Degron Protease Blockade Sensor to Image Epigenetic Histone Protein Methylation in Cells and Living Animals

Abstract

Lysine methylation of histone H3 and H4 has been identified as a promising therapeutic target in treating various cellular diseases. The availability of an in vivo assay that enables rapid screening and preclinical evaluation of drugs that potentially target this cellular process will significantly expedite the pace of drug development. This study is the first to report the development of a real-time molecular imaging biosensor (a fusion protein, [FLuc2]-[Suv39h1]-[(G4S)3]-[H3-K9]-[cODC]) that can detect and monitor the methylation status of a specific histone lysine methylation mark (H3-K9) in live animals. The sensitivity of this sensor was assessed in various cell lines, in response to down-regulation of methyltransferase EHMT2 by specific siRNA, and in nude mice with lysine replacement mutants. In vivo imaging in response to a combination of methyltransferase inhibitors BIX01294 and Chaetocin in mice reveals the potential of this sensor for preclinical drug evaluation. This biosensor thus has demonstrated its utility in the detection of H3-K9 methylations in vivo and potential value in preclinical drug development.

Epigenetic mechanisms underlying nervous system diseases

“…In fact, they are quite challenging to study because of their myriad complexities, dynamic nature, and functional interrelationships (outlined above). Recent scientific and technologic advances – such as those related to analyzing single cells and single molecules (Hyun et al, 2015), synthetic biology and genome editing (Han et al, 2014), optogenetics (Konermann et al, 2013), chemical biology (Baud et al, 2014), and molecular imaging (Sekar et al, 2015a, b) – are starting to offer more sophisticated tools and techniques for interrogating the epigenome with a higher degree of temporal and spatial resolution as well as cell type and genomic site specificity. These and other emerging strategies will undoubtedly deliver novel insights into the major epigenetic mechanisms and uncover additional roles for epigenetic processes.…”

Coordinated histone modifications and chromatin reorganization in a single cell revealed by FRET biosensors

“…Genetically encoded biosensors based on FRET, by allowing the visualization and quantification of biochemical signals in single live cells with high spatiotemporal resolutions, have revolutionized research in cell biology. Several FRET and bioluminescence biosensors have been reported to detect histone methylation, acetylation, and phosphorylation (15,16,27,32,43,44). However, the limited sensitivity of these genetically encoded FRET biosensors, particularly for histone methylations, has hindered their broad application.…”

“…Recent genome-wide analysis of H3K9me3 during cell cycle (12) and the observation of histone K9 demethylation KDM4C on mitotic chromatin point to an overall decrease of H3K9me3 in the nucleus during mitosis (13). This paradox in observations may be attributed to the epigenetic heterogeneity across different individual cells, as well as the lack of precise and sensitive detecting tools in monitoring the dynamic H3K9me3 signals at subcellular regions (14)(15)(16).…”

Live-cell imaging probes to track chromatin modification dynamics

“…By injecting cells that express the reporters into mice, the luciferase activity can be monitored in vivo . A degron blockade methylation sensor was also developed for luciferase-mediated methylation sensing [ 82 ]. In this case, a full-length firefly luciferase is fused with the H3 N-terminal amino acids, a linker, Suv39h1 CD and a degron protease recognition sequence.…”