PALI1 as a physical bridge between two master-regulator histone methyltransferase complexes
Melanie Murray, Dr. Qi Zhang and A/Prof. Chen Davidovich
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia.
PALI1 is a transcriptional regulator essential for mouse development and can be found in two types of chromatin modifier complexes: G9A and PRC2. G9A and PRC2 are two histone methyltransferase complexes essential for mouse development through the deposition of the H3K9me2 and H3K27me3 repressive marks, respectively. G9A and PRC2 are frequently dysregulated in cancer and the discovery of new functional centres in them would provide targets for new therapeutics. PRC2 and G9A share a portion of their genomic targets, although the mechanism is unknown. PALI1 directly interacts with PRC2 and allosterically activates PRC2, but the way PALI1 regulates G9A is unknown. Although some correlative observations permit hypothesizing that PALI1 bridges G9A and PRC2, there is no direct evidence to support this hypothesis and it is not even known if PALI1 directly interacts with G9A.
Using rational design with limited proteolysis, we constructed, expressed, and purify a soluble, monodispersed and enzymatically active 300 kDa G9A-GLP-PALI1 complex. The G9A-GLP-PALI1 complex includes the nearly intact G9A and GLP methyltransferase subunits and their binding region from PALI1. Through cross-linking mass spectrometry (XL-MS), we mapped the binding sites between PALI1 to G9A-GLP in a free solution or in the context of chromatin. Electrophoretic mobility shift assay (EMSA), using fluorescently labelled human nucleosome constructs, indicates that linker DNA is required for high-affinity interactions between the G9A-GLP complex to nucleosomes. Importantly, EMSA and histone methyltransferase assays using nucleosome constructs indicate that G9A-GLP retains its normal chromatin-binding and enzymatic activities in the presence of PALI1. Overall, our results indicate that PALI1 is sufficient for direct interactions with the G9A-GLP complex, while allowing G9A-GLP to retain its normal enzymatic and chromatin-binding activities. Our data support a model where PALI1 predominantly functions as a physical bridge to link between G9A to PRC2.