Transcriptional Down-regulation Of Smurf1/2Ubiquitin Ligase By Upstream Stimulate Factor2(USF2)

Objective:To express USF2is a negative transcription regulator of Smurfl and Smurf2gene expressions, which can repress Smurf1/2protein and mRNA levels. And further study the mechanisms of this regualtion. Identify the functional region down regulating Smurf1/2transcriptional level.Methods:1. Construction of eukaryotic expression vectors of the USF2and its truncated genesThe reverse primers were designed for the USF2and its two truncated genes,USF2(1-235aa) and USF2(236-346aa). Using polymerase chain reaction and gene recombination techniques, USF2gene and two fragments deletions were amplified and cloned into pCMV-Myc vector respectively. The positive clones were identified by restrictive enzymes assay and then transfected into MCF7cells to detect their expressions.2. Transcriptional Down-regulation of Smurfl/2Ubiquitin Ligase by USF2(1) Effect of Smurfl/2on regulation of the USF2steady-state levels.MCF7cells seeded in12-well plate were transfected with increasing amounts (0μg,0.8μg,1.4μg,2μg) of Flag-Smurf1/2. Cell lysates were immunoblotted with anti-Flag-USF2, and-GAPDH antibodies. Coimmunoprecipitation of endogenous USF2and endogenous Smurfs with each other from MCF7cells. Western blot analysis of whole-cell lysate and the indicated immunoprecipitates used anti-USF2and anti-Smurfl/2antibodies.(2) Reach on USF2negatively regulating Smurfl/2at the transcriptional level.Cells seeded in24-well plate were transfected with increasing amounts (0μg,0.8μg,1.4μg,2μg) of Myc-USF2. Cell lysates were immunoblotted with anti-Myc,-Smurfl,-Smurf2, and-GAPDH antibodies. Total RNA was subjected to real-time RT-PCR. Transfecting with USF2-siRNA (10nM) or control-siRNA (10nM) using siRNA transfection reagent in293T for72h. After treatment, cells were harvested. Total RNA was subjected to real-time RT-PCR. Cell lysates were immunoblotted with anti-Smurfl,-Smurf2,-USF2, and-GAPDH antibodies.cells were transfected with USF2or mock vectorc, and Smurfl/2promoter activity was assayed. Cells were transfected with USF2-specific siRNA or non-targeted control, Smurf1/2promoter activity was assayed.(3) USF2binds to the Smurf1/2promoters in vivo and vitro.The recovered DNA in MCF7cells was analyzed by PCR with primers amplifying a region of the Smurf1/2promoters. The PCR products were stained with ethidium bromide after electrophoresis on a1%agarose gel. Quantitative ChIP assays by real-time PCR.The purified GST-tagged USF2protein expressed by E.coli was evaluated by Western Blot with anti-GST antibody. EMSA was performed using probes representing Smurfl/2promoter regions with GST-USF2protein or GST-tag. Cell lysates were obtained and electrophoretic mobility shift assays performed as described in the Materials and Methods.Competition EMSA experiment with addition of10-fold or20-fold excess of unlabelled cold probes or the mutant Smurf1/2probes. Mut, mutant. Supershift assay as a control using an USF2antibody (10ug) to demonstrate that USF2is involved in the complex.3. The investigation of the functional region down regulating Smurfl/2transcriptional levelTransfecting Myc-USF2,Myc-USF2(1-235aa) or Myc-USF2(236-346aa) into MCF7cells to detect their expressions. The region that inhibit Smurf1/2transcription was explored by Western Blot and real-time PCR techniques.Results:1. Constructing eukaryotic expression vectors of theUSF2and its truncated genes successfullySuccessfully cloned USF2and its truncated genes into relevant vector. Through Western Blot and real-time PCR, the236-346aa region at the C terminal of USF2could inhibit the transcriptional level of Smurfl/2.2. Transcriptional down-regulation of Smurfl/2ubiquitin ligase by USF2(1). Smurfl/2do not regulate the steady-state levels of USF2.Overexpression of Smurfl/2had no obvious effect on the steady-state levels of USF2. The endogenous Co-IP assay was carried out to investigate whether USF2protein could associate with Smurfl/2. Smurfl/2protein could not be detected in USF2immunoprecipitate. All these results imply that USF2might not be a substrate of Smurfl/2.(2). USF2negatively regulates Smurfl/2at the transcriptional level.We went on to check whether it can regulate the expression of Smurfs. Smurfl and Smurf2proteins were obviously reduced upon ectopic expression of USF2. We further confirmed that the regulation of Smurfl/2by USF2was at the transcription level, as Smurfl/2transcripts significantly decreased when USF2was ectopically expressed. In order to further validate that USF2represses Smurfl/2, we used USF2-siRNA to knockdown the expression of USF2. The decrease of USF2expression can resulted in elevated protein and mRNA levels of Smurfl/2. Our data collectively demonstrated that USF2negatively regulates Smurfl/2at the transcriptional level.To check whether USF2modulates the transcriptional activity of Smurfl/2upon binding to E-box, we co-transfected Myc-USF2with luciferase reporter plasmids pG13s that contain the Smurfl/2promoters into H1299cells. ectopic expression of USF2remarkably inhibited the activity of Smurf1/2promoters. While Knockdown of endogenous USF2by siRNA significantly enhanced Smurfl and Smurf2promoter activity in H1299cells Collectively these results support that USF2can bind to Smurfl/2promoters and efficiently repress their transcriptional activity.(3) USF2binds to the Smurfl/2promoter specificallyTo demonstrate the interaction of USF2with the Smurf1/2promoters, we assessed the ability of USF2to bind to the Smurfl/2E-boxes in vivo by ChIP. The USF2antibody precipitated both the Smurfl and Smurf2E-box to a much larger extent than a control anti-IgG antibody. The quantitative ChIP also assayed by real-time PCR. Thus,USF2appears to bind to the Smurfl/2promoters in vivo.An EMSAs was performed using purified GST-USF2protein with double-stranded oligonucleotides containing the E-box motifs as probes. In this assay, the formation of a DNA-protein complex was identified on the gel.To characterize the specificity of the DNA-protein interactions, and to establish the role of the E-box element present in the Smurf1/2promoters, EMSAs competition experiments were performed. The labeled DNA-protein complexes were dissociated upon addition of excess competitors. Furthermore, when mutated E-box probes were used in these experiments,. No DNA-protein complex was detected. Thus, mutating the E-box in the probe abolished its binding to USF2.To further affirm the presence of USF2proteins in the binding complexes, we performed supershift EMSAs with an antibody against USF2. A clear shifting of the original complexes was detected with addition of the antibody.3. The investigation of the functional region down regulating Smurf1/2transcriptional levelBy transfecting the eukaryotic expression vectors of the USF2and its truncated genes, and then assayed by Western Blot and real-time PCR, the Myc-USF2the Myc-USF2(236-346aa) could inhibit the transcriptional level of Smurf1/2.Conclusions:USF2as a negative transcription factor of Smurf1/2, decreased Smurf1/2mRNA and their protein level by combining with the E-box motif in Smurf1/2promoter region. As far as we know this is the first evidence to reveal a negative transcription factor for Smurf1/2. And we demonstrated that USF2suppresses Smurf1/2 transcriptional level through the236-346aa region at the C terminal.