Fig 2: DCAF1 controls T-cell response during viral infection and autoimmunity.(a,b) 1 × 106 of CD4+ T cells isolated from Cd4Cre;Dcaf1fl/+;SMARTA mice (CD45.2+) and Cd4Cre;Dcaf1fl/fl;SMARTA mice (CD45.1+CD45.2+) were mixed and transferred into syngeneic wild-type recipient mice (CD45.1+) followed by LCMV infection (+LCMV) or remain uninfected (-LCMV). The percentages (a) and the numbers (b) of donor cells of different genotypes were determined by flow-cytometry 5 days after infection. Representative results and means±s.d. of 10 mice of 2 experiments are shown (*P<0.05 by Student's t-test). (c,d) Wild-type (WT) (n=8) and Cd4Cre;Dcaf1fl/fl (n=6) mice were injected with MOG/CFA to elicit autoimmune encephalomyelitis (EAE). The disease clinic scores were recorded at indicated time points. Means±s.d. are shown (c) (*P<0.05 by Mann–Whitney's U-test). The numbers of spinal cord infiltrating CD4+ T cells were assessed 22 days post EAE elicitation. Means±s.d. are shown (d) (*P<0.05 by Student's t-test).

Fig 3: DCAF1 deficiency leads to p53 protein stabilization.(a,b) The protein expression of p53, p21 and c-Myc detected by immunoblotting (a) and the mRNA expression of p21 and Bax detected by quantitative reverse transcription–PCR (qRT–PCR) (b) in CD4+ naive T cells of different genotypes at indicated time points after being activated with anti-CD3 and anti-CD28. (c,d) CD4+ naive T cells of various genotypes were activated with anti-CD3 and anti-CD28 in the presence of 4-hydroxy-tamoxifen. At indicated time points after activation, p53 and c-Myc protein expression was determined by immunoblotting (c), and p21 and Bax mRNA expression was assessed by qRT–PCR (d). (e,f) The protein expression of p53 by immunoblotting (e) and mRNA expression of p21 and Bax by qRT–PCR (f) in the quiescent and IL-2-re-stimulated effector T cells, which were generated in the presence of 4-hydroxy-tamoxifen as described in Fig. 4e,f. (g) The protein expression of p53 in CD4+ naive T cells of different genotypes at indicated time points after being activated with anti-CD3 and anti-CD28 in the presence and absence of proteasome inhibitor MG132. (h) p53 protein half-life in anti-CD3- and anti-CD28-activated (for 24 h) CD4+ T cells of different genotypes, determined by immunoblotting for p53 in the presence of translation inhibitor cycloheximide (CHX). (i) Human osteosarcoma U2OS cells were transduced (GFP+) by lentiviruses expressing CRISPR only (Control), CRISPR-Dcaf1-1 (sgRNA) or CRISPR-Dcaf1-2 (sgRNA) and then transfected by the plasmids expressing Flag-tagged Mdm2 and HA-tagged Ubiquitin (HA-Ub) as indicated. The poly-ubiquitination of immunoprecipitated (IP) p53 was detected by immunoblotting for HA tag. In this figure, results of immunoblotting are representative of three experiments. For quantitative reverse transcription–PCR assays, means±s.d. of triplicate done in one experiment representative of three are shown (*P<0.05 by Student's t-test). See also Supplementary Figs 4 and 5 and Supplementary Table 2.

Fig 4: DCAF1 interacts with COP9 signalosome.(a) The proteins preferentially immunoprecipitated by anti-DCAF1 over anti-IgG in TCR-stimulated CD4+ T cells were identified by mass spectrometry. COP9 signalosome components and CRL4 subunits identified were listed in the table (a), including the number of unique peptides (Unique Peptides) detected and the percentage of sequence coverage (Sequence Coverage). Results were obtained from three experiments. (b) Co-immunoprecipitation of CSN1, CSN2 with DCAF1 in TCR-stimulated CD4+ T cells. (c) The protein–protein interaction network of DCAF1 containing CRL4–COP9 mega complex was generated using the STRING database (Version 9.1, http://string-db.org/), based on the proteins identified in a. (d,e) The protein expression of CSN1, CSN2, DDB1, CUL4A and DCAF1 was determined in CD4+ T cells of different genotypes before (0 h) and 24 h after TCR stimulation. All results are representative of three independent experiments. See also Supplementary Table 1.

Fig 5: NONO blocks the ubiquitination-mediated degradation of SAMHD1 by DCAF1.A, B Overexpression or silencing of DCAF1 decreased or increased SAMHD1 expression, respectively. A HL60 cells were transfected with increasing concentrations of HA-tagged DCAF1 24 h, and the levels of the indicated proteins were determined by western blotting. B THP-1 cells were transfected with DCAF1 siRNAs for 24 h, and the levels of the indicated proteins were then determined by western blotting. C DCAF1 interacts with SAMHD1. 293 T cells were transfected with HA-tagged DCAF1 and Myc-tagged SAMHD1 for 24 h. Co-IP was performed with anti-Myc agarose, and the interactions were examined by western blotting. D Overexpression of DCAF1 increased the ubiquitination level of SAMHD1. Myc-tagged SAMHD1 was cotransfected with HA-tagged DCAF1, Flag-ubiquitin or empty vector into 293 T cells for 24 h, and the ubiquitination level of SAMHD1 was determined using IP followed by western blotting. E NONO blocked DCAF1-mediated degradation of SAMHD1. Myc-tagged SAMHD1 and HA-tagged DCAF1 were cotransfected into 293 T cells with or without overexpression of HA-tagged NONO for 24 h. The levels of the indicated proteins were determined by western blotting. F Overexpression of NONO inhibited DCAF1-mediated ubiquitination of SAMHD1. Myc-tagged SAMHD1, HA-tagged DCAF1, and Flag-tagged ubiquitin were cotransfected into 293 T cells with or without overexpression of HA-tagged NONO for 24 h. The ubiquitination of SAMHD1 was evaluated by IP followed by western blotting. G NONO inhibits the interaction of DCAF1 and SAMHD1. Myc-tagged SAMHD1 and HA-tagged DCAF1 were cotransfected into 293 T cells with or without overexpression of Myc-tagged NONO for 24 h. Cells were subsequently subjected to co-IP followed by western blotting with the indicated antibodies. H Silencing of NONO increases the interaction of DCAF1 with SAMHD1. THP-1 cells silencing NONO for 48 h were lysed and were subsequently subjected to co-IP followed by western blotting with the indicated antibodies. For respective immunoblots, the protein levels were quantified by ImageJ software.