Exploration Of The Upstream Regulation Mode Of ZNF366 In M2 Macrophages

Background&PurposeZNF366 was first isolated from monocyte-derived dendritic cells in 2006,so ZNF366 is also known as DC-SCRIPT.Zinc finger protein 366 has a finger-like domain that regulates transcription and translation of genes by binding to itself or other zinc finger proteins.Studies have found that ZNF366 can act as a co-regulator of multiple nuclear receptors,helping to regulate the expression of downstream genes.As an important biomolecule,ZNF366 has many researches on its downstream regulation,but research on its upstream regulation mode is still rare.By analyzing the M0/M2-mRNAs chip,the team first found that ZNF366 is highly expressed in M2 macrophages compared with M0,suggesting that ZNF366 may be involved in the differentiation of macrophages.Therefore,this paper predicts,analyzes and verifies the promoter region of ZNF366,and explores its upstream regulation mode,which lays a foundation for further research on the biological process of ZNF366 regulating M2 macrophages.Methods&Results1.mRNAs chip analysis of ZNF366 expression in M2 macrophagesThe literature was used to establish an induction program,and PBMC were induced to form M0-type and M2-type macrophages by cytokines,and the gene expression differences between the two were compared by mRNA chip analysis.2.Analysis of ZNF366 promoter gene sequenceThe upstream～2.0 kb sequence was taken as the promoter region to be studied by NCBI,UCSC and other authoritative websites.The GC content and CpG island formation ability were analyzed using tools such as CpG-Plot,and the binding of histone modification and transcription factor was analyzed in JASPAR.It revealed that the promoter region is likely to be regulated by the following transcription factors:GATA2,PU.1 and CTCF.3.Quantitative PCR detects the expression of ZNF366 after siRNAs interfere with transcription factorsMultiple siRNAs were designed for GATA2,SPI1 and CTCF.The siRNAs were transfected into M2 macrophages by liposome.The transfection efficiency was detected by qPCR.After the expression of transcription factors was down-regulated,the mRNA and protein of the target gene ZNF366 were analyzed.4.Dual-luciferase assay for analysis of transcriptional activity of promoter fragmentsThe promoter region of transcription factor binding was segmented into(0～1000bp),(0～-1300bp)and(0～-2000bp),and constructed recombinant vectors containing promoter fragments.The recombinant vectors containing flyfire luciferase was co-transfected with the pRL-TK vector containing renilla luciferase into the cell line by liposome technique,and the transcriptional activity of each fragment was detected.5.Chromatin immunoprecipitation to detect the binding of transcription factors to ZNF366 promoter regionThe chromatin of M2 type macrophages was enriched by transcription factor antibody,and the binding between transcription factors and promoter regions and transcription factors was detected by PCR and WB,respectively.Conclusions1.mRNAs microarray was used to detect gene expression changes in M0/M2 macrophages.The results showed that ZNF366 was highly expressed in M2 macrophages.2.Analysis of the ZNF366 promoter region revealed no CpG island formation but was able to bind to the transcription factors GATA2,PU.1 and CTCF.3.The results of siRNAs-qPCR interference showed that transcription factors such as SPI1 and CTCF could significantly down-regulate the expression of ZNF366 in M2 macrophages,while GATA2 down-regulated its protein expression,but had no significant effect on mRNA levels.4.The results of dual luciferase assay showed that the transcriptional activities of(0～-1kb)and(0～-1.3kb)were significantly up-regulated,but there was no significant change in the transcriptional activity of the(0～-2kb)fragment.5.Chromatin immunoprecipitation showed that PU.1 binds to the ZNF366 promoter region and binds to the transcription factor GATA2.