Chromosome-wide analysis of protein-DNA interactions

• US 6,410,243 B1
• Filed: 09/01/2000
• Issued: 06/25/2002
• Est. Priority Date: 09/01/1999
• Status: Expired due to Term

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Ren, Bing, Robert, Francois, Wyrick, John, Young, Richard A.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Taylor, Janell E.
• Application Number:
  US09/654,409
• Time in Patent Office: 662 Days
• Field of Search: 435/6, 435/7.1, 435/7.8, 435/91.1, 435/91.2, 435/320.1, 435/440, 364/496, 364/497, 364/578, 702/127, 702/30, 514/12, 514/44, 530/350, 536/24.1
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2539/101 Subtraction analysis
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

The present invention relates to a method of identifying a region (one or more) of a genome of a cell to which a protein of interest binds. In the methods described herein, DNA binding protein of a cell is linked (e.g., covalently crosslinked) to genomic DNA of a cell. The genomic DNA to which the DNA binding protein is linked is removed and combined or contacted with DNA comprising a sequence complementary to genomic DNA of the cell under conditions in which hybridization between the identified genomic DNA and the sequence complementary to genomic DNA occurs. Region(s) of hybridization are region(s) of the genome of the cell to which the protein of binds.

Claims

1. A method of identifying a region of a genome of a living cell to which a protein of interest binds, comprising the steps of:

a) crosslinking DNA binding protein in the living cell to genomic DNA of the living cell, thereby producing DNA binding protein crosslinked to genomic DNA;

b) generating DNA fragments of the genomic DNA crosslinked to DNA binding protein in a), thereby producing a mixture comprising DNA fragments to which DNA binding protein is bound;

c) removing a DNA fragment to which the protein of interest is bound from the mixture produced in b);

d) separating the DNA fragment identified in c) from the protein of interest;

e) amplifying the DNA fragment of d);

f) combining the DNA fragment of e) with DNA comprising a sequence complementary to genomic DNA of the cell, under conditions in which hybridization between the DNA fragment and a region of the sequence complementary to genomic DNA occurs; and

g) identifying the region of the sequence complementary to genomic DNA of f) to which the DNA fragment hybridzes,

whereby the region identified in g) is the region of the genome in the cell to which the protein of interest binds.

2. The method of claim 1 wherein the cell is a eukaryotic cell.

3. The method of claim 1 wherein the protein of interest is selected from the group consisting of:

a transcription factor and an oncogene.

4. The method of claim 1 wherein the DNA binding protein of the cell is crosslinked to the genome of the cell using formaldehyde.

5. The method of claim 1 wherein the DNA fragment of c) to which is bound the protein of interest is identified using an antibody which binds to the protein of interest.

6. The method of claim 1 wherein the DNA fragment of e) is amplified using ligation-mediated polymerase chain reaction.

7. The method of claim 1 wherein the complement sequence of the genome of f) is a DNA microarray.

8. The method of claim 1 further comprising:

h) comparing the region identified in g) with a control.

9. A method of identifying a region of a genome of a living cell to which a protein of interest binds, comprising the steps of:

a) formaldehyde crosslinking DNA binding protein in the living cell to genomic DNA of the living cell, thereby producing DNA binding protein crosslinked to genomic DNA;

b) generating DNA fragments of the genomic DNA crosslinked to DNA binding protein in a), thereby producing DNA fragments to which DNA binding protein is bound;

c) immunoprecipitating the DNA fragment produced in b) to which the protein of interest is bound using an antibody that specifically binds the protein of interest;

d) separating the DNA fragment identified in c) from the protein of interest;

e) amplifying the DNA fragment of d) using ligation-mediated polymerase chain reaction;

f) fluorescently labeling the DNA fragment of e);

g) combining the labeled DNA fragment of e) with a DNA microarray comprising a sequence complementary to genomic DNA of the cell, under conditions in which hybridization between the DNA fragment and a region of the sequence complementary to genomic DNA occurs;

h) identifying the region of the sequence complementary to genomic DNA to which the DNA fragment hybridizes by measuring the fluorescence intensity; and

i) comparing the fluorescence intensity measured in h) to the fluorescence intensity of a control,

whereby fluorescence intensity in a region of the genome which is greater than the fluorescence intensity of the control in the region indicates the region of the genome in the cell to which the protein of interest binds.

10. A method of determining a function of a protein of interest which binds to a genome of a living cell, comprising the steps of:

a) crosslinking DNA binding protein in the living cell to genomic DNA of the living cell, thereby producing DNA binding protein crosslinked to genomic DNA;

b) generating DNA fragments of the genomic DNA crosslinked to DNA binding protein in a), thereby producing a mixture comprising DNA fragments to which DNA binding protein is bound;

c) removing the DNA fragment to which the protein of interest is bound from the mixture produced in b);

d) separating the DNA fragment identified in c) from the protein of interest;

e) amplifying the DNA fragment of d);

f) combining the DNA fragment of e) with DNA comprising a sequence complementary to genomic DNA of the cell, under conditions in which hybridization between the DNA fragment and a region of the sequence complementary to genomic DNA occurs;

g) identifying the region of the sequence complementary to genomic DNA of f) to which the DNA fragment hybridzes; and

h) characterizing the region identified in g),

wherein the characteristics of the region of h) indicates a function of the protein of interest which binds to the genome of the cell.

11. A method of determining whether a protein of interest which binds to the genome of a living cell functions as a transcription factor, comprising the steps of:

a) crosslinking DNA binding protein in the living cell to the genomic DNA of the living cell, thereby producing DNA binding protein crosslinked to genomic DNA;

b) generating DNA fragments of the genomic DNA crosslinked to DNA binding protein in a), thereby producing a mixture comprising DNA fragments to which DNA binding protein is bound;

c) removing the DNA fragment to which the protein of interest is bound from the mixture produced in b);

d) separating the DNA fragment identified in c) from the protein of interest;

e) amplifying the DNA fragment of d);

f) combining the DNA fragment of e) with DNA comprising a sequence complementary to genomic DNA of the cell, under conditions in which hybridization between the DNA fragment and a region of the sequence complementary to genomic DNA occurs; and

g) identifying the region of the sequence complementary to genomic DNA of f) to which the DNA fragment hybridzes,

wherein if the region of the sequence complementary to genomic DNA of g) is a regulatory region, then the protein of interest is a transcription factor.