Fruit fly FTZ-Fl, silkworm BmFTZ-Fl, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor superfamily, which recognizes the same sequence, 5'-PyCAAGG PyCPu-3'. Among these proteins, a 30-amino-acid basic region abutting the C-terminal end of the zinc finger motif, designated the FTZ-Fl box, is conserved. Gel mobility shift competition by various mutant peptides of the DNA-binding region revealed that the FTZ-Fl box as well as the zinc finger motif is involved in the high-affinity binding of FTZ-Fl to its target site. Using a gel mobility shift matrix competition assay, we demonstrated that the FTZ-Fl box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also showed that the DNA-binding region of FTZ-Fl recognizes and binds to DNA as a monomer. Occurrence of the FTZ-Fl box sequence in other members of the nuclear hormone receptor superfamily raises the possibility that these receptors constitute a unique subfamily which binds to DNA as a monomer.The FTZ-F1 protein is a positive regulator of the fushi tarazu gene in blastoderm-stage embryos of Drosophila melanogaster (22). The BmFTZ-F1 protein, a factor present in the silkworm, Bombyx mon, is biochemically similar to . The embryonal long terminal repeat-binding protein (ELP), a mouse factor that is present in undifferentiated murine embryonal carcinoma cells but not in differentiated cells, represses transcription from the promoter of the long terminal repeat of Moloney leukemia virus (17). Molecular cloning of cDNAs for these three proteins revealed that they are members of the nuclear hormone receptor superfamily with Cys2-Cys2-type zinc fingers as DNA-binding domains (4,13,18). FTZ-F1, BmFTZ-F1, and ELP recognize the 9-bp sequence 5'-PyCAAGGPyCPu-3', which apparently does not have a direct or inverted repeat (16)(17)(18)(20)(21)(22). In contrast, other members of the nuclear hormone receptor superfamily usually bind to repeated sequences (1,6,24). Nevertheless, the FTZ-F1, BmFTZ-F1, and ELP proteins have high affinities to the binding-site DNA (23). These results indicate that the mechanism of binding of these proteins to DNA is somewhat different from that of other members of the nuclear hormone receptor superfamily.Using various mutant peptides in the DNA-binding domain of FTZ-F1, we demonstrated that in addition to the zinc finger motif, the basic region abutting the C-terminal end of the zinc finger motif is involved in sequence-specific DNA binding and that the 9-bp binding site is recognized by a single polypeptide. MATERIALS AND METHODSPlasmid construction. To express peptides carrying various lengths of the DNA-binding region of FIZ-F1 in Escherichia coli, three DNA fragments coding for amino acids 507 to 622, 507 to 581, and 507 to 593 preceded by methionine as the translational start site were made by using * Corresponding author.polymerase chain reactions (8) with FTZ-F1 cDNA as the template. Amplification with the N-...

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Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ESlike cells (E-1 and E-2) appear to maintain a normal diploid karyotype inde¢nitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-speci¢c embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells di¡erentiated to neural precursor cells in the presence of basic ¢broblast growth factor (bFGF), epidermal growth factor and platelet-derived growth factor. We also developed a protocol that resulted in the di¡er-entiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.

A mouse cDNA that encodes a DNA-binding protein was identified by yeast two-hybrid screening, using activating transcription factor-2 (ATF-2) as the bait. The protein contained a bZIP (basic amino acid-leucine zipper region) domain and its amino acid sequence was almost identical to that of rat Jun dimerization protein 2 (JDP2). Mouse JDP2 interacted with ATF-2 both in vitro and in vivo via its bZIP domain. It was encoded by a single gene and various transcripts were expressed in all tested tissues of adult mice, as well as in embryos, albeit at different levels in various tissues. Furthermore, mouse JDP2 bound to the cAMP-response element (CRE) as a homodimer or as a heterodimer with ATF-2, and repressed CRE-dependent transcription that was mediated by ATF-2. JDP2 was identified as a novel repressor protein that affects ATF-2-mediated transcription. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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A p21Cip1/Waf1/Sdi1 is known to act as a negative cellcycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a twohybrid system. Two splicing isoforms of TOK-1, TOK-1␣ and TOK-1␤, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1␣, but not TOK-1␤, directly bound to the C-terminal proximal region of p21, and both were expressed at the G 1 /S boundary of the cell cycle. TOK-1␣ also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1␣ enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1␣ is thus thought to be a new type of CDK2 modulator.Cell-cycle movement is known to be coordinately regulated by several combinations of cyclin-cyclin-dependent kinase (CDK) 1 complex and their inhibitors. INK4 family proteins, including p15, p16, p18, and p19, inhibit CDK4/CDK6, and Cip/Kip family proteins, including p21, p27, and p57, inhibit all of the CDKs (see recent reviews, Refs. 1-3 and references therein). A cDNA of p21Cip1/Waf1/Sdi1 was cloned independently by different three procedures: Cip1 (4), a cyclin-dependent kinase 2 (CDK2)-binding protein; Waf1, p53-inducible protein (5); and Sdi1, a senescent-inducible protein (6). The p21 gene is induced dependently or independently by p53 in cells after various stresses. p53-dependent p21 induction occurs by x-ray or UV irradiation, which causes DNA damage, and by heat shock or osmotic shock to cells (5). Nutrition starvation, contact inhibition, terminal differentiation, or aging of cells triggers p53-independent p21 induction that is brought about by transcription factors, including STAT family protein (7), C/EBP␣ (8), MyoD (9, 10), and vitamin D3 receptor (11). In any case, p21 induces cell cycle arrest, thus inhibiting CDK activity necessary for Rb inactivation.In addition to binding of CDK-cyclin to the N-terminal region of p21 (12-16), a variety of proteins were found to bind to the C-proximal region of p21. DNA replication activity of DNA polymerase ␦ is inhibited by p21 by binding to a proliferating cell nuclear antigen (PCNA), indicating that p21 also has the function of stopping the cell cycle during the S phase (12,13,15,(17)(18)(19)(20). Furthermore, SET/TAF1 (21), human papilloma virus E7 (22, 23), c-Myc (24), and calmodulin (25) are reported to bind to the C-terminal region of p21. The molecular mechanisms explaining the versatile functions of p21, however, remain to be determin...

The transcription factor FTZ-F1 is a member of the nuclear hormone receptor superfamily and is transiently expressed during the mid-and late prepupal periods in Drosophila melanogaster. A putative pupal cuticle gene, EDG84A, is expressed slightly following FTZ-F1 expression during the prepupal period and carries a strong FTZ-F1 binding site between bases 100 and 92 upstream of its transcription start site. In this study, EDG84A mRNA was found to be prematurely expressed upon heat induction of FTZ-F1 in prepupae carrying the heat shock promoter-FTZ-F1 cDNA fusion gene construct. Transgenic fly lines having the 0.8-kb region of the EDG84A promoter fused to lacZ expressed the reporter gene in a tissue-and stage-specific manner. Base substitutions in the FTZ-F1 binding site within the 0.8-kb promoter abolished expression of lacZ. These results strongly suggest that the EDG84A gene is a direct target of FTZ-F1. Deletion studies of the cis-regulatory region of the EDG84A gene revealed that space-specific expression in imaginal disc-derived epidermis is controlled by the region between bp ؊408 and ؊104 from the transcription start site. The region between bp ؊408 and ؊194 is necessary to repress expression in a posterior part of the body, while the region between bp ؊193 and ؊104 carries a positive element for activation in an anterior part of the body. These results suggest that FTZ-F1 governs expression of the EDG84A gene in conjunction with putative tissue-specific regulators.

Jun dimerization protein-2 (JDP2) is a component of the AP-1 transcription factor that represses transactivation mediated by the Jun family of proteins. Here, we examine the functional mechanisms of JDP2 and show that it can inhibit p300-mediated acetylation of core histones in vitro and in vivo. Inhibition of histone acetylation requires the N-terminal 35 residues and the DNA-binding region of JDP2. In addition, we demonstrate that JDP2 has histone-chaperone activity in vitro. These results suggest that the sequence-specific DNA-binding protein JDP2 may control transcription via direct regulation of the modification of histones and the assembly of chromatin.