- Email: [email protected]
Nonredundant Roles of the Elongation Factor MEN in Postimplantation Development
Biochemical and Biophysical Research Communications 279, 563–567 (2000) doi:10.1006/bbrc.2000.3970, available online at http://www.idealibrary.com on
Nonredundant Roles of the Elongation Factor MEN in Postimplantation Development Kinuko Mitani,* ,1 Tetsuya Yamagata,* Chika Iida,* Hideaki Oda,† Kazuhiro Maki,* Motoshi Ichikawa,* Takashi Asai,* Hiroaki Honda,* Mineo Kurokawa,* and Hisamaru Hirai* ,2 *Department of Hematology and Oncology and †Department of Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
Received November 8, 2000
The MEN/ELL gene was cloned as a fusion partner of the MLL gene in the t(11;19)(q23;p13.1) translocation, which is found in adult myeloid leukemia. MEN belongs to a family of RNA polymerase II elongation factors and dysregulated production of MEN through the MLL promoter could cause malignant transformation of myeloid cells. To pursue the physiological role and determine the requirement of the MEN gene product in mouse development, we generated knockout mice (MENⴚ/ⴚ) by gene targeting in embryonic stem cells. After intercrossing heterozygous mice to generate homozygous mutants, we identified no homozygotes (MENⴚ/ⴚ) even at E9.5, as well as after birth, by Southern analysis. Moreover, histological examinations revealed degenerative changes in nearly onefourth of E6.5 embryos, which were gradually resorbed by E8.5. Our findings demonstrated that MENⴚ/ⴚ mice are embryonic lethal, and die before E6.5 and after implantation. MEN should play a nonredundant role in postimplantation development of mice. © 2000 Academic Press Key Words: MEN/ELL; elongation factor; knockout mice; embryonic lethality; leukemia; MLL; 11q23 translocation.
MEN is a member of a family of RNA polymerase II elongation factors (1), which consist of P-TEFb, SII, TFIIF, Elongin (SIII), MEN, and ELL2. P-TEFb catalyzes the conversion of early, termination-prone transcription complexes into productive complexes (2). SII expedites elongation by preventing RNA polymerase II from terminating transcription prematurely at a variety of transcription impediments (3). The remaining 1 Present address: Department of Hematology, Dokkyo University School of Medicine, Tochigi, Japan. 2 To whom correspondence should be addressed at Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. Fax: ⫹81-3-5689-7286. E-mail: [email protected].
four support transcriptional elongation by suppressing transient pausing of polymerase reaction at many sites along the template DNA (1, 4 –9). MEN, as well as ELL2, which is 49% identical and 66% similar to MEN, exhibit the elongation enhancing activity conferred by their N-terminal regions (10, 11). MEN also possesses an RNA polymerase II interaction domain in its most N-terminal area, which is capable of negatively regulating the polymerase activity in promoter-specific transcription initiation (11). The MEN gene, mapped to 19p13.1, was originally cloned as the MLL/MEN chimeric cDNAs generated by the t(11;19)(q23;p13.1) translocation (12, 13) which is specifically found in adult myeloid leukemia. The MLL gene located at 11q23 is commonly involved in the 11q23 translocations, which are the most frequently observed chromosomal abnormalities in human leukemias (14, 15). The MLL gene product holds functional domains of transcription factors, including AT-hooks in its N-terminal portion, two zinc finger domains in its middle, and the TRX homology domain in its C-terminal region (16, 17) and positively regulates the expression of the Hoxa-7, Hoxc-9 (18, 19) and ARP1 (20) genes. While MLL⫺/⫺ mice are reported to be embryonic lethal, MLL⫹/⫺ mice display a slight anemia and thrombocytopenia, and axial skeletal and sternal malformations (19). Upon the translocation, the MLL gene is disrupted between the AT-hooks and the zinc finger domains, and the resulting N-terminal 1406 amino acids are fused with the entire coding region of MEN except for its N-terminal 45 amino acids. There are two known mechanisms in leukemogenesis by the t(11;19)(q23;p13.1) translocation. The first one is that the MLL/MEN fusion protein, in which the N-terminal RNA polymerase II interaction domain of MEN has been lost, could show full activation in elongation and cause leukemogenesis. Secondly, MEN itself is a multifunctional protein. Besides transcriptional elongation activities, it also stimulates AP-1 activity by increasing the Fos expression (21, 22) and inhibits transcriptional
563
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 279, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. MEN⫺/⫺ mice are embryonic lethal. (a) Targeting strategy. The homologous recombination event inserted a TK-Neo cassette into the exon 1 of the MEN gene. H, HindIII; probe 1, 5⬘ external probe; probe 2, 3⬘ external probe. (b) Generation of targeted ES cells. G418-resistant ES clones were screened by Southern blot analysis with 5⬘ external (left panel), 3⬘ external (middle panel), and the neo gene (right panel) probes using HindIII digests. Expected fragments hybridized with these probes as depicted in a. The fragment sizes of the wild-type and mutant alleles hybridized with probe 1 were 20 kb and 15 kb, respectively. Those with probe 2 were 20 kb and 6 kb, respectively. The mutant fragment of 6 kb detected with probe 2 was also hybridized with the neo gene probe. Two appropriately targeted clones, 209 and 242, were obtained. ⫹/⫹, wild type; ⫹/⫺, targeted ES cells (209). (c) Results of intercrossing heterozygotes. Embryos were genotyped by Southern blot analysis as in b. No homozygous embryos (⫺/⫺) were found. ⫹/⫹, wild type; ⫹/⫺, heterozygotes.
activity of the p53 tumor suppressor gene (23, 24), leading to the increased colony formation of Rat1 cells. Therefore, the MLL/MEN chimeric protein driven by the MLL promoter might show a stronger oncogenic function through both enhancing the AP-1 activity and suppressing the p53 function. The question whether the MEN protein has redundant activities with other elongation factors, especially with ELL2, could be of great interest. To address the question, we introduced a targeted mutation into the MEN gene by homologous recombination in mouse embryonic stem (ES) cells. Loss of MEN function demonstrated that MEN is indispensable for early embryogenesis. Thus, MEN is a critical elongation factor in embryonic development. MATERIALS AND METHODS Construction of the targeting vector. Murine MEN genomic sequences were isolated from a 129/SvJ strain FIXII library (Stratagene, La Jolla, CA) with a 60-bp EcoRI/PmaCI MEN cDNA probe encoding its N-terminal 17 amino acids in the exon 1. A 10-kb EcoRI fragment in inserts from positive phage was subcloned into pBluescript SK plasmid (Stratagene), and subjected to restriction enzyme mapping. The 1.1-kb TK-Neo cassette derived from pMC1Neo vector
(Stratagene) was inserted into the PmaCI site within the exon 1. The resulting EcoRI fragment of 11.1-kb was then fused with EcoRIdigested pBluescript SK plasmid containing a 1.1-kb MC1-DTA cassette at the SalI site in its multicloning site to generate the final targeting construct. Disruption of the exon 1 resulted in stop codons in all reading frames. Gene targeting in ES cells and generation of mutant mice. The MEN targeting construct was linearized with NotI digestion and electroporated into 129/SvJ ES cells. A 5⬘ flanking 0.3 kb EcoRI/ BamHI fragment (probe 1 in Fig. 1a), a 3⬘ flanking 0.1 kb EcoRI/KpnI fragment (probe 2 in Fig. 1a), and the neo gene probe were used to identify appropriately targeted ES cells by Southern blot analysis with HindIII digests. Aggregation with the targeted ES cells and C57BL/6 derived-morula was performed by Genomsystems Inc. to generate chimeras. One of the resulting chimeras, which exhibited 95% contribution from the ES cells on the basis of agouti coat color, was shown to contribute to the germline, by mating with C57BL/6 mice and subsequent genotype examinations on their pups by Southern analysis. Heterozygous mice were intercrossed to generate homozygous mutants. Neonates and embryos were also genotyped by Southern blot analysis. Histological analysis. To determine the stage of embryonic lethality, we performed histological analysis of the E6.5, 7.5, and 8.5 embryos after mating of heterozygous mice. Pregnant mice were sacrificed at each embryonic day and the whole uteri were fixed in 10% neutral buffered formaldehyde, embedded in paraffin, serially sectioned (8 m), and stained with hematoxylin and eosin. Sections
564
Vol. 279, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
MEN Homozygotes Die before E9.5 Stage
No. of litters
No. of embryos
⫹/⫹
⫹/⫺
Resorbed
E9.5 E10.5 E12.5 E14.5 Neonate
11 3 2 5 13
92 32 13 27 77
23 11 3 12 25
69 20 10 15 52
38 9 5 9 —
(60 –200 pieces per embryo) were carefully examined with a microscope.
RESULTS AND DISCUSSION To inactivate the MEN gene, the TK-Neo cassette was inserted into the exon 1 (Fig. 1a). Of 96 G418resistant ES clones, two independent clones, 209 and 242, showed expected mutant bands of 15 kb and 6 kb hybridized with probes 1 and 2, respectively, in addition to the wild-type fragments of 20 kb (Fig. 1b). The mutant fragment of 6 kb hybridized with probe 2 was also detected with the neo gene probe, indicating that homologous recombination correctly occurred only at the MEN gene locus. These two clones, which contained the appropriately targeted MEN locus, were aggregated with C57BL/6-derived morula and one (209) of them led to germline transmission of the mutation. F 1 mice heterozygous for disruption of the MEN gene appeared normal in size, fertility, and overall development (data not shown). Heterozygous mice were intercrossed to generate homozygotes (MEN⫺/⫺). Of the 77 neonates genotyped, no homozygotes were identified, suggesting that loss of MEN is incompatible with normal embryonic development (Table 1). We sacrificed pregnant females at various days postcoitus to establish the time and cause of death. At E14.5, 12.5, 10.5, and 9.5, we found no live MEN-lacking embryos by Southern analysis (Table 1 and Fig. 1c). Instead, resorbed embryos were detected at a ratio of approximately one-fourth among total implanted portions. These data suggested that MEN⫺/⫺ mice are lethal by E9.5. We next microscopically analyzed the morphological changes of the embryos to precisely identify the stage where MEN⫺/⫺ embryos turn lethal. At E6.5, 4 out of 20 implanted portions in the uteri showed degenerative embryos (Fig. 2a). These composed of almost necrotic cells with scattered condensed nuclei and obscure cytoplasmic margins, accompanied with hemorrage. The cells showed loose contact with one another and we observed no differentiation to ectoderm or entoderm. On the other hand, the other 16 portions contained embryos with normal growth, showing embryonic ectoderm around the proamniotic cavity (Fig. 2b). At E7.5, 3 out of 16 implanted portions examined
showed similar degenerative findings to those at E6.5 (Fig. 2c). One implanted portion revealed empty cavity without embryonic tissues. The other 12 portions contained normally developed embryos with gastrulation (Fig. 2d). At E8.5, 3 out of 12 implanted portions were empty (Fig. 2e). Maternal decidual proliferation, which is evidence for implantation, was observed, but no definite mass was found in the cavity even after observation of all the serial sections. The other nine implanted portions in the uteri showed morphologically normal embryos, in which cephalic neural fold and notochord were well developed (Fig. 2f). We could not clarify whether the degenerative or resorbed embryos observed were actually homozygotes, because they died too early. However, it is reasonable to speculate that MEN⫺/⫺ mice die before E6.5, because approximately one-fourth of embryos were already degenerative at E6.5, and gradually resorbed by E8.5. We also could not specify the reason of death. This is the first report describing the animals deficient in a transcriptional elongation factor. Because MEN is essential for early embryonic development, it might play a non-redundant role to maintain early embryogenesis through regulation of general transcription. We could propose two possible mechanisms in embryonic lethality of MEN⫺/⫺ mice. First, no or less effective transcriptional elongation might occur in the absence of MEN. Considering that MEN is ubiquitously expressed in adult tissues (12, 13) and also diffusely detected in E7.5 embryos (25), it seems that MEN has a fundamental role in keeping life. Second, MEN might exhibit its function on specific target genes, which have a pivotal role in development. It is likely that MEN⫺/⫺ ES cells are viable, because MEN⫺/⫺ embryos die after implantation, as is evident by the presence of maternal decidual tissues. Using differential display or DNA tips with MEN⫺/⫺ ES cells, we could answer the question whether MEN is a key elongation factor for transcription or has specific target genes. Moreover, making chimeric mice with MEN⫺/⫺ ES cells might permit direct studies on organ-specific requirement of MEN. Thirman et al. reported that MEN/ELL is expressed diffusely throughout embryos between E7.5 and E14.5, but specific expression in the liver and gastrointestinal tract becomes prominent by E16.5 (25). If MEN exerts its nonredundant function in such specific organs at the embryonic stage, MEN⫺/⫺ ES cells would not contribute to those organs. In contrast, if no transcription is activated in any organs without MEN, MEN⫺/⫺ ES cells could not be detected in any organs or tissues. Conditional gene targeting technologies, which regulate temporal and cell type-specific inactivation of target genes with Cre/loxP recombination system, are also a powerful tool to determine the cell-specific functions of genes, when their knockout mice are lethal in early development. It will be useful to clarify the roles of MEN,
565
Vol. 279, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6.
7.
8.
9.
10.
11.
FIG. 2. MEN⫺/⫺ mice die before E6.5. (a and b) Magnification ⫻20, transverse section, E6.5; (c and d) magnification ⫻10, transverse section, E7.5; (e and f) magnification ⫻4, transverse section, E8.5. (a, c, and e) Degenerative (a and c) or resorbed (e) embryos, which were expected to be homozygotes because they were observed at a frequency of almost one-fourth. In contrast, normally developed embryos are shown in b, d, and f.
especially in hematopoietic cells, to consider the mechanisms in leukemogenesis by the t(11;19)(q23;p13.1) translocation. Its non-redundant functions among several transcriptional elongation factors might suggest a critical role of dysregulated general transcription in development of hematological malignancies.
12.
13.
14. 15. 16.
ACKNOWLEDGMENTS 17. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and from the Ministry of Education, Science, and Culture of Japan.
18.
REFERENCES 1. Shilatifard, A., Lane, W. S., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1996) An RNA polymerase II elongation factor encoded by the human ELL gene. Science 271, 1873–1876. 2. Marshall, N. F., and Price, D. H. (1995) Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J. Biol. Chem. 270, 12335–12338. 3. Reines, D. (1994) Nascent RNA cleavage by transcription elongation complex. In Transcription Mechanisms and Regulation (Conaway, R. C., and Conaway, J. W., Eds.), pp. 263–278, Raven Press, New York, NY. 4. Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) RNA polymerase II elongation factor SIII. I. Identification, purification, and properties. J. Biol. Chem. 268, 25587–25593. 5. Bradsher, J. N., Tan, S., McLaury, H.-J., Conaway, J. W., and Conaway, R. C. (1993) RNA polymerase II elongation factor SIII.
19.
20.
21.
22.
566
II. Functional properties and role in RNA chain elongation. J. Biol. Chem. 268, 25594 –25604. Kephart, D. D., Wang, B. Q., Burton, Z. F., and Price, D. H. (1994) Functional analysis of Drosophila factor 5 (TFIIF), a general transcriptional factor. J. Biol. Chem. 269, 13536 –13543. Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1989) Dynamic interaction between a Drosophila transcription factor and RNA polymerase II. Mol. Cell. Biol. 9, 1465–1475. Tan, S., Aso, T., Conaway, R. C., and Conaway, J. W. (1994) Roles for both the RAP30 and RAP74 subunits of transcription factor IIF in transcription initiation and elongation by RNA polymerase II. J. Biol. Chem. 269, 25684 –25691. Tan, S., Conaway, R. C., and Conaway, J. W. (1995) Dissection of transcription factor TFIIF functional domains required for initiation and elongation. Proc. Natl. Acad. Sci. USA 92, 6042– 6046. Shilatifard, A., Duan, D. R., Haque, D., Florence, C., Schubach, W. H., Conaway, J. W., and Conaway, R. C. (1997) ELL2, a new member of an ELL family of RNA polymerase II elongation factors. Proc. Natl. Acad. Sci. USA 94, 3639 –3643. Shilatifard, A., Haque, D., Conaway, R. C., and Conaway, J. W. (1997) Structure and function of RNA polymerase II elongation factor ELL. Identification of two overlapping ELL functional domains that govern its interaction with polymerase and the ternary elongation complex. J. Biol. Chem. 272, 22355–22363. Mitani, K., Kanda, Y., Ogawa, S., Tanaka, T., Inazawa, J., Yazaki, Y., and Hirai, H. (1995) Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11;19)(q23;p13.1) translocation. Blood 85, 2017–2024. Thirman, M. J., Levitan, D. A., Kobayashi, H., Simon, M. C., and Rowley, J. D. (1994) Cloning of ELL, a gene that fuses to MLL in a t(11;19)(q23;p13.1) in acute myeloid leukemia. Proc. Natl. Acad. Sci. USA 91, 12110 –12114. Rowley, J. D. (1990) Recurring chromosome abnormalities in leukemia and lymphoma. Semin. Hematol. 27, 122–136. Rubnitz, J. E., Behm, F. G., and Downing, J. R. (1996) 11q23 rearrangements in acute leukemia. Leukemia 10, 74 – 86. Gu, Y., Nakamura, T., Alder, H., Prasad, R., Canaani, O., Cimino, G., Croce, C. M., and Canaani, E. (1992) The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell 71, 701–708. Tkachuk, D. C., Kohler, S., and Cleary, M. L. (1992) Involvement of a homolog of Drosophila Trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 71, 691–700. Joh, T., Hosokawa, Y., Suzuki, R., Takahashi, T., and Seto, M. (1999) Establishment of an inducible expression system of chimeric MLL-LTG9 protein and inhibition of Hox a7, Hox b7 and Hox c9 expression by MLL-LTG9 in 32Dcl3 cells. Oncogene 18, 1125–1130. Yu, B. D., Hess, J. L., Horning, S. E., Brown, G. A. L., and Korsmeyer, S. J. (1995) Altered Hox expression and segmental identity in Mll-mutant mice. Nature 378, 505–508. Arakawa, H., Nakamura, T., Zhadanov, A. B., Fidanza, V., Yano, T., Bullrich, F., Shimizu, M., Blechman, J., Mazo, A., Canaani, E., and Croce, C. M. (1998) Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene. Proc. Natl. Acad. Sci. USA 95, 4573– 4578. Kanda, Y., Mitani, K., Tanaka, T., Tanaka, K., Ogawa, S., Yazaki, Y., and Hirai, H. (1997) Subcellular localization of the MEN, MLL/MEN and truncated MLL proteins expressed in leukemic cells carrying the t(11;19)(q23;p13.1) translocation. Int. J. Hematol. 66, 189 –195. Kanda, Y., Mitani, K., Kurokawa, M., Yamagata, T., Yazaki, Y., and Hirai, H. (1998) Overexpression of the MEN/ELL protein, an RNA polymerase II elongation factor, results in transformation
Vol. 279, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Rat1 cells with dependence on the lysine-rich region. J. Biol. Chem. 273, 5248 –5252. 23. Maki, K., Mitani, K., Yamagata, T., Kurokawa, M., Kanda, Y., Yazaki, Y., and Hirai, H. (1999) Transcriptional inhibition of p53 by the MLL/MEN chimeric protein found in myeloid leukemia. Blood 93, 3216 –3224. 24. Shinobu, N., Maeda, T., Aso, T., Ito, T., Kondo, T., Koike, K., and
Hatakeyama, M. (1999) Physical interaction and functional antagonism between the RNA polymerase II elongation factor ELL and p53. J. Biol. Chem. 274, 17003–17010. 25. Thirman, M. J., Diskin, E. B., Bin, S. S., Ip, H. S., Miller, J. M., and Simon, M. C. (1997) Developmental analysis and subcellular localization of the murine homologue of ELL. Proc. Natl. Acad. Sci. USA 94, 1408 –1413.
567
Nonredundant Roles of the Elongation Factor MEN in Postimplantation Development Kinuko Mitani,* ,1 Tetsuya Yamagata,* Chika Iida,* Hideaki Oda,† Kazuhiro Maki,* Motoshi Ichikawa,* Takashi Asai,* Hiroaki Honda,* Mineo Kurokawa,* and Hisamaru Hirai* ,2 *Department of Hematology and Oncology and †Department of Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
Received November 8, 2000
The MEN/ELL gene was cloned as a fusion partner of the MLL gene in the t(11;19)(q23;p13.1) translocation, which is found in adult myeloid leukemia. MEN belongs to a family of RNA polymerase II elongation factors and dysregulated production of MEN through the MLL promoter could cause malignant transformation of myeloid cells. To pursue the physiological role and determine the requirement of the MEN gene product in mouse development, we generated knockout mice (MENⴚ/ⴚ) by gene targeting in embryonic stem cells. After intercrossing heterozygous mice to generate homozygous mutants, we identified no homozygotes (MENⴚ/ⴚ) even at E9.5, as well as after birth, by Southern analysis. Moreover, histological examinations revealed degenerative changes in nearly onefourth of E6.5 embryos, which were gradually resorbed by E8.5. Our findings demonstrated that MENⴚ/ⴚ mice are embryonic lethal, and die before E6.5 and after implantation. MEN should play a nonredundant role in postimplantation development of mice. © 2000 Academic Press Key Words: MEN/ELL; elongation factor; knockout mice; embryonic lethality; leukemia; MLL; 11q23 translocation.
MEN is a member of a family of RNA polymerase II elongation factors (1), which consist of P-TEFb, SII, TFIIF, Elongin (SIII), MEN, and ELL2. P-TEFb catalyzes the conversion of early, termination-prone transcription complexes into productive complexes (2). SII expedites elongation by preventing RNA polymerase II from terminating transcription prematurely at a variety of transcription impediments (3). The remaining 1 Present address: Department of Hematology, Dokkyo University School of Medicine, Tochigi, Japan. 2 To whom correspondence should be addressed at Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. Fax: ⫹81-3-5689-7286. E-mail: [email protected].
four support transcriptional elongation by suppressing transient pausing of polymerase reaction at many sites along the template DNA (1, 4 –9). MEN, as well as ELL2, which is 49% identical and 66% similar to MEN, exhibit the elongation enhancing activity conferred by their N-terminal regions (10, 11). MEN also possesses an RNA polymerase II interaction domain in its most N-terminal area, which is capable of negatively regulating the polymerase activity in promoter-specific transcription initiation (11). The MEN gene, mapped to 19p13.1, was originally cloned as the MLL/MEN chimeric cDNAs generated by the t(11;19)(q23;p13.1) translocation (12, 13) which is specifically found in adult myeloid leukemia. The MLL gene located at 11q23 is commonly involved in the 11q23 translocations, which are the most frequently observed chromosomal abnormalities in human leukemias (14, 15). The MLL gene product holds functional domains of transcription factors, including AT-hooks in its N-terminal portion, two zinc finger domains in its middle, and the TRX homology domain in its C-terminal region (16, 17) and positively regulates the expression of the Hoxa-7, Hoxc-9 (18, 19) and ARP1 (20) genes. While MLL⫺/⫺ mice are reported to be embryonic lethal, MLL⫹/⫺ mice display a slight anemia and thrombocytopenia, and axial skeletal and sternal malformations (19). Upon the translocation, the MLL gene is disrupted between the AT-hooks and the zinc finger domains, and the resulting N-terminal 1406 amino acids are fused with the entire coding region of MEN except for its N-terminal 45 amino acids. There are two known mechanisms in leukemogenesis by the t(11;19)(q23;p13.1) translocation. The first one is that the MLL/MEN fusion protein, in which the N-terminal RNA polymerase II interaction domain of MEN has been lost, could show full activation in elongation and cause leukemogenesis. Secondly, MEN itself is a multifunctional protein. Besides transcriptional elongation activities, it also stimulates AP-1 activity by increasing the Fos expression (21, 22) and inhibits transcriptional
563
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 279, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. MEN⫺/⫺ mice are embryonic lethal. (a) Targeting strategy. The homologous recombination event inserted a TK-Neo cassette into the exon 1 of the MEN gene. H, HindIII; probe 1, 5⬘ external probe; probe 2, 3⬘ external probe. (b) Generation of targeted ES cells. G418-resistant ES clones were screened by Southern blot analysis with 5⬘ external (left panel), 3⬘ external (middle panel), and the neo gene (right panel) probes using HindIII digests. Expected fragments hybridized with these probes as depicted in a. The fragment sizes of the wild-type and mutant alleles hybridized with probe 1 were 20 kb and 15 kb, respectively. Those with probe 2 were 20 kb and 6 kb, respectively. The mutant fragment of 6 kb detected with probe 2 was also hybridized with the neo gene probe. Two appropriately targeted clones, 209 and 242, were obtained. ⫹/⫹, wild type; ⫹/⫺, targeted ES cells (209). (c) Results of intercrossing heterozygotes. Embryos were genotyped by Southern blot analysis as in b. No homozygous embryos (⫺/⫺) were found. ⫹/⫹, wild type; ⫹/⫺, heterozygotes.
activity of the p53 tumor suppressor gene (23, 24), leading to the increased colony formation of Rat1 cells. Therefore, the MLL/MEN chimeric protein driven by the MLL promoter might show a stronger oncogenic function through both enhancing the AP-1 activity and suppressing the p53 function. The question whether the MEN protein has redundant activities with other elongation factors, especially with ELL2, could be of great interest. To address the question, we introduced a targeted mutation into the MEN gene by homologous recombination in mouse embryonic stem (ES) cells. Loss of MEN function demonstrated that MEN is indispensable for early embryogenesis. Thus, MEN is a critical elongation factor in embryonic development. MATERIALS AND METHODS Construction of the targeting vector. Murine MEN genomic sequences were isolated from a 129/SvJ strain FIXII library (Stratagene, La Jolla, CA) with a 60-bp EcoRI/PmaCI MEN cDNA probe encoding its N-terminal 17 amino acids in the exon 1. A 10-kb EcoRI fragment in inserts from positive phage was subcloned into pBluescript SK plasmid (Stratagene), and subjected to restriction enzyme mapping. The 1.1-kb TK-Neo cassette derived from pMC1Neo vector
(Stratagene) was inserted into the PmaCI site within the exon 1. The resulting EcoRI fragment of 11.1-kb was then fused with EcoRIdigested pBluescript SK plasmid containing a 1.1-kb MC1-DTA cassette at the SalI site in its multicloning site to generate the final targeting construct. Disruption of the exon 1 resulted in stop codons in all reading frames. Gene targeting in ES cells and generation of mutant mice. The MEN targeting construct was linearized with NotI digestion and electroporated into 129/SvJ ES cells. A 5⬘ flanking 0.3 kb EcoRI/ BamHI fragment (probe 1 in Fig. 1a), a 3⬘ flanking 0.1 kb EcoRI/KpnI fragment (probe 2 in Fig. 1a), and the neo gene probe were used to identify appropriately targeted ES cells by Southern blot analysis with HindIII digests. Aggregation with the targeted ES cells and C57BL/6 derived-morula was performed by Genomsystems Inc. to generate chimeras. One of the resulting chimeras, which exhibited 95% contribution from the ES cells on the basis of agouti coat color, was shown to contribute to the germline, by mating with C57BL/6 mice and subsequent genotype examinations on their pups by Southern analysis. Heterozygous mice were intercrossed to generate homozygous mutants. Neonates and embryos were also genotyped by Southern blot analysis. Histological analysis. To determine the stage of embryonic lethality, we performed histological analysis of the E6.5, 7.5, and 8.5 embryos after mating of heterozygous mice. Pregnant mice were sacrificed at each embryonic day and the whole uteri were fixed in 10% neutral buffered formaldehyde, embedded in paraffin, serially sectioned (8 m), and stained with hematoxylin and eosin. Sections
564
Vol. 279, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
MEN Homozygotes Die before E9.5 Stage
No. of litters
No. of embryos
⫹/⫹
⫹/⫺
Resorbed
E9.5 E10.5 E12.5 E14.5 Neonate
11 3 2 5 13
92 32 13 27 77
23 11 3 12 25
69 20 10 15 52
38 9 5 9 —
(60 –200 pieces per embryo) were carefully examined with a microscope.
RESULTS AND DISCUSSION To inactivate the MEN gene, the TK-Neo cassette was inserted into the exon 1 (Fig. 1a). Of 96 G418resistant ES clones, two independent clones, 209 and 242, showed expected mutant bands of 15 kb and 6 kb hybridized with probes 1 and 2, respectively, in addition to the wild-type fragments of 20 kb (Fig. 1b). The mutant fragment of 6 kb hybridized with probe 2 was also detected with the neo gene probe, indicating that homologous recombination correctly occurred only at the MEN gene locus. These two clones, which contained the appropriately targeted MEN locus, were aggregated with C57BL/6-derived morula and one (209) of them led to germline transmission of the mutation. F 1 mice heterozygous for disruption of the MEN gene appeared normal in size, fertility, and overall development (data not shown). Heterozygous mice were intercrossed to generate homozygotes (MEN⫺/⫺). Of the 77 neonates genotyped, no homozygotes were identified, suggesting that loss of MEN is incompatible with normal embryonic development (Table 1). We sacrificed pregnant females at various days postcoitus to establish the time and cause of death. At E14.5, 12.5, 10.5, and 9.5, we found no live MEN-lacking embryos by Southern analysis (Table 1 and Fig. 1c). Instead, resorbed embryos were detected at a ratio of approximately one-fourth among total implanted portions. These data suggested that MEN⫺/⫺ mice are lethal by E9.5. We next microscopically analyzed the morphological changes of the embryos to precisely identify the stage where MEN⫺/⫺ embryos turn lethal. At E6.5, 4 out of 20 implanted portions in the uteri showed degenerative embryos (Fig. 2a). These composed of almost necrotic cells with scattered condensed nuclei and obscure cytoplasmic margins, accompanied with hemorrage. The cells showed loose contact with one another and we observed no differentiation to ectoderm or entoderm. On the other hand, the other 16 portions contained embryos with normal growth, showing embryonic ectoderm around the proamniotic cavity (Fig. 2b). At E7.5, 3 out of 16 implanted portions examined
showed similar degenerative findings to those at E6.5 (Fig. 2c). One implanted portion revealed empty cavity without embryonic tissues. The other 12 portions contained normally developed embryos with gastrulation (Fig. 2d). At E8.5, 3 out of 12 implanted portions were empty (Fig. 2e). Maternal decidual proliferation, which is evidence for implantation, was observed, but no definite mass was found in the cavity even after observation of all the serial sections. The other nine implanted portions in the uteri showed morphologically normal embryos, in which cephalic neural fold and notochord were well developed (Fig. 2f). We could not clarify whether the degenerative or resorbed embryos observed were actually homozygotes, because they died too early. However, it is reasonable to speculate that MEN⫺/⫺ mice die before E6.5, because approximately one-fourth of embryos were already degenerative at E6.5, and gradually resorbed by E8.5. We also could not specify the reason of death. This is the first report describing the animals deficient in a transcriptional elongation factor. Because MEN is essential for early embryonic development, it might play a non-redundant role to maintain early embryogenesis through regulation of general transcription. We could propose two possible mechanisms in embryonic lethality of MEN⫺/⫺ mice. First, no or less effective transcriptional elongation might occur in the absence of MEN. Considering that MEN is ubiquitously expressed in adult tissues (12, 13) and also diffusely detected in E7.5 embryos (25), it seems that MEN has a fundamental role in keeping life. Second, MEN might exhibit its function on specific target genes, which have a pivotal role in development. It is likely that MEN⫺/⫺ ES cells are viable, because MEN⫺/⫺ embryos die after implantation, as is evident by the presence of maternal decidual tissues. Using differential display or DNA tips with MEN⫺/⫺ ES cells, we could answer the question whether MEN is a key elongation factor for transcription or has specific target genes. Moreover, making chimeric mice with MEN⫺/⫺ ES cells might permit direct studies on organ-specific requirement of MEN. Thirman et al. reported that MEN/ELL is expressed diffusely throughout embryos between E7.5 and E14.5, but specific expression in the liver and gastrointestinal tract becomes prominent by E16.5 (25). If MEN exerts its nonredundant function in such specific organs at the embryonic stage, MEN⫺/⫺ ES cells would not contribute to those organs. In contrast, if no transcription is activated in any organs without MEN, MEN⫺/⫺ ES cells could not be detected in any organs or tissues. Conditional gene targeting technologies, which regulate temporal and cell type-specific inactivation of target genes with Cre/loxP recombination system, are also a powerful tool to determine the cell-specific functions of genes, when their knockout mice are lethal in early development. It will be useful to clarify the roles of MEN,
565
Vol. 279, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6.
7.
8.
9.
10.
11.
FIG. 2. MEN⫺/⫺ mice die before E6.5. (a and b) Magnification ⫻20, transverse section, E6.5; (c and d) magnification ⫻10, transverse section, E7.5; (e and f) magnification ⫻4, transverse section, E8.5. (a, c, and e) Degenerative (a and c) or resorbed (e) embryos, which were expected to be homozygotes because they were observed at a frequency of almost one-fourth. In contrast, normally developed embryos are shown in b, d, and f.
especially in hematopoietic cells, to consider the mechanisms in leukemogenesis by the t(11;19)(q23;p13.1) translocation. Its non-redundant functions among several transcriptional elongation factors might suggest a critical role of dysregulated general transcription in development of hematological malignancies.
12.
13.
14. 15. 16.
ACKNOWLEDGMENTS 17. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and from the Ministry of Education, Science, and Culture of Japan.
18.
REFERENCES 1. Shilatifard, A., Lane, W. S., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1996) An RNA polymerase II elongation factor encoded by the human ELL gene. Science 271, 1873–1876. 2. Marshall, N. F., and Price, D. H. (1995) Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J. Biol. Chem. 270, 12335–12338. 3. Reines, D. (1994) Nascent RNA cleavage by transcription elongation complex. In Transcription Mechanisms and Regulation (Conaway, R. C., and Conaway, J. W., Eds.), pp. 263–278, Raven Press, New York, NY. 4. Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) RNA polymerase II elongation factor SIII. I. Identification, purification, and properties. J. Biol. Chem. 268, 25587–25593. 5. Bradsher, J. N., Tan, S., McLaury, H.-J., Conaway, J. W., and Conaway, R. C. (1993) RNA polymerase II elongation factor SIII.
19.
20.
21.
22.
566
II. Functional properties and role in RNA chain elongation. J. Biol. Chem. 268, 25594 –25604. Kephart, D. D., Wang, B. Q., Burton, Z. F., and Price, D. H. (1994) Functional analysis of Drosophila factor 5 (TFIIF), a general transcriptional factor. J. Biol. Chem. 269, 13536 –13543. Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1989) Dynamic interaction between a Drosophila transcription factor and RNA polymerase II. Mol. Cell. Biol. 9, 1465–1475. Tan, S., Aso, T., Conaway, R. C., and Conaway, J. W. (1994) Roles for both the RAP30 and RAP74 subunits of transcription factor IIF in transcription initiation and elongation by RNA polymerase II. J. Biol. Chem. 269, 25684 –25691. Tan, S., Conaway, R. C., and Conaway, J. W. (1995) Dissection of transcription factor TFIIF functional domains required for initiation and elongation. Proc. Natl. Acad. Sci. USA 92, 6042– 6046. Shilatifard, A., Duan, D. R., Haque, D., Florence, C., Schubach, W. H., Conaway, J. W., and Conaway, R. C. (1997) ELL2, a new member of an ELL family of RNA polymerase II elongation factors. Proc. Natl. Acad. Sci. USA 94, 3639 –3643. Shilatifard, A., Haque, D., Conaway, R. C., and Conaway, J. W. (1997) Structure and function of RNA polymerase II elongation factor ELL. Identification of two overlapping ELL functional domains that govern its interaction with polymerase and the ternary elongation complex. J. Biol. Chem. 272, 22355–22363. Mitani, K., Kanda, Y., Ogawa, S., Tanaka, T., Inazawa, J., Yazaki, Y., and Hirai, H. (1995) Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11;19)(q23;p13.1) translocation. Blood 85, 2017–2024. Thirman, M. J., Levitan, D. A., Kobayashi, H., Simon, M. C., and Rowley, J. D. (1994) Cloning of ELL, a gene that fuses to MLL in a t(11;19)(q23;p13.1) in acute myeloid leukemia. Proc. Natl. Acad. Sci. USA 91, 12110 –12114. Rowley, J. D. (1990) Recurring chromosome abnormalities in leukemia and lymphoma. Semin. Hematol. 27, 122–136. Rubnitz, J. E., Behm, F. G., and Downing, J. R. (1996) 11q23 rearrangements in acute leukemia. Leukemia 10, 74 – 86. Gu, Y., Nakamura, T., Alder, H., Prasad, R., Canaani, O., Cimino, G., Croce, C. M., and Canaani, E. (1992) The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell 71, 701–708. Tkachuk, D. C., Kohler, S., and Cleary, M. L. (1992) Involvement of a homolog of Drosophila Trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 71, 691–700. Joh, T., Hosokawa, Y., Suzuki, R., Takahashi, T., and Seto, M. (1999) Establishment of an inducible expression system of chimeric MLL-LTG9 protein and inhibition of Hox a7, Hox b7 and Hox c9 expression by MLL-LTG9 in 32Dcl3 cells. Oncogene 18, 1125–1130. Yu, B. D., Hess, J. L., Horning, S. E., Brown, G. A. L., and Korsmeyer, S. J. (1995) Altered Hox expression and segmental identity in Mll-mutant mice. Nature 378, 505–508. Arakawa, H., Nakamura, T., Zhadanov, A. B., Fidanza, V., Yano, T., Bullrich, F., Shimizu, M., Blechman, J., Mazo, A., Canaani, E., and Croce, C. M. (1998) Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene. Proc. Natl. Acad. Sci. USA 95, 4573– 4578. Kanda, Y., Mitani, K., Tanaka, T., Tanaka, K., Ogawa, S., Yazaki, Y., and Hirai, H. (1997) Subcellular localization of the MEN, MLL/MEN and truncated MLL proteins expressed in leukemic cells carrying the t(11;19)(q23;p13.1) translocation. Int. J. Hematol. 66, 189 –195. Kanda, Y., Mitani, K., Kurokawa, M., Yamagata, T., Yazaki, Y., and Hirai, H. (1998) Overexpression of the MEN/ELL protein, an RNA polymerase II elongation factor, results in transformation
Vol. 279, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Rat1 cells with dependence on the lysine-rich region. J. Biol. Chem. 273, 5248 –5252. 23. Maki, K., Mitani, K., Yamagata, T., Kurokawa, M., Kanda, Y., Yazaki, Y., and Hirai, H. (1999) Transcriptional inhibition of p53 by the MLL/MEN chimeric protein found in myeloid leukemia. Blood 93, 3216 –3224. 24. Shinobu, N., Maeda, T., Aso, T., Ito, T., Kondo, T., Koike, K., and
Hatakeyama, M. (1999) Physical interaction and functional antagonism between the RNA polymerase II elongation factor ELL and p53. J. Biol. Chem. 274, 17003–17010. 25. Thirman, M. J., Diskin, E. B., Bin, S. S., Ip, H. S., Miller, J. M., and Simon, M. C. (1997) Developmental analysis and subcellular localization of the murine homologue of ELL. Proc. Natl. Acad. Sci. USA 94, 1408 –1413.
567