern of human DIO3OS We used human DIO3OS cDNA 1 as a probe to analyze several human multiple-tissue Northern blots. The results are shown in Fig. 2 and reveal that the human DIO3OS gene is widely expressed in many tissues. A complex pattern of transcripts is observed. The most prominent bands are approximately 4.8, 3.7, 2.6, and 1.4 kb in size and are detected in most tissues. Other bands, some of them very small and some of them appearing to be doublets, are also observed, e.g., in pancreas (Figs. 2A, lane 8, and 2C, lane 1), heart (2B, lane 3), and muscle (2B, lane 8). A weaker band corresponding to a very large transcript (approx 8 kb) is observed in fetal lung (2D, lane 3). DIO3OS transcripts appear to be most abundant in testis and adrenal cortex (2A, lanes 4 and 5); prostate, bladder, and uterus (2B, lanes 1, 4, and 7); placenta (2C, lane 6); and fetal lung (2D, lane 3). Due to the splicing complexity, it is very difficult to establish which of the isolated cDNAs correspond to a specific band in the Northern blot, but an estimation of size suggests that most of these cDNAs are missing between 500 and 1500 bp of 5V sequence. In addition, probing of the human fetal Northern blot with cDNA 4 (3 kb in length, see Fig. 1), which comprises most of the ‘‘distal’’ genomic sequence, resulted in the identification of a single band in fetal lung of approximately 8 kb (data not shown) that coincides in size with that observed in Fig. 2D (lane 3). Taken together, these results suggest that the distal polyadenylation site is used to generate larger and less abundant transcripts, not easily detectable by Northern analysis. Exon – intron structure of the mouse Dio3os gene Exons in mouse Dio3os are structurally similar to human exons 1 to 4. Sequencing of mouse Dio3os cDNAs also reveals multiple splicing variants and their structures are summarized in Fig. 3. Interestingly, the first exon of mouse
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Dio3os cDNA 6 (see Fig. 3) includes a portion of the mouse Dio3 gene promoter and exon, oriented in the opposite direction. Despite the similarities in exon– intron structure between mouse and human, the exonic sequence is not highly conserved (less than 60% homology), except for exon 1 (96%), which is located in the highly conserved G+C region. Considering the interspecies similarities in the antisense gene structure between human and mouse, it is reasonable to expect that an open reading frame would also show some conservation and be shared—if only partially— by different cDNAs. No open reading frame meeting these criteria was found. However, a 408-bp open reading frame encompassing all or parts of exons 2, 3, and 4 is found in one of the human cDNAs deposited with GenBank (Accession No. AF305836). Multiple transcripts for human DIO3 A 2.1-kb mRNA coding for the D3 protein has been noted in human placenta [10], in different mouse and rat tissues, and in cell culture models [2,21]. A transcript of this size is expected given the location of the known mouse promoter [15] and the length of the cDNA. However, larger, less abundant transcripts have been detected by Northern blot analysis in hyperthyroid rat brain when a rat Dio3 probe is used [16]. Interestingly, the largest human cDNA for DIO3 isolated by Salvatore et al. [11] contains 180 bp that lay upstream of the expected transcription start site (as mapped previously for the mouse gene) and includes the TATA box and other promoter elements. We have obtained a similar cDNA fragment using 5VRACE and human fetal lung RNA (data not shown). These results in rat and human suggest the existence of larger transcripts for DIO3. To address further this issue of multiple DIO3 transcripts, we hybridized the same multiple-tissue human Northern blots with the larger human D3 cDNA mentioned
Fig. 2. Tissue expression pattern of the human DIO3OS. Commercially available multiple-tissue human Northern blots were probed with an antisense cDNA (GenBank Accession No. AI290455). Blots were autoradiographed for 24 h and then hybridized with a cyclophilin cDNA to provide a control for the amount of mRNA loaded per lane. (A) Lanes 1, stomach; 2, small intestine; 3, thymus; 4, testis; 5, adrenal cortex; 6, thyroid; 7, adrenal medulla; 8, pancreas. (B) Lanes 1, prostate; 2, stomach; 3, heart; 4, bladder; 5, small intestine; 6, colon; 7, uterus; 8, muscle. (C) Lanes 1, pancreas; 2, kidney; 3, muscle; 4, liver; 5, lung; 6, placenta; 7, brain; 8, heart. (D) Lanes 1, fetal kidney; 2, fetal liver; 3, fetal lung; 4, fetal brain. A, B, and C are aligned with the same RNA ladder indicated at the left side. Ladder for D is indicated at the right.
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Fig. 3. Structure of the mouse Dio3os gene. The alternative splicing of six cDNAs for the mouse Dio3os gene is shown. Exonic sequences are depicted as thick bars and splicing donor and acceptor sites are connected by lines. The cDNA number is displayed at the same level as the vertices of the corresponding splicing lines. These cDNAs were isolated in our laboratory, though the splicing pattern of some of them is compatible with that of some of the ESTs deposited in GenBank databases. Accession numbers are listed under Materials and methods.
above [11], which includes promoter elements. The results (Fig. 4) show a strong 2.1-kb band in placenta (Fig. 4C, lane 6), fetal liver (4D, lane 2), and uterus (4B, lane 7). In addition, two other bands, approximately 4.8 and 3.2 kb, were found in several other tissues. The 4.8-kb band was the most abundant in heart (4B, lane 3, and 4C, lane 8) and skeletal muscle (lane 8 in 4A and 4B), while the 3.2-kb band was predominant in testis (4A, lane 4), bladder, and uterus (4B, lanes 4 and 7). All or some of these three bands were present in other tissues as well, such as adrenal cortex (4A, lane 5), thyroid (4A, lane 6), prostate and stomach (4B, lanes 1 and 2), pancreas (4C, lane 1), and fetal lung (4D, lane 3). We also probed a different human Northern blot with a DIO3 cDNA fragment comprising only the last 200 bp of the coding region plus all the 3Vuntranslated region. In this case we detected again bands of 3.2 and 2.1 kb in size, plus
an additional 2.5-kb band in heart (Fig. 4E). However, no 4.8-kb transcript was detected, suggesting that this larger band is not a true D3-coding transcript, since it hybridizes only with the most 5V region of the larger D3 cDNA. Interestingly, the other human transcripts detected (2.5 and 3.2 kb in size) match the size of the larger Dio3 transcripts previously detected in hyperthyroid rat brain [16], indicating that they could be conserved D3-coding transcripts that contain an additional 5Vuntranslated region and are possibly transcribed from an alternative promoter. Mouse and human D3 promoters are conserved As noted, the G+C-rich region upstream of the DIO3 gene spans about 1.5 kb and is 83% homologous between mouse and human. A finer comparative analysis of the sequence shows that there are three smaller regions, 240,
Fig. 4. Expression pattern of human DIO3 transcripts. (A – D) Multiple-tissue human Northern blot analysis using as a probe the human D3 cDNA isolated by Salvatore et al. [11], which contains 180 bp of the 5Vproximal promoter region. The same blots from Fig. 2 were stripped and hybridized. Blots were autoradiographed for 3 days. Blots and lanes are the same as in Fig. 2. (E) A multiple-tissue human Northern blot was probed with a partial 1.1 kb DIO3 cDNA that resulted from PstI/ApaI digestion of the DIO3 cDNA and includes the end of the coding region and almost all of the 3V-UTR. The blot was autoradiographed for 3 days. It was then hybridized with a cyclophilin cDNA to provide a control of the amount of mRNA loaded per lane. Lanes: 1, brain; 2, heart; 3, kidney; 4, spleen; 5, liver; 6, colon.
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420, and 290 bp in size and designated A, B, and C (Fig. 5), that are conserved to an even higher degree (86, 92, and 96% homology, respectively). Regions A and C (Fig. 5A) coincide, respectively, with the previously characterized mouse Dio3 promoter [15] and exon 1 of the Dio3os gene. Comparison of the mouse and human basal promoter regions (240 bp of 5V flanking region) shows conservation of several promoter elements (Fig. 5), including the TATA box, which is critical for activity since we have previously demonstrated that its deletion results in loss of 98% of the Dio3 promoter activity [15].
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Two different human genomic fragments containing the conserved promoter region show potent promoter activity (500- to 2000-fold increase over that of the empty vector) when subcloned in front of a luciferase reporter gene and transfected into three different cell lines (Fig. 5B). These activities are similar to those obtained from a mouse promoter construct under the same conditions (Fig. 5B), though some differences between cell lines are observed. Stronger promotion of transcription is observed in cell lines expressing endogenous Dio3 (XTC-2 and BVS-1 cells) and certain promoter specificity is observed between species.
Fig. 5. (A) Homology between mouse and human D3 genes and promoter regions. The DIO3 exon is shown as a thick bar and the open reading frame (ORF) is shown in black. Highly conserved sequences within approximately 1.7 kb of 5Vflanking region (designated A, B, and C) are shown in thinner, gray bars. Not all restriction sites are shown. (B) Human DIO3 promoter activity. The promoter activity of genomic fragments corresponding to the human DIO3 5Vflanking region and to the described mouse Dio3 promoter [15] was tested in different cell lines by transient transfection experiments. A control plasmid expressing hgalactosidase was cotransfected to correct for transfection efficiency. Bars represent the means F standard deviations of three different cultures from two or more different experiments.
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For example, in rat BVS-1 cells, a fragment of the human DIO3 promoter has one-third of the promoter activity shown by a mouse Dio3 promoter fragment of similar length. DIO3OS 5V-end and promoter analysis Despite several attempts to isolate and clone the 5Vend of the DIO3OS gene, we have been unable to extend the 5V sequence above exon 1 by more than 70 bp. Apparently, the extremely high G+C content of this genomic region makes this very difficult when using various RT-PCR-based methods, probably due to strong secondary structures in both the RNA and the first-strand cDNA.
To investigate further the possibility that larger Dio3 mRNAs are transcribed by an alternative upstream promoter and to locate a genomic region that may contain the promoter of the Dio3os gene, we used available software (bimas.dcrt. nih.gov/molbio/proscan/ and www.molbiol.ox.ac.uk/) to analyze 20 kb of mouse and human genomic sequence (centered at the Dio3 gene). Three regions were predicted as potential sense or antisense promoters: the CG-rich region 5V of Dio3 and two other regions located within 6 kb 3Vof the Dio3 gene. We subcloned several human genomic fragments that corresponded to these regions and analyzed them for promoter activity using a luciferase reporter vector transfected into various cells lines (Fig. 6). Several human frag-
Fig. 6. Promoter analysis of the human DIO3 locus. (A) Location and orientation of the genomic fragments that were tested for promoter activity are indicated by arrows. (B) Promoter activity of the genomic fragments indicated in A. Activities were assayed by transient transfections in the cells indicated as described under Materials and methods. Results are expressed as a percentage of the activity measured for the DIO3 promoter (fragment 1), which was used as a positive control (absolute activities of the DIO3 promoter are shown in Fig. 5B). The data represent the means F standard deviations of determinations performed in triplicate cultures from one to three different transfection experiments. The numbers correspond to the genomic fragments indicated by arrows in A. N.D., not determined.
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ments showed significant promoter activity (Fig. 6B). A genomic region approximately 6 kb 3Vof the DIO3 gene and centered around conserved KpnI and XbaI restriction sites (fragments 2 and 8) had strong sense and antisense promoter activities. These were very significant in more than one cell line, if we consider that the fragment used as positive control (the DIO3 promoter, fragment 1) induces a hundreds or thousands fold increase in promoter activity as shown in Fig. 5. Shorter fragments from this region lose their promoter activity (fragments 5, 6, and 7). Also, significant sense promoter activity is found in a short fragment (No. 3) located
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5Vof the DIO3 exon. No promoter activity was detected in COS-7 cells when a fragment containing the DIO3 promoter in the antisense orientation (No. 10) was transfected. Strong promoter activity was also detected in various mouse genomic fragments (Fig. 7). Interestingly, a fragment corresponding to the region located 3V of Dio3 (No. 9) displayed antisense promoter activity that was higher than that measured for the Dio3 promoter (No. 1) when transfected into COS-7 cells. The promoter activity of this fragment was also significant in BeWo cells. As with the human locus, a fragment (No. 2) from this region also
Fig. 7. Promoter analysis of the mouse Dio3 locus. (A) Location and orientation of the genomic fragments that were tested for promoter activity are indicated by arrows. (B) Promoter activity of the genomic fragments indicated in A. Activities were assayed by transient transfections in the cells indicated as described under Materials and methods. Results are expressed as a percentage of the activity measured for the Dio3 promoter (fragment 1), which was used as a positive control (absolute activities of the Dio3 promoter are shown in Fig. 5B). The data represent the means F standard deviations of determinations performed in triplicate cultures from one to three different transfection experiments. The numbers correspond to the genomic fragments indicated by arrows in A. N.D., not determined.
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displayed strong promoter activity in the sense orientation, whereas shorter antisense oriented fragments (Nos. 8 and 10) did not promote transcription. A genomic fragment located upstream of the Dio3 exon (No. 4) showed strong promoter activity in the sense orientation in COS-7 and BeWo cells, but none in the D3-expressing BVS-1 and XTC-2 cells. No antisense promoter activity was detected in a fragment (No. 7) corresponding to the Dio3 5V flanking region. Taken together these results support the data obtained by Northern analysis in this and previous work [16] and suggest that there could be an upstream alternative promoter for Dio3. These results also suggest the possibility of an antisense promoter in the region located 3Vof Dio3 that may be the origin of the Dio3os transcripts. Analysis of this region revealed two groups of sequences, 170 and 220 bp in length, that were homologous (84 and 81%) between mouse and human (regions P1 and P2 in Fig. 8A). Region P1 contains putative AP-1 and serum-response elements, while region P2 contains a putative TATA box. Preliminary results indicate that
the serum-response element in P1 is functional. P1 and P2 regions are not able to promote transcription independently (see promoter activity of mouse fragments 3, 8, and 10 in Fig. 7 and human fragments 5, 6, and 7 in Fig. 6), but together they display robust promoter activity when oriented in either direction (mouse fragments 2 and 9 in Fig. 7 and human fragments 2 and 8 in Fig. 6). No ESTs have been found to match genomic sequences between the DIO3 gene and the presumed antisense promoter, nor has it been possible to isolate a partial cDNA for the antisense gene by connecting exon 1 with sequences for which the strong antisense promoter activity was found. This is probably due to the difficulties in performing reverse transcription and PCR through the highly C+G-enriched DIO3 5Vflanking region. However, we performed Northern analysis of RNA isolated from Dio3-expressing BVS-1 cells with three mouse genomic fragments. The first was a BamHI/KpnI 3.5-kb restriction fragment (Fig. 8B, top left) that comprised most of the known exon – intron structure of
Fig. 8. (A) Conserved sequences in the region with antisense promoter activity. The region displaying antisense promoter activity is expanded; conserved regions are shown in gray and conserved sequences are boxed. Arrows point to a conserved putative serum-response element (SRE) and a putative TATA box. (B) Northern blots of RNA from BVS-1 cells probed with the three different genomic fragments indicated in the map. Arrows point to two antisense transcripts hybridizing with probes 1 and 3 that, based on this analysis, include sequences that are located at both sides of the Dio3 gene.
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the antisense gene, the second fragment contained the Dio3 coding region, and the third was a 0.75-kb BglII restriction fragment located 3Vof the Dio3 gene (Fig. 8B, top). The first probe showed three major transcripts for the antisense gene in these cells, approximately 3.5, 1.6, and 1.2 kb in size. (Note that the relative abundance of these transcripts may not be accurately judged from this blot since the genomic probe used likely favors hybridization with larger, lessspliced transcripts.) As expected, the 2.2-kb transcript for the Dio3 mRNA is observed when using the second probe. Interestingly, the third probe revealed two weak transcripts that matched the size of those obtained with the first probe, suggesting that short exonic sequences for at least some of the antisense transcripts are located downstream of the Dio3 gene. This supports further the hypothesis that antisense exonic and promoter sequences are located 3Vof Dio3.
Discussion The DIO3 gene, located on human chromosome 14q32, codes for the type 3 deiodinase, an enzyme highly expressed in the pregnant uterus, the placenta, and the fetus. We have previously characterized the mouse Dio3 gene, demonstrated that all the coding and 3V untranslated regions are contained in a single exon, and identified its promoter [15]. Here we characterize the human DIO3 gene and show that the structures of the gene and promoter elements are essentially identical to those of the mouse. In addition we describe other mouse and human ESTs that correspond to genomic sequences within the DIO3 locus. The location of the poly(A)+ tail of these ESTs indicates that they are transcribed antisense of the DIO3 gene and belong to a second gene at this locus that has been designated DIO3OS. The DIO3OS gene is expressed in most tissues and multiple transcripts are detected by Northern analysis. The structures of these ESTs and various cDNAs generated by RT-PCR and RACE techniques show the presence of at least six exons and two alternate polyadenylation sites for human DIO3OS. Exons 1 to 4 and the proximal polyadenylation site are also found in mouse Dio3os. The distal polyadenylation site in human DIO3OS is not found in the mouse, and Northern analysis using RNA from human tissues indicates that only very large and/or less abundant transcripts are derived using this site. The possibility that this humanspecific distal polyadenylation site belongs to a different gene is unlikely but cannot be entirely excluded. The sequences of the DIO3OS cDNAs demonstrate a complex pattern of alternative splicing of transcripts from this gene; up to 13 different DIO3OS cDNAs have been identified. Although the exon– intron structure is conserved between mouse and human, the exonic sequence is not conserved, except for exon 1 (96% homology), which lies in the highly conserved G+C-rich region flanking the DIO3 gene. A comparison of the sizes of the bands in the Northern and DIO3OS cDNAs indicates that additional 5Vsequence has
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yet to be identified. However, we have not been able to extend this 5V sequence, presumably due to the high G+C content of this region. An open reading frame (408 bp of length) is found in one of the human DIO3OS cDNAs (Accession No. AF305836). However, this open reading frame is not found in the corresponding mouse cDNA and includes a start codon that shows little homology to the consensus Kozak sequence [22]. Furthermore, the predicted polypeptide sequence shows no significant homology with any entries in the protein databases. These observations suggest that DIO3OS is a noncoding gene, a finding proposed for several antisense genes present in other imprinted loci [23]. Alternatively, a coding region for the DIO3OS gene might lie within the highly conserved exon 1 and additional 5V sequence yet to be identified. Although the pattern of transcripts corresponding to the antisense human gene is more complex than that of DIO3, it is worth noting that there are overall similarities in the patterns of tissue expression of both genes. The tissues in which the antisense transcripts are most abundant correspond largely to those expressing higher levels of DIO3 transcripts such as uterus, testis, and bladder. In this regard, we have found no significant promoter activity in the antisense orientation for the DIO3 promoter, and further analysis of the region suggests that the promoter for the antisense gene is likely located 3V of DIO3. This suggests that both genes are not transcribed from the same promoter. An alternative explanation for the similar tissue expression pattern is the existence of common regulatory mechanisms within the imprinted chromosomal domain. Because of the complexity of this locus, we have investigated the genomic structure and the pattern of transcripts from the DIO3 gene. We have shown that a proximal human DIO3 promoter immediately upstream of the coding region is conserved between human and mouse with regard to sequence and location. In addition, larger DIO3 transcripts, 4.8, 3.2, and 2.5 kb in size, can be demonstrated when using a DIO3 cDNA that contains the coding region and the proximal DIO3 promoter sequence. The largest transcript is not detected when using a probe corresponding to the 3V half of the D3 cDNA. This observation indicates that the 4.8-kb band is not a D3-coding mRNA. Search of the human and mouse genomic sequences downstream of the DIO3 gene and 3VRACE experiments have not detected any alternative splicing or additional 3V untranslated sequence that might correspond to the DIO3 gene (data not shown). Taken together, these results suggest that the 3.2and 2.5-kb transcripts, previously detected in the rat brain [16], are likely D3-coding mRNAs containing additional 5V untranslated sequence and transcribed from an upstream alternative promoter. In fact, herein we have shown strong promoter activity of a mouse genomic fragment located 5Vof the previously characterized proximal promoter [15]. Promoter activity is also significant in a similarly located human fragment. These fragments contain sequences corresponding to exons 1 and 2 of the antisense gene in
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both species. Along with the isolation of a mouse Dio3os cDNA that includes sequences from the Dio3 promoter (cDNA 6), these results indicate that both Dio3 and Dio3os genes overlap at least in their 5Vregions and that the overlap occurs in the G+C-rich region. The occurrence of antisense promoter activity in a region located 3V of Dio3, the conservation of DNA sequences and putative promoter features, and the detection of mRNA transcripts of the same size using genomic probes at both sides of the Dio3 gene also suggest that the Dio3 gene lies within the structure of the Dio3os gene. A genomic arrangement in which the structures of two or more genes overlap in antisense orientation is rare, but has been described with imprinted genes [24]. Since the mouse Dio3 shows preferential expression from the paternal allele [17,18], this locus might be another of several examples of imprinted genes that are associated with overlapping antisense transcripts [25 – 27]. Such antisense transcripts are usually also imprinted and noncoding [24], have been proposed to serve regulatory functions [28], and in certain cases are a critical part of the mechanisms of genomic imprinting [29]. We speculate that this may also be the case for the Dio3os gene and its human homolog. More research is needed to characterize this interesting locus fully and to determine the role of DIO3OS.
Materials and methods Genomic clones, restriction mapping, vectors, subcloning and sequencing
kit from Stratagene. Sequencing was performed using an automated sequencer with fluorescent dye terminators (PE Applied Biosystems, Foster City, CA, USA). Cell cultures and transient transfections Human choriocarcinoma BeWo and COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). BVS-1 cells are a D3-expressing cell line we have generated by subculturing precursor cells from rat brown adipose tissue [20]. Both BeWo and BVS-1 cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (Life Technologies). COS-7 cells were grown in the same medium supplemented with 5% fetal bovine serum and 5% horse serum. Xenopus laevis XTC-2 and XL-2 cell lines were kindly provided by Dr. J. Tata (National Institute for Medical Research, The Ridgeway, London, UK) and cultured in 0.6 Leivobitz-15 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Life Technologies). All cell types were plated at a density of approximately 2 105 cells per well in sixwell culture dishes. Cells were transfected the next day using the FuGene6 reagent (Roche, Indianapolis, IN, USA) with 1 – 2 Ag of plasmid DNA and 0.5 Ag of h-galactosidase pCH110 expression vector (Pharmacia – Biotech) to correct for transfection efficiency. Luciferase and h-galactosidase activities were determined in cell lysates using assay reagents from Promega Corp. (Madison, WI, USA). Light emission was quantified using an EG&G Berthold microplate luminometer LB 96V (Wallac, Gaithersburg, MD). Northern analysis
Genomic human and mouse (129SVJ strain) P1 clones for the DIO3 gene were identified using a PCR-based screening system as described [14] and purchased from Genome Systems (St. Louis, MO, USA). cDNA clones corresponding to expressed sequence tags deposited with GenBank (Accession Nos. AF305836, AI29045, R73460, AW556460, and W97869) were obtained from the same company. Additional cDNAs generated in this work were sequenced and this information was deposited with GenBank. Their accession numbers are listed later in this section (see below) along with the methods used to generate them. Restriction maps of both human and mouse genomic clones were obtained by digestion with various restriction enzymes (Life Technologies, Gaithersburg, MD, USA). Southern blotting followed standard procedures using the rat and human D3 cDNAs as probes. Genomic DNA fragments were subcloned into pBluescript SK (Stratagene, La Jolla, CA, USA) and pXP2 [19]. The pCH110 h-galactosidase expression vector was obtained from Pharmacia – Biotech (Piscataway, NJ, USA). Genomic DNA fragments used in transient transfection studies were subcloned following standard procedures into the pXP2 vector [19], which contains a luciferase reporter gene. PCR products were subcloned using the PCR Script
Multiple-tissue human Northern blots were obtained from Clontech (Palo Alto, CA, USA) and Origene Technologies (Rockville, MD, USA). Blots were prehybridized at 42jC for 8 –20 h in 50% formamide, 5 SSC, 5 Denhardt’s, 50 mM sodium phosphate, pH 6.5, and 0.2% SDS and then hybridized at 42jC overnight with the specified radioactive probe in the same buffer containing 2 Denhardt’s. Blots were washed four times for 15 min at room temperature with 2 SSC/0.1% SDS and twice for 20 min with 0.1 SSC/ 0.1% SDS at 50 to 65jC and then autoradiographed. cDNAs and genomic DNA fragments were labeled with radioactive [32P] dCTP (ICN Biochemicals, Inc., Costa Mesa, CA, USA) using the Oligolabelling Kit (Pharmacia), and probes were purified through G-50 columns (Pharmacia). Total RNA from BVS-1 cells was isolated using a guanidinium chloride-based method as previously described [8]. Generation of cDNAs by RT-PCR and RACE DIO3OS and Dio3os cDNAs were generated by 5VRACE, by PCR using different human Marathon Ready cDNA kits (Clontech), or by RT-PCR of mRNAs obtained from Clontech or in our laboratory. ExTaq polymerase (Panvera Corp.,
A. Hernandez et al. / Genomics 83 (2004) 413–424
Madison, WI, USA) was used in all PCR reactions. Reverse transcriptase (Superscript II) was obtained from Life Technologies. Primer sequences are listed below. The technique and tissue sample used, GenBank accession numbers, and primers utilized to generate the specific cDNAs described in this work and numbered in Figs. 1 and 3 are as follows: human DIO3OS cDNAs 1 (GenBank AF469199) and 2 (GenBank AF469200) were obtained from human placenta by PCR using primers hc5low1 and hc5100; human DIO3OS cDNAs 3 (GenBank AF469201), 4 (GenBank AF469202), and 5 (GenBank AF469203) were obtained by 5VRACE from human fetal lung using the primer NGas2; human DIO3OS cDNA 6 (GenBank AF469204) was obtained from fetal lung by PCR using primers NG10 and hc51500; human DIO3OS cDNA 7 (GenBank AF460205) was obtained from fetal lung by PCR using primers NG8 and hc5120s; human DIO3OS cDNAs 8 (GenBank AF469206), 9 (GenBank AF469207), and 10 (GenBank AF469208) were obtained by RT-PCR from human liver using primers hc5low1 and Ngas2. Accession numbers for mouse Dio3os cDNAs indicated in Fig. 3 are No. 1, AY077457; No. 2, AY283182; No. 3, AY283181; No. 4, W97869; No. 5, AY077458; and No. 6, AY077459.
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Primers [11]
Primers were obtained from Life Technologies and their sequences are as follows: NGas2 (antisense), 5VCTCATGCAACGGAGGCTGAG-3V; hc5-100 (antisense), 5 V- AT C T G G G C C A G C C T G G G A G - 3 V; h N G 8 , 5 VCAAGCGGAGGACACTGCGG-3V; NG10 (antisense), 5V-CTGCGGCTGTACTTTGTCCCC-3V; hc5low1 (sense), 5V-CTGAGCCACCTTCGCGGAC-3V; hc5120s (sense), 5VGTTTGCCCAGCAGACCTCCC-3V; hc51500 (sense), 5VGCCCAATAGGAAGCACCTG-3V; mST75 (sense), 5V-CAGAGAGCGGAGCATGGTGG-3V. PCRs were performed in 200-Al tubes in a PTC-200 DNA Engine thermal cycler (MJ Research, Inc., Watertown, MA, USA). Typically, a ‘‘touchdown’’ PCR protocol was used. This consisted of an initial 5 to 10 cycles that included a denaturing step (92jC for 30 s), an annealing step (starting at 72jC and going down to 66jC), and an extension step (72jC, 3 min). This was followed by an additional 25 to 30 regular cycles (using 66jC as annealing temperature) and a 5-min final extension.
[12]
[13]
[14]
[15]
[16]
Acknowledgment We thank P. Reed Larsen (Brigham and Women’s Hospital, Harvard Medical School) for the human DIO3 cDNA clone.
[17]
[18]
References [19] [1] D.L. St. Germain, V.A. Galton, The deiodinase family of selenoproteins, Thyroid 7 (4) (1997) 655 – 668. [2] V.A. Galton, E. Martinez, A. Hernandez, E.A. St. Germain, J.M.
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Bates, D.L. St. Germain, Pregnant rat uterus expresses high levels of the type 3 iodothyronine deiodinase, J. Clin. Invest. 103 (7) (1999) 979 – 987. J.M. Koopdonk-Kool, J.J. de Vijlder, G.J. Veenboer, C. Ris-Stalpers, J.H. Kok, T. Vulsma, K. Boer, T.J. Visser, Type II and type III deiodinase activity in human placenta as a function of gestational age, J. Clin. Endocrinol. Metab. 81 (6) (1996) 2154 – 2158. E. Roti, L.E. Braverman, S. Fang, S. Alex, C.E. Emerson, Ontogenesis and placental inner ring thyroxine deiodinase and amniotic fluid 3,3V,5V-triiodothyronine concentration in the rat, Endocrinology 111 (1982) 959 – 963. T. Huang, I.J. Chopra, R. Boado, D.H. Solomon, G.N. Chua Teco, Thyroxine inner ring monodeiodinating activity in fetal tissues of the rat, Pediatr. Res. 23 (1988) 196 – 199. T. Huang, I.J. Chopra, A. Beredo, D.H. Solomon, G.N. Chua Teco, Skin is an active site of inner ring monodeiodination of thyroxine to 3,3V,5V-triiodothyronine, Endocrinology 117 (1985) 2106 – 2113. T. Huang, A. Beredo, D.H. Solomon, I.J. Chopra, The inner ring (5-) monodeiodination of thyroxine (T4) in cerebral cortex during fetal, neonatal, and adult life, Metabolism 35 (1986) 272 – 277. A. Hernandez, M.J. Obregon, Presence of growth factors-induced type III iodothyronine 5-deiodinase in cultured rat brown adipocytes, Endocrinology 136 (10) (1995) 4543 – 4550. F. Courtin, P. Liva, J.M. Gavaret, D. Toru-Delbauffe, M. Pierre, Induction of 5-deiodinase activity in astroglial cells by 12-O-tetradecanoylphorbol 13-acetate and fibroblast growth factors, J. Neurochem. 56 (1991) 1107 – 1113. W. Croteau, S.L. Whittemore, M.J. Schneider, D.L. St. Germain, Cloning and expression of a cDNA for a mammalian type III iodothyronine deiodinase, J. Biol. Chem. 270 (1995) 16569 – 16575. D. Salvatore, S.C. Low, M. Berry, A.L. Maia, J.W. Harney, W. Croteau, D.L. St. Germain, P.R. Larsen, Type 3 iodothyronine deiodinase: cloning, in vitro expression, and functional analysis of the placental selenoenzyme, J. Clin. Invest. 96 (5) (1995) 2421 – 2430. D.L. St. Germain, R. Schwartzman, W. Croteau, A. Kanamori, Z. Wang, D.D. Brown, V.A. Galton, A thyroid hormone regulated gene in Xenopus laevis encodes a type III iodothyronine 5-deiodinase, Proc. Natl. Acad. Sci. USA 91 (1994) 7767 – 7771 (Erratum appears in Proc. Natl. Acad. Sci. USA 91, 11282,1994). S. Van der Geyten, J.P. Sanders, E. Kaptein, V.M. Darras, E.R. Kuhn, J.L. Leonard, T.J. Visser, Expression of chicken hepatic type I and type III iodothyronine deiodinases during embryonic development, Endocrinology 138 (12) (1997) 5144 – 5152. A. Hernandez, J.P. Park, G.J. Lyon, T.K. Mohandas, D.L. St. Germain, Localization of the type 3 iodothyronine deiodinase (DIO3) gene to human chromosome 14q32 and mouse chromosome 12F1, Genomics 53 (1) (1998) 119 – 121. A. Hernandez, G.J. Lyon, M.J. Schneider, D.L. St. Germain, Isolation and characterization of the mouse gene for the type 3 iodothyronine deiodinase, Endocrinology 140 (1) (1999) 124 – 130. H.M. Tu, G. Legradi, T. Bartha, D. Salvatore, R.M. Lechan, P.R. Larsen, Regional expression of the type 3 iodothyronine deiodinase messenger ribonucleic acid in the rat central nervous system and its regulation by thyroid hormone, Endocrinology 140 (2) (1999) 784 – 790. C. Tsai, S.P. Lin, M. Ito, N. Takagi, S. Takada, A.C. Ferguson-Smith, Genomic imprinting contributes to thyroid hormone metabolism in the mouse embryo, Curr. Biol. 12 (2002) 1221 – 1226. A. Hernandez, S. Fiering, E. Martinez, V.A. Galton, D.L. St. Germain, The gene locus encoding iodothyronine deiodinase type 3 (Dio3) is imprinted in the fetus and expresses antisense transcripts, Endocrinology 143 (11) (2002) 4483 – 4486. S.K. Nordeen, Luciferase reporter gene vectors for analysis of promoters and enhancers, Biotechniques 6 (5) (1988) 454 – 457. A. Hernandez, D.L. St. Germain, Dexamethasone inhibits growth factor-induced type 3 deiodinase activity and mRNA expression in
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A. Hernandez et al. / Genomics 83 (2004) 413–424 a cultured cell line derived from rat neonatal brown fat vascular – stromal cells, Endocrinology 143 (7) (2002) 2652 – 2658. A. Hernandez, D.L. St. Germain, M.J. Obregon, Transcriptional activation of type III inner ring deiodinase by growth factors in cultured rat brown adipocytes, Endocrinology 139 (2) (1998) 634 – 639. M. Kozak, At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells, J. Mol. Biol. 196 (1987) 947 – 950. W. Reik, J. Walter, Genomic imprinting: parental influence on the genome, Nat. Rev. Genet. 2 (1) (2001) 21 – 32. W. Reik, J. Walter, Genomic imprinting: parental influence on the genome, Nat. Rev. Genet. 2 (1) (2001) 21 – 32. C. Rougeulle, H. Glatt, M. Lalande, The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain, Nat. Genet. 17 (1) (1997) 14 – 15.
[26] C. Rougeulle, C. Cardoso, M. Fontes, L. Colleaux, M. Lalande, An imprinted antisense RNA overlaps UBE3A and a second maternally expressed transcript, Nat. Genet. 19 (1) (1998) 15 – 16. [27] J. Peters, S.F. Wroe, C.A. Wells, H.J. Miller, D. Bodle, C.V. Beechey, C.M. Williamson, G. Kelsey, A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2, Proc. Natl. Acad. Sci. USA 96 (7) (1999) 3830 – 3835. [28] T. Li, T.H. Vu, Z.L. Zeng, B.T. Nguyen, B.E. Hayward, D.T. Bonthron, J.F. Hu, A.R. Hoffman, Tissue-specific expression of antisense and sense transcripts at the imprinted Gnas locus, Genomics 69 (3) (2000) 295 – 304. [29] F. Sleutels, R. Zwart, D.P. Barlow, The non-coding Air RNA is required for silencing autosomal imprinted genes, Nature 415 (6873) (2002) 810 – 813.
Complex organization and structure of sense and antisense transcripts $ expressed from the DIO3 gene imprinted locus Arturo Hernandez, * Maria E. Martinez, Walburga Croteau, and Donald L. St. Germain Department of Medicine, Dartmouth Medical School, Lebanon, NH 03756, USA Received 25 April 2003; accepted 22 August 2003
Abstract The human DIO3 gene and its mouse homolog, Dio3, map to chromosomes 14q32 and 12F1, respectively, and code for the type 3 deiodinase, an enzyme that inactivates thyroid hormones and is highly expressed during pregnancy and development. Mouse Dio3 is imprinted and preferentially expressed from the paternal allele in the fetus. We analyzed the human DIO3 genomic region and identified a gene (DIO3OS) that is transcribed in the antisense orientation. Multiple DIO3OS transcripts are expressed in most tissues. The structure of several DIO3OS cDNAs obtained by RT-PCR-based techniques reveals the occurrence of numerous splice variants. The exon – intron structures of DIO3OS are similar in mouse and human, but the homology of the exonic sequence is very low, except for the first exon, and no conserved open reading frame is present. We also detected DIO3 transcripts containing additional 5Vuntranslated sequence and a potential alternative upstream promoter for mouse Dio3. Exonic sequence of a Dio3os cDNA overlaps with the Dio3 promoter and strong promoter activity in the antisense orientation is detected in a genomic fragment located 3Vof mouse and human DIO3 but not in the DIO3 promoter region. These results suggest that the DIO3 gene may lie within the structure of the antisense gene, a complex arrangement often observed in imprinted loci. D 2003 Published by Elsevier Inc. Keywords: DIO3 promoter; Type 3 deiodinase; Antisense gene; Genomic imprinting
The Dio3 gene codes for the type 3 deiodinase (D3), a selenoprotein that plays a central role in thyroid hormone metabolism [1]. D3 enzymatic activity results in the inactivation of thyroid hormones, since it transforms both the prohormone T4 and the active hormone T3 into metabolites that are biologically inactive [1]. D3 displays a marked developmental pattern of expression: it is highly expressed in pregnant uterus [2], placenta [3,4], and the fetus [5], while in the adult, expression is low and limited to a few selected tissues [6,7]. D3 also is strongly induced by serum, growth factors, and phorbol esters in cell culture systems [8,9]. D3 cDNAs have been isolated from several species [10 – 13] and the mouse and human D3 genes (Dio3 and DIO3, $ Sequence data from this article have been deposited with the EMBL/ GenBank Data Libraries under Accession Nos. AF469199 – AF469208, AY077457 – AY077459, AY283181, AY283182, and W97869. * Corresponding author. Department of Medicine, Dartmouth Medical School, Borwell Building, Room 720 West, One Medical Center Drive, Lebanon, NH 03756. Fax: +1-603-650-6130. E-mail address: [email protected] (A. Hernandez).
0888-7543/$ - see front matter D 2003 Published by Elsevier Inc. doi:10.1016/j.ygeno.2003.08.024
respectively) have been localized to mouse chromosome 12F1 and human 14q32 [14]. Characterization of the mouse Dio3 gene has shown that the coding and 3V-untranslated regions are contained in a single exon, approximately 1.9 kb in length (GenBank Accession No. AF426023), which is transcribed using a promoter located immediately upstream [15]. The expression of the Dio3 gene results in a transcript of 2.1 kb in size that is most abundant in those rat and mouse tissues showing the highest D3 activity, such as decidual tissue [2] and placenta [11], and in growth factorstimulated cell culture systems [8,9]. However, larger transcripts have also been detected in the brain of hyperthyroid adult rats, when a rat D3 cDNA is used as a probe [16]. Interestingly, the mouse Dio3 gene is imprinted and preferentially expressed from the paternal allele in the developing fetus [17,18]. To characterize the DIO3 and Dio3 loci further, we have identified and partially characterized both human and mouse genes that overlap with the DIO3 and Dio3 promoter regions and are transcribed in an antisense orientation. We have designated these
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human and mouse genes DIO3OS and Dio3os, respectively, and speculate that they may play a role in the maintenance of Dio3 preferential monoallelic expression.
Results Identification and structure of human DIO3OS About 10 kb of the mouse and human DIO3 5V flanking regions was subcloned from previously isolated P1 genomic clones [14] and sequenced, and their sequences were compared. The first 1.5 kb of 5V flanking region was found to be extremely G+C rich (80% of the sequence) and was highly conserved between mouse and human (83% homology, Fig. 1). Further 5V the homology is low (less than 60%), except for a 180-bp region that is 80% homologous, contains a conserved BamHI site, and is located approximately at positions
Fig. 1. Structure of the human DIO3OS gene. The exon – intron structures, compared to the genomic sequence, of 13 cDNAs for the DIO3OS gene are shown. The conserved short region containing the proximal polyadenylation site is indicated in black and circled in the genomic diagram. Exons are depicted as thick bars and introns as thin bars. The first 3 cDNAs were found in GenBank (accession numbers shown), while the other 10 (numbered from 1 to 10) were generated in these studies from RNA of different human tissues by RT-PCR and RACE methods. The specific method and human RNA samples used to generate cDNAs 1 – 10, along with their GenBank accession numbers, are listed for each cDNA under Materials and methods. The six exons most consistently observed in the DIO3OS gene are designated at the bottom as 1 through 6.
4200 and 4000 from the human and mouse D3 transcription start sites, respectively (circled in Fig. 1). Mouse and human genomic sequences containing this short, conserved region were found to match the sequences of several mouse and human expressed sequence tags deposited in GenBank (Fig. 1). All these human and mouse ESTs contained a consensus polyadenylation site (referred to as the proximal site) and a poly(A)+ tail at their 3V end. The orientation of their 3V ends indicated that they were transcribed in an orientation antisense to that of DIO3. The 180 bp of conserved sequence was found to contain and flank the consensus polyadenylation site. Further analysis identified another group of human ESTs in GenBank that matched a human genomic region located 9.25 kb upstream of the DIO3 start site (Fig. 1). This second group of ESTs also contained putative polyadenylation sites (referred to as the distal site) and their 3V ends were oriented in the same direction, that is, antisense with respect to DIO3. In the mouse, no additional ESTs were found to match a similar, distal genomic region. These data showed that at least one other gene, which we have designated in the human and the mouse DIO3OS and Dio3os, respectively (OS for opposite strand), is transcribed from this locus in an orientation that is antisense to that of DIO3 and Dio3. Three human ESTs for DIO3OS deposited with GenBank (GenBank Accession Nos. AI290455, R73460, and AF305836) that contain the proximal polyadenylation site were sequenced. Comparison of these data with the genomic sequence revealed the occurrence of alternative splicing (Fig. 1, top three cDNAs). The most 5V sequence of these cDNAs corresponds to an exon (designated as 1) that shows 96% conservation between mouse and human and is located 1 kb upstream of the DIO3 transcription start, within the G+C-enriched region (Fig. 1). To characterize the exon – intron structure of DIO3OS further and to clarify whether both human proximal and distal polyadenylation sites belong to the same gene, we performed 3V and 5V rapid amplification of cDNA ends (RACE) and RT-PCR using RNA from several human tissues. Several DIO3OS cDNAs were obtained, subcloned, sequenced, and assigned a number for use in this article. The structures of these cDNAs are shown in Fig. 1, in which, at the bottom, we have numbered the exons most consistently observed from 1 to 6. The structures of these cDNAs reveal a complex splicing pattern. The sequence of the five cDNAs at the top of Fig. 1 shows alternative splicing of introns between exons 2, 3, and 4 and within exon 4. The structures of DIO3OS cDNAs 6 to 10 demonstrate that the distal polyadenylation site is also used for transcription from the DIO3OS gene. A very short variant of exon 4 is seen in cDNA 6, while up to three different splicing variants of exon 5 are observed in cDNAs 5, 8, and 9. This variable pattern of splicing leads to a complex mix of transcripts encoded by the DIO3OS gene.
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Expression pattern of human DIO3OS We used human DIO3OS cDNA 1 as a probe to analyze several human multiple-tissue Northern blots. The results are shown in Fig. 2 and reveal that the human DIO3OS gene is widely expressed in many tissues. A complex pattern of transcripts is observed. The most prominent bands are approximately 4.8, 3.7, 2.6, and 1.4 kb in size and are detected in most tissues. Other bands, some of them very small and some of them appearing to be doublets, are also observed, e.g., in pancreas (Figs. 2A, lane 8, and 2C, lane 1), heart (2B, lane 3), and muscle (2B, lane 8). A weaker band corresponding to a very large transcript (approx 8 kb) is observed in fetal lung (2D, lane 3). DIO3OS transcripts appear to be most abundant in testis and adrenal cortex (2A, lanes 4 and 5); prostate, bladder, and uterus (2B, lanes 1, 4, and 7); placenta (2C, lane 6); and fetal lung (2D, lane 3). Due to the splicing complexity, it is very difficult to establish which of the isolated cDNAs correspond to a specific band in the Northern blot, but an estimation of size suggests that most of these cDNAs are missing between 500 and 1500 bp of 5V sequence. In addition, probing of the human fetal Northern blot with cDNA 4 (3 kb in length, see Fig. 1), which comprises most of the ‘‘distal’’ genomic sequence, resulted in the identification of a single band in fetal lung of approximately 8 kb (data not shown) that coincides in size with that observed in Fig. 2D (lane 3). Taken together, these results suggest that the distal polyadenylation site is used to generate larger and less abundant transcripts, not easily detectable by Northern analysis. Exon – intron structure of the mouse Dio3os gene Exons in mouse Dio3os are structurally similar to human exons 1 to 4. Sequencing of mouse Dio3os cDNAs also reveals multiple splicing variants and their structures are summarized in Fig. 3. Interestingly, the first exon of mouse
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Dio3os cDNA 6 (see Fig. 3) includes a portion of the mouse Dio3 gene promoter and exon, oriented in the opposite direction. Despite the similarities in exon– intron structure between mouse and human, the exonic sequence is not highly conserved (less than 60% homology), except for exon 1 (96%), which is located in the highly conserved G+C region. Considering the interspecies similarities in the antisense gene structure between human and mouse, it is reasonable to expect that an open reading frame would also show some conservation and be shared—if only partially— by different cDNAs. No open reading frame meeting these criteria was found. However, a 408-bp open reading frame encompassing all or parts of exons 2, 3, and 4 is found in one of the human cDNAs deposited with GenBank (Accession No. AF305836). Multiple transcripts for human DIO3 A 2.1-kb mRNA coding for the D3 protein has been noted in human placenta [10], in different mouse and rat tissues, and in cell culture models [2,21]. A transcript of this size is expected given the location of the known mouse promoter [15] and the length of the cDNA. However, larger, less abundant transcripts have been detected by Northern blot analysis in hyperthyroid rat brain when a rat Dio3 probe is used [16]. Interestingly, the largest human cDNA for DIO3 isolated by Salvatore et al. [11] contains 180 bp that lay upstream of the expected transcription start site (as mapped previously for the mouse gene) and includes the TATA box and other promoter elements. We have obtained a similar cDNA fragment using 5VRACE and human fetal lung RNA (data not shown). These results in rat and human suggest the existence of larger transcripts for DIO3. To address further this issue of multiple DIO3 transcripts, we hybridized the same multiple-tissue human Northern blots with the larger human D3 cDNA mentioned
Fig. 2. Tissue expression pattern of the human DIO3OS. Commercially available multiple-tissue human Northern blots were probed with an antisense cDNA (GenBank Accession No. AI290455). Blots were autoradiographed for 24 h and then hybridized with a cyclophilin cDNA to provide a control for the amount of mRNA loaded per lane. (A) Lanes 1, stomach; 2, small intestine; 3, thymus; 4, testis; 5, adrenal cortex; 6, thyroid; 7, adrenal medulla; 8, pancreas. (B) Lanes 1, prostate; 2, stomach; 3, heart; 4, bladder; 5, small intestine; 6, colon; 7, uterus; 8, muscle. (C) Lanes 1, pancreas; 2, kidney; 3, muscle; 4, liver; 5, lung; 6, placenta; 7, brain; 8, heart. (D) Lanes 1, fetal kidney; 2, fetal liver; 3, fetal lung; 4, fetal brain. A, B, and C are aligned with the same RNA ladder indicated at the left side. Ladder for D is indicated at the right.
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Fig. 3. Structure of the mouse Dio3os gene. The alternative splicing of six cDNAs for the mouse Dio3os gene is shown. Exonic sequences are depicted as thick bars and splicing donor and acceptor sites are connected by lines. The cDNA number is displayed at the same level as the vertices of the corresponding splicing lines. These cDNAs were isolated in our laboratory, though the splicing pattern of some of them is compatible with that of some of the ESTs deposited in GenBank databases. Accession numbers are listed under Materials and methods.
above [11], which includes promoter elements. The results (Fig. 4) show a strong 2.1-kb band in placenta (Fig. 4C, lane 6), fetal liver (4D, lane 2), and uterus (4B, lane 7). In addition, two other bands, approximately 4.8 and 3.2 kb, were found in several other tissues. The 4.8-kb band was the most abundant in heart (4B, lane 3, and 4C, lane 8) and skeletal muscle (lane 8 in 4A and 4B), while the 3.2-kb band was predominant in testis (4A, lane 4), bladder, and uterus (4B, lanes 4 and 7). All or some of these three bands were present in other tissues as well, such as adrenal cortex (4A, lane 5), thyroid (4A, lane 6), prostate and stomach (4B, lanes 1 and 2), pancreas (4C, lane 1), and fetal lung (4D, lane 3). We also probed a different human Northern blot with a DIO3 cDNA fragment comprising only the last 200 bp of the coding region plus all the 3Vuntranslated region. In this case we detected again bands of 3.2 and 2.1 kb in size, plus
an additional 2.5-kb band in heart (Fig. 4E). However, no 4.8-kb transcript was detected, suggesting that this larger band is not a true D3-coding transcript, since it hybridizes only with the most 5V region of the larger D3 cDNA. Interestingly, the other human transcripts detected (2.5 and 3.2 kb in size) match the size of the larger Dio3 transcripts previously detected in hyperthyroid rat brain [16], indicating that they could be conserved D3-coding transcripts that contain an additional 5Vuntranslated region and are possibly transcribed from an alternative promoter. Mouse and human D3 promoters are conserved As noted, the G+C-rich region upstream of the DIO3 gene spans about 1.5 kb and is 83% homologous between mouse and human. A finer comparative analysis of the sequence shows that there are three smaller regions, 240,
Fig. 4. Expression pattern of human DIO3 transcripts. (A – D) Multiple-tissue human Northern blot analysis using as a probe the human D3 cDNA isolated by Salvatore et al. [11], which contains 180 bp of the 5Vproximal promoter region. The same blots from Fig. 2 were stripped and hybridized. Blots were autoradiographed for 3 days. Blots and lanes are the same as in Fig. 2. (E) A multiple-tissue human Northern blot was probed with a partial 1.1 kb DIO3 cDNA that resulted from PstI/ApaI digestion of the DIO3 cDNA and includes the end of the coding region and almost all of the 3V-UTR. The blot was autoradiographed for 3 days. It was then hybridized with a cyclophilin cDNA to provide a control of the amount of mRNA loaded per lane. Lanes: 1, brain; 2, heart; 3, kidney; 4, spleen; 5, liver; 6, colon.
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420, and 290 bp in size and designated A, B, and C (Fig. 5), that are conserved to an even higher degree (86, 92, and 96% homology, respectively). Regions A and C (Fig. 5A) coincide, respectively, with the previously characterized mouse Dio3 promoter [15] and exon 1 of the Dio3os gene. Comparison of the mouse and human basal promoter regions (240 bp of 5V flanking region) shows conservation of several promoter elements (Fig. 5), including the TATA box, which is critical for activity since we have previously demonstrated that its deletion results in loss of 98% of the Dio3 promoter activity [15].
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Two different human genomic fragments containing the conserved promoter region show potent promoter activity (500- to 2000-fold increase over that of the empty vector) when subcloned in front of a luciferase reporter gene and transfected into three different cell lines (Fig. 5B). These activities are similar to those obtained from a mouse promoter construct under the same conditions (Fig. 5B), though some differences between cell lines are observed. Stronger promotion of transcription is observed in cell lines expressing endogenous Dio3 (XTC-2 and BVS-1 cells) and certain promoter specificity is observed between species.
Fig. 5. (A) Homology between mouse and human D3 genes and promoter regions. The DIO3 exon is shown as a thick bar and the open reading frame (ORF) is shown in black. Highly conserved sequences within approximately 1.7 kb of 5Vflanking region (designated A, B, and C) are shown in thinner, gray bars. Not all restriction sites are shown. (B) Human DIO3 promoter activity. The promoter activity of genomic fragments corresponding to the human DIO3 5Vflanking region and to the described mouse Dio3 promoter [15] was tested in different cell lines by transient transfection experiments. A control plasmid expressing hgalactosidase was cotransfected to correct for transfection efficiency. Bars represent the means F standard deviations of three different cultures from two or more different experiments.
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For example, in rat BVS-1 cells, a fragment of the human DIO3 promoter has one-third of the promoter activity shown by a mouse Dio3 promoter fragment of similar length. DIO3OS 5V-end and promoter analysis Despite several attempts to isolate and clone the 5Vend of the DIO3OS gene, we have been unable to extend the 5V sequence above exon 1 by more than 70 bp. Apparently, the extremely high G+C content of this genomic region makes this very difficult when using various RT-PCR-based methods, probably due to strong secondary structures in both the RNA and the first-strand cDNA.
To investigate further the possibility that larger Dio3 mRNAs are transcribed by an alternative upstream promoter and to locate a genomic region that may contain the promoter of the Dio3os gene, we used available software (bimas.dcrt. nih.gov/molbio/proscan/ and www.molbiol.ox.ac.uk/) to analyze 20 kb of mouse and human genomic sequence (centered at the Dio3 gene). Three regions were predicted as potential sense or antisense promoters: the CG-rich region 5V of Dio3 and two other regions located within 6 kb 3Vof the Dio3 gene. We subcloned several human genomic fragments that corresponded to these regions and analyzed them for promoter activity using a luciferase reporter vector transfected into various cells lines (Fig. 6). Several human frag-
Fig. 6. Promoter analysis of the human DIO3 locus. (A) Location and orientation of the genomic fragments that were tested for promoter activity are indicated by arrows. (B) Promoter activity of the genomic fragments indicated in A. Activities were assayed by transient transfections in the cells indicated as described under Materials and methods. Results are expressed as a percentage of the activity measured for the DIO3 promoter (fragment 1), which was used as a positive control (absolute activities of the DIO3 promoter are shown in Fig. 5B). The data represent the means F standard deviations of determinations performed in triplicate cultures from one to three different transfection experiments. The numbers correspond to the genomic fragments indicated by arrows in A. N.D., not determined.
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ments showed significant promoter activity (Fig. 6B). A genomic region approximately 6 kb 3Vof the DIO3 gene and centered around conserved KpnI and XbaI restriction sites (fragments 2 and 8) had strong sense and antisense promoter activities. These were very significant in more than one cell line, if we consider that the fragment used as positive control (the DIO3 promoter, fragment 1) induces a hundreds or thousands fold increase in promoter activity as shown in Fig. 5. Shorter fragments from this region lose their promoter activity (fragments 5, 6, and 7). Also, significant sense promoter activity is found in a short fragment (No. 3) located
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5Vof the DIO3 exon. No promoter activity was detected in COS-7 cells when a fragment containing the DIO3 promoter in the antisense orientation (No. 10) was transfected. Strong promoter activity was also detected in various mouse genomic fragments (Fig. 7). Interestingly, a fragment corresponding to the region located 3V of Dio3 (No. 9) displayed antisense promoter activity that was higher than that measured for the Dio3 promoter (No. 1) when transfected into COS-7 cells. The promoter activity of this fragment was also significant in BeWo cells. As with the human locus, a fragment (No. 2) from this region also
Fig. 7. Promoter analysis of the mouse Dio3 locus. (A) Location and orientation of the genomic fragments that were tested for promoter activity are indicated by arrows. (B) Promoter activity of the genomic fragments indicated in A. Activities were assayed by transient transfections in the cells indicated as described under Materials and methods. Results are expressed as a percentage of the activity measured for the Dio3 promoter (fragment 1), which was used as a positive control (absolute activities of the Dio3 promoter are shown in Fig. 5B). The data represent the means F standard deviations of determinations performed in triplicate cultures from one to three different transfection experiments. The numbers correspond to the genomic fragments indicated by arrows in A. N.D., not determined.
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displayed strong promoter activity in the sense orientation, whereas shorter antisense oriented fragments (Nos. 8 and 10) did not promote transcription. A genomic fragment located upstream of the Dio3 exon (No. 4) showed strong promoter activity in the sense orientation in COS-7 and BeWo cells, but none in the D3-expressing BVS-1 and XTC-2 cells. No antisense promoter activity was detected in a fragment (No. 7) corresponding to the Dio3 5V flanking region. Taken together these results support the data obtained by Northern analysis in this and previous work [16] and suggest that there could be an upstream alternative promoter for Dio3. These results also suggest the possibility of an antisense promoter in the region located 3Vof Dio3 that may be the origin of the Dio3os transcripts. Analysis of this region revealed two groups of sequences, 170 and 220 bp in length, that were homologous (84 and 81%) between mouse and human (regions P1 and P2 in Fig. 8A). Region P1 contains putative AP-1 and serum-response elements, while region P2 contains a putative TATA box. Preliminary results indicate that
the serum-response element in P1 is functional. P1 and P2 regions are not able to promote transcription independently (see promoter activity of mouse fragments 3, 8, and 10 in Fig. 7 and human fragments 5, 6, and 7 in Fig. 6), but together they display robust promoter activity when oriented in either direction (mouse fragments 2 and 9 in Fig. 7 and human fragments 2 and 8 in Fig. 6). No ESTs have been found to match genomic sequences between the DIO3 gene and the presumed antisense promoter, nor has it been possible to isolate a partial cDNA for the antisense gene by connecting exon 1 with sequences for which the strong antisense promoter activity was found. This is probably due to the difficulties in performing reverse transcription and PCR through the highly C+G-enriched DIO3 5Vflanking region. However, we performed Northern analysis of RNA isolated from Dio3-expressing BVS-1 cells with three mouse genomic fragments. The first was a BamHI/KpnI 3.5-kb restriction fragment (Fig. 8B, top left) that comprised most of the known exon – intron structure of
Fig. 8. (A) Conserved sequences in the region with antisense promoter activity. The region displaying antisense promoter activity is expanded; conserved regions are shown in gray and conserved sequences are boxed. Arrows point to a conserved putative serum-response element (SRE) and a putative TATA box. (B) Northern blots of RNA from BVS-1 cells probed with the three different genomic fragments indicated in the map. Arrows point to two antisense transcripts hybridizing with probes 1 and 3 that, based on this analysis, include sequences that are located at both sides of the Dio3 gene.
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the antisense gene, the second fragment contained the Dio3 coding region, and the third was a 0.75-kb BglII restriction fragment located 3Vof the Dio3 gene (Fig. 8B, top). The first probe showed three major transcripts for the antisense gene in these cells, approximately 3.5, 1.6, and 1.2 kb in size. (Note that the relative abundance of these transcripts may not be accurately judged from this blot since the genomic probe used likely favors hybridization with larger, lessspliced transcripts.) As expected, the 2.2-kb transcript for the Dio3 mRNA is observed when using the second probe. Interestingly, the third probe revealed two weak transcripts that matched the size of those obtained with the first probe, suggesting that short exonic sequences for at least some of the antisense transcripts are located downstream of the Dio3 gene. This supports further the hypothesis that antisense exonic and promoter sequences are located 3Vof Dio3.
Discussion The DIO3 gene, located on human chromosome 14q32, codes for the type 3 deiodinase, an enzyme highly expressed in the pregnant uterus, the placenta, and the fetus. We have previously characterized the mouse Dio3 gene, demonstrated that all the coding and 3V untranslated regions are contained in a single exon, and identified its promoter [15]. Here we characterize the human DIO3 gene and show that the structures of the gene and promoter elements are essentially identical to those of the mouse. In addition we describe other mouse and human ESTs that correspond to genomic sequences within the DIO3 locus. The location of the poly(A)+ tail of these ESTs indicates that they are transcribed antisense of the DIO3 gene and belong to a second gene at this locus that has been designated DIO3OS. The DIO3OS gene is expressed in most tissues and multiple transcripts are detected by Northern analysis. The structures of these ESTs and various cDNAs generated by RT-PCR and RACE techniques show the presence of at least six exons and two alternate polyadenylation sites for human DIO3OS. Exons 1 to 4 and the proximal polyadenylation site are also found in mouse Dio3os. The distal polyadenylation site in human DIO3OS is not found in the mouse, and Northern analysis using RNA from human tissues indicates that only very large and/or less abundant transcripts are derived using this site. The possibility that this humanspecific distal polyadenylation site belongs to a different gene is unlikely but cannot be entirely excluded. The sequences of the DIO3OS cDNAs demonstrate a complex pattern of alternative splicing of transcripts from this gene; up to 13 different DIO3OS cDNAs have been identified. Although the exon– intron structure is conserved between mouse and human, the exonic sequence is not conserved, except for exon 1 (96% homology), which lies in the highly conserved G+C-rich region flanking the DIO3 gene. A comparison of the sizes of the bands in the Northern and DIO3OS cDNAs indicates that additional 5Vsequence has
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yet to be identified. However, we have not been able to extend this 5V sequence, presumably due to the high G+C content of this region. An open reading frame (408 bp of length) is found in one of the human DIO3OS cDNAs (Accession No. AF305836). However, this open reading frame is not found in the corresponding mouse cDNA and includes a start codon that shows little homology to the consensus Kozak sequence [22]. Furthermore, the predicted polypeptide sequence shows no significant homology with any entries in the protein databases. These observations suggest that DIO3OS is a noncoding gene, a finding proposed for several antisense genes present in other imprinted loci [23]. Alternatively, a coding region for the DIO3OS gene might lie within the highly conserved exon 1 and additional 5V sequence yet to be identified. Although the pattern of transcripts corresponding to the antisense human gene is more complex than that of DIO3, it is worth noting that there are overall similarities in the patterns of tissue expression of both genes. The tissues in which the antisense transcripts are most abundant correspond largely to those expressing higher levels of DIO3 transcripts such as uterus, testis, and bladder. In this regard, we have found no significant promoter activity in the antisense orientation for the DIO3 promoter, and further analysis of the region suggests that the promoter for the antisense gene is likely located 3V of DIO3. This suggests that both genes are not transcribed from the same promoter. An alternative explanation for the similar tissue expression pattern is the existence of common regulatory mechanisms within the imprinted chromosomal domain. Because of the complexity of this locus, we have investigated the genomic structure and the pattern of transcripts from the DIO3 gene. We have shown that a proximal human DIO3 promoter immediately upstream of the coding region is conserved between human and mouse with regard to sequence and location. In addition, larger DIO3 transcripts, 4.8, 3.2, and 2.5 kb in size, can be demonstrated when using a DIO3 cDNA that contains the coding region and the proximal DIO3 promoter sequence. The largest transcript is not detected when using a probe corresponding to the 3V half of the D3 cDNA. This observation indicates that the 4.8-kb band is not a D3-coding mRNA. Search of the human and mouse genomic sequences downstream of the DIO3 gene and 3VRACE experiments have not detected any alternative splicing or additional 3V untranslated sequence that might correspond to the DIO3 gene (data not shown). Taken together, these results suggest that the 3.2and 2.5-kb transcripts, previously detected in the rat brain [16], are likely D3-coding mRNAs containing additional 5V untranslated sequence and transcribed from an upstream alternative promoter. In fact, herein we have shown strong promoter activity of a mouse genomic fragment located 5Vof the previously characterized proximal promoter [15]. Promoter activity is also significant in a similarly located human fragment. These fragments contain sequences corresponding to exons 1 and 2 of the antisense gene in
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both species. Along with the isolation of a mouse Dio3os cDNA that includes sequences from the Dio3 promoter (cDNA 6), these results indicate that both Dio3 and Dio3os genes overlap at least in their 5Vregions and that the overlap occurs in the G+C-rich region. The occurrence of antisense promoter activity in a region located 3V of Dio3, the conservation of DNA sequences and putative promoter features, and the detection of mRNA transcripts of the same size using genomic probes at both sides of the Dio3 gene also suggest that the Dio3 gene lies within the structure of the Dio3os gene. A genomic arrangement in which the structures of two or more genes overlap in antisense orientation is rare, but has been described with imprinted genes [24]. Since the mouse Dio3 shows preferential expression from the paternal allele [17,18], this locus might be another of several examples of imprinted genes that are associated with overlapping antisense transcripts [25 – 27]. Such antisense transcripts are usually also imprinted and noncoding [24], have been proposed to serve regulatory functions [28], and in certain cases are a critical part of the mechanisms of genomic imprinting [29]. We speculate that this may also be the case for the Dio3os gene and its human homolog. More research is needed to characterize this interesting locus fully and to determine the role of DIO3OS.
Materials and methods Genomic clones, restriction mapping, vectors, subcloning and sequencing
kit from Stratagene. Sequencing was performed using an automated sequencer with fluorescent dye terminators (PE Applied Biosystems, Foster City, CA, USA). Cell cultures and transient transfections Human choriocarcinoma BeWo and COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). BVS-1 cells are a D3-expressing cell line we have generated by subculturing precursor cells from rat brown adipose tissue [20]. Both BeWo and BVS-1 cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (Life Technologies). COS-7 cells were grown in the same medium supplemented with 5% fetal bovine serum and 5% horse serum. Xenopus laevis XTC-2 and XL-2 cell lines were kindly provided by Dr. J. Tata (National Institute for Medical Research, The Ridgeway, London, UK) and cultured in 0.6 Leivobitz-15 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Life Technologies). All cell types were plated at a density of approximately 2 105 cells per well in sixwell culture dishes. Cells were transfected the next day using the FuGene6 reagent (Roche, Indianapolis, IN, USA) with 1 – 2 Ag of plasmid DNA and 0.5 Ag of h-galactosidase pCH110 expression vector (Pharmacia – Biotech) to correct for transfection efficiency. Luciferase and h-galactosidase activities were determined in cell lysates using assay reagents from Promega Corp. (Madison, WI, USA). Light emission was quantified using an EG&G Berthold microplate luminometer LB 96V (Wallac, Gaithersburg, MD). Northern analysis
Genomic human and mouse (129SVJ strain) P1 clones for the DIO3 gene were identified using a PCR-based screening system as described [14] and purchased from Genome Systems (St. Louis, MO, USA). cDNA clones corresponding to expressed sequence tags deposited with GenBank (Accession Nos. AF305836, AI29045, R73460, AW556460, and W97869) were obtained from the same company. Additional cDNAs generated in this work were sequenced and this information was deposited with GenBank. Their accession numbers are listed later in this section (see below) along with the methods used to generate them. Restriction maps of both human and mouse genomic clones were obtained by digestion with various restriction enzymes (Life Technologies, Gaithersburg, MD, USA). Southern blotting followed standard procedures using the rat and human D3 cDNAs as probes. Genomic DNA fragments were subcloned into pBluescript SK (Stratagene, La Jolla, CA, USA) and pXP2 [19]. The pCH110 h-galactosidase expression vector was obtained from Pharmacia – Biotech (Piscataway, NJ, USA). Genomic DNA fragments used in transient transfection studies were subcloned following standard procedures into the pXP2 vector [19], which contains a luciferase reporter gene. PCR products were subcloned using the PCR Script
Multiple-tissue human Northern blots were obtained from Clontech (Palo Alto, CA, USA) and Origene Technologies (Rockville, MD, USA). Blots were prehybridized at 42jC for 8 –20 h in 50% formamide, 5 SSC, 5 Denhardt’s, 50 mM sodium phosphate, pH 6.5, and 0.2% SDS and then hybridized at 42jC overnight with the specified radioactive probe in the same buffer containing 2 Denhardt’s. Blots were washed four times for 15 min at room temperature with 2 SSC/0.1% SDS and twice for 20 min with 0.1 SSC/ 0.1% SDS at 50 to 65jC and then autoradiographed. cDNAs and genomic DNA fragments were labeled with radioactive [32P] dCTP (ICN Biochemicals, Inc., Costa Mesa, CA, USA) using the Oligolabelling Kit (Pharmacia), and probes were purified through G-50 columns (Pharmacia). Total RNA from BVS-1 cells was isolated using a guanidinium chloride-based method as previously described [8]. Generation of cDNAs by RT-PCR and RACE DIO3OS and Dio3os cDNAs were generated by 5VRACE, by PCR using different human Marathon Ready cDNA kits (Clontech), or by RT-PCR of mRNAs obtained from Clontech or in our laboratory. ExTaq polymerase (Panvera Corp.,
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Madison, WI, USA) was used in all PCR reactions. Reverse transcriptase (Superscript II) was obtained from Life Technologies. Primer sequences are listed below. The technique and tissue sample used, GenBank accession numbers, and primers utilized to generate the specific cDNAs described in this work and numbered in Figs. 1 and 3 are as follows: human DIO3OS cDNAs 1 (GenBank AF469199) and 2 (GenBank AF469200) were obtained from human placenta by PCR using primers hc5low1 and hc5100; human DIO3OS cDNAs 3 (GenBank AF469201), 4 (GenBank AF469202), and 5 (GenBank AF469203) were obtained by 5VRACE from human fetal lung using the primer NGas2; human DIO3OS cDNA 6 (GenBank AF469204) was obtained from fetal lung by PCR using primers NG10 and hc51500; human DIO3OS cDNA 7 (GenBank AF460205) was obtained from fetal lung by PCR using primers NG8 and hc5120s; human DIO3OS cDNAs 8 (GenBank AF469206), 9 (GenBank AF469207), and 10 (GenBank AF469208) were obtained by RT-PCR from human liver using primers hc5low1 and Ngas2. Accession numbers for mouse Dio3os cDNAs indicated in Fig. 3 are No. 1, AY077457; No. 2, AY283182; No. 3, AY283181; No. 4, W97869; No. 5, AY077458; and No. 6, AY077459.
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Primers [11]
Primers were obtained from Life Technologies and their sequences are as follows: NGas2 (antisense), 5VCTCATGCAACGGAGGCTGAG-3V; hc5-100 (antisense), 5 V- AT C T G G G C C A G C C T G G G A G - 3 V; h N G 8 , 5 VCAAGCGGAGGACACTGCGG-3V; NG10 (antisense), 5V-CTGCGGCTGTACTTTGTCCCC-3V; hc5low1 (sense), 5V-CTGAGCCACCTTCGCGGAC-3V; hc5120s (sense), 5VGTTTGCCCAGCAGACCTCCC-3V; hc51500 (sense), 5VGCCCAATAGGAAGCACCTG-3V; mST75 (sense), 5V-CAGAGAGCGGAGCATGGTGG-3V. PCRs were performed in 200-Al tubes in a PTC-200 DNA Engine thermal cycler (MJ Research, Inc., Watertown, MA, USA). Typically, a ‘‘touchdown’’ PCR protocol was used. This consisted of an initial 5 to 10 cycles that included a denaturing step (92jC for 30 s), an annealing step (starting at 72jC and going down to 66jC), and an extension step (72jC, 3 min). This was followed by an additional 25 to 30 regular cycles (using 66jC as annealing temperature) and a 5-min final extension.
[12]
[13]
[14]
[15]
[16]
Acknowledgment We thank P. Reed Larsen (Brigham and Women’s Hospital, Harvard Medical School) for the human DIO3 cDNA clone.
[17]
[18]
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