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

基因表达的控制需要一个蛋白质因子的工作组，这些蛋白质因子以协调和组合的方式发挥作用，以打开和关闭特定的基因。通过磷酸化、甲基化、乙酰化或添加小蛋白质如泛素对这些因子进行共价修饰，长期以来一直被认为可以增强它们的功能潜力，为转录机制提供动态可塑性。SUMO化是一种新型的修饰方式，它涉及小分子蛋白SUMO与靶蛋白的共价偶联。这种特征性较差的修饰在蛋白质-蛋白质相互作用的控制、亚细胞定位和基因表达调控中具有拟议的作用。我们最近发现一种锌指蛋白，ZNF 74，我们正在研究作为一种发育障碍的候选基因，迪乔治综合征，和它的蛋白质伴侣称为TIF 1 β被sumoylated。TIF 1蛋白（TIF 1）是锌指蛋白（如ZNF 74）和核受体（如视黄酸受体）的共调节因子，锌指蛋白属于KRAB多指蛋白家族（在人类蛋白质组中有超过300个成员）。我们假设类小泛素化调节TIF 1 s共调节因子的转录活性，从而影响核受体和KRAB多指蛋白靶基因的调节。我们的主要目标是：1）开发一种生物物理测定法，其允许活细胞中类小泛素化的定量实时检测，2）确定TIF 1类小泛素化对其亚细胞定位的影响以及对其与其转录伴侣的相互作用的影响，3）确定TIF 1类小泛素化状态对其募集到特定基因启动子和其抑制转录的能力的影响。我们的长期目标是破译sumoylation和其他修饰之间的动态相互作用，如磷酸化，甲基化，乙酰化和泛素化，发生在TIF 1 s及其相关因子参与控制基因表达。这将使我们更好地了解这些蛋白质如何有助于沉默或转录激活的启动，维持和终止。

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.