Notes: To characterize the effectiveness of LP99, a potential bromodomain inhibitor, BRD7 and BRD9 were fused with NanoLuc® luciferase and histones H3.3 and H4 were fused with HaloTag® protein for use in BRET. The two proteins were expressed in HEK 293 cells, and the histone-HaloTag® fusions were fluorescently labeled with the HaloTag® NanoBRET™ 618 Ligand. Once the NanoBRET™ Nano-Glo® Substrate was added, NanoBRET™ ratios were assessed in the presence of varying concentrations of LP99. (4567)

Notes: The University of Washington Clinical Molecular Genetics Laboratory used the Microsatellite Instability Analysis (MSI) kit to test 81 colorectal tumor specimens. (4889)

Notes: This paper introduces NanoBRET technology, which provides an improved alternative to conventional BRET protein interaction assays. NanoBRET assays combine the extremely bright NanoLuc luciferase with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The greater light intensity and improved spectral resolution of the NanoBRET assay results in increased detection sensitivity and dynamic range over current BRET technologies. Performance of the assay is demonstrated using several model systems, and the ability to image BRET in individual cells is illustrated. The  authors also demonstrate the application of NanoBRET in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells. (4575)

Notes: This paper describes a method for using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets in live cells. The authors used cell-permeable fluorescent tracers in a competitive binding assay to quantify drug engagement with target proteins fused to Nanoluc luciferase. Using this approach, they were able to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. (4587)

Notes: In this paper the authors report on the discovery, binding mode, and structure:activity relationship of the first potent, selective series of inhibitors of the BRPF1 (bromodomain and PHD finger-containing)  bromodomain.  Bromodomains are specific protein modules present in a group of chromatin-regulator proteins responsible for “reading” acetylated lysine residues. Although some bromodomain-containing proteins (BCPs), such as those in the BET subfamily, are well characterized and have been identified as potential therapeutic targets, other BCPs, including those in the BPRF subfamily, are less well understood.  These authors set out to generate selective inhibitors of the BRPF1 domain in order to better understand the functional role of this specific bromodomain region. Using an inhibitor discovery strategy based on other known compound-bromodomain interactions, a potent, selective inhibitor of the BRPF1 bromodomain was identified, synthesized, and characterized using in vitro methods.  To demonstrate the function of this compound in live cells, the NanoBRET™ assay for protein:protein interactions (PPI) was used. The NanoBRET™ PPI assay enabled the authors to demonstrate both the cell permeability of the newly identified compound and also the ability of the compound to disrupt chromatin binding of the BRPF1 domain. NanoLuc® Luciferase-tagged BRPF1 bromodomain and HaloTag®-labeled Histone H3.3 were used for the NanoBRET™ assay in HEK293 cells. Dose-response curves performed with the NanoBRET™ assay enabled calculation of the cellular IC50 of the newly identified compound. A less active control analog compound was unable to inhibit the BRPF1 bromodomain:Histone H3.3 interaction, demonstrating assay specificity. Finally, the newly identified compound was inactive in NanoBRET™ assays using a second BRPF1 isoform containing a natural insertion, a result that was consistent with the proposed compound mode of action. Confirmation that the new identified compound can enter cells and disrupt the BRPF1 bromodomain:chromatin interaction in a cellular environment suggests that it may be a useful compound for studying the physiological role and therapeutic potential of BCPs containing the BRPF1 bromodomain. (4514)

Notes: To investigate whether protein conformation of Rabin8 plays a role in autoinhibition, the authors created a Rabin8 fusion construct with NanoLuc® luciferase at the N terminus and HaloTag® protein at the C terminus so that they can use BRET as an indication of protein conformation. A t-SRARE protein, syntaxin-4 (STX4), which is known to have a closed conformation, was constructed with the same NanoLuc® luciferase-STX4-HaloTag® protein configuration for use as a positive control. Both the control STX4 protein and Rabin8 were expressed in E. coli, the NanoBRET™ Nano-Glo® Substrate added and fluorescence measured. As a negative control, TEV protease was used to cleave the HaloTag® sequence from the protein fusions, eliminating the NanoBRET™ signal. NanoBRET™ signals were determined from experiments comparing Rabin8 with a gain-of-function Rabin8 mutant, exposing Rabin8 to constitutively active ERK2 or a kinase-dead ERK2 and assessing wildtype Rabin8 versus Rabin8-4D, where the aspartates acted as phosphorylation mimics. (4566)

Notes: In this paper the authors report on the identification and characterization of small-molecule inhibitors of the histone lysine methyltransferase Enhancer of Zeste Homolog 2 (EZH2). EZH2 has been identified as a potential oncology target because of its role in histone lysine methylation and associated involvement in the manipulation of chromatin structure. EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), and recurrent mutations in EZH2 have been discovered in multiple cancer cell types resulting in aberrantly high H3 lysine 27 (H3K27) trimethylation levels. The authors identified compound CPI-360 as a potent inhibitor of EZH2 methyl transferase activity that functions on the basis of S-adenosyl-L-methionine (SAM)-competition and demonstrated that treatment of cells with CP1-360 resulted in reduced di and tri, but not mono, methylation of H3K27 in a dose-dependent manner. Using NanoBRET™ protein:protein interaction assays, the authors were able to demonstrate in cells that the inhibition of EZH2 activity was not due to disruption of the PRC2 complex and also did not affect binding of EZH2 to chromatin, presenting a novel mechanism for the inhibition of EZH2 activity. The authors further demonstrate how inhibiting EZH2 methyl transferase activity affects H3K27 methylation and transcriptional patterns in lymphoma cell lines and demonstrate the effect of EZH2 inhibitors on growth and viability of lymphoma cells in vitro using Cell Titer-Glo® Luminescent Cell Viability Assay and in in vivo xenograft models. (4562)

Notes: In this study the authors looked for molecular mechanisms underlying the role of kainite receptors in ischemic stroke. In their studies, the researchers examined binding of Src kinase to GluK2, and the site of this interaction. A GST pulldown assay confirmed a direct interaction between GluK2 and Src in vitro. Then a bioluminescence resonance energy transfer (BRET) assay was used to examine this GluK2-Src interaction in live HEK293 cells, using NanoLuc® Luciferase as the energy donor, and HaloTag-labeled GluK2 as the energy acceptor. As reported, the co-expression of NLuc and HaloTag® fusions resulted in a significant NanoBRET ratio. The authors added untagged GluK2 as a competitor of the GluK2-HaloTag and Src-NLuc interaction, which resulted in reduction of the NanoBRET ratio. These results demonstrated that GluK2 interacts directly with Src in living cells. The GloMax® Discover Detection System was used to measure the output of these assays.

Their source of NanoLuc® Luciferase and HaloTag® tags was the NanoBRET™ PPI Starter System (Cat.# N1811, N1821). (4696)

Notes: These authors set out to determine how TET2 and TET3 proteins are involved in epigenetic regulation. To characterize proteins that interact with TET, the authors expressed full-length TET1, TET2 and TET3 as HaloTag® fusion proteins and performed protein pull-downs. They identified novel interactions between all three TET proteins and O-GlcNAc transferase (OGT), which catalyzes the addition of N-acetylglucosamine (GlcNAc) to numerous transcription factors, regulatory proteins and histones to activate or inhibit the target protein or recruit additional proteins. In this paper, they focused on TET2 and TET3, which showed the strongest interaction with OGT. They mapped TET2, TET3 and OGT binding throughout  the genome by expressing these proteins as HaloTag® fusion proteins in HEK293T cells, crosslinking the proteins and DNA, then capturing the fusion proteins and associated DNA fragments and performing high-throughput sequencing to show that TET2/3 and OGT colocalize at active gene promoters and were tightly clustered near transcription start sites.

For expression of HaloTag® fusion proteins and controls, HEK-293 cells were plated at 12 ×10⁶ cells in a 150mm dish and grown to 70–80% confluency before transfection with 30µg of plasmid using the FuGENE® HD Transfection Reagent.

To assess whether TET2/3-OGT activity affects the interaction of SET1/COMPASS with chromatin, the authors used bioluminescence resonance energy transfer (BRET). They created a fusion protein consisting of the H3K4 methyltransferase SETD1A and NanoLuc® luciferase as the energy donor and a fluorescently labeled histone H3.3-HaloTag® fusion protein as the energy acceptor.  These BRET experiments confirmed that TET2/3-OGT activity is necessary for SET1/COMPASS complex function and showed that TET and OGT activities promote binding of SETD1A, a component of the SET1/COMPASS complex, to chromatin. This binding increases H3K4me3 levels. Thus, the authors’ data support a TET2/3-OGT-mediated mechanism for regulating the SET1/COMPASS complex and thus H3K4me3. (4262)