Fig 2: YY1 phase separation is required for the connection between distal enhancer elements and the FOXM1 promoter. (A) Schematic view across the FOXM1 gene locus (chr12:2 768 321–3 408 320 [hg19]) with genomic and epigenetic information. Graphic active regulatory regions were generated using the ENCODE database, and potential enhancers (E1 to E5) are shaded in yellow. Fragments digested by EcoRI or HindIII are numbered and shown below the graph. (B) Chromatin conformation capture (3C) analysis to examine direct physical interactions between the FOXM1 promoter and enhancer elements. The 3C analyses followed the protocol in Materials and Methods, using EcoRI in genomic DNA digestion. Ligation step was performed with or without T4 DNA ligase as indicated. Numbers on the top of the gel correspond to ‘EcoRI digestion site’ in the bottom row of (A). PCR to examine direct physical interactions between the FOXM1 promoter and each of EcoRI fragments used the primer sets in Supplementary Table S3. Numbers of fragments overlapping with enhancers and interacting with the FOXM1 promoter are in red text. (C) Schematic interactions between enhancers and the FOXM1 promoter on chromosome 12 (top row), and PCR product images of ligated DNA in 3C assays conducted in the absence or presence of 1% of 1,6-hexanediol (bottom row). (D) PCR product images of ligated DNA in 3C assays in MDA-MB-231 and MCF-7 cells infected by lentivirus carrying an empty vector, or expressing Flag-YY1 WT or its (H-A) mutant. (E) Reporter assays to examine the effects of enhancers on the FOXM1 promoter. Sectionalized enhancers 3, 4 or 5 were individually placed adjacent to the FOXM1 promoter to create reporter vectors, as schematically shown in the top row. R-Cont and L-Cont indicate control fragments that are located to right and left of the FOXM1 TSS, respectively. Reporter vectors were transfected into MDA-MB-231 and MCF-7 cells with or without YY1 knockdown (bottom row). Data are shown as mean ± s.d. (n = 3). (F) Reporter assays to assess the effects of JQ1. Reporter vectors with enhancer fragments were transfected into MDA-MB-231 and MCF-7 cells with or without JQ1, followed by examining Gluc activity. Data are shown as mean ± s.d. (n = 3). (G and H) DNA FISH (G) and RNA FISH (H) assays to determine the colocalization of YY1 puncta with the FOXM1 genomic locus and FOXM1 nascent hnRNA, respectively, in MDA-MB-231 and MCF-7 cells. In G and H, each inset shows a view zoomed-in from the white box in a merge image. Line graph showed the fluorescence intensity, and histogram shows the quantification of DNA-FISH (G) or RNA-FISH (H) foci colocalized with YY1 puncta in the nucleus (n = 18 cells). (I) Schematic model of an enhancer cluster formed by YY1-mediated phase separation with incorporation of coactivators and stabilization by three distal enhancers to activate FOXM1 gene transcription.

Fig 3: YY1 binds to the FOXM1 promoter and activates its expression. (A) YY1 and FOXM1 expression in mammary cell lines. Nontumorigenic MCF-10A cells, and three indicated breast cancer cell lines were analyzed by Western blot using indicated antibodies (top row). YY1 and FOXM1 mRNA levels were quantified by RT-qPCR (bottom row). (B) Effects of YY1 on FOXM1 in mammary cells. MCF-10A cells were infected by lentivirus expressing YY1, while MDA-MB-231 and MCF-7 cells were infected by lentivirus carrying sh-YY1. YY1 and FOXM1 protein and mRNA levels were analyzed by Western blot (top row) and RT-qPCR (bottom row). (C) Mapping the YY1-regulated region in the FOXM1 promoter. Conserved YY1 binding elements with a core sequence of ATGG (Jaspar Matrix ID: MA0095.2, top row). Reporters with different FOXM1 promoter lengths driving Gluc were generated. Data of reporter assays in response to ectopic YY1 in HeLa cells were examined (bottom row). Data represent mean ± s.d. (n = 3). (D) Evaluation of putative YY1 binding sites in the FOXM1 promoter. Putative YY1 binding sites S1–S5 were identified in the 1141-bps region upstream of the transcription start site (TSS) of the FOXM1 gene, with WT and mutants presented (top panel). Data of reporter assays in response to YY1 in HeLa cells are presented in the bottom row. (E) ChIP-qPCR assays to examine YY1 binding to the FOXM1 promoter. The FOXM1 promoter is presented in the top row with five qPCR primer pairs. YY1 antibody and a control IgG were used in ChIP assays. Data quantification and agarose gels of ChIP-qPCR in MDA-MB-231 and MCF-7 cells were presented in the middle and bottom rows, respectively. (F) EMSA to test YY1 binding to the FOXM1 promoter. His × 6-YY1 was incubated with Cy5-labeled S2, S4, S5 probes, and mutants (S2M, S4M and S5M) with a CDC6 promoter probe and its mutant as positive and negative controls, respectively. YY1-probe complex and free probe positions are labeled on the left. (G) Left: representative images of droplet formation by EGFP-YY1 with Cy5-labeled probes in Figure 7F and Supplementary Table S2. Right: schematic model of droplet formation promoted by DNA probes. (H) Image of live MDA-MB-231 and MCF-7 cells transfected with EGFP-YY1(?ZF) plasmid. (I) ChIP-qPCR assays to test the effects of YY1(H-A) mutant expression on coactivators’ binding to the FOXM1 promoter. MDA-MB-231 cells with endogenous YY1 knocked down by sh-YY1-3'-UTR and infected by lentivirus carrying an empty vector, or expressing Flag-YY1 WT or its (H-A) mutant were collected. EP300, BRD4 and MED1 antibodies, and a control IgG were used in ChIP assays, and qPCR primers are shown in Supplementary Table S1. Both quantification and agarose gels of ChIP-qPCR were presented. (J and K) Evaluation of YY1 mutations’ effects on the FOXM1 promoter. In (J), FOXM1 promoter reporter (pFOXM1-prmt-Gluc) and pCMV-SEAP vector were cotransfected with YY1 WT and mutant vectors into HeLa (left) and HEK-293T (right) cells in 24-well plates. Gluc activity in each well was measured and normalized against its SEAP activity. In (K), YY1 vectors were transfected into MDA-MB-231 (left) and MCF-7 (right) cells with endogenous YY1 knockdown, followed by RT-qPCR to quantify FOXM1 mRNA levels. Western blot analyses showing shRNA-mediated endogenous YY1 knockdown are presented at bottom panels.

Fig 4: Upregulation of Nedd4 by erastin was mediated by FOXM1.a, b Erastin induced the expression of FOXM1 and Nedd4. A375 cells were treated with erastin at different time points (0–12 h) or different concentrations (0–10 µM). Cell lysates were analyzed by immunoblotting with indicated antibodies. The protein levels of FOXM1 and Nedd4 were quantitated by Image J. c Structure of Nedd4 promoter showing the location of two FOXM1 binding sites (in red). d ChIP analysis shows occupancy by FOXM1 on Nedd4 promoter, as indicated, in A375 cells treated with erastin (5 µM) for 8 h. GAPDH serves as a negative control. e Dual-luciferase reporter assay showing the activity of Nedd4 promoter in response to erastin in A375 cells. The Nedd4 promoter reporters were transfected into A375 cells for 24 h, treated by erastin (5 µM) for an additional 12 h, then luciferase activity was measured. f Knockdown of FOXM1 suppressed erastin-induced Nedd4 expression and blocked VDAC2/3 degradation. A375 cells expressing control shRNA or FOXM1 shRNA constructs were treated with DMSO or erastin (5 µM) for 12 h, the protein levels of VDAC2, VDAC3, and Nedd4 were assayed by western blot. g Overexpression of Nedd4 but not the C867S mutant rescues the protein degradation of VDAC2/3 inhibited by FOMX1 knockdown. Cells expressing indicated DNA constructs were treated with DMSO or erastin (5 µM) for 12 h, the protein level of VDAC2/3 was assayed by western blot. h–j Overexpression of Nedd4 or knockdown of VDAC2/3 increased erastin-induced ferroptotic cell death which was inhibited by FOXM1 knockdown. Cells were transfected with indicated DNA constructs for 48 h, and treated with erastin (5 µM) for 12 h. Cell viability, intracellular MDA, GSH, and GSSG levels were measured (h). The lipid ROS level was assessed by flow cytometry using C11-BODIPY (i). The protein levels of indicated genes were assessed by immunoblotting (j). Data shown represent as mean ± SD from three independent experiments. Comparisons were made using Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s. not significant.

Fig 5: Effects of YY1 cIDR and H-cluster mutagenesis on its biological functions. (A) Ectopic YY1 expression with simultaneous endogenous YY1 knockdown. MDA-MB-231 cells with DOX-induced sh-YY1 targeting the 3′-UTR of the YY1 mRNA were infected by lentivirus expressing 3 × Flag-YY1 WT and mutants, or a vector. Cell lysates were analyzed by Western blot using indicated antibodies. (B) Effects of YY1 mutations on cell viability. MDA-MB-231 and MCF-7 cells expressing 3 × Flag-YY1 WT and mutants with endogenous YY1 knockdown were evaluated by WST-1 assays to determine cell viability. **P < 0.01; ***P < 0.001. (C and D) Mouse xenograft tumor formation to evaluate the function of YY1(H-A) mutant. MDA-MB-231 cells (4 × 106) carrying DOX-inducible sh-YY1-3′-UTR and infected by lentivirus carrying an empty vector, or expressing WT YY1 or YY1(H-A). In (C), the growth curves of mouse xenograft tumors were generated by measuring tumor sizes twice a week and then calculating tumor volumes. In (D), IF staining and RT-qPCR analyses of frozen xenograft tumor samples. The IF images of YY1, Ki-67 and FOXM1 antibodies and their merged images with DAPI are presented, with the quantitation shown at the upper right panel. The RT-qPCR data of YY1 and FOXM1 mRNA expression normalized to GAPDH levels at the lower right panel. (E–H) IDR fusion studies to evaluate the function of the H-cluster of YY1. In (E), the diagrams of fusion proteins by conjugating the FUS IDR (FUSIDR) and TIA1 IDR (TIA1IDR) to the N-terminus of YY1(H-A) mutant are displayed. In (F), representative fluorescence and DIC images of EGFP-conjugated proteins as labeled on top are presented. Quantification of droplet's numbers and area is shown at the low panel. In (G), live U2OS cells transfected with EGFP-conjugated proteins as labeled at left are shown. In the quantifications of (F) and (G), P values for the comparison of different groups are labeled. In (H), cell viability was determined by WST-1 assays for the MDA-MB-231 cells with endogenous YY1 knocked down by sh-YY1-3′-UTR and infected by lentivirus carrying an empty vector or expressing Flag-YY1 WT, H-A mutant, or its fusion proteins. **P < 0.01; ***P < 0.001; n.s.: not significant.