KDM4A is essential for tumor cell proliferation. (A) MTT cell proliferation assay and (B) crystal violet staining of normal immortalized RWPE-1 cell and prostate cancer cell lines LNCaP, CWR22Rv1 and C4-2B, infected with lentivirus carrying control pLKO.1 or shKDM4A constructs. The cells were infected on day 0, and MTT was added to detect viable cells every two days after infection. 8 days after lentiviral infection, the cells were fixed and stained by crystal violet. (C) Caspase 3/7 activity of the lentiviral-infected cells was detected on day 6. (D) 3 days after infection, the expression of KDM4A was examined by western blotting. (E) CWR22Rv1 xenograft tumor growth. CWR22Rv1 cells were infected by lentivirus carrying pLKO.1 or shKDM4A for 2 days, and then implanted subcutaneously into nude mice. 10 days after implantation, the tumor volumes were measured up to 3 weeks. Data are presented as mean ± SD, with the SD derived from 10 mice. (F) Ki-67 and KDM4A immunohistochemistry staining of the tumor sections isolated from the xenografts. All cell culture-based data are presented as the average of n ≥ 3 replicates ± SD. **:p< 0.01, *: p<0.05

KDM4A regulates expression of E2F1 target genes. (A) DAVID functional annotation and pathway analysis of the genes that showed two-fold alterations in KDM4A-knockdown LNCaP cells in microarray analysis. GOs that showed statistically strong enrichment of alteration with low P-values are listed. Bar graph and the numbers indicate gene counts of each pathway. % of hits indicates the percentage of genes that are altered in each GO. (B) Gene expression in LNCaP cells transduced with control or shKDM4A for 3 days was detected by qRT-PCR. The data is presented as the average of n= 3 replicates ± SD. The statistics of difference between shKDM4A and pLKO.1 is p< 0.01. (C) qRT-PCR analysis of the gene expression in 8 tumors isolated from CWR22Rv1-pLKO.1 and -shKDM4A xenografts. Color scheme indicates Max and Min expression across the 8 tumor specimens. (D) Intersection of published KDM4A (GSM831035) and E2F1 (GSM935366) ChIP-seq binding sites examined in hESC and HeLa cells, respectively.

KDM4A associates with E2F1. (A) Association of ectopically expressed KDM4A with E2F1.293T cells were co-transfected with Flag or Flag-KDM4A and HA-E2F1 followed by IP using anti-Flag antibody. The co-IP'd E2F1 was detected by anti-HA antibody with immunoblotting (IB). (B) Endogenous association of KDM4A and E2F1. LNCaP and CWR22Rv1 cell lysates were used for KDM4A IP, and the co-IP'd E2F1 was detected by anti-E2F1 antibody. Mouse normal IgG was used as negative control. (C) Flag-KDM4A truncated proteins and HA-E2F1 were co-expressed in 293T cells for co-IP as described above. Asterisks indicate positive binding. (D) In vitro GST pull-down assay of recombinant GST-E2F1 fragments with purified Flag-KDM4A protein. Asterisks indicate positive binding. DBD, DNA binding domain; LZ, leucine zipper; MB, marked box; TD, transactivation domain.

Chromatin recruitment of KDM4A with E2F1 enhances E2F1 transcriptional activity. (A) ChIP analysis of KDM4A and E2F1 occupancy on CDC2 and CCNE2 promoters in LNCaP cells using IgG, anti-KDM4A or anti-E2F1 antibodies. ChIP DNAs were analyzed by qPCR with primers that amplify genomic regions either proximal (P) or distal (D) to TSS for each gene. (B) ChIP-reChIP assay of KDM4A and E2F1 binding. LNCaP chromatin was used for the first ChIP with anti-KDM4A. The KDM4A bound chromatin was eluted and subjected to reChIP using IgG, anti-E2F1 or anti-KDM4A, followed by qPCR analysis of the target sites. (C) KDM4A and E2F1 occupancy on the target promoters during cell cycle progression. LNCaP cells were arrested at G1/S transition by double thymidine block and subsequently released to complete medium at 0 hr. Cells at the indicated time points were harvested for KDM4A and E2F1 ChIP assays. The protein levels and phospho-H3S10, indicating the stage of mitosis are shown in the western blots. (D) CDC2 and CCNE1 promoter activities. The CDC2 and CCNE1 promoter-luciferase constructs were co-expressed with empty HA or HA-E2F1, and wild type (wt) or inactive (H188A) KDM4A in PC3 cells. The luciferase activity was measured 48 hours after transfection. (E) Enrichment of E2F1, PolII and H3K9me3 on the target promoters in control and KDM4A-knockdown LNCaP cells. The level of H3K9me3 was normalized by histone H3. The western blot shows KDM4A level. All data are presented as the average of n= 3 replicates ± SD. **:p<0.01, *:p<0.05.

PDK1 and PDK3 are direct targets of KDM4A and E2F1. (A) Schematic PDK1 and PDK3 promoters. Black bars indicate putative E2F response elements and the consensus sequence. White bars indicate E2F1 binding sites identified in ENCODE ChIP-seq analysis. Grey bars indicate the qPCR amplification sites proximal to the TSS used in this study. (B) ChIP-qPCR analysis of KDM4A and E2F1 occupancy on proximal (P) or distal (D) regions of PDK1 and PDK3 promoters in LNCaP cells. The distal site for PDK1 was designed at the intergenic region downstream of PDK1. (C) ChIP-reChIP assay of KDM4A and E2F1 binding on the PDK1 and PDK3 promoters, as described in . (D) Enrichment of E2F1 and H3K9me3 on PDK1 and PDK3 promoters in control and KDM4A-knockdown LNCaP cells. The H3K9me3 level was quantified as described in . (E) RNA and protein levels of KDM4A, PDK1 and PDK3 in pLKO.1 and shKDM4A cells. LNCaP cells infected for 3 days were harvested for qRT-PCR and western blotting. All data are presented as the average of n= 3 replicates ± SD. **: p< 0.01, *:p< 0.05.

Knockdown of KDM4A, PDK1 or PDK3 induces mitochondrial metabolism. LNCaP cells were infected by lentivirus carrying pLKO.1, shKDM4A, shPDK1 or shPDK3. 3 days after infection, the cells and cultured media were harvested to measure cellular (A) PDH activity and (B) lactate secretion. The PDH activity was normalized by protein concentration of the cell lysates. (C–D) The cellular ECAR and OCR were measured on day 3 after lentiviral infection. (E) Mitochondrial ROS was detected by MitoSOX fluorescence dye 4 days after transduction. Images from randomly selected fields were taken. Scale bar: 50 µm. (F,G) Cell growth and caspase 3/7 activity in shPDK1 and shPDK3 cells were examined as described in . PDK1 and PDK3 expression is shown in the western blots. All data are presented as the average of n= 3 replicates ± SD. **: p< 0.01, *: p< 0.05.

Ectopic expression of PDK1 and PDK3 rescues the KDM4A metabolic phenotypes. Lentivirus carrying PDK1 or PDK3 overexpression constructs were used to infect LNCaP cells together with the shKDM4A virus. (A) PDH activity, (B) lactate production, (C) ECAR and (D) OCR were measured as described in 3 days after infection. (E) Total cellular ROS was labeled by CM-H2DCFDA. (F) Mitochondrial ROS was detected by MitoSOX fluorescence dye as described above. Scale bar: 50 µm. (G) KDM4A, PDK1 and PDK3 protein levels were examined 3 days after infection. All data are presented as the average of n= 3 replicates ± SD. **: p< 0.01, *: p< 0.05.