E3 Ubiquitin Ligase TRIM21 Regulates The Chromatin Remodeling Factor BAF155 Through The Ubiquitin-proteasome System

Background:The BAF(Brg/Brm-associated factors)complex is an ATPase-dependent multisubunit chromatin remodeler,which can remodel chromatin by recruiting epigenetic factors to regulate specific gene expression.Studies have shown that BAF complexes play important roles in maintaining normal biological functions,especially in neurodevelopmental processes,including self-renewal and proliferation of neural stem cells,dendrite development,and neural circuit formation.The core subunits BAF155 and BAF170 of the BAF complex maintain the integrity of the complex.When BAF155 and BAF170 are knocked out,all other known BAF subunits are dissociated from the complex and degraded by the proteasome pathway,thus affecting chromatin regulation and gene expression of nerve cells.However,the regulatory mechanism of BAF155,the core subunit in BAF complex,remains unclear.Our previous studies have shown that E3 ubiquitin ligase TRIM21 may have a potential physical interaction with BAF155.However,this interaction needs to be further confirmed and the biological significance of the interaction also needs to be further explored.We hypothesize that TRIM21 regulates BAF155 protein degradation through physical interactions,thereby further regulating the biological function of BAF155.Understanding how TRIM21 regulates the protein levels and function of the chromatin remodeling factor BAF155 will help reveal the homeostasis regulation mechanism of BAF155,which is crucial for understanding the neurosystem-related diseases involved in TRIM21 mutations and/or BAF155 mutations.Objective:The purpose of the study is to explore the interaction between E3 ubiquitin ligase tripartite motif-containing protein 21(TRIM21)and chromatin remodeling factor BAF155,and to illustrate the specific molecular mechanisms of the regulation of BAF155 homeostasis by TRIM21.Methods:1.Construct or obtain required plasmids.The mammalian expression plasmids used in the study come as following: the constructs of p CAGGSBS-BAF155、pCMV-myc-nuc-BAF155、TRIM21、HA-TRIM21、HA-TRIM21-△RING、HA-TRIM21-△B-BOX、HA-TRIM21-△SPRY、HA-TRIM21-△PRY、Control-shRNA、TRIM21-shRNA#1、TRIM21-shRNA#2、TRIM21-shRNA#3 are kind gifts from Dr.Tran Tuoc.The plasmid of pcDNA3.1-3xHA-Ub is kindly presented by Dr.Yumei Li.The constructs of pcDNA3.1-3xHA-Ub-K48 R and pcDNA3.1-3xHA-Ub-K63 R were generated via PCR-mediated mutagenesis.The plasmids of pCMV-myc-nuc-BAF155(1-956 aa)、(1-752 aa)、(1-608 aa)、(1-377 aa)、(402-1105 aa)、(596-1105 aa)、(595-95 6aa)were generated by insert the propriate PCR produced-BAF155 cDNA fragment from pCMV-myc-nuc-BAF155 into the backbone of pCMV-myc-nuc-BAF155,between the restriction enzyme sites of Sal I and Not I.The plasmid of pcDNA3.1-HA-TRIM21-LD(C16A,C31 A and H33W)was generated through PCR-mediated mutagenesis,the TRIM21-LD cDNA fragment was inserted into pcDNA3.1-HA between EcoRI and XhoI sites.The plasmid of pcDNA3.1-HA empty vector was a kind gift from Prof.Hueng-sik Choi.2.The physical interaction between TRIM21 and BAF155 was investigated by CoImmunoprecipitation technique.Human embryonic kidney cells(HEK293T)were cotransfected with the mammalian expression vectors encoding HA-TRIM21 and Myc-BAF155 proteins respectively,and the Myc-BAF155 proteins were immunoprecipitation by Myc antibody,followed by the investigation of the presence of HA-TRIM21 in the co-precipitation complex.Thus,the interaction between cellular exogenous HA-TRIM21 and Myc-BAF155 were clarified.In parallel,whole cell lysates were extracted from HEK293 T,and endogenous BAF155 protein was immunoprecipitated by BAF155 antibody.Western Blotting was used to detect whether TRIM21 protein was co-precipitated,so as to determine whether there was an interaction between endogenous TRIM21 and BAF155.3.The effect of overexpression or knockdown of TRIM21 on BAF155 protein expression in human embryonic kidney cells(HEK293T)and mouse brain neuroma cells(Neuro-2A)was detected by Western Blot(WB).After overexpression or knockdown of TRIM21 gene expression in HEK293 T and Neuro-2A cells,the protein levels of BAF155 under different treatment were conditions were detected by WB after treatment with proteasome inhibitor MG132,so as to explore the specific molecular mechanism of TRIM21’s involvement in the homeostasis regulation of BAF155.Results:1.TRIM21 interacts with BAF155When Myc-BAF155 and HA-TRIM21 were overexpressed in HEK293 T,HA-TRIM21 was co-immunoprecipitated with Myc-BAF155 by the Myc antibody,indicating exogenous BAF155 interacts with TRIM21.In parallel,when whole cell lysates of HEK293 T were extracted and BAF155 was pull-down by the specific BAF155 antibody,the protein of TRIM21 was detected in the precipitated complex,indicating endogenous BAF155 interact with TRIM21 in cells.2.Overexpression/Knockdown of TRIM21 can decrease/increase the expression level of BAF155 protein,respectively.In Neuro-2A cells,when expression plasmids encoding Myc-BAF155 and pcDNA3.1(empty vector)were transfected in the control group,while the plasmids encoding Myc-BAF155 and HA-TRIM21 were transfected in the experimental group,we found that compared with the control group,the protein expression level of Myc-BAF155 in the experimental group was decreased by the overexpression of HA-TRIM21.Similarly,Neuro-2A cells were co-transfected with Control-shRNA or TRIM21-shRNA expression plasmids,and our results showed that MycBAF155 protein levels were elevated when TRIM21 was knocked down by TRIM21-shRNA.Those data indicate that TRIM21 negatively regulates the protein level of BAF155.3.TRIM21 mediated degradation of BAF155 depends on its ubiquitin ligase activity.Co-transfected expression plasmids encoding Myc-BAF155 with pcDNA3.1/TRIM21-WT/TRIM21-LD in HEK293 T cells.Compared with the effect of overexpression of TRIM21-WT on BAF155,the ability of the TRIM21-LD mutant to mediate the degradation of BAF155 was significantly reduced,indicating the negative regulation of TRIM21 on BAF155 depends on the E3 ligase activity of TRIM21.4.Overexpression of Ubiquitin increased the ubiquitination level of BAF155,while TRIM21 significantly promoted the ubiquitination modification of BAF155.HEK293T cells were co-transfected with HA-Ub and Myc-BAF155 encoding constructs.After treatment with MG132 for 0 h,12 h and 2 4h,Myc antibody immunoprecipitation was performed,and ubiquitin antibody was used to detect the ubiquitination level of BAF155.Overexpression of ubiquitin promoted the ubiquitination level of Myc-BAF155 and coincubation of MG132 made the ubiquitination levels of Myc-BAF155 more obvious.Interestingly,overexpression of TRIM21 significantly promoted the ubiquitination level of BAF155.5.Overexpression of TRIM21 resulted in increased degradation of BAF155 due to increased ubiquitination modification,while MG132 treatment could partially restore BAF155 protein level.The WB results after co-transfection of Myc-BAF155 and HA-TRIM21 encoding constructs showed that overexpression of TRIM21 could reduce the protein levels of MycBAF155,while MG132 co-incubated for 12 h or 24 h could partially restore the decreased expression levels of Myc-BAF155 by TRIM21.6.Ub-K63 was involved in the TRIM21-mediated ubiquitination of BAF155.The expression plasmids encoding Myc-BAF155,HA-Ub WT/HA-Ub K48R/HA-Ub K63 R and HA-TRIM21 were co-transfected into HEK293 T cells.The co-precipitation experiment using Myc antibody demonstrated that the ubiquitination levels of BAF155 decreased when using HA-Ub K63 R mutants,compared with that from wild type HA-Ub..7.The RING and SPRY functional domains of TRIM21 were involved in the degradation of BAF155.The amino acid residue of K354 in BAF155 is the major ubiquitination site for TRIM21-mediated degradation.The mammalian expression plasmids for truncated mutants of Myc-BAF155 and HATRIM21 were co-transfected into HEK293 T cells.Firstly,our western Blot results demonstrated that BAF155(1-608 aa)is the minimum domain that could be degraded by TRIM21.Secondly,mammalian expression vectors for lysine mutants in BAF155(1-608 aa)were constructed.In addition,all the vectors for the mutants were co-transfected with HA-TRIM21 into HEK293 T cells as indicated in the figures,and the residue of K354 of BAF155 was identified as the main ubiquitination site for TRIM21-mediated degradation.In a parallel experiment,truncated mutatnts of HA-TRIM21 and Myc-BAF155 were co-overexpressed in HEK293 T cells.Western Blotting results showed that Myc-BAF155 was almost not degraded when the RING and SPRY domains in HA-TRIM21 were absent.Conclusion:Through a series of biochemical experiments,we demonstrated that TRIM21 can act a negative regulator on the key subunit BAF155 in the chromatin remodeling BAF complex,and it reduces the stability of BAF155 through the ubiquitin-mediated proteasome pathway.