Fig 2: TDG is enriched at enhancers and promoters genome-wide and colocalizes with the XPC complex subunit RAD23B. (A) The number of ChIP-seq peaks identified using MACS2 for each data set. Approximately 93% of biotin–TDG (Neri et al. 2015) ChIP-seq peaks overlap with RAD23B in mouse ESCs. (B) Heat map of TDG- and RAD23B-bound regions over a ±5-kb window around the TSSs of genes. Genes in each heat map are sorted according to descending levels of TDG ChIP-seq signal. (C) Integrative Genomics Viewer (IGV)-computed ChIP-seq tracks are plotted as (number of reads) × [1,000,000/(total read count)] for pluripotency genes Nanog and Pou5f1. Mock and normal IgG were used as specificity controls for the TDG and RAD23B ChIP, respectively.

Fig 3: XPC increases somatic reprogramming fidelity and iPSC self-renewal capacity. (A) Schematic diagram for human iPSC induction modified from methods in Okita et al. (2011). (B) Flow cytometry analysis of control and XPC complex (XPC–RAD23B–CETN2) gain-of-function cell populations 24 d post-induction for the late stage human iPSC marker TRA-1-60. (C) The average number of colonies obtained in the reprogramming experiment depicted in B. (D) iPSCs derived from control or XPC–RAD23B–CETN2-overexpressing HDFs were challenged with single-cell dissociation and allowed to recover for 3–4 d before staining with alkaline phosphatase (AP). The average number of AP+ colonies formed per 2.5 × 104 single cells plated. Error bars depict the standard deviation. n = 3. (*) P < 0.05, calculated by two-tailed Student's t-test.

Fig 4: XPC-mediated stimulation of TDG activity requires the C terminus of XPC and N terminus of TDG. (A) Schematic representation of human XPC (wild-type [WT]) highlighting the DNA-binding domain (DBD) and various protein–protein interaction domains. XPC truncations used in this study—ΔN (195–940 amino acids), Δ338 (del338–519 amino acids), and ΔC (1–814 amino acids)—are also shown. (B) TDG cleavage activity of a 5′-labeled T:G mismatch-containing oligonucleotide dsDNA in the presence of wild-type XPC or various XPC truncation complexes (ΔN, Δ338, and ΔC). (C) The CETN2 subunit is dispensable for stimulation of TDG-mediated base excision. TDG cleavage activity of a 5′-labeled T:G mismatch DNA substrate in the presence of the XPC–RAD23B heterodimer or a heterodimer containing ΔC XPC and RAD23B. (D) Schematic representation of full-length human TDG (FL) and truncations used in the in vitro glycosylase assay. The domains shown previously to be essential for G:U and G:T mismatch repair are indicated (Gallinari and Jiricny 1996). (E) Glycosylase activity of TDG truncations in the presence or absence of wild-type XPC was assayed using 5fC:G (left) and 5caC:G (right) substrates. The concentrations used were 0.2 µM DNA, 0.4 µM XPC complexes, and 50 nM TDG. (F) Relative global DNA methylation assayed by 5mC-specific ELISA using genomic DNA purified from HDFs overexpressing wild-type or ΔC human XPC. Error bars represent the standard deviation. n = 3. (***) P < 0.001; (**) P < 0.01; (n.s.) nonsignificant, calculated by two-way ANOVA.

Fig 5: Inhibiting the retroviral protease site of Ddi2 enhances the binding of Ddi2 to Ub conjugates but decreases binding to proteasomes.A, the design of the experiment in (B) to test the effects of the Ddi2 protease activity on its association with Ub conjugates. Lysates of HAP1 Ddi2 knockout cells were treated with 1 µM bortezomib (BTZ) for 4 h (to obtain a high content of Ub conjugates), but no Ddi2 was mixed with lysates of HAP1 Ddi2 knockout cells transfected with WT Ddi2 or the inactive mutant D252V Q256L Ddi2 (DV/QL) and treated with 10 µM of the E1 inhibitor TAK-243 (Eli) for 4 h (to obtain high levels of WT or inactive Ddi2 and no Ub conjugates). After these lysates were combined, the lysates were incubated at 37 °C for 1 h, and the changes in levels of the Ub conjugates were analyzed at different times. B, inactive Ddi2 protects Ub conjugates from deubiquitylation. Lysates containing ubiquitylated proteins but no Ddi2 were mixed with lysates containing high levels of Ddi2, but lacking Ub conjugates as described in (A). The mixed lysates (left three lanes) were incubated at 37 °C for 1 h with 1.6 µM Ub aldehyde (Ub-Al) to block DUb activity and 10 µM E1i to prevent further ubiquitylation, or 10 µM BTZ, which allows DUb function but blocks proteasomal degradation. C, inactive Ddi2 protects ubiquitylated E6AP from deubiquitylation. HAP1 Ddi2 knockout cells were transfected with WT or D252V Q256L Ddi2 and treated with 10 µM of the E1 inhibitor Eli for 4 h as in (B). Ubiquitylated GST-E6AP was added to the lysates and incubated at 37 °C for up to 3 h. D, inhibition of the proteolytic activity of Ddi2 with nelfinavir (10 µM, 30 min) increases the binding of Ddi2 and UBQLN1 but not Rad23B to ubiquitylated E6AP. Lysates of HEK293-HBTH-Rpn1 were incubated with ubiquitylated GST-E6AP on immobilized glutathione-Sepharose for 1 h at 4 °C, and immunoblots for different proteins were quantitated. E, mutation of the Ddi2 protease site increases the binding of Ddi2 to ubiquitylated E6AP. Lysates of WT Ddi2 and D252N mutant HCT116 cells were incubated as in (D) with ubiquitylated E6AP. Quantification of immunoblots of Ddi2 that bound to ubiquitylated GST-E6AP as the percent binding of proteins from mutant cells versus the WT. F, inhibition of the Ddi2 protease site with nelfinavir decreases the binding of Ddi2 to 26S proteasomes. HEK293-HBTH-Rpn11 cells were treated with 10 µM nelfinavir or with vehicle (methanol) for 30 min, and lysates were incubated with streptavidin resin to bind 26S proteasomes. The amount of Ddi2 present was then assayed. (n = 4; mean; *p < 0.05, **p < 0.01, ns). DUb, deubiquitylating enzyme; GST, glutathione-S-transferase; HEK293, human embryonic kidney 293 cell line; ns, not significant; Ub, ubiquitin.