Method and compositions for monitoring DNA binding molecules...

Details Method and compositions for monitoring DNA binding molecules... Method and compositions for monitoring DNA binding molecules...

1999-02-12

2002-09-24

Sisson, Bradley L. (Department: 1634)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S069100, C435S320100, C435S325000, C435S360000, C435S361000, C435S363000, C435S419000, C530S350000, C536S023500

Reexamination Certificate

active

06455300

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of binding of molecules such as transcription factors to regions of nucleic acids, steroid hormone usage, steroid receptors and their corresponding response elements. Reagents are provided to allow methods involving direct detection of binding of a molecule, determining response element targeting by activated steroid receptors, screening for steroid agonists and antagonists, and monitoring levels of steroid agonists and antagonists in biological samples.
2. Background Art
Steroid receptors are hormone-dependent activators of gene expression. Steroid receptors mediate the action of steroid hormones (e.g., glucocorticoids, estrogens, progestins, testosterone, mineralocorticoids and 1,25-dihydroxycholecalciferol) in human tissues. After activation with the cognate ligand, receptors bind to chromatin in the nucleus and modulate the activity of target cellular genes. The binding of receptors to these target sequences is a key step in steroid function. Currently, this interaction can only be detected by indirect methods, such as reporter assays that detect the result of transcriptional activation coupled with transfection methods that introduce DNA sequences with receptor binding sites.
It is generally accepted that the unliganded glucocorticoid receptor (GR) resides in the cytoplasm, and that hormone activation leads both to nuclear accumulation and gene activation. (Gasc, J. -M. & Baulieu, E. E. (1987) in
Steroid Hormone Receptors: Their Intracellular Localisation
, ed. Clark, C. R. (Ellis Horwood Ltd., Chichester, England), pp. 233-250; Beato, M. (1989)
Cell
56, 335-344; Carson-Jurica, M. A., Schrader, W. T. & O'Malley, B. W. (1990)
Endocr. Rev
. 11, 201-220; Gronemeyer, H. (1993) in
Steroid Hormone Action
, ed. Parker, M. G. (Oxford University Press, New York), pp. 94-117; Tsai, M. J. & O'Malley, B. W. (1994)
Annu. Rev. Biochem
. 63, 451-486; Akner, G., Wikstrom, A. C. & Gustafsson, J. A. (1995)
J. Steroid Biochem. Mol. Biol
. 52, 1-16), and references therein. However, the mechanisms involved in nuclear translocation and targeting of steroid receptors to regulatory sites in chromatin have been poorly understood. It has previously been difficult to discriminate between the ability of a given receptor mutant, or a given receptor/ligand combination, to participate in the separate processes of receptor activation, nuclear translocation, sequence-specific binding, and promoter activation.
Proteins have previously been labeled with fluorescent tags to detect their localization and sometimes their conformational changes both in vitro and in intact cells. Such labeling is essential both for immunofluorescence and for fluorescence analog cytochemistry, in which the biochemistry and trafficking of proteins are monitored after microinjection into living cells (Wang, Y. L. & Taylor, D. L., eds. (1989)
Methods Cell Biol
. 29). Traditionally, fluorescence labeling is done by purifying proteins and then covalently conjugating them to reactive derivatives of organic fluorophores. The stoichiometry and locations of dye attachment are often difficult to control, and careful repurification of the proteins is usually necessary. If the proteins are to be used inside living cells, a final challenging step is to get them across the plasma membrane via micropipet techniques or various methods of reversible permeabilization. Furthermore, in previous hormone studies broken cell preparations or antibody tags in fixed cell preparations were used, both techniques that cause enormous disruption of cell structures.
The green fluorescent protein (GFP) from the jellyfish
Aequorea victoria
is a molecule whose natural function seems to be to convert the blue chemiluminescence of the Ca
2+
-sensitive photoprotein aequorin into green emission (Ward, W. W. (1979) in
Photochemical and Photobiological Reviews
, ed. Smith, K. C. (Plenum, New York), 4:1-57). GFP's absorption bands in the blue (maximally at a wave length of 395 nm with weaker absorbance at 470 nm) and emission peak in the green (at 509 mn) do not arise from a distinct cofactor but rather from an internal p-hydroxybenzylideneimidazolidinone chromophore generated by cyclization and oxidation of a serine-tyrosine-glycine sequence at residues 56-67 (Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G. & Ward, W. W. (1993)
Biochemistry
32, 1212-1218). The gene for GFP was cloned (Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. & Cormier, M. J. (1992)
Gene
111, 229-233), and the encoded protein consists of 238 amino acid residues (molecular weight 27 kD). Heterologous expression of the gene has been done in
Escherichia coli
(Heim, R., Prasher, D. C. and Tsien, R. Y. (1994)
Proc. Natl. Acad. Sci. U.S.A
. 91,12501-12504); Inouye, S. & Tsuji, F. I. (1994)
FEBS Lett
. 341, 277-280),
Caenorhabditis elegans
(Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. (1994)
Science
263, 802-805), and
Drosophila melanogaster
(Yeh, E., Gustafson, K. & Boulianne, G. L. (1995)
Proc. Natl. Acad. Sci. U.S.A
. 92, 7035-7040; Tannahill, D., Bray, S. & Harris, W. A. (1995)
Dev. Biol
. 168, 694-697 and plants (Hu, W. & Cheng, C. L. (1995)
FEBS Lett
. 369, 331-334; Baulcombe, D. C., Chapman, S. & Santa Cruz, S. (1995)
Plant J
. 7, 1045-1053). Recently, chimeric genes encoding N- and C-terminal fusions of the Drosophila exuperantia (exu) gene product, Exu (Wang, S. and Hazelrigg, T. (1994)
Nature
369, 400-403), actin Act88F gene (Barthmaier, P. and Fyrberg, E. (1995)
Dev. Biol
. 169, 770-774), and a nuclear localization signal (Davis, I., Girdham, C. H. & O'Farrell, P. H. (1995)
Dev. Biol
. 170, 726-729); of the yeast microtubule and spindle pole associated disl gene product (Nabeshima, K., Kurooka, H., Takeuchi, M., Kinoshita, K., Nakaseko, Y., & Yanagida, M. (1995)
Genes Dev
. 9, 1572-1585) and an RNA binding protein Np13p (Corbett, A. H., Koepp, D. M., Schlenstedt, G., Lee, M. S. Hopper, A. K. & Silver, P. A. (1995)
J. Cell Biol
. 130, 1017-1026); and of a mammalian ion channel protein, NMDAR1 (Marshall, J., Molloy, R., Moss, G. W., Howe, J. R. & Hughes, T. E. (1995)
Neuron
14, 211-215), microtubule-associated protein, MAP4 (Olson, K. R., McIntosh, J. R. & Olmsted, J. B. (1995)
J. Cell Biol
. 130, 639-650), and a secretory protein, chromogranin B (Kaether, C. & Gerdes, H. H. (1995)
FEBS Lett
. 369, 267-271) have been constructed fused to GFP. However, none of these chimeric proteins have been to transcription factors or co-factors and no suggestions have been made as to the usefulness of such a fusion to study physiologically relevant interaction on an amplified DNA target. Furthermore, none of these reports indicated a successful use of GFP in mammalian cells.
Many human diseases result from aberrant steroid function, and many disease states, i.e., inflammation, are treated with glucocorticoid and other steroid derivatives. A large number of drugs have been developed whose function is based on the ability to interact with and activate steroid receptors. The identification and characterization of these compounds is a laborious, time-consuming and expensive process involving years of work. Even with a large investment of resources, the true behavior of these compounds in living cells is not understood.
The present invention allows observation for the first time of in vivo target sites within a higher eukaryotic nucleus for trans-regulatory molecules, such as transcription factors, e.g., glucocorticoid receptor (GR). The visualization of physiologically relevant in vivo target sites for any transcription factor to date has not previously been accomplished. The present invention provides a powerful method for identification of any single target site in a higher eukaryotic genome, comprising roughly 60,000-80,000 genes (Bird, A. P. (1995)
Trends Genet
. 11:94-100), using a singly fluorescently-labelled regulatory factor, which has not been considered previously. Discriminating direct

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