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

基因表达的控制需要一个由蛋白质因子组成的工作组，它们协同工作，以组合的方式打开和关闭特定的基因。这些因子通过磷酸化、甲基化、乙酰化或添加如泛素等小蛋白的共价修饰，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。相扑修饰是一种类似泛素的修饰，它涉及到小蛋白相扑与靶蛋白的共价偶联，并显示出与泛素修饰的潜在串扰。苏莫化在蛋白质-蛋白质相互作用、亚细胞定位和基因表达调控中起着重要作用。我们发现，我们正在研究的一种所谓的KRAB锌指蛋白ZNF74和它的蛋白伴侣TIF1β被求和化。我们正在研究的ZNF74是一种发育障碍--迪乔治综合征的候选基因。TIF1蛋白(TIF1)是一类被称为TRIM的蛋白质，是KRAB锌指蛋白(人类蛋白质组中约300个成员)和核受体(如视黄酸受体)的共同调节因子，后者是调节基因表达的转录因子。我们推测，苏莫化和泛素化调节TIF1s共调控因子的转录活性，从而影响KRAB锌指蛋白和核受体靶基因的调控。我们的主要目标是：1)确定TIF1s苏莫化和泛素化在基因调控中的作用；2)识别和表征TIF1s苏莫化和泛素化的调控机制。对于第二个目标，我们将利用生物物理分析，称为BRET，这是我们开发的用于定量实时检测活细胞中的苏莫化和泛素化的方法。我们的长期目标是破译相思修饰和其他修饰之间的动态相互作用，如发生在TIF1或其他TRIM上的磷酸化、甲基化和乙酰化，并参与控制基因表达。这将使我们能够更好地理解这些蛋白质如何参与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 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.