Fig 2: KDM5C is required for the activation of estrogen/ERa-target genes. A) MCF7 cells were transfected with control siRNA (siCTL) or siRNA specific against KDM5C (siKDM5C) in stripping medium for three days, and then treated with or without estrogen (E2, 10-7 m, 6 h) followed by RNA-seq. Genes induced by estrogen (E2) (fold change (FC) (siCTL (E2)/siCTL) = 1.5) as well as those dependent on KDM5C for expression (fold change (FC) (siCTL (E2)/siKDM5C (E2)) = 1.5) were shown. B,C) Heat map (B) and box plot (C) representation of the expression (FPKM, log2) for genes induced by estrogen (E2) and dependent on KDM5C as described in (A). D,E) Genome browser views of RNA-seq as described in (A) for two estrogen/ERa-target genes, TFF1 (D) and GREB1 (E), were shown. F) MCF7 cells were transfected with siCTL or siKDM5C in stripping medium for three days, and treated with or without estrogen (E2, 10-7 m, 6 h), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated. G) MCF7 cells were infected with shCTL or shKDM5C lenti-virus in stripping medium for three days, and treated with or without estrogen (E2, 10-7 m, 6 h), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated. H) WT and KDM5C (sgRNA) MCF7 cells were maintained in stripping medium for three days, and treated with or without estrogen (E2, 10-7 m, 6 h), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated. I) HEK293T cells were transfected with siCTL or siKDM5C in the presence or absence of ERa and estrogen response element (ERE)-driven luciferase reporter in stripping medium and treated with or without estrogen (E2, 10-7 m, 24 h) followed by luciferase reporter activity measurement (± SD., *** p < 0.001). J,K) Tumor samples as described in Figure 1L (J) or Figure 1N (K) were subjected to RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated (± SD, * p < 0.05, *** p < 0.001).

Fig 3: Cell cycle genes are dysregulated in KDM5 KD cells. (A) KDM5-repressed genes, KDM5-activated genes, and genes with KDM5A binding in their promoter but unchanged by KD were subjected to functional enrichment analyses. The heat map shows the enrichment (reported as -LogP) for the top 5 WikiPathways in each category. (B) mRNA levels of Cdc6 (top) and Cdc20 (bottom) during the course of adipocyte differentiation in control (blue) and KDM5 KD (red) cells. Error bars represent SD (n = 3). P-value: *P < 0.05, **P <0.01, and ***P <0.005, control compared with KDM5 KD, Student's two-tailed t-test. (C) Box plots illustrating log2 normalized gene signal (as tags per kb) during differentiation from day 0 to day 2 of induced cell cycle genes from the KEGG database annotation in control (blue) and KDM5 KD (red) cells. (D) Box plot showing log2 normalized KDM5A signal at day 2 (as tags per kb) in the KDM5A-occupied promoters of all analyzed genes (gray), as well as cell cycle genes from the KEGG database annotation that are either induced (green) or repressed (red) from day 0 to day 2 of differentiation. (E) ChIP-seq profiles of KDM5A at the Cdc6, Cdc20, E2f1, Plk1, and Ccna2 loci at day 2. C and D: P-value: # P <0.05, Wilcoxon rank sum test as indicated. (F) ChIP-qPCR of KDM5A (left) and KDM5C (right) binding at promoters of selected pro-proliferative genes in control (blue) and KDM5 KD (red) cells presented as percent recovery of input. P-value: *P <0.05, **P <0.01, and ***P <0.005, positive targets versus negative control (neg. control) as well as control versus KDM5 KD, Student's two-tailed t-test.

Fig 4: HIF, PBRM1 and KDM5C are required for the expression and activity of ISGF3.A Venn diagram showing the overlaps of genes whose expression was affected by VHL, PBRM1 or KDM5C losses in 786-O cells. The p-value significance of the overlap in a venn diagram was calculated using hypergeometric test in R; (B) Heat map of shared gene targets by VHL, PBRM1 and KDM5C; (C) Enriched gene ontology (GO) analysis of genes regulated both by VHL, PBRM1 and KDM5C; (D) Western blots with the indicated antibodies in 786-O cell SDS-solubilized whole cell lysates with HIF2a, PBRM1 or KDM5C suppressed by shRNA; (E) Examination of mRNA expression of individual genes by RT-qPCR in 786-O cells after PBRM1 or KDM5C suppression by shRNA; (F) Western blots with the indicated antibodies in Ren-02 cell SDS-solubilized whole cell lysates with HIF2a, PBRM1 or KDM5C suppressed by shRNA. The p-value significance in the bar graph was calculated with two-tailed student t test. *p<0.05; **p<0.01; ***p<0.001.

Fig 5: KD of KDM5 family members in 3T3-L1 preadipocytes inhibits adipogenesis. Pre-confluent (day -4) cells were transduced with lentivirus expressing either control luciferase shRNA (control) or shRNA targeting KDM5A, KDM5B, and KDM5C (KD). Cells were induced to differentiate 2 days post confluency (day 0) using a standard differentiation cocktail. (A) Schematic representation of the experimental setup. (B) mRNA (left) and protein (right) levels of KDM5A, KDM5B, and KDM5C at day 0 in control cells and KDM5 KD cells. (C) Representatives of phase contrast micrographs from Oil red O staining performed at day 8 in control and KDM5 KD cells. (D) mRNA levels of adipocyte marker genes (left), and preadipocyte marker genes (right) at day 6 in control cells and KDM5 KD cells. mRNA (E) and protein (F) levels of the key adipocyte transcription factors C/EBPß, PPAR?, and C/EBPa during the course of adipocyte differentiation in control and KDM5 KD cells. Error bars represent standard deviation (SD), (n = 3). *P <0.05, **P <0.01, and ***P <0.005, Student's two-tailed t-test.