DNA barcoding of designer mononucleosome and chromatin array libraries for the profiling of chromatin readers, writers, erasers, and modulators thereof

Patent metadata

Field	Value
Publication number	US-10087485-B2
Application number	US-201314405303-A
Country	US
Kind code	B2
Filing date	Jun 6, 2013
Priority date	Jun 6, 2012
Publication date	Oct 2, 2018
Grant date	Oct 2, 2018

Abstract

Compositions and methods are provided for DNA barcoding of designer mononucleosome and polynucleosome (chromatin array) libraries for use, for example, for the profiling of chromatin readers, writers, erasers, and modulators thereof.

First claim

We claim:

1. A method for identifying one or more modifications associated with one or more chromatin interactors or modifiers, comprising
dividing a library of synthetic mononucleosomes into one or more sublibraries;
incubating the one or more chromatin interactors and/or modifiers with the one or more sublibraries;
isolating one or more unique DNA barcodes from one or more modified mononucleosomes associated with one or more chromatin interactors or modifiers;
multiplexing the isolated DNA barcodes by furnishing each isolated DNA sequence with an additional DNA barcode corresponding to the one or more chromatin interactors or modifiers via polymerase chain reaction; and
identifying the one or more modifications associated with each of the one or more chromatin interactors or modifiers through barcode detection via polymerase chain reaction or DNA sequencing,
wherein the library of synthetic mononucleosomes comprises two or more synthetic mononucleosomes,
wherein each of the two or more synthetic mononucleosomes comprises a complex of:
(a) a protein octamer, comprising 2 copies of each of histones H2A, H2B, H3, and H4 and optionally a linker histone, wherein at least one of the histones is unmodified and/or wherein at least one of the histones is modified, to form a pattern of histone modifications, and
(b) a nucleosomal DNA molecule, comprising
(i) a nucleosome positioning sequence (NPS), wherein the nucleosome positioning sequence is a nucleotide sequence that forms a stable protein association with one or more of the histones,
(ii) one or more unique DNA barcode sequence(s) located at defined position(s) in the nucleosomal DNA, and
(iii) one or more DNA extensions, on the 5′- and/or 3′-end of the NPS and/or within the NPS, wherein the DNA extensions are unmodified and/or wherein at least one nucleotide in the DNA extensions is modified, to form a pattern of DNA modifications, and optionally

(c) one or more non-histone chromatin-associated proteins,
wherein for each of the two or more synthetic mononucleosomes the pattern of histone modifications and the pattern of DNA modifications form a pattern of mononucleosome modifications, and
wherein for each of the two or more synthetic mononucleosomes the sequence and position of the one or more unique DNA barcode sequence(s) in the nucleosomal DNA is indicative of the pattern of mononucleosome modifications in the synthetic mononucleosome.

2. A method for assembling a synthetic mononucleosome, comprising combining one or more modified histone proteins, complementary unmodified histones and one or more barcoded nucleosomal DNA with an affinity-tagged buffer DNA, in a predetermined ratio,
wherein the synthetic mononucleosome comprises a complex of:
(a) a protein octamer, comprising 2 copies of each of histones H2A, H2B, H3, and H4 and optionally a linker histone, wherein at least one of the histones is unmodified and/or wherein at least one of the histones is modified, to form a pattern of histone modifications, and
(b) a nucleosomal DNA molecule, comprising
(i) a nucleosome positioning sequence (NPS), wherein the nucleosome positioning sequence is a nucleotide sequence that forms a stable protein association with one or more of the histones,
(ii) one or more DNA barcode sequence(s) located at defined position(s) in the nucleosomal DNA, and
(iii) one or more DNA extensions, on the 5′- and/or 3′-end of the NPS and/or within the NPS, wherein the DNA extensions are unmodified and/or wherein at least one nucleotide in the DNA extensions is modified, to form a pattern of DNA modifications, and optionally

(c) one or more non-histone chromatin-associated proteins,
wherein for the synthetic mononucleosome the pattern of histone modifications and the pattern of DNA modifications form a pattern of nucleosome modifications, and
wherein for the synthetic mononucleosome the sequence and position of the one or more DNA barcode sequence(s) in the nucleosomal DNA is indicative of the pattern of nucleosome modifications in the synthetic mononucleosome.

3. A method for determining the specificity of the recognition patterns and affinities of one or more chromatin readers which are of recombinant origin and/or extracted from nuclear cell extracts, or for determining the specificity and chromatin modification cross-talks of one or more chromatin modifiers which are of recombinant origin and/or extracted from nuclear cell extracts of one or more cell lines, comprising
(i) contacting one or more synthetic mononucleosomes with the one or more chromatin readers or the one or more chromatin modifiers,
(iii) isolating bound and/or modified synthetic mononucleosomes, and
(iii) identifying and/or quantitating the bound or modified synthetic mononucleosomes and any added marks or removed marks,
wherein the identifying and/or quantitating the bound or modified synthetic mononucleosomes comprises the use of quantitative polymerase chain reaction or next generation sequencing,
wherein each of the one or more synthetic mononucleosomes comprises a complex of:
(a) a protein octamer, comprising 2 copies of each of histones H2A, H2B, H3, and H4 and optionally a linker histone, wherein at least one of the histones is unmodified and/or wherein at least one of the histones is modified to form a pattern of histone modifications, and
(b) a nucleosomal DNA molecule, comprising
(i) a nucleosome positioning sequence (NPS), wherein the nucleosome positioning sequence is a nucleotide sequence that forms a stable protein association with one or more of the histones,
(ii) one or more DNA barcode sequence(s) located at defined position(s) in the nucleosomal DNA, and
(iii) one or more DNA extension(s), on the 5′- and/or 3′-end of the NPS and/or within the NPS, wherein the DNA extension(s) are unmodified and/or wherein at least one nucleotide in the DNA extension(s) is modified, to form a pattern of DNA modifications, and optionally

(c) one or more non-histone chromatin-associated protein(s),
wherein for each of the one or more synthetic mononucleosomes the pattern of histone modifications and the pattern of DNA modifications form a pattern of nucleosome modifications, and
wherein for each of the one or more synthetic mononucleosomes the sequence and position of the one or more DNA barcode sequence(s) in the nucleosomal DNA is indicative of the pattern of nucleosome modifications in the mononucleosome.

4. The method of claim 2 , wherein the buffer DNA is MMTV.

5. The method of claim 1 , wherein each of the two or more synthetic mononucleosomes comprises a linker histone.

6. The method of claim 1 , wherein at least one of the two or more synthetic mononucleosomes comprises a linker histone.

7. The method of claim 1 , wherein each of the two or more synthetic mononucleosomes comprises one or more non-histone chromatin-associated proteins.

8. The method of claim 1 , wherein at least one of the two or more synthetic mononucleosomes comprises one or more non-histone chromatin-associated proteins.

9. The method of claim 2 , wherein the synthetic mononucleosome comprises a linker histone.

10. The method of claim 2 , wherein the synthetic mononucleosome comprises one or more non-histone chromatin-associated proteins.

11. The method of claim 3 , wherein each of the one or more synthetic mononucleosomes comprises a linker histone.

12. The method of claim 3 , wherein at least one of the one or more synthetic mononucleosomes comprises a linker histone.

13. The method of claim 3 , wherein each of the one or more synthetic mononucleosomes comprises one or more non-histone chromatin-associated protein(s).

14.

Assignees

• Univ Princeton

Inventors

• Muir Tom W
• Nguyen Uyen T T
• Mueller Manuel M

Classifications

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C40B30/08

  by measuring catalytic activity · CPC title

• G01N33/6875

  Nucleoproteins · CPC title

• C12Q1/48

  involving transferase · CPC title