Epigenetics refers to the regulation of genomic functions, including gene expression, by changes in DNA methylation and/or histone tail modifications. These two epigenetic mechanism work in concert, with alterations in DNA modification affecting chromatin conformation and vice versa. In part epigenetic modifications are inherited, but unlike the genome itself, they are cell specific, plastic, and responsive to environmental influences.
In the Epigenetic probe project we generate well-characterised tool compounds or ‘probes’ against key epigenetic enzymes and recognition domains involved in histone regulation of transcription and provide them freely to the scientific community to increase the knowledge of these proteins in biology and disease.
The cell assay group establishes and runs cell based assays for the different epigenetic targets to test the in vitro characterised tool compounds for cellular activity. We first focus on demonstrating the on-target effect of the characterised compounds in cells to provide a well validated compound. The characterised inhibitors will then be used to explore biology of the targets and dissect findings obtained by genetic methods. Well characterised inhibitors provide detailed knowledge regarding the role of specific domains of a protein or on catalytic versus scaffolding functions of an enzyme, complementing findings obtained using siRNA, shRNA or related methods.
The group works on cellular assays for bromodomains as well as demethylases. Fluorescence Recovery After Photobleaching (FRAP) is the most direct assay to interrogate if an inhibitor displaces binding of the bromodomain from chromatin. Chromatin associated proteins typically show low mobility due to their tight immobilization on chromosomes. Displacement of the target from chromatin due to the addition of an acetyl-lysine competitive inhibitor releases the bromodomain from chromatin resulting in increased mobility of the target protein. For demethylase inhibitors direct visualisation of the histone methyl marks in immunofluorescence (IF) assays is the method of choice to describe an on-target effect of the inhibitor. While these methods are necessary for measuring the direct on-target effect of the inhibitors, they provide no insight into the biological consequence. Complementary assays measuring genome wide changes in gene transcription (e.g. microarray studies or RNA-seq) or quantitative reverse transcriptase polymerase chain reaction assays (qPCR) that monitor expression changes of specific genes are therefore necessary. These are supplemented by reporter assays or more traditional biochemical assays to explore the involvement of a specific target in basic cellular functions like proliferation, the DNA damage response or a specific disease phenotype.