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蛋白质因子的任务组，这些蛋白质因子以协调和组合的方式起作用，以打开和关闭特定的基因。人们早就知道，通过磷酸化、甲基化、乙酰化或添加小蛋白（如泛素）对这些因子进行共价修饰，可以增强它们的功能潜力，为转录机制提供动态可塑性。泛素化是一种泛素样修饰，涉及小蛋白SUMO与靶蛋白的共价偶联，并与泛素修饰表现出潜在的串扰。Sumoylation在蛋白-蛋白相互作用、亚细胞定位和基因表达调控中发挥作用。我们发现一种所谓的KRAB锌指蛋白，ZNF74，我们正在研究它作为一种发育障碍，迪乔治综合症的候选基因，它的蛋白质伙伴，叫做tif1 β被酰化了。TIF1蛋白（TIF1s）属于TRIM一类蛋白，是KRAB锌指蛋白（在人类蛋白质组中约有300个成员）和核受体（如视黄酸受体）的共同调节剂，是调节基因表达的转录因子。我们假设sumo化和泛素化可以调节TIF1s共调控因子的转录活性，从而影响靶基因对KRAB锌指蛋白和核受体的调控。我们的主要目标是：1)确定sumo酰化和泛素化在TIF1s基因调控中的作用；2)鉴定和表征TIF1s sumo酰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，我们为活细胞中的sumo化和泛素化开发了定量实时检测。我们的长期目标是破译sumo化和其他修饰（如磷酸化、甲基化和乙酰化）之间的动态相互作用，这些修饰发生在TIF1s或其他TRIM上，并参与控制基因表达。这将使我们更好地理解这些蛋白质如何参与沉默或转录激活的起始、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.