Dynamics of sumoylation and ubiquitination of TIF1 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 a new type of modification which involves the covalent coupling of a small protein SUMO to target proteins. This poorly characterised modification has a proposed role in the control of protein-protein interactions, subcellular localization and regulation of gene expression. We recently discovered that a zinc finger protein, ZNF74, that we were studying as a candidate gene for a developmental disorder, the DiGeorge syndrome, and its protein partners called TIF1beta were sumoylated. TIF1 proteins(TIF1s) are co-regulators of zinc finger proteins like ZNF74 belonging to a family called KRAB multifinger proteins (> 300 members in the human proteome) and of nuclear receptors (e.g. retinoic acid receptors). We hypothesize that sumoylation modulates the transcriptional activity of TIF1s co-regulators and thus affects the regulation of the target genes for nuclear receptors and KRAB multifinger proteins. Our main objectives are to: 1) develop a biophysical assay that allows the quantitative real-time detection of sumoylation in living cells, 2) determine the effect of the TIF1s sumoylation of their subcellular localization and on their interactions with their transcriptional partners, 3)determine the effect of the sumoylation status of the TIF1s on their recruitment to specific gene promoters and on their ability to repress transcription. Our longer term objective is to decipher the dynamic interplay between sumoylation and other modifications such as phosphorylation, methylation, acetylation and ubiquitination that occur on TIF1s and their associated factor involved in controlling gene expression. This should allow us to better understand how these proteins contribute to the initiation, maintenance and termination of silencing or transcriptional activation.