Biologically-active DNA-binding sites and related methods

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.

2. The method of claim 1 , wherein the regions of genomic DNA comprise promoter regions.

3. 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.

4. The method of claim 1 , wherein the candidate DNA-binding site is less than 20 bp in length.

5. The method of claim 1 , wherein the DNA-sequence motif is degenerate in at least one position.

6. 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.

7. The method of claim 1 , wherein the set of biologically-active DNA-binding sites comprises one or more biologically-active DNA-binding sites.

8. 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.

9. The method of claim 1 , wherein the cell is an eukaryotic cell.

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.

11. 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.

12. The method of claim 11 , wherein the pathway is a gene expression pathway.

13. 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.

14. 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.

15. 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.

16. 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.

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.