USP26 functions as a negative regulator of cellular reprogramming by stabilising PRC1 complex components

Despite much progress in the comprehension of the complex process of somatic cell reprogramming, many questions regarding the molecular mechanism of regulation remain to be answered. At present, the knowledge on the negative regulation of reprogramming process is indeed poor in contrary to the identification of positive regulators. Here we report for the first time that ubiquitin-specific protease 26 negatively regulates somatic cell-reprogramming process by stabilizing chromobox (CBX)-containing proteins CBX4 and CBX6 of polycomb-repressive complex 1 through the removal of K48-linked polyubiquitination. Thus, accumulated CBX4 and CBX6 repress the expression of pluripotency genes, such as Sox2 and Nanog, through PRC1 complexes to ubiquitinate histone H2A at their promoters. In all, our findings have revealed an essential role for ubiquitin-specific protease 26 in cellular reprogramming through polycomb-repressive complex 1.

. Characterization of Usp26 shRNA knockdown in iPSCs, related to Figure 1 in the main text (a) qPCR analysis of Usp26 shRNA knockdown efficiency, ** p<0.01 compared to control shRNA. (b) Immunofluorescence microscopic images of Nanog in Usp26 knockdown iPSCs. (c)& (d) Quantification of Nanog + (c) and SSEA1 (d) colonies after 12 days of OSKM induction in MEFs transduced with control or Usp26 shRNA, ** p<0.01 compared to control shRNA, n=4. (e) Microscopic images of iPSC morphology and GFP immunofluorescence after Usp26 knockdown. (f) Gross morphology and histology of Usp26 knockdown iPSC teratoma assay. The data are presented as means ± SD from three independent experiments; (a, c, d) Unpaired two-tailed Student's t-test.

Supplementary Figure 2.
Usp26 knockout delays RA-induced ESC differentiation and maintains ESC morphology, related to Figure 2 in the main text (a) Brightfield microscopic images of ESCs during LIF withdrawal (LIF-) and RA-induced (RA+) differentiation in wild-type (WT) and Usp26 knockout (KO) ES cells. WT or Usp26 KO ESCs were cultured in ES differentiation medium (LIF withdrawal with 1 μM RA). Individual colonies were tracked and taken pictures at days 0, 2, 4, 6, 8 and 10 under microscope. (b) Quantification of ESC colonies transduced with Dox inducible GFP-tagged mUsp26 overexpression or with GFP-tagged empty vector, **** p<0.0001 compared to empty vector. (c) Quantification of ESC colonies during LIF withdrawal (LIF-) and RA-induced (RA+) differentiation in wild-type (WT) and Usp26 knockout (KO) ES cells, ** p<0.01, **** p<0.0001 compared to WT. (d) Western blot analysis of protein levels of pluripotency markers in wild-type (WT) or Usp26 knockout (KO) ES cells differentiated by LIF withdrawal (LIF-) and RA-induction (RA+). (e) Western blot analysis of CRISPR/Cas9 knockout for USP26 in MEF cells. (f) TIDE assay of CRISPR/Cas9 knockout in Usp26 in MEF cells. The data are presented as means ± SD from three independent experiments, (b, c) Two-way ANOVA for multiple comparisons.
Supplementary Figure 3. USP26 interacts with CBX4 and CBX6, related to Figure 4 in the main text (a) 293T cells were transfected with HA-tagged EZH1, EED, EZH2, PHC1, and KDM2B plus FLAG-hUSP26 (+) or empty vector (-). Following IP with anti-FLAG beads, specific proteins were analyzed by western blotting using anti-HA antibody. (b) Wild-type (WT) or RING1A knockout (KO) 293T cells were transfected with HA-tagged CBX4 or CBX6 plus FLAG-hUSP26. Following IP with anti-FLAG beads, specific proteins were analyzed by western blotting using anti-HA antibody. (c) Western blot analysis of CRISPR/Cas9 knockout of CBX4 in 293T cells. (d) Western blot analysis of CRISPR/Cas9 knockout of CBX6 in 293T cells. (e) CBX4 knockout 293T cells were transfected with HA-tagged RING1A, CBX4, CBX6, CBX7, or PCGF2 plus FLAG-hUSP26. Following IP with anti-FLAG beads, specific proteins were analyzed by western blotting using anti-HA antibody. (f) CBX6 knockout 293T cells were transfected with HA-tagged RING1A, CBX4, CBX6, CBX7, or PCGF2 plus FLAG-hUSP26. Following IP with anti-FLAG beads, specific proteins were analyzed by western blotting using anti-HA antibody. Figure 4. USP26 specifically removes K48-linked polyubiquitin chain and blocks degradation of CBX4 and CBX6, related to Figure 5 in the main text (a) MEFs were transduced with mouse Usp26 shRNA or control shRNA lentivirus, cell extracts were harvested, and mRNA levels were analyzed by qPCR. The data are presented as means ± SD from three independent experiments. (b) MEFs were transduced with mouse Usp26 shRNA or control shRNA lentivirus, cell extracts were harvested, protein levels were analyzed by western blotting with specific antibodies as indicated (c) 293T cells were transfected with different amounts of FLAG-USP26 (0, 0.2 μg, 0.5 μg, and 1 μg). Cell extracts were harvested and analyzed by western blotting with specific antibodies, as indicated.

Supplementary
(d) Mouse ES cells were infected with or without RA treatment, and cell extracts were harvested at different time points, protein levels were analyzed by western blotting using specific antibodies, as indicated. (e) 293T cells were treated with DMSO, MG132 or Lactacystin. Specific proteins were analyzed by western blotting using anti-CBX4 and anti-CBX6 antibodies. β-actin was used as a loading control. (f) MEF cells with or without Usp26 lentivirus transduction were treated with DMSO, MG132 or Lactacystin. Specific proteins were analyzed by western blotting using anti-CBX4, anti-CBX6 and anti-USP26 antibodies. β-ACTIN was used as a loading control. (g) 293T cells were transfected with HA-tagged WT-, K6-, K11-, K27-, K29-, K33-, K48-, and K63-linked Ub, FLAG-CBX4 or FLAG-CBX6 with (+) or without (-) Myc-tagged USP26. Following IP with anti-FLAG beads, ubiquitination of CBX4 or CBX6 was analyzed by western blotting using anti-HA antibody. (a) Unpaired two-tailed Student's t-test. Figure 5. Accumulated CBX4 and CBX6 bind to pluripotent gene promoters during ESC differentiation, related to Figure 6 in the main text (a) ChIP-PCR analysis of Sox2, Nanog, Oct4, Cbx7, Cbx4, and Cbx6 promoters in ESC differentiation upon LIF withdrawal and treatment with RA, ** p<0.01 compared to day 0. (b) qPCR analysis of mouse Cbx4 and Cbx6 mRNA expression in ESCs transduced with mouse Usp26 or empty vector (EV), ** p<0.01 compared to EV at day 6. (c) qPCR analysis of mouse Cbx4 and Cbx6 mRNA expression in ESCs induced with RA or without RA, ** p<0.01 compared to RA-treatment at day 6. (d) ChIP-PCR analysis of Usp26 promoters in ESC differentiation upon LIF withdrawal and treatment with RA, ** p<0.01 compared to day 0.

Supplementary
(e-f) ChIP-PCR analysis of Sox2, Nanog and Cbx7 promoters in control shRNA, Cbx4 shRNA Cbx6 shRNA or Cbx4 and Cbx6 shRNA transduced ESCs, which were cultured upon LIF withdrawal and treatment with RA, * p<0.5, ** p<0.01 compared to day 0. (g) Luciferase assay of Oct4, Sox2, Nanog, and Cbx7 promoters with empty vector (EV), Usp26, or Usp26 and Cbx4 or Cbx6 co-transfection, ** p<0.01 compared to EV. Red arrows indicate ChIP-PCR targets and black arrows indicate transcription start sites (TSSs) at the Usp26 promoters. The data are presented as means ± SD from three independent experiments (a-f) Unpaired two-tailed Student's t-test; (g) Two-way ANOVA for multiple comparisons. (b) AP staining of iPSC colonies after OSKM induction in MEF cells infected with Usp26, Cbx4, Cbx6, or control shRNA lentivirus. OSKM transgenic MEF cells were transduced with GIPZ lentivirus-based shRNAs targeting Cbx4, Cbx6, or Cbx4 and Cbx6 and selected with puromycin for 2 days. 1000 puromycin selected OSKM transgenic MEF cells were reseeded onto feeder cells in 6-well plates. The next day, modified iSF1 medium containing 2 µg/ml Dox was added and replenished every day. The efficiency of iPSC formation was calculated based on the number of AP + iPSC colonies. The data are presented as means ± SD from four independent experiments (** p<0.01 compared to control shRNA). (c) Microscopic images of GFP and morphology, using brightfield (BF), of Usp26 KO ESCs at day 1 and day 7 after transfection with Dox inducible GFP-mCbx4, GFP-mCbx6, or empty GFP vector (EV). The data are presented as means ± SD from three independent experiments, (a) Unpaired twotailed Student's t-test; (b) Two-way ANOVA for multiple comparisons.