The present invention concerns a nuclear receptor (NR) transcriptional mediator. More specifically, isolated nucleic acid molecules are provided encoding transcriptional intermediary factor-2 (TIF2). Recombinant methods for making TIF2 polypeptides are also provided as are TIF2 antibodies. Screening methods are also provided for identifying agonists and antagonists of the activation function AF-2 of nuclear receptors, for identifying agonists and antagonists of the AD1 activation domain activity of TIF2, and for identifying agonists and antagonists of the AD2 activation domain activity of TIF2.

FIELD OF THE INVENTION
 The present invention relates to a nuclear receptor (NR) transcriptional
 mediator. More specifically, isolated nucleic acid molecules are provided
 encoding transcriptional intermediary factor-2 (TIF2). Recombinant methods
 for making TIF2 polypeptides are also provided as are screening methods
 for identifying agonists and antagonists of the activation function AF-2
 of nuclear receptors, as well as TIF2 antibodies. Also provided are
 screening methods for identifying agonists and antagonists of TIF2 AD1
 activation domain activity, as are provided screening methods for
 identifying agonists and antagonists of TIF2 AD2 activation domain
 activity.
 BACKGROUND OF THE INVENTION
 Activators that enhance the initiation of transcription by RNA polymerase B
 (II) are composed of at least two functional domains: a DNA binding domain
 and an activating domain (M. Ptashne, Nature 335:683-689 (1988); P. J.
 Mitchell e al., Science 245:371-378 (1989)). These two domains are
 generally separable functional units and each can actually be interchanged
 with the complementary region of an unrelated activator, thereby creating
 functional chimeric activators (S. Green et al., Nature 325:75-78 (1987)).
 A number of structure-function analyses of eukaryotic transcriptional
 activators have been performed, focussing primarily on the yeast GAL4 and
 GCN4 proteins and on members of the nuclear receptor family. GAL4 and GCN4
 proteins activate transcription by binding to specific upstream activation
 sequence, which have many of the characteristics of higher eukaryotic
 enhancer elements (K. Struhl, Cell 49:295-297 (1987)). The herpes simplex
 activator VP16 represents another type of activator, which activates
 transcription by binding to the DNA-bound octamer transcription factor
 rather than binding to the DNA directly (T. Gerster et al., Proc. Natl.
 Acad. Sci. USA 85:6347-6351 (1988)).
 The nuclear receptor family, which includes receptors for steroid hormones,
 thyroid hormones, vitamin D, and the vitamin A derivative retinoic acid,
 are also transcriptional enhancer factors which bind DNA directly in the
 presence of their cognate ligand by recognition of specific enhancer
 elements, i.e., hormone- or ligand-responsive elements (R. M. Evans, Cell
 240:889-895 (1988)). These cognate ligands tend to be small, hydrophobic
 molecules, including steroid hormones such as estrogen and progesterone,
 thyroid hormone, vitamin D, and various retinoids (S. Halachmi et al.,
 Science 264:1455-1458 (1994); Gronemeyer, H. and Laudet, V., Protein
 Profile 2:1173-1308 (1995)).
 Despite their small size and apparently simple structure, however, the
 cognate ligands associated with NRs are known to elicit a wide range of
 physiological responses. Adrenal steroids for example, such as cortisol
 and aldosterone, widely influence body homeostasis, controlling glycogen
 and mineral metabolism, have widespread effects on the immune and nervous
 systems, and influence the growth and differentiation of cultured cells.
 The sex hormones (progesterone, estrogen and testosterone) provoke the
 development and determination of the embryonic reproductive system,
 masculinize/feminize the brain at birth, control reproduction and related
 behavior in adults and are responsible for development of secondary sex
 characteristics. Vitamin D is necessary for proper bone development and
 plays a critical role in calcium metabolism and bone differentiation.
 Significantly, aberrant production of these hormones has been associated
 with a broad spectrum of clinical disease, including cancer and similar
 pathologic conditions.
 All NRs display a modular structure, with five to six distinct regions,
 termed A-F. The N-terminal A/B region contains the activation function
 AF-1, which can activate transcription constitutively. Region C
 encompasses the DNA binding domain (DBD), which recognizes cognate
 cis-acting elements. Region E contains the ligand-binding domain (LBD), a
 dimerization surface and the ligand-dependent transcriptional activation
 function AF-2 (reviewed in Mangelsdorft, D. J. et al., Cell 83:835-839
 (1995a); Mangelsdorft & Evans, Cell 83:841-850 (1995b); Beato, M. et al.,
 Cell 83:851-857 (1995); Gronemeyer & Laudet, "Transcription Factors 3:
 Nuclear Receptors", in Protein Profile, vol. 2, Academic Press (1995);
 Kastner, P. et al., EMBO J. 11:629-642 (1992); Chambon, P., FASEB J
 10:940-954 (1996)).
 Several classes of domains in activators are capable of mediating
 transcriptional activation. Yeast activators GAL4 and GCN4 and herpes
 simplex VP16 all contain activation domains that are composed of acidic
 stretches of amino acids, which may act by forming amphipathic a helices
 (I. A. Hope et al., Cell 46:885-894 (1986); J. Ma et al., Cell 48:847-853
 (1987); E. Giniger et al., Nature 330:670-672 (1987); S. J. Triezenberg et
 al, Genes Dev. 2:718-729 (1988)). The activation functions of human Sp1
 and CTF/NFI proteins contain glutamine- and proline-rich areas,
 respectively (A. J. Courey et al., Cell 55:887-898 (1988); N. Mermod et
 al., Cell 58:741-753 (1989)). Studies with steroid hormone receptors have
 shown that both the N-terminal A/B domain and the C-terminal hormone
 binding domain (HBD) contain transcription activation functions (AFs) (M.
 T. Bocquel et al., Nucl. Acids Res., 17:2581-2595 (1989); L. Tora et al.,
 Cell 59:477-487 (1989)). The AFs of the human estrogen receptor (hER) do
 not contain stretches of acidic amino acids (S. Halachmi et al., Science
 264:1455-1458 (1994)). Conversely, however, the human glucocorticoid
 receptor (hGR) contains two activation functions, .tau.-1 (located in the
 A/B domain) and .tau.-2 (located in the N-terminal region of the HBD),
 both of which are acidic (S. M. Hollenberg et al., Cell 55:899-906
 (1988)).
 From the results of studies on transcriptional interference/squelching
 between nuclear receptors and on homo- and heterosynergistic stimulation
 of initiation of transcription from minimal promoters by the activation
 functions present in hER (AF-1 and AF-2) and the acidic activator VP16, it
 has been proposed that AFs may activate transcription by interacting with
 different components of the basic initiation complex (Bocquel et al.,
 Nucl. Acids. Res. 17:2581-2595 (1989); Meyer et al., Cell 57:433-442
 (1989); L. Tora et al., Cell 59:477-487 (1989)). Studies of the
 transcriptional interference/squelching properties of AADs, hER AF-1 and
 hER AF-2, however, showed that both hER AF-1 and AF-2 can squelch acidic
 activators, such as VP16, but that the converse was not true, i.e., AADs
 do not squelch hER AF-1 or AF-2. Moreover, hER AF-1 and AF-2, which are
 clearly distinguished by their synergistic properties, nevertheless
 squelch each other (D. Tasset et al., Cell 62:1177-1187 (1990)).
 Based on these results, it was proposed that a string of transcriptional
 intermediary factors (TIFs) exists, interposed between enhancer factors
 and the basic transcriptional factors. For example, AF-1 and AF-2 have
 been suggested to contact the string of TIFs at functionally equivalent
 points, while AADs are believed to interact at an earlier point in the
 series (D. Tasset et al., Cell 62:1177-1187 (1990)).
 Several putative coactivator TIFs for NR AF-2s have been characterized (see
 Chambon, P., FASEB J 10:940-954 (1996); Glass, C. K. et al., Current Opin.
 Cell Biol. 9:222-232 (1997); Horwitz, K. B. et al., Mol. Endocrinol.
 10:1167-1177 (1996) for recent reviews). In particular, LeDouarin, B. et
 al., EMBO J. 15:6701-6715 (1996) have demonstrated that a 10-amino acid
 fragment of TIF1.alpha. is necessary and sufficient to mediate interaction
 with RXR in a ligand- and AF-2 integrity-dependent manner. Notably, within
 this TIF1.alpha. fragment, they identified a LxxLLL (SEQ ID NO:13) motif,
 termed NR box, whose integrity is required for interaction with nuclear
 receptors, and pointed out that this motif is conserved in several other
 putative coactivators (LeDouarin, B. et al., EMBO J. 15:6701-6715 (1996))
 Whereas TIF1.alpha. and several other putative coactivators do not, or
 only very poorly, stimulate transactivation by NRs in transiently
 transfected mammalian cells, the TIF2/SRC-1 family (Onate, S. A. et al.,
 Science 270:1354-1357 (1995); Voegel, J. J. et al., EMBO J. 15:3667-3675
 (1996)), the CBP/p300 family (Kamei, Y. et al., Cell 85:403-414 (1996);
 Chakravarti, D. et al., Nature 5:99-103 (1996); Hanstein, B., et al.,
 Proc. Natl. Acad. Sci USA 93:11540-11545 (1996); Smith, C. L. et al.,
 Proc. Natl. Acad. Sci. USA 93:8884-8888 (1996); for recent reviews see
 Eckner, R. , Biol. Chem. 377:685-688 (1996); Janknecht &Hunter, Current
 Biol. 6:951-954 (1996b); Shikama, N. et al., Trends in Cell Biol.
 7:230-236 (1997)) and the androgen receptor coactivator ARA70 (Yeh &
 Chang, Proc. Natl. Acad Sci. USA 93:5517-5521 (1996)) have been
 unequivocally shown to enhance AF-2 activity.
 In addition to binding NRs, CBP/p300 can also interact directly with SRC-1
 (Kamei, Y. et al., Cell 85:403-414 (1996); Yao, T. P. et al., Proc. Natl.
 Acad. Sci. USA 93:10626-10631 (1996)) and both factors have been shown to
 exert histone acetyltransferase activity (Bannister & Kouzarides, Nature
 384:641-643 (1996); Ogryzko, V. V. et al., Cell 87:953-959 (1996)).
 Moreover, CBP/p300 can recruit p/CAF which is itself a nuclear histone
 acetyltransferase (Yang, X. J. et al., Nature 382:319-324 (1996)).
 However, apart from interacting with coactivators in a ligand-dependent
 manner, NRs have also been shown to interact, often in a
 ligand-independent fashion, directly or indirectly with components of the
 transcriptional machinery, suds as TFIIB, TBP, TAFs, or TFIIH (Baniahmad
 et al., (1993)); Jacq, X. et al., Cell 79:107-117 (1994); Schulman, I G.
 et al., Mol. Cell. Biol. 16:3807-3813 (1996); May, M. et al., EMBO J.
 15:3093-3104 (1996); Mengus, G. et al., Genes & Dev. 11:1381-1395 (1997)).
 Hong, H. et al., Proc. Natl. Acad. Sci. USA 93:4948-4952 (1996) originally
 described a partial cDNA of the mouse homologue of TIF2, named GRIP 1, and
 recently reported the isolation of a full length GRIP1 cDNA (Hong, H. et
 al., Mol. Cell. Biol. 17:2735-2744 (1997)). Using the yeast Saccharomyces
 cerevisiae as a model system, they have shown that transcriptional
 activation by TR, RAR and RXR, could also be stimulated by GRIP1
 coexpression, which suggests that TIF2/GRIP1 could be a general
 coactivator for NRs (Hong, H. et al., Mol. Cell. Biol. 17:2735-2744
 (1997)).
 The overall picture emerging from multiple recent studies on the mechanisms
 by which nuclear receptors modulate target gene transcription involves
 three subsequent steps, (i) the ligand-induced transconformation of the NR
 LBD, which results in (ii) the dissociation of corepressors and formation
 of TIFs/coactivator complexes, which themselves (iii) through interaction
 with additional downstream factors (e.g., CBP, p300) modulate the
 acetylation status of core histones and, thus, chromatin
 condensation/decondensation. Histone acetylation on its own is, however,
 insufficient for transcription activation (Wong et al., (1997)), and a
 simultaneous or subsequent fourth event comprises the direct and/or
 indirect recruitment of elements of the transcription machinery (e.g.,
 TFIB, TBP, TAFs, TFIIH; Jacq, X. et al., Cell 79:107-117 (1994); Schulman,
 IG. et al., Mol. Cell. Biol. 16:3807-3813 (1996); May, M. et al., EMBO J.
 15:3093-3104 (1996); Mengus, G. et al., Genes & Dev. 11:1381-1395 (1997)).
 Note that such interactions do not need to be ligand-dependent, if the
 primary function of the liganded LBD (AF-2) is to regulate DNA
 accessibility through chromatin remodeling. Indeed, several of the
 reported interactions between NRs and general transcription factors occur
 in a ligand-independent manner. Accordingly, there is a need in the art
 for the isolation and characterization of transcriptional intermediary
 factors.
 SUMMARY OF THE INVENTION
 By screening 340,000 clones of a human placenta cDNA expression library
 with an estradiol-bound estrogen receptor probe, the present inventors
 have identified a cDNA clone containing the gene encoding transcriptional
 intermediary factor 2 (TIF2). By the invention, TIF2 has been shown to
 exhibit all the properties expected for a TIF/mediator of AF-2: it
 interacts directly with the LBDs of several NRs in an agonist- and
 AF-2-integrity-dependent manner in vitro and in vivo, harbours an
 autonomous AF, relieves NR autosquelching, and enhances the activity of NR
 AF-2s when overexpressed in mammalian cells.
 Thus, in one aspect, the present invention provides isolated nucleic acid
 molecules comprising a polynucleotide encoding TIF2 whose amino acid
 sequence is shown in FIG. 1 (SEQ ID NO:2) or a fragment thereof. In
 another aspect, the invention provides isolated nucleic acid molecules
 encoding TIF2 having an amino acid sequence as encoded by the cDNA
 deposited as ATCC Deposit No. 97612.
 The invention further provides an isolated nucleic acid molecule that
 hybridizes under stringent conditions to the above-described nucleic acid
 molecules. The present invention also relates to variants of the nucleic
 acid molecules of the present invention, which encode fragments, analogs
 or derivatives of the TIF2 protein, e.g., polypeptides having at least one
 biological activity that is substantially similar to at least on
 biological activity of the TIF2 protein.
 The present invention is further directed to isolated nucleic acid
 molecules that encode a cytoplasmic TIF2 polypeptide. Methods for
 generating nucleic acid molecules that encode a cytoplasmic TIF2
 polypeptide include mutating or deleting the NLSs-coding N-terminal region
 of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1). Preferably,
 nucleic acid molecules encoding a cytoplasmic TIF2 polypeptide will be
 fragments having a deletion in all or part of the N-terminal NLSs coding
 region. By the invention, the cytoplasmic TIF2 polypeptides described
 herein display at least one biological activity that is substantially
 similar to at least one biological activity of TIF2.
 Further embodiments of the invention include isolated nucleic acid
 molecules comprising a polynucleotide having a nucleotide sequence at
 least 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or
 99% identical to the above described nucleic acid molecules.
 The present invention also relates to vectors which contain the
 above-described isolated nucleic acid molecules, host cells transformed
 with the vectors and the production of TIF2 polypeptides by recombinant
 methods.
 The present invention further provides isolated TIF2 polypeptides having
 the amino acid sequence shown in FIG. 1 (SEQ ID NO:2). In a further
 aspect, isolated TIF polypeptides are provided having an amino acid
 sequence as encoded by the cDNA deposited as ATCC Deposit No. 97612.
 Screening methods are also provided for identifying agonists and
 antagonists of nuclear receptor AF-2 function, for identifying agonists
 and antagonists of TIF2 AD1 activity, and for identifying agonists and
 antagonists of TIF2 AD2 activity. Also provided are TIF2 antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 The present invention provides isolated nucleic acid molecules comprising a
 polynucleotide encoding transcriptional intermediary factor-2 (TIF2) whose
 amino acid sequence is shown in FIG. 1 (SEQ ID NO:2). The TIF2 protein of
 the present invention shares sequence homology with the human steroid
 receptor coactivator SRC-1 (SEQ ID NO:3) (FIG. 3). The nucleotide sequence
 shown in FIG. 1 (SEQ ID NO:1) was obtained by sequencing a cDNA clone
 which was deposited on Jun. 14, 1996 at the ATCC and given accession
 number 97612.
 Nucleic Acid Molecules
 In one embodiment of the present invention, isolated nucleic acid molecules
 are provided which encode the TIF2 protein. Sequence similarities between
 TIF2 and SRC-1 (Onate et al, Science 270:1354 (1995)) indicate the
 existence of a novel gene family of NR transcriptional mediators. Using
 information provided herein, such as the nucleotide sequence in FIG. 1
 (SEQ ID NO:1) or the above-described deposited clone, a nucleic acid
 molecule of the present invention encoding a TIF2 polypeptide may be
 obtained using standard cloning and screening procedures. Illustrative of
 the invention, the nucleic acid molecule described in FIG. 1 (SEQ ID NO:1)
 was discovered in a cDNA expression library from human placenta tissue.
 The TIF2 cDNA of the present invention encodes a protein of about 159 kDa
 (1,464 amino acids), which includes N-terminal nuclear localization
 signals (NLSs), one Gln- and three Ser/Thr-rich regions, and two charged
 clusters (FIG. 3). TIF2 is widely expressed, since the corresponding
 transcript was found in several human tissues, including pancreas, kidney,
 muscle, liver, lung, placenta, brain and heart (FIG. 2c).
 Isolated nucleic acids of the present invention may be in the form of RNA,
 such as mRNA, or in the form of DNA, including, for instance, cDNA and
 genomic DNA obtained by cloning or produced synthetically. The DNA may be
 double-stranded or single-stranded. Single-stranded DNA or RNA may be the
 coding strand, also known as the sense strand, or it may be the non-coding
 strand, also referred to as the anti-sense strand.
 By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule,
 DNA or RNA, which has been removed from its native environment. For
 example, recombinant DNA molecules contained in a vector are considered
 isolated for purposes of the present invention. Additional illustrative
 examples of isolated DNA molecules include recombinant DNA molecules
 maintained in heterologous host cells and purified (partially or
 substantially) DNA molecules in solution. Isolated RNA molecules include
 in vitro RNA transcripts of the DNA molecules of the present invention as
 well as partially or substantially purified mRNA molecules. Isolated
 nucleic acid molecules according to the present invention further include
 such molecules produced synthetically.
 Isolated nucleic acid molecules of the present invention include DNA
 molecules comprising an open reading frame (ORF) with an initiation codon
 at position 163-165 of the nucleotide sequence shown in FIG. 1 (SEQ ID
 NO:1); and DNA molecules which comprise a sequence substantially different
 than that described above but which, due to the degeneracy of the genetic
 code, still encode the TIF2 protein. Of course, the genetic code is well
 known in the art. Thus, it would be routine for one skilled in the art to
 generate the degenerate variants described above.
 In another aspect, the invention provides isolated nucleic acid molecules
 encoding the TIF2 polypeptide having an amino acid sequence as encoded by
 the cDNA clone deposited as ATCC Deposit No. 97612 on June 14, 1996
 (American Type Culture Collection, (ATCC) 10801 University Boulevard,
 Manassas, Va. 20110-2209). The invention further provides an isolated
 nucleic acid molecule having the nucleotide sequence shown in FIG. 1 (SEQ
 ID NO:1) or the nucleotide sequence of the TIF2 cDNA contained in the
 above-described clone, or a nucleic acid molecule having a sequence
 complementary to one of the above sequences. Such isolated nucleic acid
 molecules, preferably DNA molecules, are useful as probes for gene mapping
 by in situ hybridization with chromosomes and for detecting expression of
 the TIF2 gene in human tissue, for instance, by Northern blot analysis.
 In another aspect, the invention provides an isolated nucleic acid molecule
 that hybridizes under stringent conditions to the above-described nucleic
 acid molecules. As used herein "stringent conditions" is intended to mean,
 as a non-limiting example, overnight incubation at 42.degree. C. in a
 solution comprising 50% formamide, 5.times.SSC (150 mM NaCl, 15 mM
 trisodium citrate), 50 mM sodium phosphate (pH7.6), 5.times.Denhardt's
 solution, 10% dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon
 sperm DNA, followed by washing the filters in 0.1.times.SSC at about
 65.degree. C. Preferably, such "an isolated nucleic acid molecule that
 hybridizes under stringent conditions" will be at least 15 bp, preferably
 at least 20 bp, more preferably at least 30 bp, and most preferably, at
 least 50 bp in length.
 As used herein, "fragments" of an isolated DNA molecule having the
 nucleotide sequence of the deposited cDNA or the nucleotide sequence as
 shown in FIG. 1 (SEQ ID NO:1) is intended to mean DNA fragments at least
 15 bp, more preferably at least 20 bp, and most more preferably at least
 30 bp in length which are useful as diagnostic probes and primers as
 discussed above and in more detail below. Larger DNA fragments, up to, for
 example, 500 bp in length, are also useful as probes according to the
 present invention. A fragment of at least 20 bp in length, for example, is
 intended to mean fragments which include 20 or more contiguous bases from
 the nucleotide sequence of the deposited cDNA or the nucleotide sequence
 as shown in FIG. 1 (SEQ ID NO:1). As indicated, such fragments are useful
 diagnostically either as a probe according to conventional DNA
 hybridization techniques or as primers for amplification of a target
 sequence by the polymerase chain reaction (PCR).
 Since the gene has been deposited and the nucleotide sequence shown in FIG.
 1 (SEQ ID NO:1) is provided, generating such DNA fragments would be
 routine to the skilled worker in the relevant art. Restriction
 endonuclease cleavage or shearing by sonication, for example, may easily
 be used to generate fragments of various sizes. Alternatively, the DNA
 fragments of the present invention can be generated synthetically
 according to the methods and techniques known and available to those
 skilled in the art. Ten expressed sequence tags with homology to part of
 the TIF-2 nucleoide sequence were identified by the inventors in GenBank:
 GenBank Accession numbers T77249, R77864, T77464, R77770, R08880, T85560,
 R25318, T85561, R08986 and R26517.
 The present invention further relates to variants of the nucleic acid
 molecules of the present invention, which encode for fragments, analogs or
 derivatives of the TIF2 protein, e.g., polypeptides having biological
 activity substantially similar to the TIF2 protein. Variants may occur
 naturally, such as isoforms and allelic variants. Non-naturally occurring
 variants may be produced using any of the mutagenesis techniques known and
 available to those skilled in the art.
 Such variants include those produced by nucleotide substitutions, deletions
 or additions. The substitutions, deletions or additions may involve one or
 more nucleotides. The variants may be altered in coding or non-coding
 regions or both. Alterations in the coding regions may produce
 conservative or non-conservative amino acid substitutions, deletions or
 additions. Especially preferred among these are silent substitutions,
 additions and deletions, which do not alter the properties and activities
 of the TIF2 protein or fragment thereof Also especially preferred in this
 regard are conservative substitutions.
 The present invention is further directed to isolated nucleic acid
 molecules that encode a cytoplasmic TIF2 polypeptide. Full-length TIF2 is
 a nuclear protein due to the presence of N-terminal nuclear localization
 signals (NLSs) (FIG. 3). By a "cytoplasmic TIF2 polypeptide", is intended
 a TIF2 polypeptide that is essentially found in the cytoplasm after being
 recombinantly expressed in mammalian cells. Methods for generating nucleic
 acid molecules that encode a cytoplasmic TIF2 polypeptide include mutating
 or deleting the NLSs-coding N-terminal region of the nucleotide sequence
 shown in FIG. 1 (SEQ ID NO:1). Examples of NLS sequences include amino
 acids 13-20 and 31-39 and nucleotides 199-222 and 253-279 of FIG. 1 (See
 also, FIG. 3). Suitable mutations to the NLSs-coding N-terminal region
 include substitutions, deletions and insertions which result in a nucleic
 acid molecule that encodes a TIF2 polypeptide lacking the nuclear
 localization function. Methods for generating such mutations will be
 readily apparent to the skilled artisan and are described, for instance,
 in Molecular Cloning, A Laboratory Manual, 2nd edition, edited by
 Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), Cold Spring Harbor
 Laboratory Press.
 Preferably, nucleic acid molecules encoding a cytoplasmic TIF2 polypeptide
 will be fragments having a deletion in all or part of the N-terminal NLSs
 coding region. Methods for generating such fragments are described below.
 According to the present invention, such nucleic acid fragments further
 include N-terminal deletions extending beyond the NLSs coding region and
 may also include C-terminal deletions. For example, the present inventors
 have generated a nucleic acid molecule encoding the cytoplasmic TIF2.1
 polypeptide (amino acids 624 to 1287 in FIGS. 1 and 3 (SEQ ID NO: 2)),
 which, like the nuclear full-length TIF2, interacts in an
 agonist-dependent manner with the nuclear receptors and enhances nuclear
 receptor-mediated transcriptional activation. The present inventors have
 also generated a nucleic acid molecule encoding the cytoplasmic TIF2.5
 polypeptide (amino acids 624-869 in FIGS. 1 and 3 (SEQ ID NO:2)), which
 interacts with the NID domain of nuclear receptors, but does not enhance
 transcription. Also generated were nucleic acids encoding the cytoplasmic
 TIF2.8 and TIF 2.12 polypeptides (amino acids 1010-1179 and amino acids
 940-1131, respectively, in FIGS. 1 and 3 (SEQ ID NO:2), which enhance
 transcription, but do not bind to nuclear receptors. Thus, by the
 invention, nucleic acid molecules are provided encoding cytoplasmic TIF2
 polypeptides that interact in an agonist-dependent manner with nuclear
 receptors and enhance nuclear receptor-mediated transcriptional
 activation. Also provided are cytoplasmic TIF2 polypeptides that bind to
 nuclear receptors, but do not enhance transcription as are provided
 cytoplasmic TIF2 polypeptides that enhance transcription, but do not bind
 to nuclear receptors. As the skilled artisan will recognize, the length of
 such nucleic acid molecules can vary.
 Further embodiments of the invention include isolated nucleic acid
 molecules comprising a polynucleotide having a nucleotide sequence at
 least 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or
 99% identical to: (a) the nucleotide sequence of the cDNA deposited as
 ATCC 97612; (b) the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1); (c)
 the nucleotide sequence of the cDNA deposited as ATCC 97612 which encodes
 the full-length TIF2 protein; (d) the nucleotide sequence shown in FIG. 1
 (SEQ ID NO:1), which encodes the full-length TIF2 protein; (e) the
 nucleotide sequence of the cDNA deposited as ATCC 97612, which encodes the
 functional coactivator TIF2.1 protein; (f) the nucleotide sequence shown
 in FIG. 1 (SEQ ID NO:1), which encodes the functional coactivator TIF2.1
 protein; (g) the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) which
 encodes the TIF2.0 polypeptide; (h) the nucleotide sequence shown in FIG.
 1 (SEQ ID NO:1) which encodes the TIF2.2 polypeptide; (i) the nucleotide
 sequence shown in FIG. 1 (SEQ ID NO:1) which encodes the TIF2.3
 polypeptide; (j) the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1)
 which encodes the TIF2.4 polypeptide; (k) the nucleotide sequence shown in
 FIG. 1 (SEQ ID NO:1) which encodes the TIF2.5 polypeptide; (l) the
 nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) which encodes the TIF2.6
 polypeptide; (m) the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1)
 which encodes the TIF2.7 polypeptide; (n) the nucleotide sequence shown in
 FIG. 1 (SEQ ID NO:1) which encodes the TIF2.8 polypeptide; (o) the
 nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) which encodes the TIF2.9
 polypeptide; (p) the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1)
 which encodes the TIF2.10 polypeptide; (q) the nucleotide sequence shown
 in FIG. 1 (SEQ ID NO:1) which encodes the TIF2.12 polypeptide; and (r) a
 nucleotide sequence complementary to any of the nucleotide sequences in
 (a-q).
 Whether any two nucleic acid molecules have nucleotide sequences that are
 at least 90%, 95%, 96%, 97%, 98%, or 99% "identical" can be determined
 conventionally using known computer algorithms such as the "fastA" program
 using, for example, the default parameters (Pearson and Lipman, Proc.
 Natl. Acad. Sci. USA 85:2444 (1988)). The present application is directed
 to such nucleic acid molecules having a nucleotide sequence at least 90%,
 95%, 96%, 97%, 98%, 99%, identical to the nucleotide sequence of the
 above-recited nucleic acid molecules irrespective of whether they encode a
 polypeptide having TIF2 activity. This is because, even where a particular
 nucleic acid molecule does not encode a polypeptide having TIF2 activity,
 one of skill in the art would still know how to use the nucleic acid
 molecule as a probe. Uses of the nucleic acid molecules of the present
 invention that do not encode a polypeptide having TIF2 activity include,
 inter alia, (1) isolating the TIF2 gene or allelic variants thereof in a
 cDNA library; (2) in situ hybridization (FISH) to metaphase chromosomal
 spreads to provide precise chromosomal location of the TIF2 gene as
 described in Verma et al., Human Chromosomes: a Manual of Basic
 Techniques, Pergamon Press, New York (1988), and Northern Blot analysis
 for detecting TIF2 mRNA expression in specific tissues, such as placenta
 tissue.
 Preferred, however, are nucleic acid molecules having a nucleotide sequence
 at least 90%, and preferably at least 95%, 96%, 97%, 98%, or 99% identical
 to the nucleotide sequence of the above-described nucleic acid molecules
 which do, in fact, encode a polypeptide having at least one TIF2 protein
 activity. As used herein, "a polypeptide having a TIF2 protein activity"
 is intended to mean polypeptides exhibiting similar, but not necessarily
 identical, activity as at least one biological activity of the TIF2
 protein as measured in a particular biological assay. For example, the
 TIF2 protein of the present invention interacts directly in an
 agonist-dependent manner with the ligand binding domains of several
 nuclear receptors. Moreover, when recombinantly expressed in mammalian
 cells, the TIF2 protein of the present invention enhances transcription
 via CBP-dependent and CBP-independent routes.
 Thus, "a polypepticle having a TIF2 protein activity" includes polypeptides
 having one or more of the following activities: interaction with the LBD
 of one or more NRs in an agonist-dependent manner; enhancement of
 CBP-dependent transcriptional activation; or enhancement of
 CBP-independent transcriptional activation.
 Screening assays for determining whether a candidate polypeptide has TIF2
 protein activity are described in detail in Examples 1, 3, 4, and 6 below.
 For example, by performing such assays, the present inventors have
 discovered that the functional coactivator fragment TIF2.1 (amino acids
 624 to 1287 in FIGS. 1 and 3 (SEQ ID NO: 2)) is "a polypeptide having a
 TIF2 protein activity." The present inventors have also discovered that
 the fragment TIF2.5 (amino acids 624-869) binds to the LBD of NRs without
 activating transcription, and is "a polypeptide having a TIF2 protein
 activity." Also discovered was the fragment TIF2.2 (amino acids 1288-1464
 as shown in FIG. 1 (SEQ ID NO:2)), which enhances CBP-independent
 transcription. Thus, TIF2.2 is "a polypeptide having a TIF2 protein
 activity." Another fragment discovered by the inventors, TIF 2.8 (amino
 acids 1010-1179 as shown in FIG. 1 (SEQ ID NO:2)) is a "polypeptide having
 a TIF2 protein activity" as it activates CBP-dependent transcription.
 Due to the degeneracy of the genetic code, one of ordinary skill in the art
 will immediately recognize that a large number of the nucleic acid
 molecules having a nucleotide sequence at least 90%, preferably at least
 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence of the
 above-described nucleic acid molecules will encode "a polypeptide having a
 TIF2 protein activity." In fact, since degenerate variants all encode the
 same polypeptide, this will be clear to the skilled artisan even without
 performing the above described screening assays. It will be further
 recognized by those skilled in the art that, for such nucleic acid
 molecules that are not degenerate variants, a reasonable number will also
 encode a polypeptide having a TIF2 protein activity. This is because the
 skilled artisan is fully aware of possible amino acid substitutions that
 are either less likely or not likely to significantly affect protein
 function (e.g., replacing one aliphatic amino acid with a second aliphatic
 amino acid).
 Guidance concerning how to make phenotypically silent amino acid
 substitutions is provided, for example, in J. U. Bowie et al.,
 "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid
 Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate
 that there are two main approaches for studying the tolerance of an amino
 acid sequence to change. The first method relies on the process of
 evolution, in which mutations are either accepted or rejected by natural
 selection. The second approach uses genetic engineering to introduce amino
 acid changes at specific positions of a cloned gene and selections or
 screens to identify sequences that maintain functionality. As the authors
 state, these studies have revealed that proteins are surprisingly tolerant
 of amino acid substitutions. The authors further indicate which amino acid
 changes are likely to be permissive at a certain position of the protein.
 For example, most buried amino acid residues require nonpolar side chains,
 whereas few features of surface side chains are generally conserved. Other
 such phenotypically silent substitutions are described in Bowie et al.,
 supra, and the references cited therein.
 Vectors and Host Cells
 The present invention also relates to vectors which include the isolated
 DNA molecules of the present invention, host cells which are genetically
 engineered with the recombinant vectors, and the production of TIF2
 polypeptides or fragments thereof, such as TIF2.1, by recombinant
 techniques.
 Recombinant constructs may be introduced into host cells using well known
 techniques such infection, transduction, transfection, transvection,
 electroporation and transformation. The vector may be, for example, a
 phage, plasmid, viral or retroviral vector. Retroviral vectors may be
 replication competent or replication defective. In the latter case, viral
 propagation generally will occur only in complementing host cells.
 The polynucleotides may be joined to a vector containing a selectable
 marker for propagation in a host. Generally, a plasmid vector is
 introduced in a precipitate, such as a calcium phosphate precipitate, or
 in a complex with a charged lipid. If the vector is a virus, it may be
 packaged in vitro using an appropriate packaging cell line and then
 transduced into host cells.
 Preferred are vectors comprising cis-acting control regions to the
 polynucleotide of interest. Appropriate trans-acting factors may be
 supplied by the host, supplied by a complementing vector or supplied by
 the vector itself upon introduction into the host.
 In certain preferred embodiments in this regard, the vectors provide for
 specific expression, which may be inducible and/or cell type-specific.
 Particularly preferred among such vectors are those inducible by
 environmental factors that are easy to manipulate, such as temperature and
 nutrient additives.
 Expression vectors useful in the present invention include chromosomal-,
 episomal- and virus-derived vectors, e.g., vectors derived from bacterial
 plasmids, bacteriophage, yeast episomes, yeast chromosomal elements,
 viruses such as baculoviruses, papova viruses, vaccinia viruses,
 adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and
 vectors derived from combinations thereof, such as cosmids and phagemids.
 The DNA insert should be operatively linked to an appropriate promoter,
 such as the phage lambda PL promoter, the E. coli lac, trp and tac
 promoters, the SV40 early and late promoters and promoters of retroviral
 LTRs, to name a few. Other suitable promoters will be known to the skilled
 artisan. The expression constructs will further contain sites for
 transcription initiation, termination and, in the transcribed region, a
 ribosome binding site for translation. The coding portion of the mature
 transcripts expressed by the constructs will preferably include a
 translation initiating AUG at the beginning and a termination codon (UAA,
 UGA or UAG) appropriately positioned at the end of the polypeptide to be
 translated.
 As indicated, the expression vectors will preferably include at least one
 selectable marker. Such markers include dihydrofolate reductase or
 neomycin resistance for eukaryotic cell culture and tetracycline or
 ampicillin resistance genes for culturing in E. coli and other bacteria.
 Representative examples of appropriate hosts include, but are not limited
 to, bacterial cells, such as E. coli, Streptomyces and Salmonella
 typhimurium cells; fungal cells, such as yeast cells; insect cells such as
 Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, Cos and
 Bowes melanoma cells; and plant cells. Appropriate culture mediums and
 conditions for the above-described host cells are known in the art.
 Illustrative examples of vectors preferred for use in bacteria include, but
 are not limited to, pA2, pQE70, pQE60 and pQE-9, available from Qiagen;
 pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a,
 pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3,
 pKK233-3, pDR540, pRIT5 available from Pharmacia. Preferred eukaryotic
 vectors include, but are not limited to, pWLNEO, pSV2CAT, pOG44, pXT1 and
 pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available
 from Pharmacia. Other suitable vectors will be readily apparent to the
 skilled artisan.
 Among known bacterial promoters suitable for use in the present invention
 include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the
 gpt promoter, the lambda PR and PL promoters and the trp promoter.
 Suitable eukaryotic promoters include the CMV immediate early promoter,
 the HSV thymidine kinase promoter, the early and late SV40 promoters, the
 promoters of retroviral LTRs, such as those of the Rous sarcoma virus
 ("RSV"), and metallothionein promoters, such as the mouse
 metallothionein-1 promoter.
 Introduction of the construct into the host cell can be effected by calcium
 phosphate transfection, DEAE-dextran mediated transfection, cationic
 lipid-mediated transfection, electroporation, transduction, infection or
 other methods. Such methods are described in many standard laboratory
 manuals, such as Davis et al., Basic Methods in Molecular Biology (1986).
 Transcription of the DNA encoding the polypeptides of the present invention
 by higher eukaryotes may be increased by inserting an enhancer sequence
 into the vector. Enhancers are cis-acting elements of DNA, generally about
 10 to 300 bp in size, that act to increase transcriptional activity of a
 promoter in a given host cell-type. Illustrative examples of enhancers
 include, but are not limited to, the SV40 enhancer, which is located on
 the late side of the replication origin at bp 100 to 270, the
 cytomegalovirus early promoter enhancer, the polyoma enhancer on the late
 side of the replication origin, and adenovirus enhancers.
 For secretion of the translated protein into the lumen of the endoplasmic
 reticulum, into the periplasmic space or into the extracellular
 environment, appropriate secretion signals may be incorporated into the
 expressed polypeptide. The signals may be endogenous to the polypeptide or
 they may be heterologous signals.
 The polypeptide may be expressed in a modified form, such as a fusion
 protein, and may include not only secretion signals, but also additional
 heterologous functional regions. Thus, for instance, a region of
 additional amino acids, particularly charged amino acids, may be added to
 the N-terminus of the polypeptide to improve stability and persistence in
 the host cell, during purification, or during subsequent handling and
 storage. Also, peptide moieties may be added to the polypeptide to
 facilitate purification.
 The TIF2 protein or fraction thereof can be recovered and purified from
 recombinant cell cultures by well-known methods including ammonium sulfate
 or ethanol precipitation, acid extraction, anion or cation exchange
 chromatography, phosphocellulose chromatography, hydrophobic interaction
 chromatography, affinity chromatography, hydroxylapatite chromatography
 and lectin chromatography. Most preferably, high performance liquid
 chromatography ("HPLC") is employed for purification.
 Polypeptides of the present invention include, but are not limited to,
 naturally purified products, products of chemical synthetic procedures,
 and products produced by recombinant techniques from a prokaryotic or
 eukaryotic host, including, for example, bacterial, yeast, higher plant,
 insect and mammalian cells. Depending upon the host employed in a
 recombinant production procedure, the polypeptides of the present
 invention may be post translationally modified (e.g., glycosylated,
 phosphorylated, farnesylated, etc.). In addition, polypeptides of the
 invention may also include an initial modified methionine residue, in some
 cases as a result of host-mediated processes.
 TIF2 Polypeptides and Fragments
 The invention further provides an isolated TIF2 polypeptide having the
 amino acid sequence encoded by the deposited cDNA, or the amino acid
 sequence as shown in FIG. 1 (SEQ ID NO:2), or a fragment thereof Preferred
 polypeptide fragments will have a TIF2 protein activity. In order for a
 TIF2 polypeptide to interact in an agonist-dependent manner with nuclear
 receptors and to enhance nuclear receptor-mediated transcriptional
 activation, such TIF2 polypeptide fragments should at least include amino
 acid residues 624 to 113 1 as shown in FIG. 1 (SEQ ID NO:2) or amino acid
 substitutions, additions or deletions thereof that are not significantly
 detrimental to the polypeptides' ability to interact in an
 agonist-dependent manner with nuclear receptors and to enhance nuclear
 receptor-mediated transcriptional activation. In order for a TIF2
 polypeptide fragment to interact with the LBD of an NR without activating
 transcription, the TIF2 polypeptide fragments should at least include
 amino acids 624-869 as shown in FIG. 1 (SEQ ID NO:2), or amino acid
 substitutions, additions or deletions thereof that are not significantly
 detrimental to the polypeptides' ability to interact with the LBD of an
 NR. In order for a TIF2 polypeptide fragment to activate CBP-dependent
 transcription, the TIF2 polypeptide should at least include amino acid
 residues 1010-1131 as shown in FIG. 1 (SEQ ID NO:2) or amino acid
 substitutions, additions or deletions thereof that are not significantly
 detrimental to the polypeptides' ability to activate CBP-dependent
 transcription. For a TIF2 polypeptide to activate CBP-independent
 transcription, the TIF2 polypeptide should at least include amino acid
 residues 1288-1464 as shown in FIG. 1 (SEQ ID NO:2) or amino acid
 substitutions, additions or deletions thereof that are not significantly
 detrimental to the polypeptides' ability to activate CBP-indepentent
 transcription.
 Exemplary TIF2 polypeptide fragments according to the present invention
 include cytoplasmic TIF2 polypeptides having at least one mutation or
 deletion in a N-terminal NLS region that interferes with the nuclear
 localization function. Methods for generating cytoplasmic TIF2
 polypeptides are described above.
 As used herein, an "isolated" polypeptide or protein is intended to mean a
 polypeptide or protein removed from its native environment, such as
 recombinantly produced polypeptides and proteins expressed in host cells
 and native or recombinant polypeptides which have been substantially
 purified by any suitable technique (e.g., the single-step purification
 method disclosed in Smith and Johnson, Gene 67:31-40 (1988), which is
 incorporated by reference herein). Isolated polypeptides or proteins
 according to the present invention further include such compounds produced
 synthetically.
 The present inventors have discovered that the full-length TIF2 protein is
 an about 1464 amino acid residue protein with a deduced molecular weight
 of about 160 kDa. It will be recognized by those skilled in the art that
 some amino acid sequence of the TIF2 protein can be varied without
 significant effect on the structure or function of the protein. If such
 differences in sequence are contemplated, it should be remembered that
 there will be critical areas on the protein which determine activity, such
 as the region described above which has been determined by the inventors
 as being critical to the protein's ability to enhance nuclear
 receptor-mediated transcriptional activation. In general, it is often
 possible to replace residues which form the tertiary structure, provided
 that residues performing a similar function are used. In other instances,
 the type of residue may be completely unimportant if the alteration occurs
 at a non-critical region of the protein.
 Thus, the present invention further includes variations of the TIF2
 polypeptide which show substantial TIF2 polypeptide activity or which
 include regions of TIF2 protein such as the protein fragments discussed
 below. Such mutants include deletions, insertions, inversions, repeats,
 and type substitutions (for example, substituting one hydrophilic residue
 for another, but not strongly hydrophilic for strongly hydrophobic as a
 rule). Small changes or such "neutral" amino acid substitutions will
 generally have little effect on activity.
 Typically seen as conservative substitutions are the replacements, one for
 another, among the aliphatic amino acids Ala, Val, Leu and lie;
 interchange of the hydroxyl residues Ser and Thr, exchange of the acidic
 residues Asp and Glu, substitution between the amide residues Asn and Gln,
 exchange of the basic residues Lys and Arg and replacements among the
 aromatic residues Phe and Tyr.
 As indicated in detail above, further guidance concerning which amino acid
 changes are likely to be phenotypically silent (i.e., not likely to have a
 significant deleterious effect on a function) can be found in Bowie et
 al., "Deciphering the Message in Protein Sequences: Tolerance to Amino
 Acid Substitutions," Science 247:1306-1310 (1990)).
 The polypeptides of the present invention include polypeptides having an
 amino acid sequence as encoded by the deposited cDNA, an amino acid
 sequence as shown in SEQ ID NO:2, as well as an amino acid sequence at
 least 80% identical, more preferably at least 90% identical, and most
 preferably at least 95%, 96%, 97%, 98%, or 99% identical, to the amino
 acid sequence encoded by the deposited cDNA, to the amino acid sequence as
 shown in SEQ ID NO:2, or to the amino acid sequence of a polypeptide
 fragment described above. Whether two polypeptides have an amino acid
 sequence that is at least 80%, 90% or 95% identical can be determined
 using a computer algorithm as described above.
 As described in detail below, the nucleic acid molecules and polypeptides
 of the present invention are useful in screening assays for identifying
 agonist and antagonist of NR AF2-mediated transactivation. For example, in
 Halachmi, S., et al., Science 264: 1455 (1994), the authors show that
 tamoxifen, which has growth inhibitory effects in breast cancer, disrupts
 the formation of a complex that includes the estrogen receptor and
 ERAP160. Accordingly, the nucleic acid molecules and polypeptides of the
 present invention are useful in assays for identifying drugs capable of
 enhancing or inhibiting nuclear receptor-mediated pathways.
 The nucleic acid molecules and polypeptides of the present invention are
 useful in screening assays for identifying agonists and antagonists of
 TIF2 AD 1 activity, and in screening assays for identifying agonist and
 antagonists of TIF2 AD2 activity, as described in detail below.
 Screening Methods
 Nuclear receptors (NRs) are members of a superfamily of ligand-inducible
 transcriptional regulatory factors that include steroid hormone, thyroid
 hormone, vitamin D3 and retinoid receptors (Leid, M., et al., Trends
 Biochem. Sci. 17:427-433 (1992); Leid, M., et al., Cell 68:377-395 (1992);
 and Linney, E. Curr. Top. Dev. Biol., 27:309-350 (1992)). NRs exhibit a
 modular structure which reflects the existence of several autonomous
 functional domains. Based on amino acid sequence similarity between the
 chicken estrogen receptor, the human estrogen and glucocorticoid
 receptors, and the v-erb-A oncogene, Krust, A., et al, EMBO J. 5:891-897
 (1986), defined six regions, A, B, C, D, E and F (see FIG. 6), which
 display different degrees of evolutionary conservation amongst various
 members of the nuclear receptor superfamily. The highly conserved region C
 contains two zinc fingers and corresponds to the core of the DNA-binding
 domain (DBD), which is responsible for specific recognition of the cognate
 response elements. Region E is functionally complex, since in addition to
 the ligand-binding domain (LBD), it contains a ligand-dependent activation
 function (AF-2) and a dimerization interface. An autonomous
 transcriptional activation function (AF-1) is present in the non-conserved
 N-terminal A/B regions of the steroid receptors. Interestingly, both AF-1
 and AF-2 of steroid receptors exhibit differential transcriptional
 activation properties which appear to be both cell type and promoter
 context specific (Gronemeyer, H. Annu. Rev. Genet. 25:89-123 (1991)).
 The all-trans (T-RA) and 9-cis (9C-RA) retinoic acid signals are transduced
 by two families of nuclear receptors, RAR .alpha., .beta. and .gamma. (and
 their isoforms) are activated by both T-RA and 9C-RA, whereas RXR .alpha.,
 .beta. and .gamma. are exclusively activated by 9C-RA (Allenby, G. et al,
 Proc. Natl. Acad. Sci. USA 90:30-34 (1993)). The three RAR types differ in
 their B regions, and their main isoforms (.alpha.1 and .alpha.2,
 .beta.1-4, and .gamma.1 and .gamma.2) have different N-terminal A regions
 (Leid, M. et al, Trends Biochem. Sci. 17:427-433 (1 992)). Similarly, the
 RXR types differ in their A/B regions (Mangelsdorf, D. J. et al, Genes
 Dev. 6:329-344 (1992)).
 The E-region of RARs and RXRs has also been shown to contain a dimerization
 interface (Yu, V. C. et al, Curr. Opin. Biotechnol 3:597-602 (1992)). Most
 interestingly, it was demonstrated that RAR/RYR heterodimers bind much
 more efficiently in vitro than homodimers of either receptor to a number
 of RA response elements (RAREs) (Yu, V. C. et al, Cell 67:1251-1266
 (1991); Berrodin, T. J. e al., Mol. Endocrinol 6:1468-1478 (1992); Bugge,
 T. H. et al, EMBO J. 11:1409-1418 (1992); Hall, R. K. et al, Mol. Cell.
 Biol. 12: 5527-5535 (1992); Hallenbeck, P. L. et al, Proc. Natl. Acad.
 Sci. USA 89:5572-5576 (1992); Husmann, M. et al., Biochem. Biophys. Res.
 Commun. 187:1558-1564 (1992); Kliewer, S. A. et al, Nature 355:446-449
 (1992b); Leid, M. et al, Cell 68:377-395 (1992); Marks, M. S. et al, EMBO
 J. 11: 1419-1435 (1992); Zhang, X. K. et al, Nature 355:441-446 (1992)).
 RAR and RXR heterodimers are also preferentially formed in solution in
 vitro (Yu, V. C. et al, Cell 67:1251-1266 (1991); Leid, M. et al., Cell
 68:377-395 (1992); Marks, M. S. et al, EMBO J. 11:1419-1435 (1992)),
 although the addition of 9C-RA appears to enhance the formation of RXR
 homodimers in vitro (Lehman, J. M. et al, Science 258:1944-1946 (1992);
 Zhang, X. K. et al., Nature 358:587-591 (1992b)). That RAR-RXR
 heterodimers, rather than the corresponding homodimers, preferentially
 bind to RAREs in cultured cells in vivo has been strongly supported by
 experiments described in Durand, B. et al, Cell 71:73-85 (1992).
 As retinoic acid is known to regulate the proliferative and differentiative
 capacities of several mammalian cell types (Gudas, L. J. et al. (1994) In
 Sporn, M. B., Roberts, A. B. and Goodman, D. S. (eds), The Retinoids. 2nd
 edition, Raven Press, New York, pp. 443-520), retinoids are used in a
 variety of chemopreventive and chemotherapeutic settings. The prevention
 of oral, skin and head and neck cancers in patients at risk for these
 tumors has been reported (Hong, W. K. et al., N. Engl. J. Med.
 315:1501-1505 (1986); Hong, W. K. et al., N. Engl. J. Med. 323:795-801
 (1990); Kraemer, K. H. et al, N. Engl. J. Med. 318:1633-1637 (1988);
 Bollag, W. et al., Ann. Oncol. 3:513-526 (1992); Chiesa, F. et al., Eur.
 J. Cancer B. Oral Oncol. 28:97-102 (1992); Costa, A. et al., Cancer Res.
 54:Suppl. 7, 2032-2037 (1994)), and retinoids are used in the therapy of
 acute promyelocytic leukemia (Huang, M. E. et al., Blood 72:567-572
 (1988); Castaigne, S. et al., Blood 76:1704-1709 (1990); Chomienne, C. et
 al., Blood 76:1710-1717(1990); Chomienne, C. et al., J. Clin. Invest.
 88:2150-2154 (1991); Chen Z. et al., Leukemia 5:288-292 (1991); Lo Coco,
 F. et al., Blood 77:165701659(1991); Warrell, R. P., Jr. et al., N. Engl.
 J. Med. 324:1385-1393 (1991)), squamous cell carcinoma of the cervix and
 the skin (Verma, A. K., Cancer Res. 47:5097-5101 (1987); Lippman S. M. et
 al, J. Natl Cancer Inst. 84:235-241 (1992); Lippman S. M. et al., J. Natl
 Cancer Inst. 84:241-245 (1992)) and Kaposi sarcoma (Bonhomme, L. et al.,
 Ann. Oncol. 2:234-235 (1991)).
 For example, in Chen, J -Y et al., EMBO J. 14(6):1187-1197 (1995), a number
 of dissociating synthetic retinoids are characterized that selectively
 induce AF-2 activation function present in the LBD of RAR.beta.
 (.beta.AF-2). The authors also report that these synthetic retinoids, like
 RA, can inhibit the anchorage-independent growth of oncogene-transformed
 3T3 cells. Further, the promoter of the human interleukin-6 (IL-6) gene,
 whose product is involved in the regulation of hematopoiesis, immune
 responses and inflammation (Kishimoto, T. et al., Science 258:593-597
 (1992)), is induced by RA but not by the `dissociating` retinoids which
 repressed its activity.
 In addition to the retinoid receptors, compounds with agonistic and
 antagonistic properties on functions of the steroid receptors have also
 been reported. For example, in Meyer, M -E. et al., EMBO J. 9(12):
 3923-3932 (1990), a transient expression/gel retardation system was used
 to study the effects of RU486 and R5020 on glucocorticoid and progesterone
 receptor function. Further, in Halachimi, S., et al., Science
 264:1455-1458 (1994), tamoxifen is shown to competitively inhibit
 estradiol-induced ERAP160 binding to the estrogen receptor, suggesting a
 mechanism for its growth-inhibitory effects in breast cancer. Accordingly,
 due to their clinical importance, there is great interest in developing
 screening methods capable of identifying agonist and antagonist of nuclear
 receptor transactivation.
 As indicated, the present inventors have cloned a gene encoding TIF2 and
 have shown that TIF2 and a cytoplasmic fragment thereof bind, in an
 agonist-dependent manner, to all nuclear receptors analyzed--RAR, RXR, ER,
 TR, VDR, GR and AR. Further, the present inventors have shown that TIF2
 polypeptides are transcriptional mediators of the nuclear receptor AF-2.
 Thus, the present invention further provides a screening method for
 identifying a nuclear receptor (NR) antagonist, which involves: (a)
 providing a host cell containing recombinant genes which express a
 polypeptide comprising a NR ligand binding domain (LBD) and a polypeptide
 comprising transcriptional intermediary factor-2 (TIF-2) or a
 TIF-2-fragment, wherein, in the presence of an agonist, said TIF-2 and
 said TIF-2-fragment bind said NR LBD; (b) administering a candidate
 antagonist to said cell; and (c) determining whether said candidate
 antagonist reduces either: (1) TIF-2- or TIF-2-fragment-binding to the
 AF-2 of said NR LBD as compared to said binding in the absence of said
 candidate antagonist; or (2) TIF-2- or TIF-2-fragment-stimulated NR LBD
 AF-2-mediated transactivation as compared to said transactivation in the
 absence of said candidate antagonist.
 In a further aspect, a screening method is provided for identifying a
 nuclear receptor (NR) agonist, which involves: (a) providing a host cell
 containing recombinant genes which express a polypeptide comprising a NR
 ligand binding domain (LBD) and a polypeptide comprising transcriptional
 intermediary factor-2 (TIF-2) or a TIF-2-fragment, wherein, in the
 presence of an agonist, said TIF-2 and said TIF-2-fragment bind said NR
 LBD; (b) administering a candidate agonist to said cell; and (c)
 determining whether said candidate agonist enhances either: (1) TIF-2- or
 TIF-2-fragment-binding to the AF-2 of said NR LBD as compared to said
 binding in the absence of said candidate agonist; or (2) TIF-2- or
 TIF-2-fragment-stimulated NR LBD AF-2-mediated transactivation as compared
 to said transactivation in the absence of said candidate agonist.
 By "a host cell containing recombinant genes" is intended host cells into
 which one or more of the recombinant constructs described herein have been
 introduced or a progeny of such host cells.
 Candidate antagonist and agonist according to the present invention include
 `dissociating` ligands for nuclear receptors such as those described in
 Chen et al., EMBO J. 14:1187-1197 (1995) and Ostrowski et al., Proc. Natl.
 Acad. Sci. USA 92:1812-1816 (1995). Progesterone and glucocorticoid
 receptor agonist and antagonist are described in Meyer et al., EMBO J. 9
 (12): 3923-3932 (1990). An estrogen receptor antagonist is described in
 Halachmi et al., Science 264:1455-1458 (1994). Thus, methods are known in
 the art for developing candidate nuclear receptor agonist and antagonist
 for screening according to the present invention. For example, the crystal
 structure of the ligand binding domains of certain nuclear receptors have
 been described. In particular, the crystal structure of the RXR LBD is
 described in Bourguet et al., Nature 375:377-382 (1995); the crystal
 structure of the RAR LBD is described in Renaud et al., Nature 378:681-689
 (1995); and the crystal structure of a thyroid hormone receptor is
 described in Wagner et al., Nature 378:690-697 (1995). Using information
 from the crystal structure of a nuclear receptor, computer programs are
 available for designing the structure of candidate agonist and antagonist
 which would likely bind to the ligand binding domain. Suitable computer
 program packages for this purpose include WHAT IF (Vriend, G., J. Mol.
 Graphics8:52-56 (1990)), and GRID (Goodford, J. Med. Chem. 28:849-857
 (1985)).
 Recombinant genes encoding a polypeptide comprising TIF2 or a TIF2-fragment
 capable of binding nuclear receptors in an agonist -dependent manner are
 described above. Recombinant genes encoding a polypeptide comprising a NR
 LBD have been described in great detail in the art. Methods for
 determining whether a candidate agonist or antagonist enhances or
 interferes with TIF-2 or TIF-2-fragment binding to a NR are known in the
 art. For example, the effect of a candidate agonist or antagonist on TIF2-
 or TIF-2-fragment-binding to a NR LBD can be studied using
 glutathione-S-transferase (GST) interaction assays by tagging NR LBDs with
 GST as described in detail in the Experimental section below and in Le
 Douarin et al., EMBO J 14:2020-2033 (1995).
 Where the effect of a candidate agonist or antagonist on NR AF-2
 transactivation is to be assayed, preferably, the recombinant genes will
 encode a chimeric polypeptide comprising a NR LBD fused to a DNA binding
 domain from a transactivator protein. In a further preferred embodiment,
 the host cell expressing the recombinant genes will also express a
 reporter gene. For example, in Chen e al., EMBO J. 14(6):1187-1197 (1995),
 three `reporter` cell lines have been established in which RAR.alpha.,
 RAR.beta., or RAR.gamma. agonists induce luciferase activity that can be
 measured in the intact cells using a single-photon-counting camera. These
 cell lines stably express chimeric proteins containing the DNA binding
 domain of the yeast transactivator GAL4 fused to the EF regions (which
 contain that LBD and the AF-2 activation function) of RAR.alpha.
 (GAL-RAR.alpha.), RAR.beta. (GAL-RAR.beta.) or RAR.gamma.
 (GAL-RAR.gamma.), and a luciferase reporter gene driven by a pentamer of
 the GAL4 recognition sequence (`17m`) in front of the .beta.-globin
 promoter (17mx5-G-Luc). This reporter system is insensitive to endogenous
 receptors which cannot recognize the GAL4 binding site. Further examples
 of reporter genes and reporter expression vectors for use according to the
 present invention to screen candidate agonist and antagonist of retinoid
 receptors are provided in FIG. 6.
 The ER expression vectors HE0, HE19 and HE15, the GR expression vectors HG1
 and HG3 and the PR expression cPR1 and cPR3 are described in Kumar e al.,
 Cell 51:941-951 (1987) and Gronemeyer et al., EMBO J. 6:3985-3994 (1987).
 The GR expression vector HG8 and the PR expression vector cPR5A are
 described in Bocquel et al., Nucl. Acids Res. 17:2581-2595 (1989).
 Reporter genes for the above described ER, GR and PR expression vectors
 include MMTV-CAT (in the case of PR and GR; Cato et al., EMBO J.
 5:2237-2240 (1986)) and vit-tk-CAT (in the case of ER; Klein-Hitpass et
 al., Cell 41:1055-1061 (1986)).
 The TR expression vector LexA-TR is described in Lee et al., Nature
 374:91-94(1995), which also describes using the yeast two hybrid system to
 identify compounds that affect TR transactivation.
 Still further references disclosing reporter plasmids containing a reporter
 gene and expression vectors encoding a NR LBD include Meyer et al., Cell
 57:433-442 (1989); Meyer et al., EMBO J. 9(12):3923-3932 (1990); Tasset et
 al., Cell 62:1177-1187 (1990); Gronemeyer, H. and Laudet, V., Protein
 Profile 2:1173-1308 (1995); Webster et al., Cell 54:199-207 (1988);
 Strahle et al., EMBO J. 7:3389-3395 (1988); Seipel et al., EMBO J.
 11:4961-4968 (1992); and Nagpal et al., EMBO J. 12:2349-2360 (1993). In a
 particularly preferred embodiment, the effect of a candidate agonist or
 antagonist on NR AF-2-mediated transactivation is assayed according to the
 method described in the legend to FIG. 5 above.
 The present inventors have identified an activation domain of TIF2, AD1
 (amino acids 1010-1131 as shown in FIG. 1 (SEQ ID NO:2)), which mediates
 the CBP-dependent transcriptional activation function of TIF2. Further,
 the present inventors have shown that polypeptides containing this
 activation domain, when fused to a DNA-binding domain of a transcriptional
 activator, is capable of activating transcription via a CBP-dependent
 pathway. Accordingly, the present invention further provides a screening
 method of identifying an agonist of TIF2 AD1 activation domain activity,
 which involves: (a) providing a host cell containing a recombinant gene or
 genes which express a polypeptide comprising a transcriptional activator
 DNA-binding domain (DBD) and a TIF-2 AD1 activation domain 1; (b)
 administering a candidate agonist to said cell; and (c) determining
 whether said candidate agonist enhances TIF2 AD1 activation domain
 activity.
 The invention further provides for a screening method for identifying an
 antagonist of TIF2 AD1 activation domain activity, which comprises: (a)
 providing a host cell containing a recombinant gene or genes which express
 a polypeptide comprising a transcriptional activator DNA-binding domain
 (DBD) and a TIF-2 AD1 activation domain 1; (b) administering a candidate
 antagonist to said cell; and (c) determining whether said candidate
 antagonist inhibits TIF2 AD 1 activation domain activity.
 By "transcriptional activator" it is meant molecules that enhance the
 initiation of transcription by RNA polymerase B (II). Transcriptional
 activators include yeast transcriptional activators, such as GAL4 and
 GCN4; the herpes simplex activator, VP16; and members of the nuclear
 receptor family, which includes RAR, RXR, ER, TR, VDR, GR, and AR.
 Recombinant genes encoding a polypeptide comprising a TIF2 AD1 activation
 domain are described below. Recombinant genes encoding a polypeptide
 comprising a transcriptional activator DBD are well known in the art.
 Methods for determining whether a candidate agonist or antagonist enhances
 or interferes with transcription are well known in the art. For example,
 the effect of a candidate agonist or antagonist of TIF2 AD1 activation
 domain activity can be determined using CAT assays as described below and
 in Gronemeyer et al. (1987) and Bocquel et al., Nucl. Acids Res. (1989).
 Where the effect of a candidate agonist or antagonist of TIF2 AD1
 activation domain activity is to be determined, preferably, recombinant
 genes will encode a chimeric polypeptide comprising a transcriptional
 activator DBD fused to a TIF2 polypeptide comprising the AD1 activation
 domain. In a further embodiment, the host cell expressing the recombinant
 genes will also express a reporter gene. Examples of reporter genes are
 described above. In a particularly preferred embodiment, the effect of a
 candidate agonist or antagonist of TIF2 AD1 activation domain function
 will be determined as described in the legend to FIG. 7(c).
 The present inventors have also identified a second activation domain of
 TIF2, AD2 (amino acids 1288-1464 as shown in FIG. 1 (SEQ ID NO:2)), which
 mediates CBP-independent transcriptional activation. Further, the present
 inventors have shown that polypeptides containing this activation domain,
 when fused to a DNA-binding domain of a transcriptional activator, are
 capable of activating transcription via a CBP-independent pathway.
 Accordingly, the present invention further provides a screening method for
 identifying an agonist of TIF2 AD2 activation domain activity, which
 comprises: (a) providing a host cell containing a recombinant gene or
 genes which express a polypeptide comprising a transcriptional activator
 DNA-binding domain (DBD) and a TIF-2 AD2 activation domain; (b)
 administering a candidate agonist to said cell; and (c) determining
 whether said candidate agonist enhances TIF2 AD2 activation domain
 activity.
 The invention further provides for a screening method for identifying an
 antagonist of TIF2 AD2 activation domain activity, which comprises: (a)
 providing a host cell containing a recombinant gene or genes which express
 a polypeptide comprising a transcriptional activator DNA-binding domain
 (DBD) and a TIF-2 AD2 activation domain; (b) administering a candidate
 antagonist to said cell; and (c) determining whether said candidate
 antagonist inhibits TIF2 AD2 activation domain activity.
 Recombinant genes encoding a polypeptide comprising a TIF2 AD2 activation
 domain are described below. Recombinant genes encoding a polypeptide
 comprising a transcriptional activator DBD are well known in the art.
 Methods for determining whether a candidate agonist or antagonist enhances
 or interferes with transcription are known in the art.
 Where the effect of a candidate agonist or antagonist of TIF2 AD2
 activation domain activity is to be determined, preferably, recombinant
 genes will encode a chimeric polypeptide comprising a transcriptional
 activator DBD fused to a TIF2 polypeptide comprising the AD2 activation
 domain. Transcriptional activators are described above. In a further
 embodiment, the host cell expressing the recombinant genes will also
 express a reporter gene. Examples of reporter genes are described above.
 In a particularly preferred embodiment, the effect of a candidate agonist
 or antagonist of TIF2 AD2 activation domain activity will be determined as
 described in the legend to FIG. 7(c).
 TIF-2 Antibodies and Methods
 TIF2 antibodies are also provided by the present invention, as specific for
 a TIF2 protein, a TIF2 polypeptide, a TIF2 protein fragment or a TIF2
 polypeptide fragment. The term "antibody" is meant to include polyclonal
 antibodies, monoclonal antibodies (mAbs), chimeric antibodies,
 anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in
 soluble or bound form, as well as fragments thereof provided by any known
 technique, such as, but not limited to enzymatic cleavage, peptide
 synthesis or recombinant techniques. Polyclonal antibodies are
 heterogeneous populations of antibody molecules derived from the sera of
 animals immunized with an antigen. A monoclonal antibody contains a
 substantially homogeneous population of antibodies specific to antigens,
 which population contains substantially similar epitope binding sites.
 MAbs may be obtained by methods known to those skilled in the art. See,
 for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Pat. No.
 4,376,110; Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
 Greene Publishing Assoc. and Wiley Interscience, N.Y., (1987-1996); and
 Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor
 Laboratory (1988); Colligan et al., eds., Current Protocols in Immunology,
 Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992-1996), the
 contents of which references are incorporated entirely herein by
 reference.
 Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE,
 IgA, GILD and any subclass thereof. A hybridoma producing a mAb of the
 present invention may be cultivated in vitro, in situ or in vivo.
 Production of high titers of mAbs in vivo or in situ makes this the
 presently preferred method of production.
 Chimeric antibodies are molecules different portions of which are derived
 from different animal species, such as those having variable region
 derived from a murine mAb and a human immunoglobulin constant region,
 which are primarily used to reduce immunogenicity in application and to
 increase yields in production, for example, where murine mAbs have higher
 yields from hybridomas but higher immunogenicity in humans, such that
 human/murine chimeric mAbs are used. Chimeric antibodies and methods for
 their production are known in the art (Cabilly et al., Proc. Natl. Acad.
 Sci. USA 81:3273-3277 (1984); European Patent Application 125023
 (published Nov. 14, 1984); Neuberger et al., Nature 314:268-270 (1985);
 Taniguchi et al., European Patent Application 171496 (1985); Morrison et
 al., European Patent Application 173494 (1986); Neuberger et al., PCT
 Application WO 86/01533, (1986); Kudo et al., European Patent Application
 184187 (1986); Morrison et al., European Patent Application 173494 (1986);
 Robinson et al., PCT Publication PCT/US86/02269 (1987); Liu et al., Proc.
 Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad.
 Sci. USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988);
 and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor
 Laboratory (1988)). These references are entirely incorporated herein by
 reference.
 An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique
 determinants generally associated with the antigen-binding site of an
 antibody. An Id antibody can be prepared by immunizing an animal of the
 same species and genetic type (e.g., mouse strain) as the source of the
 mAb with the mAb to which an anti-Id is being prepared. The immunized
 animal will recognize and respond to the idiotypic determinants of the
 immunizing antibody by producing an antibody to these idiotypic
 determinants (the anti-Id antibody). See, for example, U.S. Pat. No.
 4,699,880, which is herein entirely incorporated by reference.
 The anti-Id antibody may also be used as an "immunogen" to induce an immune
 response in yet another animal, producing a so-called anti-anti-Id
 antibody. The anti-anti-Id may be epitopically identical to the original
 mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic
 determinants of a mAb, it is possible to identify other clones expressing
 antibodies of identical specificity. The anti-Id mAbs thus have their own
 idiotypic epitopes, or "idiotopes" structurally similar to the epitope
 being evaluated, such as GRB protein-.alpha..
 The term "antibody" is also meant to include both intact immunoglobulin
 molecules as well as fragments thereof, such as, for example, Fab and
 F(ab').sub.2, which are capable of binding antigen. Fab and F(ab').sub.2
 fragments lack the Fc fragment of intact antibody, clear more rapidly from
 the circulation, and may have less non-specific tissue binding than an
 intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). It will be
 appreciated that Fab and F(ab').sub.2 and other fragments of the
 antibodies useful in the present invention may be used for the detection
 and quantitation of a TIF2 according to the methods disclosed herein for
 intact antibody molecules. Such fragments are typically produced by
 proteolytic cleavage, using enzymes such as papain (to produce Fab
 fragments) or pepsin (to produce F(ab').sub.2 fragments).
 An antibody is said to be "capable of binding" a molecule if it is capable
 of specifically reacting with the molecule to thereby bind the molecule to
 the antibody. The term "epitope" is meant to refer to that portion of any
 molecule capable of being bound by an antibody which can also be
 recognized by that anti-body. Epitopes or "antigenic determinants" usually
 consist of chemically active surface groupings of molecules such as amino
 acids or sugar side chains and have specific three dimensional structural
 characteristics as well as specific charge characteristics.
 An "antigen" is a molecule or a portion of a molecule capable of being
 bound by an antibody which is additionally capable of inducing an animal
 to produce antibody capable of binding to an epitope of that antigen. An
 antigen may have one, or more than one epitope. The specific reaction
 referred to above is meant to indicate that the antigen will react, in a
 highly selective manner, with its corresponding antibody and not with the
 multitude of other antibodies which may be evoked by other antigens.
 The antibodies, or fragments of antibodies, useful in the present invention
 may be used to quantitatively or qualitatively detect a TIF2 protein,
 polypeptide, or fragment, in a sample or to detect presence of cells which
 express a TIF2 of the present invention. This can be accomplished by
 immunofluorescence techniques employing a fluorescently labeled antibody
 (see below) coupled with light microscopic, flow cytometric, or
 fluorometric detection.
 The antibodies (of fragments thereof) useful in the present invention may
 be employed histologically, as in immunofluorescence or immunoelectron
 microscopy, for in situ detection of a TIF2 protein, polypeptide, or
 fragment, of the present invention. In situ detection may be accomplished
 by removing a histological specimen form a patient, and providing a
 labeled antibody of the present invention to such a specimen. The antibody
 (or fragment) is preferably provided by applying or by overlaying the
 labeled antibody (or fragment) to a biological sample. Through the use of
 such a procedure, it is possible to determine not only the presence of a
 TIF2 protein, polypeptide, or fragment, but also its distribution on the
 examined tissue. Using the present invention, those of ordinary skill will
 readily perceive that any of wide variety of histological methods (such as
 staining procedures) can be modified in order to achieve such in situ
 detection.
 Such assays for a TIF2 protein, polypeptide, or fragment, of the present
 invention typically comprises incubating a biological sample, such as a
 biological fluid, a tissue extract, freshly harvested cells such as
 lymphocytes or leukocytes, or cells which have been incubated in tissue
 culture, in the presence of a detectably labeled antibody capable of
 identifying a TIF2 protein, polypeptide, or fragment, and detecting the
 antibody by any of a number of techniques well-known in the art.
 The biological sample may be treated with a solid phase support or carrier
 such as nitrocellulose, or other solid support or carrier which is capable
 of immobilizing cells, cell particles or soluble proteins. The support or
 carrier may then be washed with suitable buffers followed by treatment
 with a detectably labeled TIF2-specific antibody. The solid phase support
 or carrier may then be washed with the buffer a second time to remove
 unbound antibody. The amount of bound label on said solid support or
 carrier may then be detected by conventional means.
 By "solid phase support", "solid phase carrier", "solid support", "solid
 carrier", "support" or "carrier" is intended any support or carrier
 capable of binding antigen or antibodies. Well-known supports or carriers,
 include glass, polystyrene, polypropylene, polyethylene, dextran, nylon
 amylases, natural and modified celluloses, polyacrylamides, gabbros, and
 magnetite. The nature of the carrier can be either soluble to some extent
 or insoluble for the purposes of the present invention. The support
 material may have virtually any possible structural configuration so long
 as the coupled molecule is capable of binding to an antigen or antibody.
 Thus, the support or carrier configuration may be spherical, as in a bead,
 or cylindrical, as in the inside surface of a test tube, or the external
 surface of a rod. Alternatively, the surface may be flat such as a sheet,
 test strip, etc. Preferred supports or carriers include polystyrene beads.
 Those skilled in the art will know many other suitable carriers for
 binding antibody or antigen, or will be able to ascertain the same by use
 of routine experimentation.
 The binding activity of a given lot of anti-TIF2 antibody may be determined
 according to well known methods. Those skilled in the art will be able to
 determine operative and optimal assay conditions for each determination by
 employing routine experimentation. Other such steps as washing, stirring,
 shaking, filtering and the like may be added to the assays as is customary
 or necessary for the particular situation.
 One of the ways in which a TIF2-specific antibody can be detectably labeled
 is by linking the same to an enzyme and use in an enzyme immunoassay
 (EIA). This enzyme, in turn, when later exposed to an appropriate
 substrate, will react with the substrate in such a manner as to produce a
 chemical moiety which can be detected, for example, by spectrophotometric,
 fluorometric or by visual means. Enzymes which can be used detectably
 label the antibody include, but are not limited to, malate dehydrogenase,
 staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
 dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate
 isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,
 glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,
 glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
 The detection can be accomplished by colorimetric methods which employ a
 chromogenic substrate for the enzyme. Detection may also be accomplished
 by visual comparison of the extent of enzymatic reaction of a substrate in
 comparison with similarly prepared standards.
 Detection may be accomplished using any of a variety of other immunoassays.
 For example, by radioactivity labeling the antibodies or antibody
 fragments, it is possible to detect R-PTPase through the use of a
 radioimmunoassay (RIA). A good description of RIA maybe found in
 Laboratory Techniques and Bio chemistry in Molecular Biology, by Work, T.
 S. et al., North Holland Publishing Company, NY (1978) with particular
 reference to the chapter entitled "An Introduction to Radioimmune Assay
 and Related Techniques" by Chard, T., incorporated by reference herein.
 The radioactive isotope can be detected by such means as the use of a
 .gamma. counter or a scintillation counter or by autoradiography.
 It is also possible to label an anti-TIF2 antibody with a fluorescent
 compound. When the fluorescently labeled antibody is exposed to light of
 the proper wave length, its presence can be then be detected due to
 fluorescence. Among the most commonly used fluorescent labeling compounds
 are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
 allophycocyanin, o-phthaldehyde and fluorescamine.
 The antibody can also be detectably labeled using fluorescence emitting
 metals such as .sup.152 EU, or others of the lanthanide series. These
 metals can be attached to the antibody using such metal chelating groups
 as diethylenetriamine pentaacetic acid (EDTA).
 The antibody also can be detectably labeled by coupling it to a
 chemiluminescent compound. The presence of the chemiluminescent-tagged
 antibody is then determined by detecting the presence of luminescence that
 arises during the course of a chemical reaction. Examples of particularly
 useful chemiluminescent labeling compounds are luminol, isoluminol,
 theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
 Likewise, a bioluminescent compound may be used to label the antibody of
 the present invention. Bioluminescence is a type of chemiluminescence
 found in biological systems in which a catalytic protein increases the
 efficiency of the chemiluminescent reaction. The presence of a
 bioluminescent protein is determined by detecting the presence of
 luminescence. Important bioluminescent compounds for purposes of labeling
 are luciferin, luciferase and aequorin.
 An antibody molecule of the present invention may be adapted for
 utilization in a immunometric assay, also known as a "two-site" or
 "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled
 antibody (or fragment of antibody) is bound to a solid support or carrier
 and a quantity of detectably labeled soluble antibody is added to permit
 detection and/or quantitation of the ternary complex formed between
 solid-phase antibody, antigen, and labeled antibody.
 Typical, and preferred, immunometric assays include "forward" assays in
 which the antibody bound to the solid phase is first contacted with the
 sample being tested to extract the antigen form the sample by formation of
 a binary solid phase antibody-antigen complex. After a suitable incubation
 period, the solid support or carrier is washed to remove the residue of
 the fluid sample, including unreacted antigen, if any, and then contacted
 with the solution containing an unknown quantity of labeled antibody
 (which functions as a "reporter molecule"). After a second incubation
 period to permit the labeled antibody to complex with the antigen bound to
 the solid support or carrier through the unlabeled antibody, the solid
 support or carrier is washed a second time to remove the unreacted labeled
 antibody.
 In another type of "sandwich" assay, which may also be useful with the
 antigens of the present invention, the so-called "simultaneous" and
 "reverse" assays are used. A "simultaneous" and "reverse" assays are used.
 A simultaneous assay involves a single incubation step as the antibody
 bound to the solid support or carrier and labeled antibody are both added
 to the sample being tested at the same time. After the incubation is
 completed, the solid support or carrier is washed to remove the residue of
 fluid sample and uncomplexed labeled antibody. The presence of labeled
 antibody associated with the solid support or carrier is then determined
 as it would be in a conventional "forward" sandwich assay.
 In the "reverse" assay, stepwise addition first of a solution of labeled
 antibody to the fluid sample followed by the addition of unlabeled
 antibody bound to a solid support or carrier after a suitable incubation
 period is utilized. After a second incubation, the solid phase is washed
 in conventional fashion to free it of the residue of the sample being
 tested and the solution of unreacted labeled antibody. The determination
 of labeled antibody associated with a solid support or carrier is then
 determined as in the "simultaneous" and "forward" assays.
 Having generally described the invention, the same will be more readily
 understood by reference to the following examples, which are provided by
 way of illustration and are not intended as limiting.
 EXAMPLE 1
 TIF2 Cloning and Expression
 In keeping with previous reports (Halachmi, S. et al., Science 264:
 1455-1458 (1994); Cavailles, V. et al., Proc. Natl. Acad. Sci. USA
 91:10009-10013 (1994); Kurokawa, R. et al, Nature 377:451-454 (1995)), we
 observed agonist-dependent binding in vitro of a 160-kDa protein from
 .sup.35 S-labelled whole cell extracts (HeLa, Cos-1, P19.6, MCF-7) to the
 glutathione-S-transferase (GST)-tagged LBDs of retinoic acid (RAR) and
 estrogen (ER) receptors (FIG. 2a). One cDNA clone, identified by screening
 340,000 clones of a human placenta cDNA expression library with an
 estradiol (E2)-bound .sup.32 P-labelled ER(DEF) probe, encoded a protein
 fragment (TIF2.1) that interacted on Far-Western blots with three
 different .sup.32 P-labelled NR LBDs (ER, RAR, RXR) in an
 agonist-dependent manner (not shown), and could therefore correspond to
 the above 160-kDa protein. The TIF2 coding sequence (FIG. 3a), preceded by
 in-frame stop codons 5' of the initiator AUG, was obtained upon
 rescreening with a TIF2.1 cDNA probe. Human TIF2 cDNA encodes a 159,160 Da
 protein (1,464 amino acids), which includes N-terminal putative nuclear
 localization signals (NLSs), one Gln- and three Ser/Thr-rich regions, and
 two charged clusters (FIG. 3). Some regions of TIF2 show significant
 sequence similarities with the recently described (Onate, S.A. et al.,
 Science 270:1354-1357 (1995)) steroid receptor coactivator SRC-1 (FIG. 3).
 TIF2 appears to be widely expressed, since the corresponding transcript
 was found in several human tissues, albeit at a much lower level in kidney
 (FIG. 2c and not shown).
 Immunodepletion studies strongly support that TIF2 is the 160-kDa protein
 species which interacts in an agonist-dependent manner with NR LBDs (see
 above and Halachmi, S. et al., Science 264:1455-1458 (1994); Cavailles, V.
 et al., Proc. Natl. Acad. Sci. USA 91:10009-10013 (1994); and Kurokawa, R.
 et al, Nature 377:451-454 (1995)). Western blotting with a rabbit
 antiserum (p.alpha.-TIF2), raised against bacterially expressed TIF2.1,
 revealed predominantly a 160-kDa HeLa cell protein that interacted with
 agonist-bound GST-ER(DEF) (FIG. 2b, lanes 1 and 2; see also legend to FIG.
 2b). Immunodepletion with a mouse monoclonal TIF2 antibody (m.alpha.-TIF2)
 prior to affinity purification resulted in a specific decrease of TIF2,
 but not TIF1 (Le Douarin, B. et al., EMBO J. 14:2020-2033 (1995)) amounts,
 retained on E2-bound GST-ER(DEF) (FIG. 2b, compare lanes 2 with 4 and 6
 with 8). Importantly, the subsequent Far-Western analysis with an E2-bound
 .sup.32 P-GST-ER(DEF) probe revealed the 160-kDa species only in control,
 but not TIF2-immunodepleted extracts (FIG. 2b, compare lanes 10 and 12).
 Transiently expressed full-length TIF2 was nuclear and mainly associated
 with discrete bodies (FIG. 4a). Since the overexpressed TIF2.1 fragment
 was essentially cytoplasmic (supporting the above assignment of a
 N-terminal TIF2 NLS), the interaction of TIF2.1 with NRs could be studied
 in mammalian cells using nuclear cotranslocation assays. In the absence of
 ligand, TIF2.1 remained cytoplasmic and NRs were nuclear (for RAR.alpha.,
 ER and PR, see FIGS. 4b, d, g). Agonist exposure, however, resulted in all
 three cases in nuclear colocalization of TIF2.1 and NR, indicating NR-TIF2
 interaction in vivo (FIGS. 4c, e, h). Agonist-dependent interaction of
 TIF2.1 with NRs was observed for all other receptors analyzed (RXR, TR,
 VDR, GR and AR; not shown). Interestingly, no interaction was detected
 between ER and TIF2.1 in presence of the ER AF-2 antagonist
 hydroxytamoxifen (OHT) (FIG. 4f), and the PR AF-2 antagonist RU 486
 reversed the R5020-induced PR-TIF2.1 interaction (FIG. 4i).
 In agreement with the Far-Western blot experiments NRs and TIF2 directly
 interacted, as purified TIF2.1 protein bound in vitro in the presence of
 an agonist to GST-ER(DEF), GST-RAR.alpha.(DEF), GST-RXR.alpha.(DE) and
 GST-TR(DE) (FIGS. 4k, l, m, lanes 3 and 4; FIG. 4n, lanes 5 and 6). As
 expected, TIF2 binding to GST-RXR.alpha.(DE) occurred with 9cis-RA (9C-RA)
 but not all-trans-RA (T-RA) (FIG. 4n, lanes 1 and 2), and OHT prevented
 E2-dependent binding of TIF2 to GST-ER(DEF) (FIG. 4n, lanes 7-9). The
 integrity of the conserved core of the ER, RAR.alpha. and RXR.alpha. AF-2
 activating domains (AF-2 AD) which was shown to be critical for AF-2
 activity (Le Douarin, B. et al, EMBO J. 14:2020-2033 (1995); Danielian, P.
 S. et al., EMBO J. 11:1025-1033 (1992); Durand, B. et al., EMBO J.
 13:5370-5382 (1994); and Gronemeyer, H. and Laudet, V., Protein Profile
 2:1173-1308 (1995), and therein), was required for TIF2 interaction in
 vitro. Most AF-2 AD core mutants which have lost AF-2 activity (ER, FIG.
 4k, lanes 5-8; RAR.alpha., FIG. 4l, lanes 5-10; RXR.alpha., FIG. 4m, lanes
 5-8) did not detectably, or only weakly, associate with TIF2, whereas the
 GST-LBD fusion of the RXR.alpha. mutant E461Q, whose AF-2 is only
 partially impaired (Le Douarin, B. et al., EMBO J. 14:2020-2033 (1995)),
 still exhibited a significant RA-dependent interaction with TIF2.1 in
 vitro (FIG. 4m, lanes 9 and 10). No significant interaction of TIF2.1 was
 observed with either GST-VP16 (acidic activation domain), GST-TBP,
 GST-TFIIB, or a series of GST-TAFs (hTAF.sub.II 18, hTAF.sub.II 20,
 hTAF.sub.II 28 and hTAF.sub.II 55; see Jacq, X. et al, Cell 79:107-117 (1
 994); Mengus, G. et al., EMBO J. 14:1520-1531 (1995)) (not shown).
 Conceptually, a TIF capable of mediating the transcriptional activity of a
 cognate AF to the transcription machinery, could itself be an activator
 when fused to a heterologous DNA-binding domain. Interestingly, in
 transiently transfected HeLa cells, TIF2.1 fused to the GAL4 DNA-binding
 domain strongly transactivated a GAL4 reporter (FIG. 5a). Thus, TIF2 may
 correspond to one of the hypothetical limiting factor(s) previously
 proposed to be involved in NR transcriptional interference/squelching
 (Meyer, M. -E. et al., Cell 57:433-442 (1989); Bocquel, M. -T. et al.,
 Nucl. Acids Res. 17:2581-2595 (1989); Tasset, D. et al, Cell 62:1177-1187
 (1990)). Supporting this possibility, "anti-squelching" experiments showed
 that expression of TIF2.1 in ER-transfected cells could, at least
 partially, reverse the transcriptional autointerference (Bocquel, M. -T.
 et al., Nucl. Acids Res. 17:2581-2595 (1989)) generated by expressing
 increased amounts of ER (FIG. 5b; note the marked shift of the bell-shaped
 activation curve to higher ER concentrations in the presence of TIF2.1).
 At high ER expression levels, the TIF2.1-stimulated transactivation
 decreased, possibly due to squelching of other putative mediators (Jacq,
 X. et al., Cell 79:107-117 (1994); Lee, J. W. et al., Nature 374:91-94
 (1995); Le Douarin, B. et al., EMBO J. 14:2020-2033 (1995); vom Baur, E.
 et al., EMBO J. 15:110-124 (1996); Lee, J. W. et al., Endocrinology
 9:243-254 (1995); Cavailles, V. et al., EMBO J. 14:3741-3751 (1995);
 Onate, S. A. et al., Science 270:1354-1357 (1995)) and/or transcriptional
 factors.
 As expected, coexpression of TIF2.1 with antagonist-bound NR did not lead
 to any stimulation of the transactivation brought about by AF-1 in the
 presence of pure AF-2 antagonists (Berry, M. et al., EMBO J. 9:2811-2818
 (1990); Meyer, M. E. et al., EMBO J. 9:3923-3932 (1990)), further
 supporting that TIF2 is AF-2-specific (FIG. 5b and 5e for ER, OHT; FIG. 5d
 for PR, RU486). TIF2 expression also increased AF-2/agonist-mediated
 transactivation by the androgen (AR) and progesterone (PR) receptors, but
 not transactivation by GAL-VP16 and GAL-AP2 (FIG. 5c). Under similar
 conditions, transactivation by GAL-RAR, GAL-RXR, GAL-VDR, GAL-TR and
 GAL-GR were unaffected by TIF2 (FIG. 5c, and not shown), suggesting that
 for these NRs either TIF2 is not critically involved in mediating their
 AF-2 activities or endogenous TIF2 amounts are sufficient to optimally
 support transactivation, for instance, because TIF2 has a higher affinity
 for these receptors. TIF2-stimulation is to some extent affected by the
 promoter environment of the responsive gene, as the TIF2 effect on
 PR/5020-induced transactivation was greater for a complex (MMTV) than for
 a minimal (GRE-TATA) promoter, although the latter was also reproducibly
 stimulated (FIG. 5d). As expected from the distinct levels of TIF2
 transcripts in different tissues (FIG. 2c), the effect of TIF2 was cell
 type-dependent, since TIF2 had a much stronger effect on PR- and
 ER-induced transactivations in Cos-1 than in HeLa cells (FIGS. 5d, e).
 Squelching(Meyer, M. -E. et al., Cell 57:433-442 (1989); Bocquel, M. -T. et
 al., Nucl. Acids Res. 17:2581-2595 (1989); Tasset, D. et al., Cell
 62:1177-1187 (1990)) and structural studies (Bourguet, M. et al., Nature
 375:377-382 (1995); Benaud, J. -P. et al., Nature 378:681-689 (1995);
 Wagner, R. L. et al., Nature 378:690-697 (1995); Wurtz, J. -M. et al.,
 Nature Struct. Biol. 3:87-94 (1996)) have supported a model in which
 binding of the ligand to the LBD of NRs results in conformational changes
 generating the surface(s) required for interaction with transcriptional
 intermediary factors (TIFs/mediators) which transduce the AF-2 activity to
 the transcription machinery. Conceptually such mediators should exhibit
 the following properties: (i) they should bind to agonist, but not
 antagonist-bound NR LBDs, (ii) their binding should be prevented by
 mutations abolishing AF-2 activity, (iii) they should collectively exhibit
 a transactivation function(s), (iv) their expression should relieve AF-2
 autosquelching, and (v) their overexpression should stimulate the activity
 of AF-2, whenever they are present in limiting amounts. The present study
 is the first report of a bona fide mediator of NR AF-2s which exhibits all
 these properties.
 EXAMPLE 2
 Production of TIF-2 Antibodies
 The following TIF2 antibodies were made using known techniques, unless
 otherwise specified below. See, e.g., Ausubel et al, eds., CURRENT
 PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc. and Wiley
 Interscience, N.Y., (1987-1996); Harlow and Lane ANTIBODIES: A LABORATORY
 MANUAL Cold Spring Harbor Laboratory (1988); and Colligan et al., eds.,
 Current Protocols in Immunology, Greene Publishing Assoc. and Wiley
 Interscience, N.Y., (1992-1996), the contents of which references are
 incoporated entirely herein by reference.
 Polyclonal Antibodies.
 The TIF2.1 coding sequence (amino acids 624-1287) was cloned into pET15b
 and the resulting plasmid transformed into the E. coli strain BL21 (DE3).
 After overexpression of the (His).sub.6 -TIF2.1 protein the bacteria were
 lysed and the protein purified from the crude extract via affinity
 chromatography on a HiTrap chelating column (Pharmacia) as described (see
 Bourguet et al, Prot. Expr. Purif. 6:604-608 (1995) for technical
 details). Aliquots of the purified protein (50 .mu.g) in emulsion
 (Freund's Adjuvant) were injected (only once) into a New Zealand rabbit
 using a multisite intradermal injection protocol. Antisera obtained from
 serial bleeds revealed a single band of about 160 kDa on Western blots of
 extracts from Cos-1 cells transformed with a full length TIF2 expression
 vector.
 Monoclonal Antibodies.
 A 20mer amino-acid peptide of TIF2 (with an added C-terminal cysteine) was
 selected on the basis of its potential immunogenic characteristics in
 terms of hydrophilicity, flexibility, surface probability and the
 `antigenic index` according to software programmes Plot and
 Peptidestructure from the GCG package.
 The chosen peptide corresponds to the N terminal fragment encoded by the
 TIF2 partial cDNA initially isolated (which encodes amino acids 624 to
 1287) and corresponds to amino acids 624 to 643 of the total protein (SEQ
 ID NO:2):
 ##STR1##
 The peptide was coupled to ovalbuinin via the additional cysteine using the
 MBS heterobifunctional crosslinker. Injections were performed in BALB/c
 mice intraperitonally and intravenously.
 Spleen cells from the immunized mice were fused to the Sp2/0 Ag14 myeloma.
 Growing hybridomas were first screened by ELISA using the recombinant
 TIF2.1 protein and the free peptide. Positive cultures were then tested by
 immunocytofluorescence on Cos cells transfected with TIF2.1 (in the pSG5
 vector) as well as by western blot using the transfected Cos cell extracts
 and HeLa nuclear extract. The positive cultures were also tested for their
 ability to immunoprecipitate the TIF2.1 protein from Cos transfected
 cells.
 Cultures were cloned twice on soft agar. 5 hybridomas have been
 established, 4 secreting IgG.sub.1, .kappa. and 1 IgG.sub.2a, .kappa.
 antibodies, as shown in the Table: