Effects Of SETD4 On LPS-induced IL-6 Transcription

SET domain is a conserved sequence in the carboxyl terminal of the three regulatory factors of Drosophila melanogaster proteins:Su(var)3-9,Enhancer of zeste,Trithorax.We call such proteins containing SET domain as the SET-domain containing protein.It has been shown that SET-domain containing proteins regulate gene transcription by methylating histone and non-histone.With the development of proteomics,more and more SET-domain containing proteins have been closely associated with the development of embryo development,tumor and inflammation related diseases.However,the function and mechanism of some proteins are still unclear.SETD4(SET domain-containing protein 4)is a differential protein screened by proteomics analysis in the liver of mice with endotoxic shock.Only partial functions in SETD4 have been reported,but the role of SETD4 in inflammation and its mechanism are still unclear.We found that SETD4 protein distributed in the whole cell,mainly distributed in cytoplasm,and expressed in various cells in the preliminary study.The expression of SETD4 and IL-6 mRNA were up-expression after stimulation of lipopolysaccharide(LPS),which suggests that SETD4 protein may be involved in the process of LPS-induced IL-6 transcription.Because SET family proteins play an important role in gene transcription regulation,In order to demonstrate that the main functional domain of SETD4 and whether its regulate IL-6 transcription by constructing SETD4 and its mutants plasmids.The RAW 264.7 cell line of stably expressing HA-SETD4 was constructed to provide cell model for the subsequent search for exploring SETD4 interaction proteins and the mechanism of SETD4.The experimental results have been obtained as follows:1.SETD4 and its mutantΔ SET,Δ Sub plasmids and IL-6 promoter luciferase reporter gene plasmid were constructed successfully;2.There were no effects on IL-6 promoter luciferase reporter gene in AML 12 cells transfected the expression plasmids of SETD4 and its mutantsΔ SET,Δ Sub;3.SETD4 promoted LPS-induced IL-6 mRNA in AML 12 cells,while the SET domain deletion significantly reduced the regulation,which suggested that the SET domain was the main functional structure of SETD4 protein;4.The RAW264.7 cell line of stably expressing HA-SETD4 was successfully constructed.Based on the above results,we have concluded that SETD4 enhancing IL-6 transcription mainly depend on its SET domain.The RAW264.7 cell line of stably expressing HA-SETD4 provides cell model for the subsequent search for SETD4 interacting proteins.