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. 2021 Apr 10;2(4):370–387. doi: 10.1158/2643-3230.BCD-20-0108

Figure 5.

Figure 5.

KDM5A and MYC co-occupy and activate the genes downregulated by JQKD82. A, Heatmap showing KDM5A enrichment at regions bound by MYC, CDK7, CDK9, and RNAPII in MM.1S cells, resolved by ChIP-sequencing (± 5 kb from each of the protein-bound regions are shown). B, Venn diagram depicting the overlap of genes co-occupied by KDM5A and MYC at TSS and genes downregulated by JQKD82 in MM.1S cells (top). Metagene plots showing occupancy of MYC and KDM5A at genes downregulated by JQKD82 (right) and those not downregulated by JQKD82 (left) in MM.1S cells (bottom). RPM, reads per million mapped reads. C, Representative gene tracks demonstrating enrichment of KDM5A, H3K4me3, MYC, CDK7, CDK9, and RNAPII at MYC target gene loci (CDK4 and NOLC1) in MM.1S cells. D, 293T cells were cotransfected with 50 ng of human CDK4 regulatory element-luciferase reporter and 300 ng of KDM5A expression plasmid or empty vector. After 8 hours of transfection, the cells were treated with 10 μmol/L of JQKD82 or DMSO for 16 hours and assayed for luciferase activity. RLU, relative luminescence unit. E, 293T cells were transfected with 50 ng of human CDK4 regulatory element-luciferase reporter together with 200 ng of KDM5A and/or MYC expression plasmids. After 24 hours of transfection, luciferase activities were measured. Data represent mean ± SD of triplicate samples. **, P < 0.01; ***, P < 0.001; unpaired Student t test (D and E). F, 293T cells expressing FLAG-tagged KDM5A were harvested; cell lysates were then immunoprecipitated (IP) with anti-FLAG (mouse monoclonal), and subjected to immunoblot analysis with anti-FLAG, anti-CCNT2 (rabbit polyclonal), and anti-CDK9 (rabbit monoclonal). G, 293T cells were transfected with eGFP-KDM5A together with mCherry-CCNT2. After fixation, nuclei were stained with DAPI. Localizations of KDM5A and CCNT2 were observed using confocal laser scanning microscopy. Scale bars, 10 μm. H, Cell lysates from MM.1S cells were immunoprecipitated with anti-KDM5A, anti-CCNT2, or normal rabbit IgG (control) and subjected to immunoblot analysis with anti-KDM5A or anti-CCNT2. I, Cell lysates from MM.1S cells were incubated with KDM5-C49-Biotin, free biotin, or KDM5-C49 at 100 μmol/L for 24 hours. The chemical probes were pull down with streptavidin agarose resin, and proteins associated with probes were subjected to immunoblot analysis with anti-KDM5A, anti-CDK9, or anti-CCNT2.