Fig 2: KDM6A protein levels are decreased following EMT and upregulated during MET(A) HMLE cells were transduced with either an empty vector (HMLE-Vector), or genes encoding the EMT-inducing transcription factors Snail (HMLE-Snail) or Twist (HMLE-Twist) [4]. Whole-cell lysates were analyzed by immunoblotting for KDM6A. β-actin was used as a loading control. (B-F) MCF10A cells were treated with sterile 4 mM HCl containing 0.1% bovine serum albumin (Vehicle) or TGFB for 5 days (TGFB+). In parallel, MCF10A cells were exposed to TGFB for 5 days in order to induce EMT, and subsequently subjected to TGFB-withdrawal, for a further 5 days, to elicit MET (TGFB+/-). (B-C) MCF10A cells, treated as described, were fixed and immunostained for E-cadherin (B) and N-cadherin (C). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (D) MCF10A cells, treated as described, were analyzed by immunoblotting for KDM6B and vimentin (Vim). COX IV was used as a loading control. (E) MCF10A cells, treated as described, were fixed and immunostained for KDM6A. Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. (F) Cells were analyzed by immunoblotting for KDM6A, EZH2 and RING1A. β-actin was used as a loading control.

Fig 3: Western blot analysis showing the expression of BMI1, RING1A, RING1B, EZH2, γ-H2AX, Ub-H2A, p-CHK2, p-ATM, RNF8, RNF168, MEL18, p53BP, BRCA1 proteins in MDAMB-231cells transfected with miR-15a, miR-16 or both miR-15a/16 under etoposide-induced DNA damage conditions. Actin served as a gel loading control. Blots were cropped to enhance the representation (A,B). Immunofluorescence data showing accumulation of BMI1, ϒ-H2AX (C) RING1B (D) and Ub-H2A (E) p53BP (F) in MDAMB-231 cells transfected with miR-15a miR-16 or both miR-15a/16 under etoposide- treated conditions. Bar indicates 200 μm.

Fig 4: USP7 associates with cPRC1 and is required for Polycomb target gene repression(A) Table showing spectral counts of IP (from two biological replicates) samples from untagged and CBX2 endogenously Flag-tagged HEK293FT cells. Normalized spectral counts on the right side were obtained by dividing total spectral counts by the number of amino acids (length) of each protein. The normalized spectral counts for the bait protein (CBX2, CBX4, or PCH2) were set to 1, and the counts for co-purifying proteins are presented relative to the bait.(B) Table showing spectral counts of IP (from two biological replicates) samples from untagged and PHC2 or CBX4 endogenously Flag-tagged HEK293FT cells.(C) Schematic diagram showing the composition of canonical PRC1 complex (cPRC1). In HEK293FT cells, cPRC1 contains SCML2 and USP7 in addition to its core components RING1A/B, PCGF2/4, PHC1/2/3, and CBX2/4/6/7/8 (only CBX2, 4, and 8 are expressed in HEK293FT).(D) Immunoprecipitation (IP) experiments showing the association of USP7 with cPRC1 subunits CBX2, CBX4, and RING1B in HEK293FT cells.(E and F) Venn diagrams showing overlap among upregulated (E) and downregulated (F) genes in siUSP7 with upregulated genes in EED KO and RING1A/B DKO cells in RNA-seq experiments. Hypergeometric probability p values: siUSP7 upreg vs. RING1A/B DKO upreg, 2.8 x 10−517; siUSP7 upreg vs. EED KO, 9.6 x 10−393 upreg, siUSP7 downreg vs. RING1A/B DKO upreg, 0.2; siSENP3 downreg vs. EED KO upreg, 0.4.(G) Dot plots showing RNA-seq changes (from two biological replicates) of siUSP7 compared with siControl (siCtrl) in the EED KO-upregulated and -downregulated sets of genes in RNA-seq experiments in HEK293FT cells. Data are presented as mean values +/− SEM. p value is from the two-tailed Wilcoxon test.(H and I) Average distribution of the indicated ChIP-seq reads (log2) (from two biological replicates) for genes upregulated (H) and downregulated genes (I) in siUSP7 RNA-seq experiments from HEK293FT cells. Enrichment levels were normalized with reads per genome coverage. Read counts per gene were summed in 50-nt bins.(J) Genome browser snapshots of RNA-seq experiments showing expression levels of the indicated genes in siCtrl and siUSP7 cells and ChIP-seq showing enrichment of RING1B, H2AK119ub1, and H3K27me3. Enrichment levels were normalized with reads per genome coverage.

Fig 5: miR-200a, miR-200b, miR-15a, miR-429, miR-203 regulate PRC1 proteins in MDAMB-231 cells.Expression of BMI1, RING1A, RING1B and Ub-H2A in MDAMB-231(A) and BT549 (B) cells having overexpression of miR-200a, miR-200b, miR-15a, miR-429, miR-203. Expression of BMI1, Ub-H2A protein in MDAMB-231cells transfected with Anti-miR- 200a, Anti-miR-200b, Anti-miR-15a, Anti-miR-449, Anti-miR-203 (C) BMI1, RING1A localization in MDAMB-231 cells having overexpressed miR-200a, miR-200b, miR-15a, miR-449, miR-203under confocal microscopy (D, E). Cells transfected with Scramble Vector were used as controls. Bar indicates 200μm.