Fig 2: UTX alters gene expression in virus-specific CD8+ T cells(A) An illustration of the approach. WT and UTX-deficient P14+CD8+ T cells were introduced into the separate hosts, followed by CD4 depletion and infection with LCMV-A22. Donor cells from 3–4 mice were isolated by FACS at day 15 for H3K27me3 CUT&RUN or UTX CUT&Tag analyses or isolated at day 22 for RNA-seq analyses.(B) EdgeR analysis of RNA-seq data showing genes with significantly changed expression (FDR < 0.05; log2 fold change > |1.0|), comparing genes significantly downregulated in Utx-/- donor P14+ T cells (purple) to those upregulated (green). RNA-seq data are derived from FACS donor cells from 3–4 recipient mice per group.(C) GSEA of effector genes, ranked according to their relative expression in WT and Utx-/- cells. The genes in this gene set are known to be expressed in CTLs, but not naive cells.(D) Genes showing significant differences in expression were subjected to GOrilla analysis followed by REVIGO analysis. The analysis identified several biological processes that are predicted to be significantly altered between WT and Utx-/- T cells based on differential gene expression.(E) Isolated cells were subjected to H3K27me3 CUT&RUN analysis. The anti-H3K27me3-bound DNA fragments were eluted, sequenced, and quantified across the genome. The graph depicts the relative change in Utx-/- H3K27me3 density (log2 fold change) compared to WT for individual genes that were grouped based on changes in Utx-/- gene expression (RNA-seq in B). H3K27me3 changes are shown for genes having equal (black), significantly downregulated (purple), or significantly upregulated (green) RNA expression in Utx-/- P14s. The H3K27me3 CUT&RUN data are from four recipients per group.(F) UCSC genome browser images of normalized H3K27me3 tracks at the Gzma, Thy1, Cd9, Crtam, Ubash3b, and Sema7a loci, showing mean values from four independent replicates of WT (black) or Utx-/- (red). The heavy bars depict regions with significantly enriched peaks of H3K27me3 in UTX-deficient P14 cells, compared to WT cells.(G–I) UTX CUT&Tag was performed on WT CD8+ T cells at day 0 (n = 2) and day 15 post-infection (n = 4) and at day 15 for Utx-/- (KO) cells (n = 2).(G and H) The average profile of UTX coverage is plotted based on normalized read counts for all UTX peaks of enrichment that occur at transcription start sites (TSSs; G) or enhancers (H). Enhancers were annotated based on published ATAC-seq datasets (Beltra et al., 2020). Start and stop define the boundary of the UTX peaks. Line colors indicate the profile for WT uninfected P14 (D0; light blue), WT at day 15 of LCMV infection (dark blue), or Utx-/- at day 15 (black).(I) The pie graph depicts the proportion of UTX binding at TSSs, enhancers, active enhancers, and other locations (neither TSS nor enhancer) that are within 20 kb of TSSs. Active enhancer annotation was based on published H3K27ac datasets (Yao et al., 2019).(J) H3K27me3 CUT&RUN changes were correlated with UTX binding at day 15 post-infection at genes showing reduced expression in Utx-/- T cells. Utx-/- misregulated genes were categorized as UTX bound or unbound, then contrasted for the percentage of these genes that experienced increased H3K27me3 in Utx-/- P14 cells.(K and L) All H3K27me3 peaks of enrichment that failed to overlap with UTX peaks (UTX-unbound; K) are illustrated for comparison to UTX peaks near genes with reduced expression in Utx-/- cells (UTX-bound; L). The average profile of H3K27me3 coverage is plotted based on normalized read counts for WT (red) or Utx-/- P14 (dark red) samples.(M and N) The UTX-bound H3K27me3 profiles in (L) were further subdivided into UTX peaks that overlap TSSs (M) or putative enhancers (N).(O and P) Profiles of ATAC-seq normalized reads (Beltra et al., 2020) identify the open chromatin status of enhancers in “progenitor exhausted„ (Texprog1; O) or intermediate exhausted (Texint; P) T cells. These putative enhancers were subdivided based on overlap with UTX peaks of enrichment (UTX-bound; light blue profile) or peaks lacking UTX association (unbound; black profile).(Q) The profile of H3K27ac ChIP-seq normalized reads (Yao et al., 2019) demonstrates enhancer activation status at regions that overlap with UTX binding (pink) compared to enhancers not bound by UTX (black).See also Figure S6 and Tables S1, S2, S3, and S4.

Fig 3: JMJD3 regulates macrophage survival and is the major contributor to H3K27me3 mediated IL6 transcription. (A) GM-BMDMs and M-BMDMs were left untreated (NC), or treated with GSK-J4 for 48 hours, or transfected with UTX siRNAs (si-UTX) or JMJD3 siRNAs (si-JMJD3) for 48 hours. The culture media were collected and the LDH concentration in the media were determined. (B) Representative images from M-BMDMs prepared as in (A) and stained with DAPI, white scale bar represents 50 µm. (C) Quantification of slides prepared as in (B) (5 slides/treatment group). (D, E) GM-BMDMs and M-BMDMs were treated as in (A), and the IL6 concentrations in the media were detected by ELISA. (F) GM-BMDMs were treated as in (A), and cell lysates were used to detect the protein levels of UTX, JMJD3 and H3K27me3. (G, H) GM-BMDMs or M-BMDMs were treated with PBS for 48 hours (PBS), or PBS 48 hours followed by LPS 1 hour (PBS+LPS), or transfected with UTX siRNAs (si-UTX) or JMJD3 siRNAs (si-JMJD3) for 48 hours. The cell nuclei lysates were used for H3K27me3 ChIP assays. After de-crosslinking, the H3K27me3 binding DNA fragment were examined for IL6 promoter (-952 to -1008) enrichment. (I) IPA analyzed RNA-seq data of si-JMJD3 transfected GM-BMDMs, and summarized the top upstream regulator, the detailed information of IL6 was shown. (J) IPA analyzed the activation scores of different signaling pathways in LPS (100 ng/ml for 1 hour) stimulated, si-JMJD3 transfected GM-BMDMs or M-BMDMs. Error bars indicate SD, p < 0.05 (two-tailed Student’s T-test) is indicated by * for comparison with si-UTX vs. si-JMJD3, # for comparison with NC vs. LPS, $ for comparison with si-JMJD3 vs. si-JMJD3 and UTX.

Fig 4: GSK-J4 inhibits IL6 expression via regulating H3K27me3 activity in macrophages. (A) Levels of epigenetic modifications (H3K27Ace, H3K4me3, H4K20me, H3K9me3, H4K20me3, H3K27me3) were detected in GM-BMDMs and M-BMDMs. (B) BMDMs were stimulated with 100 ng/ml LPS for 2 hours, the expression levels of EZH2, JMJD3 and UTX were determined by Western Bot. (C, D) After exposing to DZNeP and GSK-J4 for 48 hours, qPCR determines IL6 mRNA level changes upon LPS stimulation in GM-BMDMs (C) and M-BMDMs (D). (E) GM-BMDMs and M-BMDMs were stimulated with LPS (100 ng/ml) for 6 hours, the concentration of IL6 in the media were measured by ELISA. ChIP experiments were performed with anti-RNAPII (F), anti-H3K27me3 (G) and anti-H3K4me3 (H), the abundance of IL6 promoter fragment (-1000 to -900 bps relative to TSS) was determined by qPCR. p < 0.05 (two-tailed Student’s T-test) is indicated by * for comparison with LPS+GSK-J4 vs. LPS, # for comparison with NC vs. LPS, $ for comparison DZNeP vs. NC under LPS stimulation, error bars indicate SD.

Fig 5: KDM6A enhances cell viability upon imatinib treatment independently of its enzymatic activity. (A) K562 cells were treated with different concentration of GSK-J4 inhibitor as indicated or DMSO vehicle control for 24 h. Histones were acid extracted as described in Methods before Western blotting analysis against H3K27me3. (B) Cell viability of K562 cells exposed to 0.5 µM imatinib and/or 100 nM GSK-J4 inhibitor determined using the CCK-8 assay. Mean ± s.d. are given for three independent experiments. Unpaired, two-tailed Student's t-test; n.s., not significant. (C) Reconstitution of K562 KDM6A KO#2 cells with Flag-wild-type KDM6A(WT) or the enzymatic-dead mutant (ED) was confirmed by Western blotting against Flag and KDM6A. (D, E) Imatinib dose-response curves for control (parental) K562 versus KDM6A KO#2 cells and KDM6A-reconstituted cells from (C) determined after 48 hours using the CCK-8 assay. (E) Comparison of IC50 values for imatinib in K562 control versus KDM6A KO#2 cells reconstituted with vector (KO+vector), KDM6A (KO+WT) or KDM6A-ED (KO+ED). Mean ± s.d. are given for three independent experiments. Unpaired, two-tailed Student's t-test; **p < 0.01. (F) Western blotting analysis showing increased cleaved PARP in KDM6A KO cells treated with 1µM imatinib for 48 h but not in KDMA6A or KDM6A-ED reconstituted cells.