Tethered Conformation Capture

• US 20110287947A1
• Filed: 05/18/2010
• Published: 11/24/2011
• Est. Priority Date: 05/18/2010
• Status: Active Application

• Current Assignee: University of Southern California
• Original Assignee: University of Southern California
• Inventors: Chen, Lin, Kalhor, Reza
• Application Number:
  US12/782,616
• Publication Number:
  US 20110287947A1
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

Disclosed are methods and systems for determining the three-dimensional structure of chromatin in eukaryotic cells. More specifically, disclosed are methods and systems for obtaining chromatin structural information by surface immobilization, i.e tethering crosslinked protein:DNA complexes and/or ligated DNA complexes to media such as beads, gels, and or matrices during the conformation capture assay. In general, the method includes contacting a cell with a cross-linking reagent to cross-link DNA and protein in the cell; lysing the cell, producing cross-linked protein:DNA complexes by cutting the chromatin using a chemical, physical or enzymatic method, substantially immobilizing the cross-linked protein:DNA complexes, ligating the cross-linked protein:DNA complexes intramolecularly such that the ligated protein:DNA complexes represent structural organization of the chromatin; characterizing the ligated DNA by sequencing or other methods; and identifying any structural organization of the chromatin. The structural organization preferably includes information relating to interacting loci of the chromatin.

Claims

1. A method of determining the three-dimensional arrangement of chromatin in a cell, comprising:

Contacting a cell with a cross-linking reagent to cross-link DNA and protein in the cell such that the structural organization of the chromatin or other protein;

DNA complexes is preserved;

lysing the cell;

producing cross-linked protein;

DNA complexes by cutting the chromatin using a chemical, physical or enzymatic method;

substantially immobilizing the cross-linked protein;

DNA complexes;

connecting the cross-linked protein;

DNA complexes intramolecularly such that the connected protein;

DNA complexes represent structural organization of the chromatin;

characterizing the DNA of the protein;

DNA complexes by sequencing or other methods; and

identifying any structural organization of the chromatin.

2. The method of claim 1, further comprising, denaturing the chromatin.

3. The method of claim 1, wherein the protein:

DNA complexes are cut by restriction digestion;

4. The method of claim 1, wherein the protein:

DNA complexes are substantially immobilized by tethering the protein;

DNA complexes to one or more media.

5. The method of claim 4, where the media is selected from the group consisting of beads, chip, colloids, matrix, and gel.

6. The method of claim 1, wherein the protein:

DNA complexes are substantially immobilized by a covalent or non-covalent (streptavidin-biotin bonding is not covalent but is very strong) bond between the side-chains of the amino acids of the proteins of chromatin and a reactive chemical group on the surface or inside of one or more media selected from the group consisting of beads, chip, colloids, matrix, and gel.

7. The method of claim 1, wherein the protein:

DNA complexes are substantially immobilized through modifying the proteins of the chromatin so to anchor the modified protein;

DNA complexes to the surface or inside of one or more media selected from the group consisting of beads, colloids, matrix, and gel.

8. The method of claim 1, wherein the protein:

DNA complexes are substantially immobilized by biotinylating the proteins of the chromatin so to anchor the biotinylated protein;

DNA complexes to a biotin binding surface

9. The method of claim 8, wherein the protein:

DNA complexes are substantially immobilized by biotinylating the thiol groups of the proteins of the chromatin so to anchor the biotinylated protein;

DNA complexes to a biotin binding surface.

10. The method of claim 8, wherein the protein:

DNA complexes are substantially immobilized by biotinylating the cysteine residues of the proteins of the chromatin so to anchor the biotinylated protein;

DNA complexes to a biotin binding surface.

11. The method of claim 8, wherein the protein:

DNA complexes are substantially immobilized by biotinylating the N-termini and lysine residues of the proteins of the chromatin so to anchor the biotinylated protein;

DNA complexes to a biotin binding surface.

12. The method of claim 8, wherein the protein:

DNA complexes are substantially immobilized by biotinylating the glutamate or aspartate residues of the proteins of the chromatin so to anchor the biotinylated protein;

DNA complexes to a biotin binding surface.

13. The method of claim 9, wherein thiol groups are added to the proteins of chromatin through a chemical reagent.

14. The method of claim 12, wherein thiol groups are added to the proteins of chromatin through reacting the proteins of chromatin with an aminothiol and a crosslinking reagent.

15. The method of claim 14, wherein thiol groups are added to the lysines of the proteins of chromatin by reacting them with a cross-linking reagent and cysteamine.

16. The method of claim 14 wherein thiol groups are added to the lysines of the proteins of chromatin by reacting them with formaldehyde and cysteamine.

17. The method of claim 8, wherein the substrate is streptavidin-coated chips or magnetic beads.

18. The method of claim 1, wherein the cells are denatured with Sodium Dodecyl Sulfate.

19. The method of claim 4, wherein the chromatin is digested with a restriction enzyme that produces a 5′

overhang of at least two non-identical bases.

20. The method of claim 19, wherein the 5′

overhang is blunted.

21. The method of claim 20, wherein the connection of the protein:

DNA complexes intramolecularly is done by blunt-ended ligation using DNA ligase.

22. The method of claim 20, wherein blunting is done with nucleotide analogues.

23. The method of claim 20, wherein a biotinylated nucleotide is used for blunting.

24. The method of claim 20, wherein a nuclease resistant nucleotide analogue is used in blunting.

25. The method of claim 20, wherein a 2-deoxy-nucleoside-5′

-(alpha-thio)-triphosphate is used in blunting.

26. The method of claim 1, wherein after the connecting step, protein:

DNA complexes that have not undergone connection are removed.

27. The method of claim 1, wherein the sequencing is massively parallel or ultrahigh-throughput sequencing.

28. The method of claim 1, wherein the structural organization is interacting loci in the nucleus of the cell.

37. The method of claim 1, wherein protein:

DNA complexes are substantially immobilized by a covalent or non-covalent bond between the DNA of chromatin and a reactive chemical group on the surface or inside of one or more media selected from the group consisting of beads, chip, colloids, matrix, and gel.

38. The method of claim 1, wherein connection of the DNA of the crosslinked Protein:

DNA complexes intramolecularly is done by ligation using DNA ligase.

39. The method of claim 1, wherein the protein:

DNA complexes are substantially immobilized relative to each other.

29. An improved method for determination of the structural organization of chromatin having less noise and higher resolution, said improved method comprising:

providing chromatin having DNA cross-linked to protein such that the structural organization of the chromatin is preserved;

producing cross-linked protein;

DNA complexes by cutting the chromatin with a restriction enzyme;

substantially immobilizing the cross-linked protein;

DNA complexes on a surface and removing non-crosslinked DNA generated by digesting the chromatin;

connecting the cross-linked protein;

DNA complexes intramolecularly and removing DNA molecules without a connection;

30. The method of claim 29, further comprising sequencing the DNA of the connected protein:

DNA complexes.

31. The method of claim 29, wherein the chromatin is digested with a restriction enzyme that produces a 5′

overhang of at least two non-identical bases

32. The method of claim 29, wherein the protein:

DNA complexes are substantially immobilized by tethering the protein;

DNA complexes on the surface of one or more media selected from the group consisting of beads, matrix, and gel. (see claim 3)

33. The method of claim 29, wherein the non-crosslinked DNA generated by digesting the chromatin is removed by washing.

34. The method of claim 29, wherein the immobilizing the cross-linked protein:

DNA complexes reduces the frequency of formation of inter-molecular connections.

35. The method of claim 29, wherein DNA molecules without a connection junction are removed by an exonuclease.

36. A kit for determining the three-dimensional arrangement of chromatin in a cell, comprising:

A cross-linking reagent for cross-linking the DNA and proteins of the chromatin;

a lysing reagent;

a denaturing reagent;

a restriction enzyme for producing cross-linked protein;

DNA complexes;

(or any other chemical, physical, or enzymatic method for cutting DNA),

a substrate for substantially immobilizing the cross-linked protein;

DNA complexes; and

one or more connecting reagents for connecting the protein;

DNA complexes intramolecularly.