Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-09
2001-02-06
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S325000, C435S372300
Reexamination Certificate
active
06183965
ABSTRACT:
BACKGROUND OF THE INVENTION
Each of the roughly 100,000 genes encoded in the human genome is subject to individual dosage control. The systems that regulate gene expression respond to a wide variety of developmental and environmental stimuli, thus allowing each cell type to express a unique and characteristic subset of its genes, and to adjust the dosage of particular gene products as needed. The importance of dosage control is underscored by the fact that targeted disruption of key regulatory molecules in mice often results in drastic phenotypic abnormalities [Johnson, R. S., et al.,
Cell,
71:577-586 (1992)], just as inherited or acquired defects in the function of genetic regulatory mechanisms contribute broadly to human disease.
The regulatory mechanisms controlling the transcription of protein- coding genes by RNA polymerase II have been extensively studied. RNA polymerase II and its host of associated proteins are recruited to the core promoter through non-covalent contacts with sequence-specific DNA binding proteins [Tjian, R. and Maniatis, T.,
Cell,
77:5-8 (1994); Stringer, K. F.,
Nature
(London), 345:783-786 (1990)]. An especially prevalent and important subset of such proteins, known as transactivators, typically bind DNA at sites outside the core promoter and activate transcription through space contacts with components of the transcriptional machinery, including chromatin remodeling proteins [Tjian, R. and Maniatis, T.,
Cell,
77:5-8 (1994); Stringer, K. F.,
Nature
(London), 345:783-786 (1990); Bannister, A. J. and Kouzarides, T.,
Nature,
384:641-643 (1996); Mizzen, C. A., et al.,
Cell,
87:1261-1270 (1996)]. The DNA-binding and activation functions of transactivators generally reside on separate domains whose operation is portable to heterologous fusion proteins [Sadowski, I., et al.,
Nature,
335:563-564 (1988)]. Though it is believed that activation domains are physically associated with a DNA-binding domain to attain proper function, the linkage between the two need not be covalent [Belshaw, P. J., et al.,
Proc. Natl. Acad. Sci. USA,
93:4604-4607 (1996); Ho, S. H., et al.,
Nature
(London), 382:822-826 (1996)]. In many instances, the activation domain does not appear to contact the transcriptional machinery directly, but rather through the intermediacy of adapter proteins known as coactivators [Silverman, N., et al.,
Proc. Natl. Acad. Sci. USA,
91:11005-11008 ((1994); Arany, Z., et al.,
Nature
(London), 374:81-84 (1995)].
The importance of controlled gene expression in human disease and the information available to date relating to the mechanisms of gene regulation have fueled efforts aimed at discovering means of overriding endogenous regulatory controls or of creating new signaling circuitry in cells [Belshaw, P. J., et al.,
Proc. Natl. Acad. Sci. USA,
93:4604-4607 (1996); Ho, S. H., et al.,
Nature
(London), 382:822-826 (1996); Rivera, V. M., et al.,
Nat. Med.,
2:1028-1032; Spencer, D. M., et al.,
Science,
262:1019-1024 (1993)]. Of particular interest in this regard are small, membrane-permeant molecules designed to modulate gene transcription in living cells [Belshaw, P. J., et al.,
Proc. Natl. Acad. Sci. USA,
93:4604-4607 (1996); Ho, S. H., et al.,
Nature
(London), 382:822-826 (1996); Rivera, V. M., et al.,
Nat. Med.,
2:1028-1032; Spencer, D. M., et al.,
Science,
262:1019-1024 (1993)]. All such efforts involved genetic engineering of transcriptional modulatory protein domains such as naturally-occurring VP16. The present invention takes a significant departure from such art by relying upon chemical rather than biological means to harness the transcriptional machinery.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the remarkable discovery that small molecular weight (e.g., <5kD), membrane-permeant compounds are capable of acting as transcriptional modulators. In preferred embodiments, the compounds of the invention, also referred to herein as transcriptional modulators, include at least one selected ligand linked, e.g., covalently linked, to at least one transcriptional modulating portion (TMP). The TMPs of the present invention can be chemical moieties, e.g., non-peptidyl, small molecules.
Accordingly, in one aspect, this invention pertains to methods and compositions for identifying novel transcriptional modulators. The method can be performed in a cell, e.g. cell-based, or in a reaction mixture, e.g., cell-free. In a cell based method, a cell is provided which has (a) a genetic construct encoding a chimeric protein and (b) a target gene under the control of at least one transcriptional regulatory element which is recognized by the DNA-binding domain of the chimeric protein. The chimeric protein includes at least one ligand-binding domain (which binds to a selected ligand) and a heterologous DNA-binding domain. The cell is contacted with a test compound under conditions which allow transcription to occur and any changes in transcriptional activity of the target gene in the presence of the test compound relative to that detected in the absence of the test compound are detected. A change in the level of transcription activity, e.g., an increase or decrease, of the target gene detected in the presence of the test compound relative to that detected in the absence of the test compound indicates that the test compound is a transcriptional modulator.
In preferred embodiments, the test compound(s) include a selected ligand linked to a test-transcriptional modulating portion (test-TMP). The identified transcriptional modulator(s) include a selected ligand linked to a TMP.
The present invention further provides a cell-free method for identifying a transcriptional modulator. The method involves providing a reaction mixture including a chimeric protein and a target gene under the control of at least one transcriptional regulatory element which is recognized by the DNA-binding domain of the chimeric protein. The reaction mixture further includes a cell-free transcription system and a test compound. Preferably, the cell-free transcription system is selected from a group consisting of a cell lysate, e.g., a HeLa cell extract, or a reconstituted protein mixture, e.g., a mixture of components of the transcriptional apparatus. The reaction mixture is provided under conditions which allow transcription to occur and any changes in transcriptional activity of the target gene in the presence of the test compound relative to that observed in the absence of the test compound are detected. In this assay, a change, e.g., an increase or a decrease, in the level of transcriptional activity of the target gene detected in the presence of the test compound relative to that detected in the absence of the test compound indicates that the test compound is a transcriptional modulator.
In yet another aspect, the invention features a method for identifying a transcriptional modulator from a plurality of test compounds. The method includes: providing cells, e.g., genetically engineered cells, which contain (i) a genetic construct encoding a chimeric protein which comprises at least one ligand-binding domain and a DNA-binding domain which is heterologous thereto, wherein the ligand-binding domain binds to a selected ligand; (ii) a target gene under the expression control of at least one transcriptional regulatory element which is recognized by the DNA-binding domain of the chimeric protein. These cells are contacted with one or more test compounds, each of which contain the selected ligand linked to at least one of a plurality of test-TMPs; and changes in transcription activity in the presence of the test compound are detected relative to that detected in the absence of the test compound. In this assay, a change, e.g., an increase or a decrease, in the level of transcriptional activity of the target gene detected in the presence of the test compound relative to that detected in the absence of the test compound indicates that the test compound is
Nyanguile Origene
Verdine Gregory L.
Clauss Isabelle M.
Foley Hoag & Eliot LLP
President and Fellows of Harvard College
Schwartzman Robert A.
Vincent Matthew P.
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