Phospho-PHF8 directly activates key initiators of cell-intrinsic immune response. (A) Profile plot of PHF8 genome occupancy (ChIP-seq) in THP1 PHF8 AA compared with THP1 PHF8 DD cells. (B) Volcano graph depicts all regulated genes in THP1 PHF8 DD cells. Differential expression is represented as LogFC in (x-axis) vs PHF8 ChIP-seq MACS analysis presented as peakScore in (y-axis). Upregulated genes with significance higher than log 0.5 (>log 0.5) are colored in violet. (C) Scatterplot of the highly significantly (>log 0.5) upregulated genes in the transcriptome of PHF8 DD cells (logFC) and PHF8 DD targets resulting from PHF8 ChIP sequencing (peakScore). (D) Protein network analysis of the correlation of 16 highly significantly (>log 0.5) upregulated genes (cell intrinsic immune response inducers) in the transcriptome of PHF8 DD cells and PHF8 DD targets resulting from ChIP sequencing. Pink color represents higher confidence based on experimental evidence. The protein-protein interaction network was established using STRING database and visualized in Cytoscape version 3.9.1. (E) ChIP-qPCR analysis of histone mark H3K9me2 on the targeted promoter of the indicated genes in THP1 PHF8 AA and THP1 PHF8 DD cells. ChIP signals are presented as percentage of input. Error bars indicate SD of 3 independent experiments. Sidak multiple comparison test (2-way ANOVA, ∗∗∗∗P < .0001). (F) RT-qPCR time-course analysis (left) of the expression of specified human genes. The relative expression of each gene was independently analyzed in 2 cell groups: PHF8 AA and PHF8 DD. Each group consisted of noninduced cells and cells induced with tamoxifen (4-OHT). Noninduced samples within each group were used as references for assessing the relative expression of the genes at each time point. Error bars indicate SD of 3 independent experiments. Bar charts (right) of RT-qPCR analysis at 24 hours after 4-OHT induction. Expression levels are relative only to the PHF8 AA 0mM (4-OHT) cells. Error bars indicate SD of 3 independent experiments; Tukey multiple comparison test (2-way ANOVA, ∗∗P < .0023; ∗∗∗∗P < .0001). (G) RT-qPCR time-course analysis of the expression of RIG-I and IFN-β human genes. The relative expression of each gene was independently analyzed in 2 cell groups: PHF8 AA and PHF8 DD. Each group consisted of noninduced cells and cells induced with tamoxifen (4-OHT). Noninduced samples within each group were used as references for assessing the relative expression of the genes at each time point. Error bars indicate SD of 3 independent experiments; Tukey multiple comparison test (2-way ANOVA, ∗∗∗∗P < .0001). (H-I) ChIP-qPCR analysis of (left) PHF8 or (middle) histone mark H3K9me2 on CMPK2 or IFIT5 targeted promoter in THP1 cells (Control) or THP1 cells after 12 hours of OKA/ATRA treatment. ChIP signals are presented as percentage of input. Error bars indicate SD of 3 independent experiments; Sidak multiple comparison test (2-way ANOVA, ∗∗P < .023; ∗∗∗P < .0009; ∗∗∗∗P < .0001). RT-qPCR time-course analysis (right) of the expression of CMPK2 or IFIT5. The relative expression of each gene was independently analyzed in nontreated and treated groups of cells. Nontreated samples were used as references for assessing the relative expression of the genes at each time point. Error bars indicate SD of 3 independent experiments; Tukey multiple comparison test (2-way ANOVA, ∗∗∗∗P < .0001). (J-K) Bar charts represent normalized colony numbers of human THP1 AML cells cotransduced with nontargeting empty vector control (Control empty) or lenti-CRISPR-V2 guide targeting the indicated endogenous genes (CMPK2 sgRNA 7, CMPK2 sgRNA 9 or IFIT5 sgRNA 1, IFIT5 sgRNA 2) as well as PHF8 vs empty vector (Control), respectively. Nontargeting empty vector control cells were taken as the reference for normalizing the colony numbers in each group. Error bars indicate SD of 3 independent experiments; Tukey multiple comparison test (2-way ANOVA, ∗∗P < .0033; ∗∗∗P < .0007, ns); (right panels) representative western blot analyses of the indicated proteins. MACS, Model based analysis for ChIP-seq; RT-qPCR, Real time quantitative PCR.

Phospho-PHF8 directly activates key initiators of cell-intrinsic immune response. (A) Profile plot of PHF8 genome occupancy (ChIP-seq) in THP1 PHF8 AA compared with THP1 PHF8 DD cells. (B) Volcano graph depicts all regulated genes in THP1 PHF8 DD cells. Differential expression is represented as LogFC in (x-axis) vs PHF8 ChIP-seq MACS analysis presented as peakScore in (y-axis). Upregulated genes with significance higher than log 0.5 (>log 0.5) are colored in violet. (C) Scatterplot of the highly significantly (>log 0.5) upregulated genes in the transcriptome of PHF8 DD cells (logFC) and PHF8 DD targets resulting from PHF8 ChIP sequencing (peakScore). (D) Protein network analysis of the correlation of 16 highly significantly (>log 0.5) upregulated genes (cell intrinsic immune response inducers) in the transcriptome of PHF8 DD cells and PHF8 DD targets resulting from ChIP sequencing. Pink color represents higher confidence based on experimental evidence. The protein-protein interaction network was established using STRING database and visualized in Cytoscape version 3.9.1. (E) ChIP-qPCR analysis of histone mark H3K9me2 on the targeted promoter of the indicated genes in THP1 PHF8 AA and THP1 PHF8 DD cells. ChIP signals are presented as percentage of input. Error bars indicate SD of 3 independent experiments. Sidak multiple comparison test (2-way ANOVA, ∗∗∗∗P < .0001). (F) RT-qPCR time-course analysis (left) of the expression of specified human genes. The relative expression of each gene was independently analyzed in 2 cell groups: PHF8 AA and PHF8 DD. Each group consisted of noninduced cells and cells induced with tamoxifen (4-OHT). Noninduced samples within each group were used as references for assessing the relative expression of the genes at each time point. Error bars indicate SD of 3 independent experiments. Bar charts (right) of RT-qPCR analysis at 24 hours after 4-OHT induction. Expression levels are relative only to the PHF8 AA 0mM (4-OHT) cells. Error bars indicate SD of 3 independent experiments; Tukey multiple comparison test (2-way ANOVA, ∗∗P < .0023; ∗∗∗∗P < .0001). (G) RT-qPCR time-course analysis of the expression of RIG-I and IFN-β human genes. The relative expression of each gene was independently analyzed in 2 cell groups: PHF8 AA and PHF8 DD. Each group consisted of noninduced cells and cells induced with tamoxifen (4-OHT). Noninduced samples within each group were used as references for assessing the relative expression of the genes at each time point. Error bars indicate SD of 3 independent experiments; Tukey multiple comparison test (2-way ANOVA, ∗∗∗∗P < .0001). (H-I) ChIP-qPCR analysis of (left) PHF8 or (middle) histone mark H3K9me2 on CMPK2 or IFIT5 targeted promoter in THP1 cells (Control) or THP1 cells after 12 hours of OKA/ATRA treatment. ChIP signals are presented as percentage of input. Error bars indicate SD of 3 independent experiments; Sidak multiple comparison test (2-way ANOVA, ∗∗P < .023; ∗∗∗P < .0009; ∗∗∗∗P < .0001). RT-qPCR time-course analysis (right) of the expression of CMPK2 or IFIT5. The relative expression of each gene was independently analyzed in nontreated and treated groups of cells. Nontreated samples were used as references for assessing the relative expression of the genes at each time point. Error bars indicate SD of 3 independent experiments; Tukey multiple comparison test (2-way ANOVA, ∗∗∗∗P < .0001). (J-K) Bar charts represent normalized colony numbers of human THP1 AML cells cotransduced with nontargeting empty vector control (Control empty) or lenti-CRISPR-V2 guide targeting the indicated endogenous genes (CMPK2 sgRNA 7, CMPK2 sgRNA 9 or IFIT5 sgRNA 1, IFIT5 sgRNA 2) as well as PHF8 vs empty vector (Control), respectively. Nontargeting empty vector control cells were taken as the reference for normalizing the colony numbers in each group. Error bars indicate SD of 3 independent experiments; Tukey multiple comparison test (2-way ANOVA, ∗∗P < .0033; ∗∗∗P < .0007, ns); (right panels) representative western blot analyses of the indicated proteins. MACS, Model based analysis for ChIP-seq; RT-qPCR, Real time quantitative PCR.

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