Assays for nucleosome remodeling activity

1. A functionalized recombinant mononucleosome comprising:

(a) a histone octamer comprising recombinant historic H2A, H2B, H3 and H4 proteins; and

(b) a DNA template comprising a nucleosome positioning sequence (NPS) that effectively positions the histone octamer and a signal site;

wherein the signal site is a protein interaction site that is shielded in the functionalized recombinant mononucleosome;

wherein the protein interaction site is a restriction enzyme site or other nuclease recognition site; and

wherein the protein interaction site is not a transcription factor binding site.

2. The functionalized recombinant mononucleosome of claim 1 , wherein one or more of the histories is a synthetic or naturally-occurring histone variant.

3. The functionalized recombinant mononucleosome of claim 1 , wherein one or more of the histories comprises one or more post-translational modification (PTM).

4. The functionalized recombinant mononucleosome of claim 2 , wherein one or more of the histones or histone variants comprises one or more PTMs.

5. The functionalized recombinant mononucleosome of claim 1 , wherein the DNA template comprises an acceptor sequence located 5′ or 3′ of the NPS.

6. The functionalized recombinant mononucleosome of claim 5 , wherein the acceptor sequence is 10-1000 basepairs.

7. The functionalized recombinant mononucleosome of claim 1 , wherein the protein interaction site is a chaperone binding site.

8. The functionalized recombinant mononucleosome of claim 1 , wherein the mononucleosome comprises a functional tag.

9. A method for quantifying enzymatic activity of a remodeling enzyme, comprising:

(a) contacting the functionalized recombinant mononucleosome of claim 1 with the remodeling enzyme and a restriction enzyme or other nuclease that interacts with the signal site; and

(b) measuring a signal from the signal site;

thereby quantifying the enzymatic activity of the remodeling enzyme.

10. The method of claim 9 , further comprising measuring a signal from the signal site of a negative control.

11. The method of claim 9 , wherein the functionalized recombinant mononucleosome is immobilized on a surface.

12. The method of claim 11 , wherein the DNA template comprises biotin and the surface comprises streptavidin.

13. The method of claim 9 , which is a fluorescence polarization (FP) assay,

wherein the DNA template comprises a restriction enzyme site or other nuclease recognition site and a fluorophore;

the functionalized recombinant mononucleosome is contacted with the remodeling enzyme and the restriction enzyme or other nuclease;

and the enzymatic activity is quantified by measuring relative changes in anisotropy.

14. The method of claim 9 , which is a time-resolved fluorescence resonance energy transfer (TR-FRET) assay,

wherein the DNA template comprises a fluorophore;

the mononucleosome comprises a histone conjugated to a fluorophore or quencher molecule;

and the enzymatic activity is quantified by measuring changes in FRET.

15. The method of claim 9 , wherein the remodeling enzyme is selected from Switch/Sugar Non-fermentable (SWI/SNF), Imitation Switch (ISWI), Nucleosome Remodeling Deacetylase (NuRD)/Mi-2/Chromodomain Helicase DNA Binding (CDH), inositol autotrophy 80 (INO80), Swi2/Snf2 related 1 (SWR1), and any combination thereof.

16. A method for identifying a compound that modulates remodeling enzyme activity, comprising:

(a) contacting the functionalized recombinant mononucleosome of claim 1 with the remodeling enzyme and a restriction enzyme or other nuclease that interacts with the signal site; in the presence and absence of the compound; and

(b) measuring a signal from the signal site in the presence and absence of the compound;

wherein a difference in the signal in the presence and absence of the compound identifies the compound as one that modulates the remodeling enzyme activity.

17. The method of claim 16 , wherein the functionalized recombinant mononucleosome is immobilized on a surface.

18. The method of claim 17 , wherein the DNA template comprises biotin and the surface comprises streptavidin.

19. The method of claim 16 which is a FP assay,

wherein the DNA template comprises a restriction enzyme site or other nuclease recognition site and a fluorophore;

the functionalized recombinant mononucleosome is contacted with the remodeling enzyme and the restriction enzyme or other nuclease in the presence and absence of the compound;

and the enzymatic activity is quantified by measuring relative changes in anisotropy.

20. The method of claim 16 which is a TR-FRET assay,

wherein the DNA template comprises a fluorophore;

the mononucleosome comprises a histone conjugated to a fluorophore or quencher molecule;

and the enzymatic activity is quantified by measuring changes in FRET.

21. The method of claim 16 , wherein the remodeling enzyme is selected from Switch Sugar Non-fermentable (SWI/SNF), Imitation Switch (ISWI), Nucleosome Remodeling Deacetylase (NuRD)/Mi-2/Chromodomain Helicase DNA Binding (CDH), inositol auxotrophy 80 (INO80), Swi2/Snf2 related 1 (SWR1), and any combination thereof.