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2 ticle/8/12/1667/2714924 by guest on 10 J
ticle/8/12/1667/2714924 by guest on 10 June 2021 Downloaded from https://academic.oup.com/mend/article/8/12/1667/2714924 by guest on 10 June 2021 Downloaded from https://academic.oup.com/mend/article/8/12/1667/2714924 by guest on 10 June 2021 1994 Vol8No.12 The cell type-specific expression of TAKl was further examined in several tissues and in particular the testis. The interstitium and the seminiferous tubules are the two major compartments in the testis (14, The Leydig cell is the key cell type in the interstitium whereas the seminiferous tubules are the site for sper- matogenesis and contain the Sertoli cells and germ cells. Spermatogenesis is a unique process of differ- entiation that occurs in three major phases, a mitotic phase, meiosis, and postmeiotic phase in which hap- loid germ cells undergo extensive remodeling to pro- duce spermatozoa (14, Cloning and Characterization of the Orphan Receptor TAKl 1669 a. b. RACE probe S-primer- TAKl cDNA T109 TAG xpai qsm’ ?.TlO Nsil , hT7 Xbal Nsil I 1 (AM Xbal NSII XT9 I ,’ , Bsml Nsil AT0 1 I 1 3’UTR probe 185 1234567 bp Fig. 1

3 . Molecular Cloning of the Orphan Recept
. Molecular Cloning of the Orphan Receptor TAKl a, cDNA clones representing the complete coding region of TAKl. Strategy used in the construction of cDNA for TAKl. The clones XT7, -8, and -10 were obtained by screening a human testis cDNA library. Clone region I in Fig. 3a) where 82% of 66 amino acid residues are identical (Fig. 3b) and in two distinct regions (shown as II and Ill in Fig. 3a) in the ligand-binding domain. The amino acid residues in regions II and Ill between TAKl and TR2-11 are, respectively, 81% and 91% identical (Fig. 3, c and d). Outside these three regions homology between TAKl and TR2-11 is much less. The N-termi- nal region and the region between the DNA- and ligand- binding domains exhibit only a 36% and 42% identity, respectively (Fig. 3a). Although the similarity in situ hybridiza- tion. When sections probed with antisense TAKl cRNA were examined at low magnification, a ring of silver grains was observed residing in the seminiferous tu- bules (Fig. 5, a b). No specific hybridization signal was observed when sections were probed with sense TAKl cRNA (Fig. 5~). At higher

4 magnification the ring of silver grains
magnification the ring of silver grains was observed to overlie the spermato- cytes (Fig. 5, d e). The number of silver grains associated with spermatogonia, spermatids, sperma- tozoa, and Sertoli cells was slightly above background levels (average right (Fig. 5, d e) a weak hybridization signal represents stage ll-lll of the cycle and, in contrast to the stage VIII-IX tubule, contained few primary spermatocytes. After observing many tu- bules in various stages, we concluded that the expres- sion of TAKl mRNA is highest around stages VIII-IX and lowest around stages ll-lll. This expression pat- resembles that of ferT mRNA, which encodes a testis-specific tyrosine kinase (36). Expression of TAKl mRNA in Vitamin A-Deficient Rat Testis Vitamin A deficiency has been reported to block sper- matogenesis at an early stage. The seminiferous tu- bules in the testis of vitamin A-deficient rats contain spermatogonia, a few preleptotene spermatocytes, and Ser-toli in hybridi- zation (Fig. 6). The normal rat testis exhibited a similar pattern of hybridization as was observed in mouse testis (Fig. 6, a b) bu

5 t testis of vitamin A-deficient rats did
t testis of vitamin A-deficient rats did not hybridize to the TAKl probe (Fig. 6, c and d). (The signal observed in the interstitial region in Fig. 6d is not significant since no silver grains are observed in the same region in Fig. 6c.) In addition, analysis of poly(A)’ RNA isolated from testes of normal and vitamin A-deficient rats by rt-PCR confirmed the absence of TAKl expression in vitamin A-deficient testis side of the sequence. The putative initiation ccdon is at nucleotide (nt) 241. An in-frame termination codon in the 5’-UTR is underlined. Region C and E/F are underlined, and the location of region A-E/ F is indicated on the right side of the sequence. The termination ccdon (nt 2029) at the end of TAKl amino acid sequence is indicated as END. The polyadenylation signal (ATTAAA) is underscored. The sequence of the extended 3’-UTR (nt 2218-3360) is derived from clone XT8 (Fig. la) and shown at the bottom. Asterisk, Site of 3’-UTR extension. hP.xRa(270) hRARa~230) hER (348) hAR (705) d. TAKl (482) TR2 (489) COUP ,304) hRXRa(357) hRARa(316, hER (435) hAR (7

6 93) Fig. 3. Schematic Comparison betwee
93) Fig. 3. Schematic Comparison between the Amino Acid Sequence of TAKl and Several Members of the Steroid Hormone Receptor Superfamily a, Alignment of amino acid sequences identified three regions with high similarity, shown as I, and Ill. The numbers above each box indicate the TAKl is expressed almost ubiquitously as shown by Northern blot analysis (Fig. 4), we examined the cell- type specific expression of TAKl in several other tis- sues by in situ hybridization. In the brain, TAKl expres- sion was not uniform but occurred in very distinct regions. In the cerebral cortex, high levels of TAKl transcripts were detected in the external granular layer (Fig. 8, a b). In the hippocampus, a strong 9. In mouse spleen TAKl expression is associated with the red pulp rather than the white pulp (Fig. 8, g h). In the femoral muscle (musculus biceps femoris), which is one of the tissues where TAKl is most highly expressed, TAKl transcripts were Cloning and Characterization of the Orphan Receptor TAKl 1673 b 9.5- 7.5- \ .o\ _\ ,: 4.4 - :.:i 2.4 - \ __, .),.) ,:, ‘. 1.35- ‘. .‘. ,.,.\ 9

7 .5 - 7.5 - 4.4 - 2.4 - 1.35- d 2.4- F
.5 - 7.5 - 4.4 - 2.4 - 1.35- d 2.4- Fig. 4. Tissue Specificity of TAKl expression Poly(A)+RNA (2 pg) isolated from different human (a and since it can bind the sequence AAAGGTCA and activate promotor activity without ligand binding (44). Whether any transcriptional activating function of TAKl is de- pendent on the binding of a specific ligand has yet to be determined. r&expression of a chimeric receptor containing the DNA-binding domain of the estrogen receptor and the ligand-binding domain of appear related, their tissue-specific expression is quite different. The expression of TR2-11 is much more restricted: TR2-11 is most highly expressed in the prostate and liver and present at low levels in the testis. In human and mouse tissues TAKl exists as two tran- scripts, 9.4 (7.8 kb in mouse) and 2.8 kb in size. The larger mRNA appears to be present almost ubiquitously whereas the testis. The generation of multiple and unique transcripts in the testis is not limited to TAKl but has been reported for a number of other genes (34, These transcripts may be generated by alternative splicing, the t

8 estis- specific use of alternative promo
estis- specific use of alternative promoter or transcription start sites, or the selection of alternative polyadenylation signals (34, 35, 46). The isolation of in situ hybridization analysis indicates that TAKl is expressed in a cell type-specific manner. In the testis, TAKl mRNA is differentially expressed during sper- matogenesis. The highest level of TAKl mRNA is as- sociated with spermatocytes. In situ hybridization analy- MOL ENDO. 1994 Vol8No.12 1674 Fig. 5. In Situ Localization of TAKl mRNA in Adult Mouse Seminiferous Tubules Bright-field (a) and dark-field (b) photomicrographs of a section in hybridization with a radiolabeled antisense TAKl RNA probe and stained with hematoxylin. c, Dark-field photomicrograph of adjacent section hybridized with a of vitamin A-deficient rat testis and t-t-PCR and Northern blot analysis of mRNA prepared from in situ hybridization analyses of TAKl expression in several other tissues. These results show that the distribution of TAKl tran- scripts is not uniform but Cloning and Characterization of the Orphan Receptor TAKl 1675 Fig. 6. In Situ L

9 ocalization of TAKl mRNA in Seminiferous
ocalization of TAKl mRNA in Seminiferous Tubules from Normal and Vitamin A-Deficient Adult Rats Bright-field (a and c) and dark-field (b and in situ hybridization with a radiolabeled antisense TAKl RNA probe and stained with hematoxylin. Bar, 50 pm. kinase Hox-7.4 (48), megl (49) hsp 70.2 (50) and ~53 (51), have been reported to be up-regulated during the meiotic phase of sper- matogenesis. Some of these genes may be candidates for regulation by TAKl. The Zfp- 29, Zfp-35, and CTfirt57 (19, 20, 52-54) encoding proteins with zinc finger motifs, are also induced during meiosis. However, the expression pattern of these genes is different from that TAKl since they MOL END0.1994 1676 Vol8No. 12 a. 5 lo 20 (days) TAKl SGP-1 C. 10 (days) 7.6- TAKl 2.6- SGP-1 2.6-m b. Fig. 7. Developmental Onset of TAKl mRNA Synthesis in Rat Testis Poly(A)+ RNA was isolated from testes taken from rats at various ages (days of age denoted above each lane or below each bar) and was subjected to rt-PCR analysis to determine the relative level of TAKl mRNA (a). The amplified products were separated on gel

10 and hybridized to a 32P-end-labeled olig
and hybridized to a 32P-end-labeled oligonucleotide probe that is complimentary to a sequence in the meiotic phase during oogenesis has yet to be estab- lished. Our results show that MATERIALS AND METHODS PCR Amplification A set of degenerate primers were designed according to the most highly conserved sequence of the DNA-binding domain of members of the nuclear receptor family (13). The sense strand primer R-P1 was 5’-T-G-(T/C)-G-A-(G/A)G-G-(G-A-T- C)-T-G-(T/C)-A-A-(G/A)-G-G-(T/C)-T-T-(T/C)-T-T-3’ (CEGCK- GFF). The antisense strand primer R-P2 was 5’-(G/A)-C-A-(T/ C~T-T-C-T-(GIT)-(G/AIT/C)-C-G-(T/C)-C-A-G-T-A-(T/C~T-G- (G/A)-C-A-3’ (CQYCRL(K/Q)KC). A lymphoblastoma Raji cell Cloning and Characterization of the Orphan Receptor TAKl 1677 Fig. 8. In Situ Localization of TAKl mRNA in Several Tissues Bright field (a, c, e, i, and k) and dark field (b, d, f, h, j, and I) in hybridization with a radiolabeled antisense TAKl RNA probe and stained with hematoxylin. a b, Mouse cerebral cortex; c and d, mouse Gyrus dentatus and hippocampus; e f, mouse cerebellar folia; Bars, 100

11 pm except panels i and j (bar, 50 pm).
pm except panels i and j (bar, 50 pm). Preparation of Vitamin A-Deficient Rats Vitamin A-deficient male in hybridization experiments. In Situ Hybridization Adult (4-month-old) C3H mouse or adult Sprague-Dawley rats (normal and vitamin A-deficient) were fixed by perfusion through the left ventricle with 0.07 M sodium cacodylate- buffered 4% paraformaldehyde, x 1 O* cpm/pg) were generated by in vitro transcrip- tion from the T3 and T7 RNA polymerase promoter in the presence of uridine 5’-[&?S]thiotriphosphate (Amersham). Probes were subjected to limited alkaline hydrolysis to reduce the size of MOL ENDO. 1994 differences in the hybridization patterns were observed be- tween these two temperatures. The slides were immersed in NTB2 emulsion (Kodak, Rochester, NY) for autoradiography, exposed for 2 weeks at 4 C, and then developed and coun- terstained with hematoxylin. Reverse-Transcriptase PCR rt-PCR was used to determine the initial appearance of TAKl mRNA during development according to the method of Murak- awa et al. (62). Poly(A)+ RNA (1 pg) samples prepared from developmentally stage

12 d testes were treated with DNase to elim
d testes were treated with DNase to eliminate contaminating genomic DNA and were then used as templates for the synthesis of single strand cDNA with Acknowledgments We thank Drs. E. M. Eddy, D. A. O’Brien, B. Sato, and N. A. Saunders for their advice during this study; Drs. E. M. Eddy and D. J. Dix for their comments on the manuscript; and Kyoko Hirose with help in preparing the manuscript. Received February 25, 1994. Re-revision received August 25, 1994. Accepted August 29, 1994. Address requests for reprints to: Dr. Anton M. Jetten, Cell Biology l TAKl was submitted to EMBL/GenBank Database Librar- ies under accession No. U10990 Note Added in Proof During review of this manuscript, Vol8 No. 12 almost the same orphan receptor sequence (3’ UTR sequence was not included) was published by C. Chang et a/. 1994 in Proc Natl Acad Sci USA 91:6040-6044. REFERENCES 1. 2. 10. O’Malley BW, Roop DR, Lai EC, Nordstorm JL, Catterall JF, Swaneck GE, Colbert Tsai M-J, Dugaiczyk A, Woo SLC 1979 The ovalbumin gene: structure, organi- zation, transcription and regulation. Recent Prog Horm Res

13 351-42 Yamamoto 561335-344 Miller J, M
351-42 Yamamoto 561335-344 Miller J, McLachlan AD, Klug A 1985 Repetitive zinc- binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J 4:1609-1614 Evans RM, Hollenberg SM 1988 Zinc fingers: guilt by association. Cell 52:1-3 Carlstedt-Duke J, Stromstedt PE, Wrange 0, Bergman T, Gustafsson JA, Jornvall H 1987 Domain structure of the 8414437-4440 Forman BM, Yang C, Au M, Casanova J, Ghysdael J, Samuels HH 1989 A domain containing leucine-zipper-like motifs mediate novel in viva interactions between the thyroid hormone and retinoic acid receptors. Mol Endo- crinol 3:161 O-l 626 Fawell SE, Lees JA, White R. Parker MG 1990 Charac- terization and colocalization of steroid binding and dimer- ization activities in the mouse estrogen receptor. Cell 60:953-962 Tasset D, Tora L, Fromental C, Scheer E, Chambon P 1990 Distinct classes of transcriptional activating domains function by different mechanisms. Cell 62:1177-l 187 Laudet V, Hanni C, Coll J, Catzeflis F, Stehelin D 1992 Evolution of the nuclear receptor gene superfamily. EMBO J 11:1003-1013 Russell LD, Ettlin TAKI: Mo

14 lecular Cloning and Characterization of
lecular Cloning and Characterization of a New Member of the Nuclear Receptor Superfamily* Takahisa Hirose, Wataru Fujimoto, Tomoichiro Yamaai, Kwan Hee Kim, Hironori Matsuura, and Anton M. Jetten Cell Biology Section (T.H., A.M.J) Laboratory of Pulmonary Pathobiology National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709 Department of Using polymerase chain reaction and two degener- ate primers whose designs were based on the two best conserved regions of the DNA-binding domain of the nuclear receptor superfamily, we identified and cloned a novel orphan receptor, named TAKl. The In situ hybridization using other mouse and rat tissues re- vealed cell type-specific expression of TAKl in sev- eral tissues. Our observations suggest a role for this putative transcription factor in the regulation of gene expression in specific cell types. In the testis, TAKl appears to control gene expression during sperma- osse9910/94$03.00/0 Molecular Endocrinology Copyright 0 by The Endocrine Society togenesis, particularly during the meiotic phase. (Molecular Endocrinol