Genome wide location and function of DNA binding proteins
View Patent ↗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. A method of identifying a set of genes where cell cycle regulator binding correlates with gene expression and of identifying genomic targets of cell cycle transcription activators in living cells is also encompassed.
1. A method comprising:
a) crosslinking DNA binding protein of a cell to genomic DNA of the cell, thereby producing crosslinked genomic DNA;
b) sonicating said crosslinked genomic DNA to produce a population of genomic fragments;
c) separating DNA fragments to which a protein of interest is bound from said population of genomic fragments; and
d) amplifying said DNA fragments by:
i. blunting said DNA fragments to produce blunt ends;
ii. ligating adaptors to said blunt ends; and
iii. amplifying said DNA fragments using a primer that binds to said adaptors;
to produce an amplified population of nucleic acids.
2. The method of claim 1 , further comprising analyzing said amplified population of nucleic acids to identify a site to which said protein of interest binds.
3. The method of claim 1 , further comprising labeling said amplified population of nucleic acids, either during or after said amplifying step d) to produce labeled DNA fragments.
4. The method of claim 1 , further comprising: e) contacting said labeled DNA fragments with a nucleic acid array under conditions in which nucleic acid hybridization occurs.
5. The method of claim 4 , further comprising reading said nucleic acid array to determine a binding pattern.
6. The method of claim 3 , wherein said amplified population of DNA fragments are labeled during said amplifying.
7. The method of claim 3 , wherein said amplified population of DNA fragments are labeled after said amplifying.
8. The method of claim 1 , wherein said blunting employs T4 DNA polymerase.
9. The method of claim 1 , wherein said genomic DNA is genomic DNA of a mammalian cell.
10. The method of claim 1 , wherein said crosslinking step a) comprises crosslinking DNA binding protein of a living cell to genomic DNA of the living cell.
11. The method of claim 1 , wherein said crosslinking step a) does not comprise crosslinking DNA binding protein of a living cell to genomic DNA of the living cell.