Identification And Characterization Of A Transcriptional Corepressor Complex For The Zinc Finger Protein ZBTB20

The transcription factor ZBTB20 is a member of zinc finger protein POK subfamily,which contains two conserved domains.One of them is BTB domain in the N-terminal,the other is the zinc finger domain in the C-terminal.ZBTB20 global knockout mice display a stark phenotype characterized by postnatal growth retardation,metabolic dysfunction,and lethality.Through the construction and analysis of tissue-specific ZBTB20 knockout mice model,our laboratory found that ZBTB20 plays an important role in the physiological processes of insulin secretion,glycogen storage,de novo lipogenesis,maintenance of circadian rhythm,innate immune response.ZBTB20 also participates in the regulation of differentiation and development of various tissues and organs,including pituitary prolactin cells,hippocampal CA1 cells,neocortical glial cells,plasma cells and cartilage.As a transcription factor,ZBTB20 binds to the promoter regions of AFP,FBP1,Brn2 and I? to exert transcriptional inhibition effect.It also directly mediates the transcription activation of some genes such as Ch REBP ?,Prl and Prokr2.It is noted that some other cofactors are required to involved in the process of the regulation of ZBTB20.ZBTB20 mediates protein polymerization through its N-terminal BTB domain and DNA binding through its C-terminal zinc finger domain.Now some studies have found that zinc finger protein can also mediate protein binding through its zinc finger domain.ZBTB7 and BCL-6,members of the same family of ZBTB20,can play a broader biological effect by recruiting transcriptional inhibition complexes.Nowadays,there are an increasing number of researches on the function of ZBTB20 and its role in human diseases.However,it is still unclear about the cofactors involved in the transcription regulation of ZBTB20 and their regulatory mechanisms.In order to identify and characterize the cofactors of ZBTB20,we established the co-immunoprecipitation and mass spectrometry analysis system of ZBTB20,and found that ZBTB20 protein binds to nucleosome remodeling and deacetylase complex(NuRD)in the mice liver tissue.We optimized the expression and purification conditions of GST fusion protein of ZBTB20,and successfully purified the full-length ZBTB20-GST fusion protein.Then we verified the combination of ZBTB20 and GATAD2 A in vitro through GST-pull down assay and found that GATAD2 A binds to the zinc finger domain of ZBTB20.And ZBTB20 specifically binds to multiple subunits(GATAD2A/B,MBD2,RBBP4/7,HDAC1,MTA1/3)in NuRD.In summary,we identified and characterized ZBTB20 transcriptional corepressor complex NuRD by establishing and optimizing the ZBTB20 related protein interaction research system,and found the binding regions between ZBTB20 and NuRD by using the ZBTB20 truncated proteins.This study contributes important clue for revealing the transcriptional inhibition mechanism of ZBTB20 protein.