Multiple Arkadia/RNF111 structures coordinate its Polycomb body association and transcriptional control. Mus musculus

The RING domain protein Arkadia/RNF111 is a ubiquitin ligase in the transforming growth factor beta (TGFβ) pathway. We previously identified Arkadia as a small ubiquitin-like modifier (SUMO)-binding protein with clustered SUMO-interacting motifs (SIMs) that together form a SUMO-binding domain (SBD). However, precisely how SUMO interaction contributes to the function of Arkadia was not resolved. Through analytical molecular and cell biology, we found that the SIMs share redundant function with Arkadia's M domain, a region distinguishing Arkadia from its paralogs ARKL1/ARKL2 and the prototypical SUMO-targeted ubiquitin ligase (STUbL) RNF4. The SIMs and M domain together promote both Arkadia's colocalization with CBX4/Pc2, a component of Polycomb bodies, and the activation of a TGFbeta pathway transcription reporter. Transcriptome profiling through RNA sequencing showed that Arkadia can both promote and inhibit gene expression, indicating that Arkadia's activity in transcriptional control may depend on the epigenetic context, defined by Polycomb repressive complexes and DNA methylation [Sun, Liu, and Hunter (2014) Mol Cell Biol 34(16):2981-2995]. Overall design: To determine the role of Arkadia in TGFβ signaling at the transcriptome level, the profiles of TGFβ-stimulated gene expression were examined in Ark+/+ (Ark_WT), Ark-/- (Ark_null), and Arkadia (WT and sim mutant)-reconstituted Ark-/- MEFs. RNA sequencing was carried out using poly(A)-enriched RNA samples from unstimulated cells and cells treated with TGFβ for 1h, 4h, or 16h as indicated. Two batches of sequencing data for a total of 16 independent samples were submitted.

RING结构域蛋白Arkadia/RNF111是转化生长因子β（transforming growth factor beta，TGFβ）通路中的泛素连接酶。我们此前将Arkadia鉴定为一类带有聚集型SUMO相互作用基序（SUMO-interacting motifs，SIMs）的小泛素样修饰蛋白（small ubiquitin-like modifier，SUMO）结合蛋白，这些基序共同构成一个SUMO结合结构域（SUMO-binding domain，SBD）。然而，SUMO相互作用究竟如何调控Arkadia的功能仍未明确。通过分子与细胞生物学分析，我们发现SIMs与Arkadia的M结构域存在功能冗余——该结构域是Arkadia与其旁系同源蛋白ARKL1/ARKL2以及经典SUMO靶向泛素连接酶（SUMO-targeted ubiquitin ligase，STUbL）RNF4的区分区域。SIMs与M结构域共同促进Arkadia与Polycomb小体组分CBX4/Pc2的共定位，以及TGFβ通路转录报告基因的激活。通过RNA测序（RNA sequencing，RNA-seq）进行的转录组分析显示，Arkadia既可促进也可抑制基因表达，这表明Arkadia在转录调控中的活性可能取决于由Polycomb抑制复合物与DNA甲基化所定义的表观遗传背景[Sun, Liu, and Hunter (2014) Mol Cell Biol 34(16):2981-2995]。实验整体设计：为在转录组层面解析Arkadia在TGFβ信号通路中的作用，我们检测了TGFβ刺激下的基因表达谱，实验对象包括Arkadia野生型（Ark+/+，Ark_WT）、Arkadia敲除型（Ark-/-，Ark_null）以及回补了野生型或sim突变型Arkadia的Ark-/-小鼠胚胎成纤维细胞（mouse embryonic fibroblasts，MEFs）。我们对未刺激细胞、经TGFβ分别处理1h、4h或16h的细胞的poly(A)富集RNA样本进行了RNA测序。共提交两批测序数据，涵盖16个独立样本。