The overarching goal is to gain a deeper understanding of how sumoylation regulates transcription through the action of the Arkadia SUMO-binding ubiquitin ligase (STUbL), and the role that this plays in TGF�nduced gene expression regulated by epigenetic marks in normal and cancer cells. First, the functional differences between Arkadia and the prototypical STUbL, RNF4, will be analyzed, focusing on the roles that the unique M domain in Arkadia, which separates the SIMs and the RING domain, and the SUMO-binding domain play in Arkadia's canonical nuclear function in the TGFsignaling pathway that leads to induction of gene expression. The M domain has been shown to be essential for Arkadia to enhance TGF�eporter gene expression, and proteins interacting with the M domain will be identified using mass spectrometry, and functionally validated. Likewise, proteins that bind to Arkadia in a SUMO-dependent manner via SUMO-interacting motifs (SIMs) will be identified. RNA-seq analysis on wild-type and Arkadia-null mouse embryo fibroblasts has been used to define a list of TGF�nduced genes that are Arkadia dependent. This approach will be extended to identify TGF�nduced genes that are dependent on the Arkadia SIMs and on the M domain. How TGF�ignals are transmitted to Arkadia in the nucleus will be investigated by studying TGF�nduced post-translational modifications in Arkadia. Second, sumoylated proteins that are recognized by Arkadia in a SIM-dependent manner and, as a result, are ubiquitylated and degraded will be identified using SIM affinity purification. Sumoylated proteins will be analyzed as candidate substrates for Arkadia ubiquitylation in cells expressing wild-type and RING mutant Arkadia upon TGF�reatment. Two other methods to identify Arkadia substrates will be used: ubiquitylation assays with Arkadia precipitates combined with MS analysis of tagged ubiquitylated proteins, and a global ubiquitome screen comparison of wild-type and Arkadia-null cells. Candidates most likely to be involved in transcriptional regulation will be validated and analyzed to determine if their degradation is promoted by TGF�and depends on sumoylation. These studies expect to identify targets for Arkadia that are ubiquitylated in a SUMO-dependent fashion and involved in TGF�ranscriptional regulation. Third, lab data shows that Arkadia is localized to Polycomb bodies, which are sites of gene repression, dependent on both its SIMs and the M domain, suggesting Arkadia may play a role in de-repressing silenced genes in response to TGF�To pursue this idea, ChIP-seq analysis will be carried out to identify Arkadia-associated genomic loci, and the results correlated with available ChIP-seq data for H3K27me3, a repressive histone mark, CpG methylation status, and Arkadia-dependent TGF�arget genes as defined by RNA-seq to see if Arkadia affects repression status in response to TGF�These experiments should provide insights into how Arkadia can facilitate expression of genes regulated by two epigenetic events in response to TGF� PUBLIC HEALTH RELEVANCE: The studies proposed in this application seek to elucidate at the molecular level how the attachment of the small protein SUMO to other proteins allows a protein called Arkadia to control the transcription of genes in the nucleus in response to transforming growth factor �a secreted protein that has an important role in suppressing the formation of tumors, including colorectal cancer.