Histone lysine and DNA methylation: dynamic marks in the chromatin

Ten-eleven translocation (TET) enzymes are epigenetic enzymes that are responsible for the iterative oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) on genomic DNA using a catalytic 2-oxoglutarate (2OG) dependent oxygenase domain. These epigenetic marks are associated with transcriptional activation and silencing depending on the modification and context. Furthermore, 5fC and 5caC participate in active demethylation pathways to revert back to cytosine (C) creating a dynamic cycle. TET enzymes play an essential role in shaping the epigenome and loss of TET2’s function has frequently been described in haematopoietic malignancies with notable examples being myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). To investigate the functional role of TET enzymes in a cellular context in greater detail, chemical probes can aid in understanding the effect of TET inhibition and can elicit a biological response. Here, two robust orthogonal high-throughput screening technologies were developed for kinetic analysis of TET enzymes and inhibitor profiling. Examination of a focused small-molecule library revealed differences in potency amongst TET isoforms and 2OG-dependent oxygenases. These results provide the initial scaffolds as a starting point for further inhibitor development. A myriad of epigenetic modifications is found on histones in nucleosome complexes. A common post translational modification is Nε-lysine methylation which is linked to transcriptional silencing or activation depending on the location within the nucleosome. The methylation levels are under dynamic control of histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs) while readout is performed by chromatin binding modules. In order to study the biological role of lysine methylation and participation in orchestrating the epigenetic landscape, chemical probes can be used. However, the relative high number of structurally related proteins and corresponding domains found in humans consisting of KMTs (SET: ~25), KDMs (JumonjiC domain: ~25) and chromatin binding modules (tudor domain (TD): ~25; plant homeodomain (PHD): >100) makes it challenging to produce both potent and specific inhibitors. The development of inhibitors for Nε-trimethyllysine chromatin binding modules to date, have been plagued with relatively weak inhibition but also with off-target effects. To aid in the development of inhibitors, epigenetic tools were developed, and their effectiveness was explored against a panel of chromatin binding modules. The tools were then applied in probing the PHD of multiple KDMs including KDM5s (JARID1s) and KDM7B (PHF8). The collective results show that these new tools can be utilized for the development of potent and selective inhibitors of chromatin binding modules that may in the future be used for targeted therapies.