The Analysis Of BRWD3 Protein Complex Involved In Transcriptional Regulation

Previous studies have shown that BRWD3(bromodomain And WD Repeat Domain Containing 3)mutation may lead to intellectual disability(ID),but as an important chromatin modifying molecule and transcription factor,its mechanism is unknown.BRWD3 is composed of two important domains,WD40 and bromodomain,respectively.It can specifically identify and bind acetylated lysine to specific chromatin targets with bromodomain.In order to study the role of BRWD3 in the transcriptional regulation process,we explored the composition of BRWD3 protein complex based on yeast two hybrid system and biolayer interferometry technology(BLI),and then,used ChIP-seq,RNAi,Luciferase Assay to analysis that BRWD3 has an transcriptional regulation effect on the downstream ID related gene.The results indicated:(1)Using the bromodomain of BRWD3 as bait,we identified 12 interacting proteins in the cDNA library of human brain embryonic through yeast two hybrid system combined with sequencing.Subsequently,we firstly selected NUFIP1 for further study.(2)Firstly,we respectively constructed the sequence of BRWD3 bromodomain region and NUFIP1 encoding region into the prokaryotic expression vector.Then,we explored the optimum conditions for the expression of prokaryotic protein,induced and purificated protein.Finally,we detected the interaction of BRWD3 and NUFIP1 by BLI and noticed that the binding signal of BRWD3 constantly enhanced along with the increase of concentration of NUFIP1.The affinity constant K_D(9.12E-08,R~2=0.967)doesn,t change with the variety of concentration.The interaction between BRWD3 and NUFIP1 was confirmed in vitro.(3)In order to screen the regulation downstream genes of BRWD3,we transfected pcDNA3.1-2Brd into cell line,and obtained 5874 downstream genes of BRWD3 with the method of ChIP-seq.Then,for the sake of understanding the effect of BRWD3 mutation on ID,we analyzed and screened the only 19 ID releated genes.(4)To analysis the interaction of BRWD3 and NUFIP1 involved in the transcription of ID releated gene,we detected that the mRNA relative expression of SETD5,SMARCB1,RLIM,SOBP,THOC2 and MEF2 C were significantly downregulated,while ELP2(p<0.05)was significantly upregulated by Q-PCR,after respectly knocking down the expression of BRWD3 and NUFIP1.Subsequently,we noticed that BRWD3 and NUFIP1 maybe combine with MEF2 C promoter region to regulate transcription with the assay of ChIP-seq,Q-PCR,Luciferase.In this study,we analyzed chromatin modifying factor and transcription factor BRWD3 and NUFIP1 possibly regulated the transcription of MEF2 C gene and provided some clues to understand the mechanism of ID induced by BRWD3 mutation.The limitation is that BRWD3 may interact with other proteins to affect neuronal activity,nervous system development and maturation by regulating downstream gene transcription.Therefor,it is still a need for further research on how BRWD3 affects the development of the nervous system.