Final Report Summary - K-METHYLATION (The Role of SETD6 Methyltransferase in the Modulation of Oncogenic Processes) We have recently identified and characterized the enzymatic activity, substrate specificity, crystal structure and cellular and physiological functions of the novel PKMT SETD6. This enzyme, whose expression is dramatically down-regulated in a wide range of human cancers, is a key regulator of proliferation and inflammatory processes. However, knowledge of the overall mode of action of SETD6 in cancer, including the identification and characterization of additional endogenous SETD6 substrates and interacting proteins, is still limited. We hypothesize that SETD6 function and SETD6-mediated methylation of key cellular substrates modulates oncogenic signaling pathways. In Aim 1 of the research proposal we decided to define the mode of action of SETD6 in cancer cells. We established various SETD6 knock-out cancer cell line using the CRISPR-CAS9 system. The system allows exploring the properties of SETD6 in relevant cancerous properties such as apoptosis, proliferation, invasion and migration. We already obtained very promising results linking SETD6 to oncogeniss. Depletion of SETD6 for different cancer cell lines led to reduce proliferation. We also found an increase in the level of apoptosis in the knock-out SETD6 cells compare to wildtype. Now that all the systems are up-running in the lab, we will continue to characterize SETD6 oncogenic cellular phenotypes. This will also include experiments to elucidate the role of SETD6 in transcriptional regulation by RNA-Seq and the proteomic studies to define SETD6 methylome in different primary and cancer cell lines using the ProtoArray platform we established in my lab. I believe that combination of the genomic experiment to understand SETD6 role in transcription regulation together with the characterization of the SETD6 methylome will provide significant insight to our understanding of SETD6 biology and will enable us to potentially translate the knowledge for diagnostic and prognostic purposes. Furthermore, defining the SETD6 methylome in specific cancer-relevant contexts will be a powerful strategy for understanding how lysine methylation influences oncogenesis and will be considered a major success. Since we recently identified PAK4—a serine/threonine kinase that is overexpressed and genetically amplified in many types of cancer—as a SETD6 substrate, Aim 2 of the research proposal was devoted to elucidating the molecular mechanism and physiological relevance of SETD6-mediated methylation of PAK4. In this project we have started to characterize the methylation cross-talk between SETD6 and PAK4. We have strong evidences that SETD6 binds and methylates PAK4 both in-vitro and in cells and that this interaction take place at the chromatin fraction. Depletion of SETD6 using the CRISPR/CAS9 system in various cell lines leads to a dramatic reduction in the expression of beta-catenin target genes under basal condition and in response to activation of the WNT signaling. A major milestone for this aim was to establish a direct molecular link between the role of SETD6 in cancer and SETD6 mediated methylation of PAK4. We show that in response to stimulation the presence of SETD6 on chromatin stabilizes the direct physical interaction between PAK4 and beta-catenin at chromatin and elevation of transcription. The data we obtained so far uncover a new regulatory layer for the WNT/beta-catenin signaling and give new insight on SETD6 biology. The results summarizing these findings were recently published in few papers.In Aim 3 we aimed to identify new SETD6 interaction networks that regulate cellular signaling pathways linked to cancer. For this, we proposed to leverage our experience in protein microarray technologies, in combination with classical cellular and biochemical approaches, to identify novel SETD6 interacting proteins in cancer cells. We have Immunoprecipitated SETD6 from cells followed by mass spectrometry analysis and identified more than 100 SETD6 interacting proteins, including DJ1, an oxidative stress sensor required for nuclear factor erythroid-derived 2 (Nrf2) transcription of anti-oxidative stress target genes. In a couple of papers, we published the results obtained from these proteomics screens. we also utilized our protoarray system and identify dozen of new SETD6 interacting proteins and few projects which we are currently further characterizing. I believe that this set of experiments could provide a seminal contribution in understanding the role of SETD6 in the regulation of oncogenic signaling cascades. In addition, a success in this aim would provide new directions of study and will set the stage for additional scientific publications.
