Super-enhancers and methods of use thereof
The present invention relates in some aspects to super-enhancers and related compositions, methods, and agents that are useful for modulating expression of cell type-specific genes that are required for maintenance of cell identity (e.g., embryonic stem cell identity) or maintenance of a disease state (e.g., cancer).
1. A method of identifying a super-enhancer in a sample comprising chromatin, comprising: obtaining chromatin from a cell wherein said chromatin has been cross-linked such that chromosomal nucleic acid in the chromatin is cross-linked to a component selected from the group consisting of BRD4, a Mediator component and H3K27Ac with which the chromosomal nucleic acid is associated to form a cross-linked complex; contacting said cross-linked complex with a ligand having affinity for the component, said ligand selected from the group consisting of an antibody to BRD4, an antibody to a Mediator component, and an antibody to H3K27Ac, to form a complex between the cross-linked complex and the ligand;
determining an amount of ligand bound to each enhancer in the cross-linked complex in the cell; and
utilizing the determined amount of ligand bound to each enhancer to identify a super-enhancer,
wherein the identified super-enhancer is bound to at least 10-fold more ligand than the median amount of ligand bound to enhancer within the cell.
2. The method of claim 1 , wherein the component is BRD4 and the ligand is an antibody to BRD4.
3. The method of claim 1 , wherein the component is a Mediator component and the ligand is an antibody to a Mediator component.
4. The method of claim 1 , wherein the component is H3K27Ac and the ligand is an antibody to H3K27Ac.
5. The method of claim 1 , comprising fragmenting the chromosomal nucleic acid of the cross-linked complex prior to the step of determining the amount of ligand bound.
6. The method of claim 5 , comprising fragmenting the chromosomal nucleic acid after forming the complex between the cross-linked complex and the ligand.
7. The method of claim 1 , wherein the cross-linking of the cross-linked complex comprises covalent cross-linking.
8. The method of claim 1 , further comprising identifying a gene associated with said super-enhancer.
9. The method of claim 8 , wherein said associated gene is identified by proximity to the super-enhancer.
10. The method of claim 8 , wherein said associated gene is identified using high throughput chromatin conformation capture data.
11. The method of claim 8 , wherein the gene associated with said super-enhancer is an endogenous gene within the cell.
12. The method of claim 1 , wherein the component is endogenous to the cell.
13. The method of claim 1 , wherein determining an amount of ligand bound to each enhancer in the cell is achieved by ChiP-Seq.
14. The method of claim 1 , wherein at least a portion of the chromosomal nucleic acid is sequenced after contacting the cross-linked complex with the ligand.
15. The method of claim 1 , wherein utilizing the determined amount of ligand bound to each enhancer to identify a super-enhancer comprises:
identifying a portion of the chromosomal nucleic acid that is bound to at least 10-fold more ligand than the median amount of ligand bound to enhancers within the cell.
16. The method of claim 1 , wherein utilizing the determined amount of ligand bound to each enhancer to identify a super-enhancer comprises:
determining the median amount of ligand bound to enhancers within the cell;
identifying a portion of the chromosomal nucleic acid that is bound to at least 10-fold more ligand than the median as a super-enhancer.
17. A method of identifying a super-enhancer in a sample comprising chromatin, comprising:
obtaining chromatin from a cell wherein said chromatin has been cross-linked such that chromosomal nucleic acid in the chromatin is cross-linked to a component selected from the group consisting of BRD4, a Mediator component and H3K27Ac with which the chromosomal nucleic acid is associated to form a cross-linked complex;
contacting said cross-linked complex with a ligand having affinity for the component, said ligand selected from the group consisting of an antibody to BRD4, an antibody to a Mediator component, and an antibody to H3K27Ac, to form complex between the cross-linked complex and the ligand;
determining an amount of ligand bound to each enhancer in the cross-linked complex in the cell; and
utilizing the determined amount of ligand to each enhancer to identify a super-enhancer, wherein the identified super-enhancer has an amount of ligand bound that is above the point where the slope of the tangent is 1 in a rank-ordered graph of the amount of ligand bound to each of the enhancers in the cell.
18. The method of claim 17 , wherein utilizing the determined amount of ligand bound to each enhancer to identify a super-enhancer comprises:
rank ordering the enhancers according to the amount of ligand bound;
identifying, with respect to the amount of ligand bound, the point where the slope of the tangent is 1 which would occur if said rank order were graphed; and
identifying any enhancer that falls above that point as a super-enhancer.
19. The method of claim 17 , wherein utilizing the determined amount of ligand bound to each enhancer to identify a super-enhancer comprises:
graphing, in rank order of the amount of ligand bound, the enhancer and the amount of ligand bound to said enhancer;
identifying the point of the graph where the slope of the tangent is 1; and
identifying any enhancer that falls above that point as a super-enhancer.
20. The method of claim 17 , wherein the component is BRD4 and the ligand is an antibody to BRD4.
21. The method of claim 17 , wherein the component is a Mediator component and the ligand is an antibody to the Mediator component.
22. The method of claim 17 , wherein the component is H3K27Ac and the ligand is an antibody to H3K27Ac.
23. The method of claim 17 , comprising fragmenting the chromosomal nucleic acid of the cross-linked complex prior to the step of determining the amount of ligand bound.
24. The method of claim 23 , comprising fragmenting the chromosomal nucleic acid after forming the complex between the cross-linked complex and the ligand.
25. The method of claim 17 , wherein the cross-linking of the cross-linked complex comprises covalent cross-linking.
26. The method of claim 17 , further comprising identifying a gene associated with said super-enhancer.
27. The method of claim 26 , wherein said associated gene is identified by proximity to the super-enhancer.
28. The method of claim 17 , wherein the component is endogenous to the cell.
29. The method of claim 17 , wherein determining an amount of ligand bound to each enhancer in the cell is achieved by ChiP-Seq.
30. The method of claim 17 , wherein at least a portion of the chromosomal nucleic acid is sequenced after contacting the cross-linked complex with the ligand.