Fig 2: PHF6 has distinct binding patterns within lineage-specific genes that result in drastic changes in nucleosome occupancy upon genetic deletion. (A) Heat maps comparing gene expression of curated CD19+ B-cell (left) and CD4+ T-cell (right) gene sets between Phf6WT (left) and Phf6KO (right) cells as determined by RNA-seq. Scale corresponds to row-wise standardized log2-transformed expression values for each gene. Each column represents a replicate sample. (B) Motif enrichment analysis for DNA regions that undergo significant changes in chromatin accessibility in Phf6KO cells. (Top) DNA motifs with decreased chromatin accessibility upon loss of Phf6. (Bottom) DNA motifs with increased chromatin accessibility upon loss of Phf6. (C) Metagene analysis of nucleosome positions (top) and fold enrichment of PHF6 binding (bottom) plotted for global analysis (left), the CD19+ B-cell gene set (middle), and the CD4+ T-cell gene set (right) assessed at TSSs ±1-kb genomic regions. (Top) Nucleosome positions are shown for Phf6WT (solid line) and Phf6KO (dotted line) cells, called by the NucleoATAC algorithm and normalized for batch effects using the chromVAR package. (Bottom) Metagene tracks of PHF6 ChIP-seq signal averaged over the TSSs ±1 kb in Phf6WT cells. (−1/+1) +1/−1 nucleosomes flanking the TSS; (NFR) nucleosome-free region. Gray bars indicate major changes in nucleosome positioning that correspond to enriched PHF6 binding. Shaded regions around average tracks denote estimates of 95% CI of the metagene average signals based on resampling.

Fig 3: The absence of Phf6 promotes an altered gene expression program in B-cell leukemia. (A) Heat map showing differentially expressed genes (fold change >4, false discovery rate [FDR] <0.05) in pairwise comparisons between Phf6WT (left) and Phf6KO (right) cells as determined by RNA-seq. Each column represents a replicate sample. The scale corresponds to row-wise standardized log2-transformed expression values for each gene. (B) The top gene ontology (GO) and PANTHER terms found to be enriched in Phf6KO cells. The P-value for each term is plotted as −log10(P-value). (C) GSEA plot depicting significant (P < 0.001) changes in pre-B lymphocyte signature genes upon Phf6 deletion, as compared with Phf6WT cells. (NES) Normalized enrichment score. (D) Quantitative PCR (qPCR) analysis of Phf6WT (blue) and Phf6KO (red) cells transduced with empty vector (EV; solid) or a vector expressing Phf6 cDNA (cDNA; dotted). Relative mRNA levels for B-cell-associated genes are shown: Phf6, Cd22, Cd74, Il4ra, Lyn, Ly86, and Blk. (E) Schematic representation of ICA used to identify differential expression signatures (independent components [ICs]) in the integrated RNA-seq data set comprised of Phf6WT, shPhf6, and Phf6KO cells. Hinton diagram representation of ICA-derived signatures. Columns denote signatures, and rows denote samples. Colors denote relative directionality of gene expression ([red] up-regulation; [green] down-regulation), and the size of each square represents the magnitude of the contribution of each sample to the respective IC. Each signature is two-sided. Vertical boxes denote statistically significant (P = 0.01, Mann-Whitney test) independent components. IC2 identified a Phf6KO-specific gene signature. (F) GSEA plot depicting (P = 0.08) enrichment in T-cell signal transduction signature upon Phf6 deletion, as compared with Phf6WT cells. (NES) Normalized enrichment score. Data represent the mean ± SD. Statistics for these data were calculated with two-sided Student's t-test. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001; (n.s.) not significant.

Fig 4: Loss of Phf6 results in decreased dependence on the driving oncogene BCR–ABL1 in vivo. (A) GSEA plots depicting significant changes in the targets of the BCR–ABL1 fusion signature (P = 0.0129) and dasatinib resistance signature (P = 0.0056) upon Phf6 deletion, as compared with Phf6WT cells. (NES) Normalized enrichment score. (B) Kaplan-Meier survival analysis of mice injected with 106 B-ALL cells of the indicated genotypes with subsequent mock or ponatinib treatment (30 mg/kg daily for four consecutive days). The number (n) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P-value is shown for the comparison. (C) Kaplan-Meier survival analysis of mice injected with 106 B-ALL cells of the indicated genotypes with mock or 10 mg/kg doxorubicin (single immunoprecipitation dose) treatment. The number (n) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P-value is shown for the comparison.

Fig 5: PHF6 exerts transcriptional regulation by interacting with histones rather than binding sequence-specific DNA sites. (A) Pie chart showing the distribution of 77,749 PHF6-binding sites across genomic regions in B-ALL cells. (TTS) Transcription termination site; (UTR) untranslated region. (B) De novo DNA sequence motifs identified in PHF6-bound regions at promoters of differentially expressed genes with their associated P-values. Shown are sequence logos of de novo position-weight matrices found by the MEME motif discovery tool (left) or those of known transcription factors whose motifs are found to be most similar to the de novo motif discovery results by Tomtom software (right). (C) Endogenous coimmunoprecipitation (co-IP) assay of TCF12, NF-κB, and PHF6 in the absence (top) or presence (bottom) of ethidium bromide (EtBr). Input is 7% of immunoprecipitation lysate. (D, left) Metagene tracks of PHF6 ChIP-seq signal averaged over all promoter–TSS tracks grouped by relative expression levels. (Red) High; (green) genomic; (purple) low. (Right) Metagene track of H3K27ac ChIP-seq signal averaged over all promoter–TSS regions. Shaded regions around average tracks denote estimates of 95% confidence interval (CI) of the metagene average signals based on resampling. (E) Metagene tracks of PHF6 ChIP-seq signal and correlation with histone marks: H3K27ac (blue), H3K4me3 (yellow; GSE66234), H3K27me3 (green). Pearson correlation of PHF6 and histone ChIP-seq signals across 10-kb regions spanning the TSS. Differentially expressed genes (solid line) and genome-wide genes (dotted line) are shown. (F) Endogenous co-IP assay of histone H3 and PHF6 in the absence (top) or presence (bottom) of EtBr. Input is 7% of immunoprecipitation lysate.