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Epidyne®-FRET for Nucleosome Remodeling Assays Nucleosome Remodeling Assay by EpiDyne®-FRET EpiDyne®-FRET allows unprecedented access to disease-relevant ATP-dependent chromatin remodeling complexes FIGURE 3 SMARCA 2 SMARCA 4 EpiDyne®-FRET nucleosomes (20 nM) were incubated with Figure 3A Figure 3B chromatin remodeling enzyme (Panel 3A, SMARCA2; panel 3B, SMARCA4) at the indicated concentration in 4.0 3.5 the presence of ATP (2 mM). 3.5 Upon ATP addition, reactions 3.0 were immediately read in 3.0 an Envision Multi-label plate 2.5 reader. Data are presented as 2.5 the mean of the Cy3-Cy5 ratio 2.0 (N=2). 2.0 1.5 Cy3/Cy5 Ratio Cy3/Cy5 1.5 Ratio Cy3/Cy5 1.0 1.0 0 1 2 3 4 5 6 7 8 9 10 0 10 20 30 40 Time, Min Time, Min nM Enzyme nM Enzyme 28 14 7 3.5 0.0 12.50 6.25 3.13 1.56 0 ORDERING INFO Chromatin Remodeling Substrate, Fluorescent Readout Enzymes EpiDyne®-FRET Nucleosome Remodeling Assay Substrate SMARCA2 Chromatin Remodeling Enzyme Catalog No. 16-4201 (Human BRM) Pack Size: 50 μg Catalog No. 15-1015 Pack Size: 100 remodeling rxns Chromatin Remodeling Substrates, Non-Fluorescent Readout SMARCA4 Chromatin Remodeling Enzyme (Human BRG1) ST601-GATC1 ST601-GATC1, 50-N-66, Biotinylated Catalog No. 15-1014 Cat. No. 16-4101 Cat. No.: 16-4114 Pack Size: 100 remodeling rxns Pack Size: 50 μg Pack Size: 50 μg ACF Chromatin Remodeling Enzyme Complex ST601-GATC1, Biotinylated ST601-GATC1,2, 50-N-66, Biotinylated Catalog No. 15-1013 Cat. No.: 16-4111 Cat. No.: 16-4115 Pack Size: 100 remodeling rxns Pack Size: 50 μg Pack Size: 50 μg Website: epicypher.com/epidyne-enzymes ST601-GATC1,2, Biotinylated ST601-GATC1,2,3, 50-N-66, Biotinylated Cat. No.: 16-4112 Cat. No.: 16-4116 Pack Size: 50 μg Pack Size: 50 μg EpiDyne® Chromatin Remodeling HTS Assay Services are available ST601-GATC1,2,3, Biotinylated Website: epicypher.com/epidyne Inquire at: [email protected] Cat. No.: 16-4113 Pack Size: 50 μg EpiDyne®-FRET , RTP, NC. All rights reserved. , RTP, ® For Nucleosome EpiCypher.com Remodeling Assays 855.374.2461 • [email protected] March, 2020 v.3.0 March, ©2020 EpiCypher Nucleosome Remodeling Assay by EpiDyne®-FRET Nucleosome Remodeling Assay by EpiDyne®-FRET EpiDyne®-FRET: a functionalized recombinant nucleosome-based assay Chromatin Remodeling Enzymes As Therapeutic Targets platform for chromatin remodeling studies Aberrant nucleosome organization can severely disrupt gene expression, DNA repair and cellular differentiation, and it also plays a major role in human disorders, including Chromatin remodeling, or the repositioning of nucleosomes, regulates DNA access and cancer, infammation, autoimmunity, schizophrenia, cardiovascular disease, and intellectual thus gene expression and genome repair. Many ATP-dependent remodeling enzyme disability. Remarkably, nearly 20% of all cancers contain mutations in subunits from the complexes are associated with human disease but are challenging study targets due to SWI/SNF family of ATP-dependent chromatin remodeling complexes. These enzyme the requirement for nucleosome-based substrates. EpiCypher® has addressed this need by complexes regulate local genome access by ‘pumping’ the DNA around histone octamers, developing the EpiDyne® platform of fully recombinant remodeling substrates to monitor thus ‘sliding’ nucleosomes. nucleosome repositioning along DNA using Fluorescence Resonance Energy Transfer (FRET) readout (Figure 1). SWI/SNF Remodeling Complex FIGURE 2 FIGURE 1 ® EpiDyne -FRET Bromodomain Schematic of SWI/SNF EpiDyne®-FRET Nucleosome remodeling complex. PBRM1* Remodeling Substrates PHF10 PHD finger Domain Yellow subunits denote consist of a Cy5-labeled DNA binding domain minimal core complex human histone octamer (H2A that recapitulates activity T120C-Cy5; shown as red SANT domain (histone tail binding) 685 nm SMARCD1 of full complex. section of octamer) wrapped SMARCB1* by 5’ Cy3-labeled DNA Nucleosome Remodeling ARID1A/B* (217bp; green ball) comprising Minimal SWI/SNF Core Complex a terminal nucleosome BCL7A-C SS18 positioning sequence (147bp Other possible subunits present Widom 601 adjacent to a SMARCA2/4* in SWI/SNF complexes TGGA-repeat region refractory BRD7* to nucleosome assembly. In its ACTL6A assembled starting state, Cy3- Cy5 FRET is at a maximum. FRET SMARCC1 SMARCC2 ACTB The activity of an ATP- SMARCE1* dependent remodeler (e.g. RSC or another SWI/SNF ATPase) is detected by a reduction in FRET signal H3K14ac as the Cy3-labeled DNA 5’ interacting end is moved away from the Cy5-labeled octamer. EpiDyne®-FRET is a one- step no-wash method 535 nm Recurrent somatic mutations in SWI/SNF subunits are observed in multiple cancers, immediately compatible with HTS applications. supporting a driver role in tumorigenesis. The mutated remodeling proteins are attractive 535 nm therapeutic targets, since further compromising their ATPase activity promotes cancer cell death but spares normal cells. TABLE 1 SUBUNIT CANCER Useful for List of cancers associated with various SWI/SNF ARID1A Ovarian, Hepatocellular, Bladder, Gastric, Endometrioid, Pancreatic, Colon, Lung, subunit mutations adapted Neuroblastoma, Burkitt Lymphoma • Inhibitor screening and development from Helming et al. Cancer ARID1B Melanoma, Neuroblastoma, Hepatocellular, Pancreatic, Liver Cell 26, 309-317 (2014). Asterisks in Figure 2 indicate • Structure-Activity Relationship assays PBRM1 Renal cell carcinoma, Breast, Gastric, Pancreatic associated cancers in Table 1. ARID2 Melanoma, Hepatocellular, Pancreatic • Biochemical profiling of ATPase family proteins SMARCA2 Lung, Colon, Breast SMARCA4 Lung, Medulloblastoma, Burkitt Lymphoma, SCCOHT SMARCB1 Rhaboid tumor, Familial Schwannomatosis SMARCE1 Spinal meningitis BRD7 Breast Nucleosome Remodeling Assay by EpiDyne®-FRET Nucleosome Remodeling Assay by EpiDyne®-FRET EpiDyne®-FRET: a functionalized recombinant nucleosome-based assay Chromatin Remodeling Enzymes As Therapeutic Targets platform for chromatin remodeling studies Aberrant nucleosome organization can severely disrupt gene expression, DNA repair and cellular differentiation, and it also plays a major role in human disorders, including Chromatin remodeling, or the repositioning of nucleosomes, regulates DNA access and cancer, infammation, autoimmunity, schizophrenia, cardiovascular disease, and intellectual thus gene expression and genome repair. Many ATP-dependent remodeling enzyme disability. Remarkably, nearly 20% of all cancers contain mutations in subunits from the complexes are associated with human disease but are challenging study targets due to SWI/SNF family of ATP-dependent chromatin remodeling complexes. These enzyme the requirement for nucleosome-based substrates. EpiCypher® has addressed this need by complexes regulate local genome access by ‘pumping’ the DNA around histone octamers, developing the EpiDyne® platform of fully recombinant remodeling substrates to monitor thus ‘sliding’ nucleosomes. nucleosome repositioning along DNA using Fluorescence Resonance Energy Transfer (FRET) readout (Figure 1). SWI/SNF Remodeling Complex FIGURE 2 FIGURE 1 ® EpiDyne -FRET Bromodomain Schematic of SWI/SNF EpiDyne®-FRET Nucleosome remodeling complex. PBRM1* Remodeling Substrates PHF10 PHD finger Domain Yellow subunits denote consist of a Cy5-labeled DNA binding domain minimal core complex human histone octamer (H2A that recapitulates activity T120C-Cy5; shown as red SANT domain (histone tail binding) 685 nm SMARCD1 of full complex. section of octamer) wrapped SMARCB1* by 5’ Cy3-labeled DNA Nucleosome Remodeling ARID1A/B* (217bp; green ball) comprising Minimal SWI/SNF Core Complex a terminal nucleosome BCL7A-C SS18 positioning sequence (147bp Other possible subunits present Widom 601 adjacent to a SMARCA2/4* in SWI/SNF complexes TGGA-repeat region refractory BRD7* to nucleosome assembly. In its ACTL6A assembled starting state, Cy3- Cy5 FRET is at a maximum. FRET SMARCC1 SMARCC2 ACTB The activity of an ATP- SMARCE1* dependent remodeler (e.g. RSC or another SWI/SNF ATPase) is detected by a reduction in FRET signal H3K14ac as the Cy3-labeled DNA 5’ interacting end is moved away from the Cy5-labeled octamer. EpiDyne®-FRET is a one- step no-wash method 535 nm Recurrent somatic mutations in SWI/SNF subunits are observed in multiple cancers, immediately compatible with HTS applications. supporting a driver role in tumorigenesis. The mutated remodeling proteins are attractive 535 nm therapeutic targets, since further compromising their ATPase activity promotes cancer cell death but spares normal cells. TABLE 1 SUBUNIT CANCER Useful for List of cancers associated with various SWI/SNF ARID1A Ovarian, Hepatocellular, Bladder, Gastric, Endometrioid, Pancreatic, Colon, Lung, subunit mutations adapted Neuroblastoma, Burkitt Lymphoma • Inhibitor screening and development from Helming et al. Cancer ARID1B Melanoma, Neuroblastoma, Hepatocellular, Pancreatic, Liver Cell 26, 309-317 (2014). Asterisks in Figure 2 indicate • Structure-Activity Relationship assays PBRM1 Renal cell carcinoma, Breast, Gastric, Pancreatic associated cancers in Table 1. ARID2 Melanoma, Hepatocellular, Pancreatic • Biochemical profiling of ATPase family proteins SMARCA2 Lung, Colon, Breast SMARCA4 Lung, Medulloblastoma, Burkitt Lymphoma, SCCOHT SMARCB1 Rhaboid tumor, Familial Schwannomatosis SMARCE1 Spinal meningitis BRD7 Breast Nucleosome Remodeling Assay by EpiDyne®-FRET EpiDyne®-FRET allows unprecedented access to disease-relevant ATP-dependent chromatin remodeling complexes FIGURE 3 SMARCA 2 SMARCA 4 EpiDyne®-FRET nucleosomes (20 nM) were incubated with Figure 3A Figure 3B chromatin remodeling enzyme (Panel
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