Dynamics of sumoylation and ubiquitination of TIF1/TRIM co-transcriptional regulators and interacting proteins

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The control of gene expression requires a task force of protein factors that function coordinately and in combinatorial fashion to turn specific genes on and off. Covalent modifications of these factors by phosphorylation, methylation, acetylation or addition of small proteins such as ubiquitin have long been known to enhance their functional potentials, providing dynamic plasticity to the trancriptional machinery. Sumoylation is an ubiquitine-like modification, which involves the covalent coupling of a small protein SUMO to target proteins and exhibits potential cross-talk with ubiquitin modification. Sumoylation has a role in the control of protein-protein interactions, subcellular localization and regulation of gene expression. We discovered that a so-called KRAB zinc finger protein, ZNF74, that we were studying as a candidate gene for a developmental disorder, the DiGeorge syndrome, and its protein partner called TIF1beta were sumoylated. TIF1 proteins(TIF1s) belong to a class of proteins called TRIM and are co-regulators of KRAB zinc finger proteins (~ 300 members in the human proteome) and nuclear receptors (e.g. retinoic acid receptors), which are transcription factors regulating gene expression. We hypothesize that sumoylation and ubiquitination modulates the transcriptional activity of TIF1s co-regulators and thus affects the regulation of the target genes for KRAB zinc finger proteins and nuclear receptors. Our main objectives are to: 1)Determine the role of sumoylation and ubiquitination on TIF1s gene regulation and 2) Identify and characterize regulatory mechanisms involved in TIF1s sumoylation and ubiquitination. For this second aim, we will take advantage of biophysical assays, called BRET, that we developed for quantitative real-time detection of sumoylation and ubiquitination in living cells. Our longer term objective is to decipher the dynamic interplay between sumoylation and other modifications such as phosphorylation, methylation, and acetylation that occur on TIF1s or other TRIM and are involved in controlling gene expression. This should allow us to better understand how these proteins contribute to initiation, maintenance and termination of silencing or transcriptional activation.