RNF4 acted as an ubiquitin E3 ligase involved in ubiquitin‐dependent degradation of Sumoylated‐Sp1.

Sp1 is a ubiquitously expressed transcription factor that plays important role in the regulation of many GC‐rich genes encoding for housekeeping proteins. The transcriptional activity of Sp1 is mainly determined by three elements: transcriptional activity, DNA binding ability and its protein stability. Our previously study revealed that modification of Sp1 by SUMO‐1 altered its localization and ubiquitination and then result in Sp1 degradation. Recently, mammalian RNF4, a Ring finger‐containing ubiquitin E3 ligase, targets sumoylated protein for ubiquitin‐dependent degradation. Our preliminary data showed that sumoylated‐Sp1 is prone to recruit RNF4 than wild‐type Sp1. Ectopic expression of RNF4 markedly decreased the protein level of Sp1 in a RNF4 dose dependent manner. By overexpressed the RNF4/CS1, a Ring domain mutant which has been reported that devoid of ubiquitin E3 ligase activity, the protein level of Sp1 did not change. In vitro pulldown assay also revealed that sumoylation increased its interaction with RNF4. Moreover, In vitro ubiquitination assay was used to determine the ubiquitination level of Sp1 in the presence of RNF4, we found that Sp1 is access to ubiquitination with RNF4. Therefore, we infer that RNF4 acted as an ubiquitin E3 ligase targets sumoylated‐Sp1 for ubiquitin‐dependent degradation.