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

ATRX Protects Cells Against Replication-Induced Genomic Instability

Expansive proliferation of neural progenitor cells (NPCs) is a prerequisite to the temporal waves of neuronal differentiation that generate the six-layered cerebral cortex. NPC expansion places a heavy burden on proteins that regulate chromatin packaging and genome integrity, which is further reflected by the growing number of developmental disorders caused by mutations in chromatin regulators. Accordingly, mutations in ATRX, a chromatin remodelling protein required for heterochromatin maintenance at telomeres and simple repeats, cause the ATR-X syndrome. Here, we demonstrate that proliferating ATRX-null cells accumulate DNA damage, while also exhibiting sensitivity to hydroxyurea-induced replication fork stalling. Specifically, PARP1 hyperactivation and replication-dependent double strand DNA breakage indicated replication fork protection defects, while DNA fiber assays confirmed that ATRX was required to protect replication forks from degradation. Interestingly, inhibition of the exonuclease MRE11 by the small molecule mirin could prevent degradation. Thus, ATRX is required to limit replication stress during NPC expansion

PHF6 Degrees of Separation: The Multifaceted Roles of a Chromatin Adaptor Protein

The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the plant homeodomain finger protein 6 (PHF6) gene. Indeed, germline mutations in PHF6 are the cause of the Börjeson–Forssman–Lehmann X-linked intellectual disability syndrome (BFLS), while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis

Methyltransferase Inhibitors: Competing with, or Exploiting the Bound Cofactor

Protein methyltransferases (PMTs) are enzymes involved in epigenetic mechanisms, DNA repair, and other cellular machineries critical to cellular identity and function, and are an important target class in chemical biology and drug discovery. Central to the enzymatic reaction is the transfer of a methyl group from the cofactor S-adenosylmethionine (SAM) to a substrate protein. Here we review how the essentiality of SAM for catalysis is exploited by chemical inhibitors. Occupying the cofactor binding pocket to compete with SAM can be hindered by the hydrophilic nature of this site, but structural studies of compounds now in the clinic revealed that inhibitors could either occupy juxtaposed pockets to overlap minimally, but sufficiently with the bound cofactor, or induce large conformational remodeling leading to a more druggable binding site. Rather than competing with the cofactor, other inhibitors compete with the substrate and rely on bound SAM, either to allosterically stabilize the substrate binding site, or for direct SAM-inhibitor interactions

HDX-MS raw data for "Structural elucidation of full-length Pfs48/45 in complex with potent mAbs isolated from a naturally exposed individual"

<p>These are the raw files of the HDX-MS data for the paper "Structural elucidation of full-length Pfs48/45 in complex with potent mAbs isolated from a naturally exposed individual."</p><p>Peptide identification files are provided in MGF/MZID, as well as in CSV format. </p&gt

Discovery of a chemical probe for PRDM9

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases