Fluorescence-based cellular assay for detection of gene regulatory activity of chromatin-associated proteins
Development of eukaryotic cells is regulated by a complex regulatory network of epigenetic signals, thus faulty epigenetic states can cause oncogenic transformation and tumor development. To analyze these networks and to identify new targets for drug development as well as to investigate the mode of action of known compounds targeting chromatin associated proteins or signal transduction pathway components a novel epi-reporter in vitro assay was developed.
Cells constantly perceive environmental signals which are integrated into a defined transcriptional output to trigger a specific response suggesting a high interconnection of signal transduction pathways with the transcriptional regulatory network especially chromatin modifying enzymes. The high interconnectivity and dynamic nature of these two regulatory networks grant high flexibility and control but also provide multiple angles to corrupt the stability of these cell type specific transcriptional programs, which can initiate pathological events such as tumorigenesis and tumor progression. Defining the mechanisms that control the establishment of a specific transcriptional program in response to an environmental stimulus is fundamental to understand how the identity of mature cells is maintained and which are the critical nodes, that if impaired, lead to tumorigenesis and tumor progression.
One challenge in research, as well as for the development of epigenetic therapies, is to identify and characterize these epigenetic (epi-)effector proteins and their (co-)regulators, as in fact many of them combine various functions and can be part of more than one complex at a time.
So far, attempts have been made to uncover epigenetic effectors by co-immunoprecipitation followed by western blot or mass spectrometry analyses.
Both approaches are complex, require specific antibodies and can identify only very abundant and extremely stable effectors. In view of the complexity of epigenetic mechanisms and their regulations, it is necessary to develop tools to significantly increase the level of knowledge about epigenetic proteins and their interconnection. This requires the development of reporter systems that allow the investigation of specific function of epigenetic proteins and direct analysis of the complex regulatory network within cells.
Scientists at the University of Stuttgart have developed a novel epi-reporter assay to identify and characterize chromatin-associated proteins, their binding partners and factors that modulate the activity of said proteins inside cells. For this, the tetracycline-controlled transcriptional activation system (Tet system) is used. Analyses can be performed by standard methods including flow cytometry and fluorescence microscopy using a fluorescent reporter gene (like GFP) as readout. Moreover, not only effectors that are directly attached to the chromatin-associated protein can be identified, but also effectors with merely functional (but no direct) connection can be detected. This will not only allow to uncover novel functional dependencies in epigenetic networks and the identification of essential components, but also enables the identification of new targets for drug development Besides this hypothesis free identification of mechanisms influencing a defined gene expression program using RNAi or CRISPR approaches, the technology allows to test the activity of inhibitors against a specific epigenetic target of interest in different cell lines and to investigate the mechanism of action of known drugs targeting chromatin associated proteins or signal transduction pathways.
The schematic representation of the fluorescent reporter system to monitor epigenetic effector complexes shows recruitment of the reverse Tet-transactivator (rtTA) fused to an epigenetic effector protein (epi-effector) to the synthetic promoter (synP) is induced by the addition of Doxycycline (DOX). The induced change in fluorescence is a direct effect of the epi-effector recruitment, actively changing the chromatin environment at the synthetic promoter. The activity of the recruited transcriptional co-repressor complex can be interrogated comprehensive functional genetic tools or small molecule libraries.
- Cellular in vitro assay
- No protein purification of chromatin-associated proteins is needed
- Hypothesis-free analysis of upstream coregulators and complex partners
- Quantification by standard methods like flow cytometry or fluorescence
- Identification of effectors which are only functionally linked to the
- Chromatin-binding protein possible
- Uncover the mechanism of epigenetic effectors
- Identification of essential coregulators of epigenetic effector proteins
- Identification of new targets for drug development
- Mode of action determination of chromatin targeted drugs
- Drug screening for particular chromatin associated complexes