Molecular regulation of PPAR?/RXRa signaling by the novel cofactor ZFP407

Cofactors interacting with PPAR? can regulate adipogenesis and adipocyte metabolism by modulating the transcriptional activity and selectivity of PPAR? signaling. ZFP407 was previously demonstrated to regulate PPAR? target genes such as GLUT4, and its overexpression improved glucose homeostasis in mice. Here, using a series of molecular assays, including protein-interaction studies, mutagenesis, and ChIP-seq, ZFP407 was found to interact with the PPAR?/RXRa protein complex in the nucleus of adipocytes. Consistent with this observation, ZFP407 ChIP-seq peaks significantly overlapped with PPAR? ChIP-seq peaks, with more than half of ZFP407 peaks overlapping with PPAR? peaks. Transcription factor binding motifs enriched in these overlapping sites included CTCF, RARa/RXR?, TP73, and ELK1, which regulate cellular development and function within adipocytes. Site-directed mutagenesis of frequent PPAR? phosphorylation or SUMOylation sites did not prevent its regulation by ZFP407, while mutagenesis of ZFP407 domains potentially necessary for RXR and PPAR? binding abrogated any impact of ZFP407 on PPAR? activity. These data suggest that ZFP407 controls the activity of PPAR?, but does so independently of post-translational modifications, likely by direct binding, establishing ZFP407 as a newly identified PPAR? cofactor. In addition, ZFP407 ChIP-seq analyses identified regions that did not overlap with PPAR? peaks. These non-overlapping peaks were significantly enriched for the transcription factor binding motifs of TBX19, PAX8, HSF4, and ZKSCAN3, which may contribute to the PPAR?-independent functions of ZFP407 in adipocytes and other cell types. Overall design: Chromatin Immunoprecipitation DNA Sequencing (ChIP-Seq) of Zfp407 in differentiated 3T3-L1 adipocytes

与过氧化物酶体增殖物激活受体γ（PPARγ）相互作用的辅因子，可通过调控PPARγ信号通路的转录活性与结合选择性，调节脂肪生成过程与脂肪细胞代谢功能。此前已有研究证实，锌指蛋白407（ZFP407）可调控PPARγ的靶基因（如葡萄糖转运蛋白4（GLUT4）），且其过表达可改善小鼠体内的葡萄糖稳态。本研究通过一系列分子生物学实验——包括蛋白质相互作用分析、定点诱变实验及染色质免疫共沉淀测序（ChIP-seq）——发现，ZFP407可在脂肪细胞核内与PPARγ/视黄醇X受体α（RXRα）蛋白复合物发生特异性相互作用。与此观测结果一致的是，ZFP407的ChIP-seq结合峰与PPARγ的ChIP-seq结合峰存在显著重叠，超过半数的ZFP407结合峰与PPARγ结合峰发生重合。在这些重叠结合位点中富集的转录因子结合基序包括CTCF、视黄酸受体α/过氧化物酶体增殖物激活受体γ（RARα/RXRγ）、TP73及ELK1，这些基序可调控脂肪细胞内的细胞发育与生理功能。对PPARγ常见的磷酸化或类泛素化修饰位点进行定点诱变，并未阻断ZFP407对其的调控作用；而对ZFP407中可能参与RXR与PPARγ结合的结构域进行诱变，则完全消除了ZFP407对PPARγ活性的调控效应。上述实验数据表明，ZFP407可调控PPARγ的转录活性，且该调控过程不依赖于翻译后修饰，极有可能通过直接蛋白结合实现，由此确定ZFP407为新发现的PPARγ辅因子。此外，ZFP407的ChIP-seq分析还鉴定出了未与PPARγ结合峰重叠的基因组区域。这些非重叠结合峰显著富集了TBX19、PAX8、HSF4及ZKSCAN3的转录因子结合基序，这些基序可能参与介导ZFP407在脂肪细胞及其他细胞类型中不依赖于PPARγ的功能调控。总体实验设计：在分化成熟的3T3-L1脂肪细胞中开展Zfp407的染色质免疫共沉淀测序（ChIP-seq）