# US 7,611,838 B2 · Biologically-active DNA-binding sites and related methods > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,611,838 B2 - Document ID: US7611838B2 - Status: Granted - Kind: B2 - Application number: 10/591,271 - Filed: 2005-03-03 - Published: 2008-02-14 - Granted: 2009-11-03 - Earliest claimed: 2004-03-04 - Assignees: Whitehead Institute for Biomedical Research - Inventors: Christopher T. Harbison; Richard A. Young; David B. Gordon; Ernest Fraenkel - Web page: https://patentgrep.com/patent/US7611838B2 ## CPC - G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites} - C12Q 2522/00: Reaction characterised by the use of non-enzymatic proteins - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The invention relates to the identification of biologically-active DNA-binding sites to which a protein of interest binds in a cell. The invention also relates to the identification of agents and conditions which alter the biologically-active DNA-binding sites to which a protein binds. One aspect of the invention also provides methods for identifying pathways that are regulated by transcriptional regulators and for modulating the activity of the pathways. ## Claims ### Claim 1 A method of identifying a set of biologically-active DNA-binding sites for a protein of interest in the genome of a cell, the method comprising (i) identifying a set of regions of genomic DNA to which the protein of interest is bound in the cell; (ii) identifying candidate DNA-binding sites in the identified regions of genomic DNA, wherein a candidate DNA-binding site comprises a sequence corresponding to a DNA-sequence motif for the protein of interest, and wherein said DNA sequence motif is enriched in said identified regions by a statistically significant amount relative to a set of genomic regions which are bound by a mutant form of the protein of interest in the cell; (iii) determining if the candidate DNA-binding sites are conserved in an equivalent genomic region in one or more species different from the species from which the cell is obtained, wherein a candidate DNA-binding site that is conserved in at least one of the different species is a biologically-active DNA-binding site. ### Claim 2 (depends on 1) The method of claim 1, wherein the regions of genomic DNA comprise promoter regions. ### Claim 3 (depends on 1) The method of claim 1, wherein a candidate DNA-binding site is conserved if the equivalent genomic region in at least one different species comprises a nucleic acid sequence that matches the DNA-sequence motif for the protein of interest. ### Claim 4 (depends on 1) The method of claim 1, wherein the candidate DNA-binding site is less than 20 bp in length. ### Claim 5 (depends on 1) The method of claim 1, wherein the DNA-sequence motif is degenerate in at least one position. ### Claim 6 (depends on 1) The method of claim 1, wherein step (iii) comprises determining if the candidate DNA-binding sites are conserved in equivalent genomic regions in two or more different species. ### Claim 7 (depends on 1) The method of claim 1, wherein the set of biologically-active DNA-binding sites comprises one or more biologically-active DNA-binding sites. ### Claim 8 (depends on 1) The method of claim 1, wherein two regions of genomic DNA are equivalent if they both comprise a sequence of at least one orthologous gene. ### Claim 9 (depends on 1) The method of claim 1, wherein the cell is an eukaryotic cell. ### Claim 10 A method of identifying an agent which alters the set of biologically-active DNA-binding sites for a protein of interest in the genome of a cell, the method comprising (i) contacting an experimental cell with a candidate agent; (ii) identifying a set of biologically-active DNA-binding sites for a protein of interest in the genome of the cell of step (i) by: (a) identifying a set of regions of genomic DNA to which the protein of interest is bound in the cell; (b) identifying candidate DNA-binding sites in the identified regions of genomic DNA, wherein a candidate DNA-binding site comprises a sequence corresponding to a DNA-sequence motif for the protein of interest, wherein the DNA sequence motif is enriched by a statistically significant amount relative to a suitable control; (c) determining if the candidate DNA-binding sites are conserved in an equivalent genomic region in one or more species different from the species from which the cell is obtained, wherein a candidate DNA-binding site that is conserved in at least one of the different species is a biologically-active DNA-binding site; thereby generating an experimental set of biologically-active DNA-binding sites; (iii) comparing (1) the experimental set of biologically-active DNA-binding sites to (2) a control set of biologically-active DNA-binding sites for the protein of interest; wherein a candidate agent is identified if the experimental set and the control set differ. ### Claim 11 (depends on 1) A method of identifying a pathway that is transcriptionally regulated by a protein of interest in a cell, the method comprising (i) identifying a set of biologically-active DNA-binding sites for a protein of interest in the genome of the cell according to the method of claim 1; and (ii) identifying at least two candidate genes likely to be regulated by binding of the protein of interest to the set of biologically-active DNA-binding sites identified in (i); wherein a pathway that is transcriptionally regulated by the protein of interest is identified if at least two candidate genes are members of the same pathway. ### Claim 12 (depends on 11) The method of claim 11, wherein the pathway is a gene expression pathway. ### Claim 13 (depends on 1) A method of identifying two sets of conditions in which a protein of interest differentially binds to the genome of a cell, the method comprising: (i) identifying, according to the method of claim 1, (1) a first set of biologically-active DNA-binding sites for the protein of interest in the genome of a cell, wherein the cell is exposed to a first set of conditions; and (2) identifying a second set of biologically-active DNA-binding sites for the protein of interest in the genome of a cell, wherein the cell is exposed to a second set of conditions; and (ii) identifying a difference in the first set of biologically-active DNA-binding sites relative to the second set of biologically-active DNA-binding sites, thereby identifying two sets of conditions in which a protein of interest differentially binds to the genome of a cell. ### Claim 14 (depends on 13) A method of identifying a property of a gene product of a gene of interest that correlates with the binding activity of a polypeptide encoded by the gene of interest to the genome of a cell, the method comprising (i) identifying two sets of conditions in which a protein of interest differentially binds to the genome of the cell according to the method of claim 13; (ii) determining a property of a gene product of the gene of interest in (a) a cell exposed to the first set of conditions; and in (b) a cell exposed to the second set of conditions; and (iii) determining if at least one property of the gene product differs in the two cells of step (ii), thereby identifying a property of said gene product that correlates with the binding activity of a said polypeptide to the genome of a cell. ### Claim 15 (depends on 10) A method of identifying a property of a gene product of a gene of interest that correlates with the binding activity of a polypeptide encoded by the gene of interest to the genome of a cell, the method comprising (i) identifying an agent which alters the set of biologically-active DNA-binding sites for a protein of interest in the genome of a cell according to the method of claim 10; (ii) determining a property of a gene product of the gene of interest in (a) a cell contacted with the agent; and in (b) a cell not contacted with the agent; and (iii) determining if at least one property of the gene product differs in the two cells of step (ii), thereby identifying a property that correlates with the binding activity of a gene of interest to the genome of a cell. ### Claim 16 (depends on 14) The method of claim 14, wherein the property is selected from the group consisting of a protein modification, expression level, enzymatic activity and intracellular localization. ### Claim 17 A method of identifying two cell genotypes in which a protein of interest differentially binds to the genome, the method comprising: (i) identifying a first set of biologically-active DNA-binding sites for the protein of interest in the genome of a cell of a first genotype; (ii) identifying a second set of biologically-active DNA-binding sites for the protein of interest in the genome of a cell of a second genotype; (iii) comparing the first set of biologically-active DNA-binding sites to the second set of biologically-active DNA-binding sites and determining if the two sets differ, thereby identifying two cell genotypes in which said protein of interest differentially binds to the genome.