Structural and Functional Elucidation of PRDM Proteins

Epigenetic signalling dictates the dynamic patterns of gene expression that are required for life. In humans, epigenetic lysine methylation is produced by chromatin-bound transcription factors that contain a SET (Su(var)3-9-E(z)-Trx-homology) domain or a Rossmann-fold domain. The PRDM (PRDI-BF1-RIZ homology domain containing) proteins are identified by an N-terminal PR/SET domain that shares the canonical SET domain fold, but with just 20-30% amino acid sequence identity. Humans possess 19 PRDM-coding genes with roles in cellular proliferation and differentiation, and dysregulated PRDM gene expression is often associated with diseases. I hypothesize that only a subgroup of PRDM proteins are active lysine methyltransferases and pursue the objective of this thesis to expand the fields of PRDM biology by revealing novel insights regarding the enzymatic and non-enzymatic properties of PRDM proteins. To address this objective, I characterized MRK-740, which is the first and only chemical probe of a PRDM protein. Here I examined the mechanism of inhibition of MRK-740 for its target PRDM9, using biophysical and structural biology techniques. I characterized specificity of MRK-740 within the PRDM family and uncovered the mechanism of inhibition. Remarkably, a structural survey of all chemical probes targeting protein methyltransferases indicated that MRK-740 functions by a previously unobserved mechanism of inhibition. Next, I examined how PRDM proteins regulate epigenetic signaling through a mechanism that is independent of intrinsic methyltransferase activity. Here I examined the PRDM paralogs, PRDM3 (also known as MECOM or MDS1-EVI1) and PRDM16 (also known as MEL1), which may lack intrinsic methyltransferase activity and identified how they directly interact with the Nucleosome Remodeling Deacetylase (NuRD) complex. Finally, I examined 13 of the 19 human PR/SET domains for their ability to bind a chemical analog of the enzyme cofactor, to identify proteins with enzymatic potential. I used nuclear magnetic resonance spectroscopy to detect binding of fluorinated S-adenosyl-L-homocysteine and identified several previously uncharacterized PR/SET domains that likely bind the cofactor. Taken together, these findings provide the impetus, tools and insights for further research into the roles of PRDM proteins in health and disease.Ph.D