Polynucleotide encoding transcriptional intermediary factor-2

Details Polynucleotide encoding transcriptional intermediary factor-2 Polynucleotide encoding transcriptional intermediary factor-2

1997-07-11

2001-07-31

Pak, Michael (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S325000, C536S023500, C536S023100

Reexamination Certificate

active

06268173

ABSTRACT:

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, &tgr;-1 (located in the A/B domain) and &tgr;-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&agr; is necessary and sufficient to mediate interaction with RXR in a ligand- and AF-2 integrity-dependent manner. Notably, within this TIF1&agr; 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&agr; and several other putative coactivators do not, or only very poor

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