C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis.

The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3's oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors.

Structural and functional profiling of the human histone methyltransferase SMYD3.

The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the "split-SET" domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 α-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20.

Cooperative signaling between oncostatin M, hepatocyte growth factor and transforming growth factor-ß enhances epithelial to mesenchymal transition in lung and pancreatic tumor models.

Epithelial to mesenchymal transition (EMT) plays a dual role in tumor progression. It enhances metastasis of tumor cells by increasing invasive capacity and promoting survival, and it decreases tumor cell sensitivity to epithelial cell-targeting agents such as epithelial growth factor receptor kinase inhibitors. In order to study EMT in tumor cells, we have characterized 3 new models of ligand-driven EMT: the CFPAC1 pancreatic tumor model and the H358 and H1650 lung tumor models. We identified a diverse set of ligands that drives EMT in these models. Hepatocyte growth factor and oncostatin M induced EMT in all models, while transforming growth factor-ß induced EMT in both lung models. We observed morphologic, marker and phenotypic changes in response to chronic ligand treatment. Interestingly, stimulation with 2 ligands resulted in more pronounced EMT compared with single-ligand treatment, demonstrating a spectrum of EMT states induced by parallel signaling, such as the JAK and PI3K pathways. The EMT changes observed in response to the ligand were reversed upon ligand withdrawal, demonstrating the 'metastable' nature of these models. To study the impact of EMT on cell morphology and invasion in a 3D setting, we cultured cells in a semisolid basement membrane extract. Upon stimulation with EMT ligands, the colonies exhibited changes to EMT markers and showed phenotypes ranging from modest differences in colony architecture (CFPAC1) to complex branching structures (H358, H1650). Collectively, these 3 models offer robust cell systems with which to study the roles that EMT plays in cancer progression.

The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing.

The coactivator-associated arginine methyltransferase CARM1 is recruited by many different transcription factors as a positive regulator. To understand the mechanism by which CARM1 functions, we sought to isolate its substrates. We developed a small-pool screening approach for this purpose and identified CA150, SAP49, SmB, and U1C as splicing factors that are specifically methylated by CARM1. We further showed that CA150, a molecule that links transcription to splicing, interacts with the Tudor domain of the spinal muscular atrophy protein SMN in a CARM1-dependent fashion. Experiments with an exogenous splicing reporter and the endogenous CD44 gene revealed that CARM1 promotes exon skipping in an enzyme-dependent manner. The identification of splicing factors that are methylated by CARM1, and protein-protein interactions that are regulated by CARM1, strongly implicates this enzyme in the regulation of alternative splicing and points toward its involvement in spinal muscular atrophy pathogenesis.