EpiDyne-FRET, recombinant nucleosome substrates for chromatin remodeling studies

Chromatin remodeling, or the repositioning of nucleosomes, regulates DNA access and thus gene expression and genome repair. Many ATP-dependent remodeling enzymes (and their multi-subunit complexes) are associated with human disease but are challenging targets to study due to the requirement for nucleosome-based substrates. EpiCypher has addressed this need by developing the EpiDyne™ platform of fully recombinant nucleosome remodeling substrates to monitor nucleosome repositioning along DNA. Here, we demonstrate the utility of EpiDyne-FRET in a homogeneous nucleosome remodeling assay using a Fluorescent Resonance Energy Transfer (FRET) readout.

Figure 1. EpiDyne-FRET Nucleosome Remodeling Substrates consist of a Cy5-labeled human histone octamer (H2A T120C-Cy5) wrapped by 5’ Cy3-labeled DNA (217bp) comprising a terminally nucleosome positioning sequence (147bp Widom 601 [REF]) adjacent to a TGGA-repeat region refractory to nucleosome assembly [REF]. In its assembled starting state, Cy3-Cy5 FRET is at a maximum. Subsequent to the action of an ATP-dependent remodeler (e.g. RSC or another SWI/SNF ATPase), FRET signal is diminished as the Cy5-labeled octamer moves away from the Cy3-labeled 5’ end of the DNA. This is a one-step no-wash method immediately compatible with HTS applications.

Chromatin Remodeling Enzymes As Therapeutic Targets

Aberrant nucleosome organization can severely disrupt gene expression, DNA repair and cellular differentiation and plays a major role in human disorders, including cancer, inflammation, autoimmunity, schizophrenia, cardiovascular disease, and intellectual disability. Remarkably, nearly 20% of all cancers contain mutations in subunits from the SWI/SNF family of ATP-dependent chromatin remodeling complexes. These enzyme complexes regulate local genome access by ‘pumping’ the DNA around histone octamers, thus ‘sliding’ nucleosomes along DNA [1]. Recurrent somatic mutations in SWI/SNF subunits are observed in multiple cancers, supporting a driver role in tumorigenesis [2]. The mutated remodeling proteins are attractive therapeutic targets, since further compromising their ATPase activity promotes cancer cell death but spares normal cells [2, 3]. This phenomenon is known as synthetic lethality and identifying inhibitors to exploit it may lead to drugs with cancer specificity [4, 5].

Chromatin Remodeling Assays Using Recombinant Mononucleosome Substrates

EpiCypher has developed EpiDyne-FRET to study the function of chromatin remodeling enzyme complexes. The nucleosome consists of a core histone octamer wrapped in ~147 bp DNA and represents the fundamental repeating unit of chromatin. EpiDyne-FRET is comprised of a terminally positioned histone octamer (H2A-T120C*Cy5) wrapped in 5’ Cy3-labelled DNA [8] (Figure 1). In its assembled start position, Cy3-Cy5 FRET is at maximum, with nucleosome remodeling detected by loss of the Cy5 signal as the histone octamer is moved towards the template DNA 3’ end or ejected.

Figure 2. RSC/ATP-dependent nucleosome remodeling reaction. EpiDyne-FRET nucleosomes (20 nM) were incubated with RSC chromatin remodeler in the presence or absence of 2 mM ATP. Upon the addition of ATP, reactions were read in an Envision Multi-label plate reader (Perkin Elmer) at times indicated. Data is presented as the mean±SD of the Cy3/Cy5 ratio (N=6).     

Figure 3. ATPγS titration with fixed ATP. EpiDyne-FRET nucleosomes (20 nM) were incubated with RSC in the presence of fixed 2 mM ATP and a titration of non-hydrolyzable ATPγS. Upon the addition of ATP, reactions were immediately read in an Envision Multi-label plate reader (Perkin Elmer). Data is presented as the mean of the Cy3/Cy5 ratio (N=2).   

Figure 4. IC50 calculation for ATPγS. The data from figure 3 were analyzed at the 20-minute time point to determine the IC50 value for ATPγS (2.29 mM) [is this comparable to literature]. Data is presented as the mean of the Cy3/Cy5 ratio vs the ATPγS concentration.  

REAGENTS AND MATERIALS

- Yeast RSC [9] (substrate also compatible with dACF [REF], dNoRC [REF] and human SMARCA2/4 (BRG1/BRM); not shown)

-  EpiDyne-FRET nucleosome remodeling substrate (EpiCypher Cat. No. 16-4201)

-  Assay Buffer (20 mM Tris pH 7.5, 50 mM KCl, 3 mM MgCl2 and 0.1mg/mL BSA (Sigma Cat. No. A3059)

-  ATP (Invitrogen Cat. No. PV3227)

- Corning 3820 384-well assay plate (Fisher Cat. No. 07-200-891)

-  16 channel multipipeter for 384-well plates

-  384-well Fluorescence microplate reader capable of Cy3/Cy5-FRET detection (Envision, Perkin Elmer)

PROCEDURE

(1)    Determine the amount of RSC (10 nM final), EpiDyne-FRET (20 nM final), and ATP (2 mM final) needed.based on the intended time points / replicates (standard is triplicate),

(2)     Prepare reactant components (room temp))

   i.  2x [Enzyme/Substrate] solution (20 nM RSC + 40 nM EpiDyne-FRET substrate in 2x assay buffer)

  ii.  2x ATP +/- Inhibitor solution(s) (4 mM ATP in ddH2O + Inhibitors (e.g. ATPγS) as required)

(3)     Add 5 µL of 2x enzyme/substrate solution to the microplate

(4)      Initiate reactions by adding 5 µL of the 2x ATP +/- inhibitor solution

(5)     At appropriate timepoints read in a 384-well plate reader capable of Cy3 (ex.531nm/em.579nm) / Cy5 (em.685nm) detection

(6)       Data is expressed as the ratio of the raw Cy3 and Cy5 emission signals at each time point [8].

References: 1. Hamiche, A., et al., ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF. Cell, 1999. 97(7): p. 833-42. 2. Vangamudi, B., et al., The SMARCA2/4 ATPase Domain Surpasses the Bromodomain as a Drug Target in SWI/SNF-Mutant Cancers: Insights from cDNA Rescue and PFI-3 Inhibitor Studies. Cancer Res, 2015. 75(18): p. 3865-78. 3. Wu, Q., et al., Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells. Oncotarget, 2016. 7(19): p. 27158-75. 4. Helming, K.C., X. Wang, and C.W. Roberts, Vulnerabilities of mutant SWI/SNF complexes in cancer. Cancer Cell, 2014. 26(3): p. 309-17. 5. Karnezis, A.N., et al., Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcaemic type. J Pathol, 2016. 238(3): p. 389-400. 6. Marinus, M.G. and N.R. Morris, Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol, 1973. 114(3): p. 1143-50. 7. Geier, G.E. and P. Modrich, Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease. J Biol Chem, 1979. 254(4): p. 1408-13. 8. Shahian, T. and G.J. Narlikar, Analysis of changes in nucleosome conformation using fluorescence resonance energy transfer. Methods Mol Biol, 2012. 833: p. 337-49. 9. Clapier, C.R., et al., Regulation of DNA Translocation Efficiency within the Chromatin Remodeler RSC/Sth1 Potentiates Nucleosome Sliding and Ejection. Mol Cell, 2016. 62(3): p. 453-61.