Participation of Transcription Elongation Factor XSII-K1 in Mesoderm-derived Tissue Development in Xenopus laevis *

We isolated a cDNA clone for a novel member of the S-II family of transcription elongation factors fromXenopus laevis. This S-II, named XSII-K1, is assumed to be the Xenopus homologue of mouse SII-K1 that we reported previously (Taira, Y., Kubo, T., and Natori, S. (1998) Genes Cells 3, 289–296). Expression of the XSII-K1gene was found to be restricted to mesoderm-derived tissues such as liver, kidney, and skeletal muscle. Contrary to the generalS-II gene, expression of the XSII-K1 gene was not detected in embryos at stages earlier than 11. The animal cap assay revealed that activin A, but not basic fibroblast growth factor, induced expression of the XSII-K1 gene and that it participated in the expression of mesoderm-specific genes such asXbra and Xα-actin. This is the first demonstration that the regulation at the level of transcription elongation is included in the development of mesoderm-derived tissues.

Transcription is a crucial step of gene expression. Various factors that participate in the process of transcription have been identified, and many of them are transcription initiation factors (1)(2)(3)(4). These factors are essential for the modulation of RNA polymerase II, so that it can recognize the transcription initiation site on a specific gene and initiate correct transcription. These transcription initiation factors are divided into two groups as follows: general initiation factors, which are commonly required for transcription, and sequence-specific initiation factors, which initiate the transcription of a specific gene(s).
Regulation of transcription elongation is assumed to be simpler than that of transcription initiation because, once RNA synthesis has begun, it is assumed to continue until RNA polymerase II reaches the transcription termination site. The number of transcription elongation factors is much lower than that of transcription initiation factors, but several have already been identified, such as S-II (TFIIS) (5-10), elongin (SIII) (11)(12)(13)(14), TFIIF (15)(16)(17)(18), P-TEF-b (19,20), and ELL (21). Among them, transcription elongation factor S-II is known to make RNA polymerase II readthrough intrinsic blocks within the transcription units of eukaryotic genes by promoting cleavage of the 3Ј-end of the nascent RNA by RNA polymerase II (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). S-II is unique in two respects. First, in various eukaryotic organisms, it forms a protein family with well conserved sequences of about 40 and 170 residues at their N and C termini, respectively, whereas each of the intervening sequence of about 50 residues is unique (10,(33)(34)(35)(36). Second, multiple S-II molecules are present in a single organism, as has been demonstrated in the mouse. One form of these molecules is general S-II (10), which is ubiquitous in various cells, and the other forms are tissue-specific (37)(38)(39)(40). The above sequence rule is applicable to all these S-II molecules.
Previously, we identified mouse SII-K1, which is expressed exclusively in heart, liver, skeletal muscle, and kidney (40). Contrary to general S-II mRNA, SII-K1 mRNA was not detected in embryos at an early developmental stage but became detectable in 15-and 17-day-old embryos, suggesting a functional difference between general S-II and SII-K1 (40). To gain further insight into the function of SII-K1 in embryonic development, we cloned the cDNA for Xenopus laevis SII-K1 and examined the expression of the Xenopus SII-K1 gene during development.

MATERIALS AND METHODS
cDNA Cloning for Xenopus SII-K1-To isolate Xenopus SII-K1 cDNA, we performed RT-PCR 1 using Xenopus kidney poly(A) ϩ RNA and two degenerate primers corresponding to Pro-178 to Asp-184 and Glu-339 to Cys-347 of SII-K1, followed by a nested PCR with degenerate primers corresponding to Asp-188 to Leu-194 and Asn-286 to Glu-293 (40). A DNA fragment of 327 bp was amplified in this way. On the basis of the sequence of this DNA fragment, we performed 5Ј-rapid amplification of cDNA ends to obtain more complete Xenopus SII-K1 cDNA, and we cloned a PCR product of 1320 bp. This cDNA contained part of the N-terminal consensus region, a subsequent unique region, and the C-terminal consensus region of the S-II family protein. To isolate the full-length Xenopus SII-K1 cDNA, we performed plaque hybridization using a DNA fragment corresponding to nucleotides ϩ2 to ϩ349 of this cDNA as a probe. This probe corresponded to the junction region of the N-terminal consensus region and the subsequent unique region. In this way, we finally isolated a full-length Xenopus SII-K1 cDNA consisting of 2,250 bp.
Northern Blot Hybridization-To detect Xenopus SII-K1 mRNA, poly(A) ϩ RNA was extracted from Xenopus embryos at stage 0, 7,8,11,13,18,21,26,33, and 39 or adult Xenopus tissues (heart, brain, spleen, lung, skeletal muscle, kidney, and testis) and subjected to Northern blot hybridization. The probe used was the PCR product corresponding to nucleotides ϩ335 to ϩ683 of the Xenopus SII-K1 cDNA (2 ϫ 10 9 cpm/ g). The same filter was rehybridized with part of the Xenopus EF1-␣ cDNA (41) to assess the efficiency of RNA extraction. Each lane con-* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation (to J. S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with accession number AB040437.
¶ To whom correspondence should be addressed. Whole Mount in Situ Hybridization-Whole mount in situ hybridization of Xenopus neurula and tail bud embryos was performed essentially as described previously (42). Embryos were fixed in MEMFA (100 mM MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO 4 , and 3.7% formalin) for 15 min at room temperature. They were then successively treated with 100, 75, 50, and 25% methanol for 5 min each time, followed by washing in a washing solution (10 mM phosphate buffer, pH 7.4, 150 mM NaCl and 0.1% Tween 20) three times for 5 min each time. The embryos were then treated with 10 g/ml proteinase K for 10 min, 0.1 M triethanolamine twice for 5 min each time, 0.25% acetic anhydride in 0.1 M triethanolamine twice for 5 min each time. The embryos were washed in the washing solution twice for 2 min each time, and then treated with 4% paraformaldehyde for 20 min, and finally washed in the washing solution five times for 5 min each time.
After hybridization, the embryos were incubated in 2ϫ SSC containing 10 mg/ml RNase A and 2 mg/ml RNase T1 for 30 min and then treated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Molecular Biochemicals) for 14 h at 4°C. Antibody bound to the probe RNA was visualized by incubating the embryos with 75 mg/ml nitro blue tetrazolium and 50 mg/ml 5-bromo-4-chloro-3-indolyl phosphate for 40 min at room temperature under dark condition. The embryos were made transparent by treating them with 100% methanol for 5 min at room temperature, followed by benzyl benzonate:benzyl alcohol, 2:1, and then photographed for examination.
Mesoderm Induction by Xenopus SII-K1-Full-length Xenopus SII-K1 mRNA was obtained by transcribing its cDNA ligated to pSP64T using a mMESSAGE kit (Ambion) and dissolved in Gurdon's solution. Stage 1 embryos were each injected with 0.2 g of Xenopus SII-K1 mRNA at their animal poles as described previously (44). When they reached stage 9, animal caps were dissected and incubated in 0.1ϫ Steinberg solution for 24 h at 20°C, as described above. Then total RNA was extracted from the animal caps, and expression of the Xbra (45) and ␣-actin genes (46) as mesoderm markers was examined by RT-PCR. The primers used to detect ␣-actin mRNA were 5Ј-GGAGTCTGCAAGCTA-TCCATGAG-3Ј and 5Ј-CACAATTGATTTTCCAGCCTCATC-3Ј.

Identification and Characterization of Xenopus SII-K1
cDNA-SII-K1 is a unique S-II family transcription factor whose expression is known to be developmentally regulated during the embryonic development of the mouse (40). To investigate the function of SII-K1 in embryonic development, we isolated a cDNA for the Xenopus homologue of SII-K1 on the basis of the sequence of mouse SII-K1. As shown in Fig. 1, this cDNA encoded a novel S-II family protein of 645 amino acid residues. The partial sequence of this cDNA was found to have been reported by Labhart and Morgan (47). When the overall structures of this S-II and mouse SII-K1 were compared, significant sequence similarities were found in the N-terminal and C-terminal conserved regions, but the sequences between these two regions were totally different.
A unique feature of this novel S-II was that it contained two repeated sequences of 18 and 50 residues in the non-conserved region ( Fig. 2A). The former sequence was repeated nine times and the latter three times (Fig. 2, B and C). These repeated sequences were novel, and no information related to them has been available in the protein data bases searched so far.
Although we isolated this cDNA on the basis of the sequence of mouse SII-K1 (40), it was difficult to conclude that this cDNA was that of the Xenopus homologue of mouse SII-K1. Therefore, by Northern blotting, we examined whether its expression was similar to that of mouse SII-K1 using RNA prepared from various tissues of Xenopus adults. As shown in Fig. 3A, an intense signal corresponding to a 2.5-kb RNA was detected in liver, skeletal muscle, and kidney, but no appreciable signal was detected in spleen, brain, and testis. This tissue-specific expression was very similar to that of mouse SII-K1 (40), except that significant expression was not detected in the heart. Signals of RNA species larger than 2.5 kb, including a relatively thick band in testis (white arrowhead), were probably those of other S-II family members. The size of XEF-1␣ mRNA in testis was a little larger than those of other tissues, suggesting that testis expresses a different subtype of XEF-1␣ family.
Another characteristic feature of the expression of the mouse SII-K1 gene is its developmental regulation. SII-K1 mRNA first becomes detectable in 15-day-old embryos and is not present in embryos at much earlier stages (40). As shown in Fig. 3B, the novel S-II mRNA was detected in embryos at stage 13 (neurula stage) or later and not in embryos at stages 0 -11, indicating that its expression was also regulated developmentally. This expression was quite different from that of general S-II. General S-II mRNA (48) was detected in embryos throughout stages 0 -39. From these results, we concluded that Expression of the XSII-K1 Gene during Embryogenesis-We located the expression of XSII-K1 by in situ hybridization using embryos at stage 13 (neurula) and stage 33 (tail bud). The XSII-K1 gene was clearly expressed in the head, somites, and tail of embryos at the neurula stage (Fig. 4A), whereas it was expressed in mesoderm-derived tissues such as head mesenchyme, pharynx (branchial arches), and somites in embryos at the tail bud stage (Fig. 4B). No significant staining was detected with the sense RNA probe. These results suggest that XSII-K1 is involved in the development of dorsal mesodermderived tissues.
During Xenopus development, activin A is known to induce dorsal mesoderm tissues (49), whereas bFGF induces ventral mesoderm tissues (50). Therefore, using the animal cap assay, we investigated whether activin A or bFGF was able to induce expression of the XSII-K1 gene. Animal caps from embryos at the neurula stage were incubated with or without 10 ng/ml activin A or bFGF. Then total RNA was extracted, and expression of the XSII-K1 gene was examined by RT-PCR. As a specific control for mesoderm tissue, we examined expression of the Xbra gene simultaneously (45). To assess the recovery of RNA, RT-PCR was performed with XEF1-␣ mRNA (41). As is evident from Fig. 5, mRNA for XSII-K1 was detected only in the activin A-treated samples, although expression of the Xbra gene was induced by both activin A and bFGF (Fig. 6). Thus, it is clear that the XSII-K1 gene is expressed in the dorsal mesoderm and not the ventral mesoderm. These results are in accord with those of in situ hybridization.
Induction of Mesoderm by Injection of XSII-K1 mRNA-It is obvious that the XSII-K1 gene is activated during the course of induction of the dorsal mesoderm by activin A. As XSII-K1 is a transcription elongation factor, it is assumed to participate in the transcription of mesoderm-specific genes, such as Xbra (45). Therefore, using animal cap assay, we examined whether XSII-K1 alone is able to activate mesoderm-specific genes. For this, we injected XSII-K1 mRNA into the animal poles of stage 1 (one-cell stage) embryos. Animal caps were dissected from embryos at the neurula stage and incubated without activin A for 24 h. Then total RNA was extracted from the embryos, and expression of the Xbra gene was examined by RT-PCR.
If XSII-K1 mRNA was injected in advance, the Xbra gene was found to be activated in the animal caps even in the absence of activin A, as shown in Fig. 6. We examined the expression of another mesoderm-specific gene, X␣-actin (46), and found that it was also activated under these conditions. These results suggested that some mesoderm-specific genes are located downstream of the XSII-K1 gene and that XSII-K1 induces their expression. This is the first report to suggest that mesoderm induction is regulated at the level of transcription elongation.

DISCUSSION
In this study, we showed that Xenopus SII-K1 participates in the development of mesoderm-derived tissues. As SII-K1 is a transcription elongation factor that belongs to the S-II family (40), this is the first clear demonstration that tissue development is regulated at the level of transcription elongation. As well as in dorsal mesoderm-derived tissues such as head mesenchyme, somites and pharynx in the neural and tail bud embryos, XSII-K1 mRNA was shown to be expressed in the adult liver, kidney, and skeletal muscle. Therefore, XSII-K1 seems to play roles in the process of tissue formation as well as tissue-specific transcription in mesoderm-derived adult tissues. In the animal cap assay, activin A was shown to activate the XSII-K1 gene. Once the XSII-K1 gene was activated in the precursor cells, the activated state seemed to be maintained even after completion of tissue formation.
We believe that XSII-K1 participates in the transcription of genes whose expression is restricted to mesoderm-derived tis-sues. As S-II family transcription elongation factor has a unique sequence between the N-terminal and C-terminal conserved regions (10, 33-36, 38, 40), this unique sequence seems to define its tissue specificity. In previous studies of mouse S-II, we demonstrated that SII-T1 and SII-K1 are expressed specifically in spermatocytes and various mesoderm-derived tissues, respectively (38 -40). However, we were unable to clarify their target genes. In the present study, we showed that the Xbra and X␣-actin genes are activated in the animal cap when XSII-K1 mRNA is directly injected into animal pole of one-cell stage embryos. These findings suggest that XSII-K1 participates in the transcription of these two genes. Therefore, these genes may contain a transcription arrest site(s) that can be read through by RNA polymerase II only in the presence of XSII-K1.
We initially intended to isolate the Xenopus homologue of SII-K1. However, XSII-K1 was quite different from SII-K1 in terms of the amino acid sequence and molecular size of the unique region. Nonetheless, we defined it as Xenopus SII-K1 because its embryonic expression and tissue-specific expression were very similar to those of SII-K1. However, we cannot exclude the possibility that another S-II family protein bearing a closer similarity to SII-K1 is present in mesoderm-derived Xenopus tissues. In the cDNA clone of XSII-K1, no in frame termination codons were identified upstream of the first Met codon. However, we assigned this codon as the first Met codon by comparison with the sequences of other S-II family proteins. Moreover, this Met codon satisfied the Kozak's rule (51).
It is noteworthy that the unique region of XSII-K1 is quite different from those of other S-II family proteins. XSII-K1 contained two repeated sequences in this region consisting of 18 and 50 amino acid residues. Although the biological significance of these sequences is unknown, we assume that there is a protein(s) that interacts with this region in mesoderm-derived tissues. Identification and characterization of such a protein(s) may be essential to elucidate the way in which XSII-K1 regulates the transcription of mesoderm-specific genes such as Xbra (45) and X␣-actin (46).