Claims
- 1. A method of determining a global gene regulatory element profile of cells, comprising:
(a) obtaining from at least one cell, or from cellular contents obtained from at least one cell, a plurality of one or more types of gene regulatory element complexes formed between cellular nucleic acid and associated protein components, said complexes comprising: (i) nucleic acid molecules and nucleic acid binding proteins; (ii) nucleic acid binding proteins and regulatory proteins; (iii) nucleic acid molecules, nucleic acid binding proteins and regulatory proteins; (iv) nucleic acid molecules, nucleic acid binding proteins, regulatory proteins and co-regulatory proteins; or (v) combinations thereof, under conditions conducive to the formation of said complexes; (b) detecting the components of the complexes that are formed; and (c) identifying one or more of the nucleic acid molecule, nucleic acid binding protein, regulatory protein, or co-regulatory protein components comprising the complexes so as to determine (i) a global gene regulatory element profile of the cells or (ii) a global analysis of transcription events occurring in the cells.
- 2. The method according to claim 1, wherein the gene regulatory element complexes are produced within the at least one cell and isolated therefrom.
- 3. The method according to claim 1, wherein the gene regulatory element complexes are produced outside of a cell by contacting a source of cellular nucleic acid sequences with a source of cellular proteins under conditions allowing for generation of the complexes.
- 4. The method according to claim 1, further comprising identifying transcribed regions regulated by the gene regulatory element complexes.
- 5. The method according to claim 1, wherein the complexes are formed inside living cells prior to isolation.
- 6. The method according to claim 1, wherein the complexes are formed under cell-free binding conditions prior to isolation.
- 7. The method according to claim 1, wherein the complexes are formed in solution.
- 8. The method according to claim 1, wherein the complexes are immobilized on localizing surfaces.
- 9. The method according to claim 1, wherein the cells are prokaryotic cells or eukaryotic cells.
- 10. The method according to claim 9, wherein the cells are selected from mammalian cells, vertebrate cells, invertebrate cells, plant cells, fungal cells, insect cells, protozoan cells, algal cells, yeast cells, Archaebacterial cells, and bacterial cells.
- 11. The method according to claim 9, wherein the cells are selected from single cells, cloned cells, homogeneous populations of cells, semi-purified cells, fully-purified cells, cells from tissues or portions thereof, cells from organs or portions thereof, or cells from whole organisms or portions thereof.
- 12. The method according to claim 11, wherein the cells comprise mixtures of different cell populations.
- 13. The method according to claim 1, wherein the nucleic acid molecules comprise one or more cis sites.
- 14. The method according to claim 13, wherein the one or more cis sites comprise associated transcribed regions.
- 15. The method according to claim 1, wherein the nucleic acid molecules comprise one or more gene regulatory sequences.
- 16. The method according to claim 13, wherein the nucleic acid molecules comprising one or more cis sites are obtained from cells, a preparation of genomic nucleic acid molecules, cloned nucleic acid sequences, or a library of synthetically prepared nucleic acid molecules.
- 17. The method according to claim 13, wherein regulatory or co-regulatory proteins are stably associated with the one or more cis sites comprising the nucleic acid molecules of the complexes.
- 18. The method according to claim 17, wherein the stable association of the regulatory or co-regulatory proteins and the cis sites comprising the nucleic acid molecules of the complexes results from one or more of chemical cross-linking, biological cross-linking, ultraviolet light cross-linking, or cleavable linker interactions.
- 19. The method according to claim 18, wherein the cross-linking is reversible.
- 20. The method according to claim 1, wherein the nucleic acid molecule and protein components comprising the complexes are obtained from a total cell extract, a nuclear extract, a cytoplasmic extract, a mitochondrial extract, a choloroplast extract, or a subcellular extract of the cells.
- 21. The method according to claim 1, further comprising performing steps (a)-(c) to determine a global gene regulatory element profile for (i) different cells, or (ii) two or more populations of cells, and comparing the profiles.
- 22. The method according to claim 1, further comprising selecting regulatory protein or nucleic acid molecule components of the complexes that bind to molecules involved in gene expression or transcription.
- 23. The method according to claim 22, wherein the molecules involved in gene expression or transcription comprise transcription factors.
- 24. The method according to claim 22, wherein the molecules involved in gene expression or transcription comprise promoter-associated factors.
- 25. The method according to claim 22, wherein the molecules involved in gene expression or transcription comprise enhancer-associated factors.
- 26. The method according to claim 1, wherein the regulatory proteins detected and identified from the complexes include general transcription factors, specific transcription factors that regulate subsets of genes, transcription-associated proteins, or co-regulatory proteins.
- 27. The method according to claim 26, wherein the transcription associated protein is polymerase.
- 28. The method according to claim 1, wherein the gene regulatory element profile provides the identification of a difference between gene expression or regulation of cells in one cellular metabolic state and gene expression or regulation of cells in a second cellular metabolic state.
- 29. The method according to claim 1, wherein the gene regulatory element profile provides the identification of a difference between gene expression or regulation of diseased cells and gene expression or regulation of non-diseased cells.
- 30. The method according to claim 1, wherein the gene regulatory element profile provides the identification of a difference between gene expression or regulation of normal cells and gene expression or regulation of abnormal cells.
- 31. The method according to claim 1, wherein the gene regulatory element profile provides the identification of a difference between gene expression or regulation of cells in one cellular physiologic state and gene expression or regulation of cells in a second cellular physiologic state.
- 32. The method according to claim 1, wherein the gene regulatory element profile provides the identification of a difference between gene expression or regulation of cells treated with an exogenous substance or agent and gene expression or regulation of untreated cells.
- 33. The method according to claim 32, wherein the exogenous substance or agent comprises a drug or chemical.
- 34. The method according to claim 13, further comprising identifying nucleic acid regulatory regions in cis site-containing nucleic acid molecules comprising the complexes by a method selected from nucleic acid amplification, nucleic acid sequencing, nucleic acid hybridization, or a combination thereof.
- 35. The method according to claim 1, further comprising identifying the nucleic acid sequences of the nucleic acid molecules bound to protein in the complexes by a method comprising the steps of:
a) denaturing the nucleic acid sequences comprising the complexes; b) binding the denatured nucleic acid sequences to detectably labeled nucleic acid molecules of known sequence; and c) identifying the nucleic acid molecules from the complex that were previously bound to protein by their binding to the detectably labeled nucleic acid molecules of known sequence.
- 36. The method according to claim 35, further comprising the step of d) sequencing the identified nucleic acid molecules.
- 37. The method according to claim 35, further comprising determining cis site motifs in the identified nucleic acid molecules associated with protein in the complexes.
- 38. The method according to claim 35, further comprising quantifying the nucleic acid molecules bound to the detectably labeled nucleic acid molecules of known sequence by determining intensity of the detectable label.
- 39. The method according to claim 38, wherein the detectable label comprises a fluorescent label, a radioactive label, an enzymatic label, or a chemiluminescent label.
- 40. The method according to claim 1, further comprising identifying the proteins comprising the complexes by a method selected from immunodetection, receptor-ligand binding, chemical methods, peptide sequencing, or array binding.
- 41. The method according to claim 40, wherein immunodetection is performed using antibodies directed toward one or more proteins of the complexes.
- 42. The method according to claim 40, wherein array binding is performed by (i) binding the proteins from the complexes onto an array comprising antibodies directed toward the protein in the complex, or (ii) binding nucleic acid from the complexes onto an array comprising nucleic acid molecules of known sequence.
- 43. The method according to claim 1, wherein identifying the nucleic acid molecules in the isolated complexes optionally comprises cloning fragments of the nucleic acid molecules into vectors, and (i) hybridizing the cloned nucleic acid molecules to nucleic acid probes of known sequence, or (ii) sequencing the cloned nucleic acid molecules.
- 44. The method according to claim 1, wherein analysis of the nucleic acid molecules in the isolated complexes comprises amplifying the nucleic acid molecules, or fragments thereof, subjecting the amplified nucleic acid molecules, or fragments thereof, to gel electrophoresis and observing amplicons of the expected size or sequences of the expected type.
- 45. The method according to claim 1, wherein analysis of the nucleic acid molecules in the isolated complexes comprises hybridizing the amplified nucleic acid molecules, or fragments thereof, to macroarrays or microarrays containing thereon known nucleic acid sequences.
- 46. The method according to claim 44 or claim 45, wherein the known nucleic acid sequences comprise cis sites, transcription regulatory regions, known transcribed regions, or predicted transcribed regions.
- 47. The method according to claim 44, wherein amplifying the nucleic acid molecules comprises a method selected from polymerase chain reaction (PCR), quantitative PCR (Q-PCR), ligation-mediated PCR (LM-PCR), transcription-mediated amplification, rolling circle amplification, or ligase chain reaction.
- 48. The method according to claim 1, further comprising directly sequencing the nucleic acid molecules, or fragments thereof, comprising the complexes, and evaluating the sequences obtained.
- 49. The method according to claim 1, wherein the nucleic acid molecules, or fragments thereof, comprising the complexes are isolated by binding to nucleic acid probes of known sequence or to arrays having bound thereto nucleic acid probes of known sequence.
- 50. The method according to claim 49, wherein the isolated nucleic acid molecules, or fragments thereof, are used as templates to synthesize a library of nucleic acid fragments comprising a selected population of nucleic acid sequences bound to protein in the complexes.
- 51. The method according to claim 50, wherein the library represents a portion of the bound sequences or sequences that are contiguous to the bound sequences.
- 52. The method according to claim 1 or claim 50, wherein the nucleic acid molecules identified or isolated from the complexes are subjected to subtractive hybridization.
- 53. The method according to claim 52, wherein the subtractive hybridization results in one or more of (i) enriching for nucleic acid sequences that are bound by a specific nucleic acid binding protein, regulatory protein, or co-regulatory protein in the complex; (ii) removing sequences common to regulatory element complexes from two or more different cells or populations of cells; (iii) enriching for sequences differentially present in regulatory element complexes from one cell or population of cells versus another cell or population of cells; (iv) enriching for sequences common to regulatory element complexes from two or more types of cells or cell populations; or (v) removing sequences present in regulatory element complexes from one cell or cell population versus another cell or cell population.
- 54. The method according to claim 1, further comprising comparing the global gene regulatory element profiles from cells comprising two or more different cell populations.
- 55. The method according to claim 54, wherein the two or more different cell populations being compared comprise different cell types within the same organism, the same cell type between different organisms, normal and diseased cells of the same type, normal and transformed cells of the same type, cells at different stages of differentiation, cells at different stages of development, treated cells and untreated cells, cells exposed to one compound and cells exposed to a second compound, cells exposed to an external condition and unexposed cells, or cells exposed to an internal condition and unexposed cells.
- 56. The method according to claim 1, wherein the complexed nucleic acid molecules comprise DNA, RNA, single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, genomic DNA, complementary DNA, DNA complementary to RNA, modified DNA, or modified RNA.
- 57. A method of determining a global gene regulatory element profile of cells, comprising:
(a) isolating from two or more different cell populations, or from cellular contents obtained from the cell populations, a plurality of one or more types of gene regulatory element complexes, said complexes formed between cellular nucleic acid molecules and associated protein components; (b) detecting the nucleic acid molecule or associated protein components of the complexes that are formed; and (c) identifying one or more of the nucleic acid molecule or associated protein components comprising the complexes so as to determine a global gene regulatory element profile of the cells or a global analysis of transcription events occurring in the cells.
- 58. The method according to claim 57, wherein the gene regulatory element complexes are produced within the cells and isolated therefrom.
- 59. The method according to claim 57, wherein the gene regulatory element complexes are produced outside of cells by contacting a source of cellular nucleic acid sequences with a source of cellular proteins under conditions allowing for generation of the complexes.
- 60. The method according to claim 1 or claim 57, wherein the global gene regulatory element profile is further combined with a technique selected from RNA analysis, proteomics analysis, transcription factor characterization, or transcription factor assay to elucidate ongoing regulatory and transcription events involving gene expression in cells.
- 61. A method of globally profiling gene regulatory element activity of cells, comprising:
(a) obtaining from cells, or from cellular contents obtained from the cells, a plurality of one or more types of gene regulatory element complexes formed between cellular nucleic acid molecules and associated protein components, said components comprising: (i) nucleic acid molecules and nucleic acid binding protein complexes; (ii) nucleic acid binding protein and regulatory protein complexes; (iii) nucleic acid molecules, nucleic acid binding protein and regulatory protein complexes; (iv) nucleic acid molecules, nucleic acid binding protein, regulatory protein and co-regulatory protein complexes; or (v) combinations thereof, under conditions conducive to the formation of said complexes; (b) isolating one or more of the protein components of the complexes using one or more affinity reagents that bind specifically to (i) the nucleic acid molecule component; (ii) the nucleic acid binding protein component; (iii) the regulatory protein component; (iv) the co-regulatory protein component; or (v) a combination thereof; and (c) identifying one or more of the nucleic acid molecule components, nucleic acid binding protein components, regulatory protein components, or co-regulatory protein components comprising the complexes so as to determine a global gene regulatory element profile of the cells.
- 62. The method according to claim 61, wherein the gene regulatory element complexes are produced within the cells and isolated therefrom.
- 63. The method according to claim 61, wherein the gene regulatory element complexes are produced outside of cells by contacting a source of cellular nucleic acid sequences with a source of cellular proteins under conditions allowing for generation of the complexes.
- 64. The method according to claim 61, wherein the one or more affinity reagents is selected from polyclonal antibodies, or binding fragments thereof, monoclonal antibodies or binding fragments thereof, intrabodies, single chain antibodies, or ligand-binding receptor proteins.
- 65. The method according to claim 61, wherein the one or more affinity reagents binds to a general transcription factor.
- 66. The method according to claim 61, wherein the one or more affinity reagents binds to a specific transcription factor.
- 67. The method according to claim 61, wherein the one or more affinity reagents binds to proteins involved in active transcription
- 68. The method according to claim 61, wherein the one or more affinity reagents binds to one or more nucleic acid molecules of the complexes.
- 69. The method according to claim 68, wherein the one or more affinity reagents is selected from nucleic acid aptamers or nucleic acid probes.
- 70. The method according to claim 61, wherein (i) the isolated nucleic acid molecule components of the complexes are identified by determining their nucleic acid sequences; (ii) the isolated protein components of the complexes are identified by determining their amino acid sequences; or (iii) a combination of (i) and (ii).
- 71. The method according to claim 70, wherein the identified nucleic acid sequences are further mapped on the appropriate genome using nucleic acid sequence databases.
- 72. The method according to claim 61, wherein the complexes of step (a) are formed in solution, on a solid support, in semi-solid medium, in gels, in column matrices, or in polymer formulations.
- 73. The method according to claim 72, wherein the solution is an aqueous solution, an organic solution, or an inorganic solution.
- 74. The method according to claim 61, wherein, following step (a), the nucleic acid molecule and associated protein complexes are separated from unbound cellular material.
- 75. A method of globally profiling gene regulatory activity of cells, comprising:
(a) obtaining nucleic acid molecule and protein complexes formed (i) within cells under conditions conducive to the formation of the complexes, or (ii) extracellularly from cellular nucleic acids and cellular proteins contacted under conditions allowing for production of the complexes; (b) isolating nucleic acid molecules from the complexes; (c) enriching the nucleic acid molecules for cell-specific transcribed nucleic acid molecules; and (d) determining the nucleic acid molecules that are specifically transcribed.
- 76. The method according to claim 75, further comprising the step of (e): identifying one or more of the proteins that comprise the complexes.
- 77. The method according to claim 75, wherein the nucleic acid comprising the complexes is DNA or RNA.
- 78. The method according to claim 75, wherein the complexes are obtained using antibodies directed against a protein comprising the complex.
- 79. The method according to claim 75, wherein the nucleic acid is identified by hybridization to nucleic acid probes, by binding to specific cis site-containing or regulatory-sequence-containing nucleic acid sequences, or by binding to nucleic acid molecules of known sequence or to immunoreactive agents arranged in an array.
- 80. The method according to claim 75, wherein the nucleic acid is isolated from the complexes using one or more of protease-digestion, phenol extraction and ethanol precipitation.
- 81. The method according to claim 75, wherein, in step (c), the cell-specific transcribed nucleic acid is enriched by subtractive hybridization to result in one or more of (i) enriching for nucleic acid sequences that are bound by a specific nucleic acid binding protein, regulatory protein, or co-regulatory protein in the complex; (ii) removing sequences common to regulatory element complexes from two or more different cells or populations of cells; (iii) enriching for sequences differentially present in regulatory element complexes from one cell or population of cells versus another cell or population of cells; (iv) enriching for sequences common to regulatory element complexes from two or more types of cells or cell populations; or (v) removing sequences present in regulatory element complexes from one cell or cell population versus another cell or cell population.
- 82. The method according to claim 75, wherein the specifically transcribed nucleic acid is determined by an amplification method selected from polymerase chain reaction (PCR), quantitative PCR (Q-PCR), ligation-mediated PCR, rolling circle amplification, transcription-mediated amplification and ligase chain reaction.
- 83. A method of globally profiling gene regulatory activity of cells, comprising:
(a) immunoprecipitating a plurality of regulatory element complexes comprising nucleic acid molecules and bound proteins from one or more cells or populations of cells; (b) analyzing the immunoprecipitated nucleic acid molecules for the presence of regulatory regions comprising cis sites or transcribed regions to obtain a global profile of gene regulatory activity; and (c) comparing the global profile of gene regulatory activity obtained in step (b) with global profiles of gene regulatory activity from different cells or cell populations to determine differences in gene expression or regulation in the different cell populations.
- 84. The method according to claim 83, wherein the regulatory element complexes are produced within the cells and isolated therefrom.
- 85. The method according to claim 83, wherein the regulatory element complexes are produced outside of the cells by contacting a source of cellular nucleic acid sequences with a source of cellular proteins under conditions allowing for generation of the complexes.
- 86. The method according to claim 83, wherein the cells are subjected to fixative prior to step (a).
- 87. The method according to claim 83, wherein cells are subjected to a cross-linking agent prior to step (a).
- 88. The method according to claim 83, wherein immunoprecipitation is performed using antibodies directed against transcription related proteins.
- 89. The method according to claim 88, wherein transcription related proteins are selected from RNA polymerase II, RNA polymerase II transcription factor B (TFIIB), RNA polymerase II transcription factor E, subunit β (TFIIEβ), acetylated histone H3 (AcH3), TATA-box binding protein (TBP) and CREB-binding protein (CBP).
- 90. The method according to claim 83, wherein the analyzing step (b) is performed using polymerase chain reaction (PCR).
- 91. The method according to claim 90, wherein the primers in the polymerase chain reaction (PCR) are specific for promoter sequences, intronic sequences, exonic sequences, enhancer sequences, sequences 5′ to promoter sequences, sequences 5′ or 3′ to genes or a combination thereof.
- 92. The method according to claim 83, further wherein the analyzing step (c) comprises using quantitative PCR (Q-PCR) to detect transcribed genes.
- 93. The method according to claim 83, further comprising identifying the protein components that are complexed with the nucleic acids.
- 94. A method for globally determining differences in gene regulatory element activity between cells, comprising:
(a) isolating from a first population of cells a plurality of gene regulatory complexes comprising nucleic acid molecule components and associated protein components; (b) analyzing (i) the nucleic acid molecule components of the complexes of step (a) to determine the presence of cis sites or regulatory regions; (ii) the protein components of the complexes of step (a) to identify the proteins as nucleic acid binding proteins, regulatory proteins, or co-regulatory proteins; or a combination of (i) and (ii); (c) isolating from a second population of cells a plurality of gene regulatory complexes comprising nucleic acid molecule components and associated protein components; (d) analyzing (i) the nucleic acid molecule components of the complexes of step (c) to determine the presence of cis sites or regulatory regions; (ii) the protein components of the complexes of step (c) to identify the proteins as nucleic acid binding proteins, regulatory proteins, or co-regulatory proteins; or a combination of (i) and (ii); and (e) comparing the components of the complexes isolated from the first and second populations of cells to determine differences in gene regulatory element activity between the cell populations.
- 95. The method according to claim 94, wherein the gene regulatory element complexes are produced within the first and second populations of cells and isolated therefrom.
- 96. The method according to claim 94, wherein the gene regulatory element complexes are produced outside of the first and second populations of cells, by contacting a source of cellular nucleic acid sequences with a source of cellular proteins under conditions allowing for generation of the complexes for each population.
- 97. The method according to claim 94, wherein the first population of cells comprises cells selected from a first cell type, physiologic state, metabolic state, disease state, or drug-treated state, and the second population of cells comprises cells selected from a second cell type, physiologic state, metabolic state, disease state, or drug-treated state.
- 98. The method according to claim 94, wherein the first and second populations of cells comprise different cell types within the same organism, the same cell type between different organisms, normal cells and diseased cells of the same types, normal and transformed cells of the same types, cells at different stages of differentiation or development, cells treated with an exogenous material and untreated cells, cells exposed to two different compounds or molecules, cells exposed to a different external or internal condition and unexposed cells, cells exposed to two different external or internal conditions, or infected cells and uninfected cells.
- 99. A method of determining a global gene regulatory element profile in cells, comprising:
(a) obtaining from two or more different cell populations a plurality of one or more types of gene regulatory element complexes formed between cis site-containing or regulatory sequence-containing nucleic acid molecules and associated protein components; and (b) detecting and analyzing one or more of the nucleic acid molecule or associated protein components comprising the complexes of the cell populations; and (c) comparing the nucleic acid molecule or protein components from the cell populations so as to determine global gene regulatory element activity in the two or more cell populations, or a global analysis of transcription events occurring in the two or more cell populations.
- 100. The method according to claim 99, wherein the gene regulatory element complexes are produced within the cells and isolated therefrom.
- 101. The method according to claim 99, wherein the gene regulatory element complexes are produced outside of cells by contacting a source of cellular nucleic acid sequences with a source of cellular proteins under conditions allowing for generation of the complexes.
- 102. The method according to claim 99, wherein cis sites contained in the nucleic acid molecules of the complexes of the cell populations are identified by isolating the nucleic acid molecules, or fragments thereof, and determining cis site-containing nucleic acid sequences.
- 103. The method according to claim 99, wherein cis sites contained in the nucleic acid molecules of the complexes of the cell populations are identified by amplifying fragments obtained from the nucleic acid molecules of the complexes and obtaining overlapping or nonoverlapping fragments, wherein the obtained fragments are further size-selected and concatamerized for cloning and sequencing.
- 104. The method according to claim 103, wherein the fragments are about 50-100 base pairs in length.
- 105. The method according to claim 99, wherein the nucleic acid molecules, or fragments thereof, of the complexes of the cell populations are hybridized to probes having known nucleic acid sequences under conditions suitable for hybrid formation, wherein the sequence of a nucleic acid molecule, or fragment thereof, is determined following the formation of hybrids.
- 106. The method according to claim 105, wherein prior to hybridization, the nucleic acid molecules, or fragments thereof, are amplified.
- 107. The method according to claim 105, further comprising a detectable label to allow detection of hybridization complexes.
- 108. The method according to claim 107, wherein the detectable label comprises a radioactive label, an enzymatic label, a fluorescent label, or a chemiluminescent label.
- 109. The method according to claim 105, wherein hybridization is performed in solution, on macroarrays, or on microarrays.
- 110. The method according to claim 105, wherein hybrid complexes are detected by autoradiography, fluorimetry, luminometry, or phosphoimage analysis.
- 111. A method for globally profiling regulatory element activity of cells, comprising:
(a) obtaining from the cells, or from cellular contents obtained from the cells, a plurality of gene regulatory element complexes comprising cis site-containing nucleic acid molecules and associated protein components selected from (i) nucleic acid molecules and nucleic acid binding proteins; (ii) nucleic acid binding proteins and regulatory proteins; (iii) nucleic acid molecules, nucleic acid binding proteins and regulatory proteins; (iv) nucleic acid molecules, nucleic acid binding proteins, regulatory proteins and co-regulatory proteins; or (v) combinations thereof, under conditions conducive to the formation of said complexes; (b) detecting the complexes; (c) identifying (i) a nucleic acid sequence of one or more cis site-containing nucleic acid molecules comprising the complexes, (ii) an amino acid sequence of one or more nucleic acid binding proteins, regulatory proteins, or co-regulatory proteins comprising the complexes, or a combination of (i) and (ii); wherein identification of the nucleic acid components of the separated complexes comprises one or more of (1) sequencing the nucleic acid molecules or a portion thereof; (2) hybridizing the nucleic acid molecules to other known nucleic acid molecules; (3) preparing a recombinant library from the isolated nucleic acid molecules or portions thereof; (4) sequencing the library or a portion thereof; or (5) amplifying the nucleic acid sequences to determine if specific nucleic acid sequences are present in the isolated nucleic acid molecules so as to globally profile gene regulatory element activity in the cells.
- 112. The method according to claim 111, wherein the gene regulatory element complexes are produced within the cells and isolated therefrom.
- 113. The method according to claim 111, wherein the gene regulatory element complexes are produced outside of cells by contacting a source of cellular nucleic acid sequences with a source of cellular proteins under conditions allowing for generation of the complexes.
- 114. The method according to claim 111, wherein the complexes of step (b) and the sequences of step (c) are compared between a first cell type, cell population, cell state, or cell treatment and at least a second cell type, cell population, cell state, or cell treatment to globally profile gene regulatory element activity in the compared cells, or to compare global gene regulatory element activity profiles between the cells or cell populations.
- 115. The method according to claim 111, wherein the detecting step (b) comprises fluorescent polarization.
- 116. The method according to claim 111, wherein the detecting step (b) comprises direct detection comprising a fluorescent label or a chemiluminescent label.
- 117. The method according to claim 111, wherein the detecting step (b) comprises separating the complexes from exogenous material.
- 118. The method according to claim 1, claim 57, claim 99, or claim 111, wherein the complexes are separated from nucleic acid molecules and proteins not comprising the complexes before the detecting step (b).
- 119. The method according to claim 118, wherein the separation is performed by one or more of the methods selected from electrophoretic mobility shift assay (EMSA), capillary electrophoresis (CE), filtration, size-exclusion filtration, affinity purification, enzyme digestion and centrifugation.
- 120. The method according to claim 111, wherein the cis site-containing nucleic acid molecules are contacted with a surface comprising a macroarray or a microarray.
- 121. The method according to claim 111, wherein the cis site-containing nucleic acid molecules are obtained from cells, genomic nucleic acid, or a library of synthetically prepared nucleic acid molecules.
- 122. The method according to claim 111, wherein the cells are selected from the group consisting of mammalian cells, vertebrate cells, invertebrate cells, plant cells, fungal cells, insect cells, protozoan cells, algal cells, yeast cells, Archaebacterial cells, and bacterial cells.
- 123. The method according to claim 111, wherein the cells are selected from single cells, cloned cells, homogeneous populations of cells, semi-purified cells, fully-purified cells, cells from tissues or portions thereof, cells from organs or portions thereof, or cells from whole organisms or portions thereof.
- 124. A method of determining a global gene regulatory element activity profile of cells, comprising:
(a) isolating from at least one cell, or from cellular contents obtained from at least one cell, a plurality of one or more types of gene regulatory element complexes formed between cellular nucleic acid and associated protein components, said complexes comprising: (i) nucleic acid molecules and nucleic acid binding proteins; (ii) nucleic acid binding proteins and regulatory proteins; (iii) nucleic acid molecules, nucleic acid binding proteins and regulatory proteins; (iv) nucleic acid molecules, nucleic acid binding proteins, regulatory proteins and co-regulatory proteins; or (v) combinations thereof, under conditions conducive to the formation of said complexes; (b) separating the one or more types of complexes from other complexes and/or from unbound components; (c) identifying (i) the nucleic acid components of the separated complexes, or (ii) the protein components of the separated complexes; and (d) combining activity information of at least two of the complexes to generate a global gene regulatory element activity profile for the cells.
- 125. The method according to claim 124, wherein the gene regulatory element complexes are produced within the cells and isolated therefrom.
- 126. The method according to claim 124, wherein the gene regulatory element complexes are produced outside of cells by contacting a source of cellular nucleic acid sequences with a source of cellular proteins under conditions allowing for generation of the complexes.
- 127. The method according to claim 124, wherein the nucleic acid molecules of the complexes are fragmented before separating the complexes.
- 128. The method according to claim 127, wherein the nucleic acid molecules are fragmented using sonication, restriction enzyme digestion, nuclease digestion, pH or elevation of temperature.
- 129. The method according to claim 124, wherein the separating at least one type or class of complexes comprises use of affinity reagents.
- 130. The method according to claim 129, wherein the affinity reagents include antibodies that recognize transcription-associated proteins.
- 131. The method according to claim 129, wherein the affinity reagents include nucleic acid probes that recognize transcription-associated nucleic acids.
- 132. The method according to claim 130, wherein the transcription-associated proteins are selected from general transcription factors, specific transcription factors that regulate subsets of genes, transcription-associated proteins, or co-regulatory proteins.
- 133. The method according to claim 132, wherein the transcription associated proteins are polymerases.
- 134. The method according to claim 130, wherein the transcription-associated proteins are selected from RNA polymerase II, RNA polymerase II transcription factor B (TFIIB), RNA polymerase II transcription factor E, subunit β (TFIIEβ), acetylated histone H3 (AcH3), TATA-box binding protein (TBP), or CREB-binding protein (CBP).
- 135. The method according to claim 124, wherein the separating step (b) comprises physical separation of the complexes based on molecular size, charge, molecular weight, or recognition of molecular moieties.
- 136. The method according to claim 124, wherein the identifying step (c) comprises quantification of the number of nucleic acid sequences comprising the complexes.
- 137. The method according to claim 124, wherein identifying the nucleic acid components of the separated complexes comprises sequencing the nucleic acid molecules or a portion thereof; hybridizing the nucleic acid molecules to other known nucleic acid molecules, amplifying the nucleic acids, or generating a recombinant library from the isolated nucleic acid molecules, or portions thereof; and (i) sequencing the library or a portion thereof, or (ii) amplifying the nucleic acids, to determine if specific nucleic acid sequences are present in the isolated nucleic acid molecules.
- 138. The method according to claim 137, wherein generating a recombinant library comprises ligating random primers to the ends of the isolated nucleic acid molecules, or portions thereof; amplifying nucleic acid sequences corresponding to the isolated nucleic acid molecules or portions thereof; size-fractionating the amplified sequences to a desired size; concatamerizing the amplified molecules into chains of about 5-30 molecules; cloning the concatamerized molecules into a suitable cloning vector, growing the clones to obtain more copies thereof, and sequencing inserts of the clones.
- 139. The method according to claim 124, wherein identifying comprises amplifying isolated nucleic acids using primers specific for specific sequences in a genome.
- 140. The method according to claim 139, wherein amplifying involves PCR, quantitative PCR, ligation-mediated PCR, rolling circle amplification, transcription-mediated amplification, and ligase chain reaction.
- 141. The method according to claim 124, wherein identifying the protein components of the separated complexes comprises immunodetection, receptor-ligand binding, chemical methods, peptide sequencing, or array binding.
- 142. The method according to claim 141, wherein immunodetection is performed using antibodies directed toward one or more proteins of the complexes.
- 143. The method according to claim 141, wherein array binding is performed by (i) binding the proteins from the complexes onto an array comprising antibodies directed toward the protein in the complex, or (ii) binding nucleic acid from the complexes onto an array comprising nucleic acid molecules of known sequence.
- 144. The method according to claim 124, wherein the components of the complexes are stably associated before said isolating step (a).
- 145. The method according to claim 144, wherein cells are subjected to a cross-linking agent prior to said isolating step (a).
- 146. The method according to claim 144, wherein stable association of the components results from one or more of chemical cross-linking, biological cross-linking, ultraviolet light cross-linking, or cleavable linker interactions.
- 147. The method according to claim 146, wherein the cross-linking is reversible.
Parent Case Info
[0001] This application is a continuation-in-part of application Serial No. PCT/IB02/00589, filed Feb. 28, 2002, the contents of which are hereby incorporated by reference in their entirety. PCT/IB02/00589 claims the benefit of application Serial No. 60/272,753, filed Mar. 1, 2001.
Provisional Applications (1)
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Number |
Date |
Country |
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60272753 |
Mar 2001 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
PCT/IB02/00589 |
Feb 2002 |
US |
Child |
10426734 |
Apr 2003 |
US |