Claims
- 1. A method for binding an exogenous molecule to a binding site, wherein the binding site is located within a region of interest in cellular chromatin, wherein the method comprises:
(a) identifying an accessible region within the region of interest; (b) identifying a target site for the exogenous molecule within the accessible region; and (c) introducing the exogenous molecule into the cell; whereby the exogenous molecule binds to the binding site.
- 2. The method according to claim 1 wherein the cellular chromatin is in a chromosome.
- 3. The method according to claim 1 wherein the accessible region is a nuclease hypersensitive region.
- 4. The method according to claim 1, further comprising the step of:
(d) testing for binding of the exogenous molecule to the binding site.
- 5. The method according to claim 4, wherein testing is by a method selected from the group consisting of chromatin immunoprecipitation and in vivo footprinting.
- 6. The method according to claim 1, wherein the exogenous molecule is a protein.
- 7. The method according to claim 6 wherein the protein performs a process selected from the group consisting of replication, recombination, integration, DNA repair, transcriptional regulation and chromatin remodeling.
- 8. The method according to claim 6 wherein the protein is used for detection of a target sequence.
- 9. The method according to claim 7, wherein the protein is a transcription factor.
- 10. The method according to claim 9, wherein the transcription factor is a zinc finger protein (ZFP).
- 11. The method according to claim 6 wherein the protein is encoded by an exogenous nucleic acid introduced into the cell.
- 12. The method according to claim 1, wherein the cell is a eukaryotic cell.
- 13. The method according to claim 12, wherein the cell is a plant cell.
- 14. The method according to claim 12, wherein the cell is a mammalian cell.
- 15. The method according to claim 14, wherein the cell is a human cell.
- 16. The method according to claim 1, wherein the binding site is in a coding region.
- 17. The method according to claim 1, wherein the binding site is in a non-coding region.
- 18. The method according to claim 10, wherein the binding site comprises the sequence 5′-NNx aNy bNz c-3′, wherein
each of (x,a), (y,b) and (z,c) is (N,N) or (G,K); and at least one of (x,a), (y,b) and (z,c) is (G,K); wherein N is any nucleotide and K is either G or T.
- 19. The method according to claim 1, wherein the exogenous molecule is introduced into the cell by a method selected from the group consisting of lipid-mediated gene transfer, electroporation, direct injection, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran mediated transfer, and viral vector-mediated transfer.
- 20. The method according to claim 11 wherein the nucleic acid is introduced into the cell by a method selected from the group consisting of lipid-mediated gene transfer, electroporation, direct injection, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran mediated transfer, and viral vector-mediated transfer.
- 21. A method for binding a ZFP transcription factor to a binding site, wherein the binding site is located within a region of interest in cellular chromatin, wherein the method comprises:
(a) identifying an accessible region within the region of interest; (b) identifying a zinc finger protein (ZFP) binding sequence within the accessible region; (c) designing a ZFP to bind to the binding sequence; and (d) introducing the ZFP into the cell; whereby the ZFP binds to the binding site.
- 22. The method according to claim 21 wherein the ZFP is introduced into the cell by introducing a DNA construct encoding the ZFP into the cell under conditions in which the construct expresses the ZFP.
- 23. The method according to claim 21 wherein the cellular chromatin is in a chromosome.
- 24. The method according to claim 21 wherein the accessible region is a nuclease hypersensitive region.
- 25. The method according to claim 21 farther comprising the step of:
(e) testing for binding of the ZFP to the binding site.
- 26. The method according to claim 25 wherein testing is by a method selected from the group consisting of chromatin immunoprecipitation and in vivo footprinting.
- 27. The method according to claim 21, wherein the binding site comprises the sequence 5′-NNx aNy bNz c-3′, wherein
each of (x,a), (y,b) and (z,c) is (N,N) or (G,K); and at least one of (x,a), (y,b) and (z,c) is (G,K); wherein N is any nucleotide and K is either G or T.
- 28. The method according to claim 21, wherein the ZFP is selected by phage display.
- 29. The method according to claim 21, wherein the ZFP is selected in vivo.
- 30. A method for identifying a binding site for an exogenous molecule, wherein the binding site is located within a region of interest in cellular chromatin, wherein the method comprises:
(a) identifying an accessible region within the region of interest; and (b) identifying a target site for the exogenous molecule within the accessible region.
- 31. The method according to claim 30, further comprising the steps of:
(c) introducing the exogenous molecule into the cell; and (d) testing for the binding of the molecule to the binding sequence.
- 32. The method according to claim 30 wherein the cellular chromatin is in a chromosome.
- 33. The method according to claim 30 wherein the accessible region is a nuclease hypersensitive region.
- 34. The method according to claim 30 wherein the exogenous molecule is a protein.
- 35. The method according to claim 34 wherein the protein is a transcription factor.
- 36. The method according to claim 35 wherein the transcription factor is a ZFP.
- 37. The method according to claim 30, wherein the binding site comprises the sequence 5′-NNx aNy bNz c-3′, wherein
each of (x,a), (y,b) and (z,c) is (N,N) or (G,K); and at least one of (x,a), (y,b) and (z,c) is (G,K); wherein N is any nucleotide and K is either G or T.
- 38. The method according to claim 31 wherein the exogenous molecule is a protein and wherein the protein is introduced into the cell by introducing a DNA construct encoding the protein into the cell under conditions in which the construct expresses the protein.
- 39. The method according to claim 31 wherein testing is by a method selected from the group consisting of chromatin immunoprecipitation and in vivo footprinting.
- 40. A complex between an exogenous molecule and a binding site, wherein the binding site is located within a region of interest in cellular chromatin and wherein the binding site is identified according to the method of claim 30.
- 41. A complex between an exogenous molecule and a binding site, wherein the binding site is located within a region of interest in cellular chromatin and wherein the binding site is identified according to the method of claim 31.
- 42. The complex according to claim 40, wherein the exogenous molecule is a protein.
- 43. The complex according to claim 42, wherein the protein performs a process selected from the group consisting of replication, recombination, integration, DNA repair, transcriptional regulation and chromatin remodeling.
- 44. The complex according to claim 42 wherein the protein is used for detection of a target sequence
- 45. The complex according to claim 43, wherein the protein is a transcription factor.
- 46. The complex according to claim 45, wherein the transcription factor is a zinc finger protein (ZFP).
- 47. The complex according to claim 40, wherein the cell is a eukaryotic cell.
- 48. The complex according to claim 47, wherein the cell is a plant cell.
- 49. The complex according to claim 47, wherein the cell is a mammalian cell.
- 50. The complex according to claim 49, wherein the cell is a human cell.
- 51. The complex according to claim 40, wherein the binding site is in a coding region.
- 52. The complex according to claim 40, wherein the binding site is in a non-coding region.
- 53. The complex according to claim 46, wherein the binding site comprises the sequence 5′-NNx aNy bNz c-3′, wherein
each of (x,a), (y,b) and (z,c) is (N,N) or (G,K); and at least one of (x,a), (y,b) and (z,c) is (G,K); wherein N is any nucleotide and K is either G or T.
- 54. A method for identifying a binding site, in a chromosome, for a ZFP transcription factor, wherein the chromosome is in a cell, wherein the method comprises:
(a) identifying a region of interest in the chromosome; (b) identifying a zinc finger protein (ZFP) binding sequence within the region; (c) designing a ZFP to bind to the binding sequence; (d) introducing the ZFP into the cell; and (e) testing for the binding of the ZFP to the binding sequence by chromatin immunoprecipitation; wherein, if binding is detected in step (e), a binding site is identified.
- 55. A method for identifying a binding site in cellular chromatin for a ZFP transcription factor, wherein the method comprises:
(a) identifying a region of interest in the cellular chromatin; (b) identifying a zinc finger protein (ZFP) binding sequence within the region; (c) designing a ZFP to bind to the binding sequence; (d) designing a DNA construct that encodes the ZFP of step(c); (e) introducing the construct into the cell; and (f) testing for the binding of the ZFP to the binding sequence by chromatin immunoprecipitation wherein, if binding is detected in step (f), a binding site is identified.
- 56. A cell comprising a complex between an exogenous molecule and a binding site, wherein the binding site is located within a region of interest in cellular chromatin and wherein the binding site is identified according to the method of claim 30.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under the provisions of 35 U.S.C. § 119 to U.S. Provisional Patent Application Ser. No. 60/200,590, filed Apr. 28, 2000; the disclosure of which is hereby incorporated by reference in its entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60200590 |
Apr 2000 |
US |