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

Figure 6.

Figure 6.

JQKD82 increases H3K4me3 and TFIID anchoring via TAF3, resulting in reduced RNAPII phosphorylation. A, Metagene plot of H3K4me3 level at the genes co-occupied by KDM5A and MYC at the TSS in MM.1S cells, and separated into groups either downregulated by JQKD82 (red) or not downregulated by JQKD82 (blue). The gene set defined in Fig. 5B was used for analysis. MM.1S cells were treated with 1 μmol/L of JQKD82 or DMSO for 48 hours. H3K4me3 level was measured by ChIP-seq normalized to reference exogenous spiked-in Drosophila chromatin (ChIP-Rx). RRPM, reference-normalized reads per million. B, Metagene plot of RNAPII phosphorylated at Ser5 (Ser5p) and Ser2 (Ser2p) ChIP-seq reads mapped to the gene loci co-occupied by KDM5A and MYC at the TSS and downregulated by JQKD82 in MM.1S cells. MM.1S cells were treated with 1 μmol/L of JQKD82 or DMSO for 48 hours. RPM, reads per million mapped reads. TES, transcription end site. C, ChIP-seq tracks of RNAPII (Ser5p), RNAPII (Ser2p), and RNAPII at representative MYC target gene loci (MYC and NOLC1) after treatment with JQKD82 in MM.1S cells. D, JQKD82 induced changes in the pausing index [the ratio of RNAPII ChIP-seq signal in the TSS region (TSSR) to the signal in the gene body] at CDK7 inhibitor (THZ1)–sensitive or –insensitive genes, and JQKD82-sensitive or -insensitive genes in MM.1S cells. Gene sensitivity status was defined by gene expression changes genome wide in MM.1S cells exposed to THZ1 (4 hours) or JQKD82 (48 hours). P values were calculated by unpaired Student t test comparing genes of different sensitivity status.E, JQKD82-induced changes in ChIP-seq signal of RNAPII (Ser5p) in the body of genes classified as sensitive or insensitive to CDK7 or JQKD82 in MM.1S cells as in D. P values were calculated by unpaired Student t test comparing genes of different sensitivity status. F, TAF3 (the subunit of TFIID with a H3K4me3 recognizing PHD domain) binding at JQKD82-downregulated genes was measured by ChIP-seq normalized to reference exogenous (ChIP-Rx) spiked-in Drosophila chromatin in MM.1S cells. Comparison is made to H3K4me3 ChIP-Rx data in MM.1S cells treated with 1 μmol/L of JQKD82 or DMSO for 48 hours. G, ChIP-seq signal of H3K4me3, CDK7, TAF3, and KDM5A at the TSS of genes either sensitive to CDK7 inhibition by THZ1 (black line) compared with THZ1-insensitive genes (gray line). H, Proposed model of KDM5A functions and the mechanism of action of JQKD82. KDM5A interacts with P-TEFb (CCNT2 and CDK9) and transactivates MYC target genes. JQKD82 or knockdown of KDM5A induces hypermethylation of H3K4me3 (hyper-H3K4me3), leading to anchoring of TFIID via TAF3 binding, which may function as a barrier to productive RNAPII phosphorylation by TFIIH (CDK7) and P-TEFb (CDK9), thereby dampening pause release and reducing MYC target gene transcription (indicated by faded RNAPII elongation complex).