Journal: Cell Death & Disease

Article Title: PHF13 epigenetically activates TGFβ driven epithelial to mesenchymal transition

doi: 10.1038/s41419-022-04940-4

Figure Lengend Snippet: A Boxplot showing TPM-normalized expression of PHF13 in normal tissues and pancreatic adenocarcinoma (PAAD). The results were generated by the online tool: GEPIA . Given that there are only 4 normal pancreatic samples in TCGA data, we combined them with the pancreatic samples in GTEx data. B Violin plot showing the expression of PHF13 at different clinical stages in patients with PAAD. The data was generated using the GEPIA tool . C Boxplot comparing the TPM-normalized expression of PHF13 between basal-like and classical subtypes. The data was generated using the GEPIA2 tool . D Boxplot showing normalized expression of PHF13 in normal pancreatic tissues (Normal), pancreatic tumors with no regional lymph node metastasis (N0), and pancreatic tumors with metastases in 1 to 3 axillary lymph nodes (N1) in the TCGA cohort. N number of patients. The result was generated from the online tool UALCAN . E Kaplan–Meier plot showing the overall survival of patients based on PHF13 expression. Data is generated from the webserver ( http://kmplot.com/analysis/ ) . HR hazard ratio, CI confidence interval.

Article Snippet: In brief, guide RNAs targeting exon 3 of the PHF13 genes were designed using the MIT CRISPR design software and further cloned into a pSpCas9(BB)-2A-GFP (PX458) plasmids from Feng Zhang lab (Addgene plasmid #48138).

Techniques: Expressing, Generated

Journal: Cell Death & Disease

Article Title: PHF13 epigenetically activates TGFβ driven epithelial to mesenchymal transition

doi: 10.1038/s41419-022-04940-4

Figure Lengend Snippet: A Heatmap showing the differential expressed genes following PHF13 depletion in pancreatic adenocarcinoma cells Panc-1. The color key was shown at the bottom of the graph. The significantly regulated genes were selected based on : the absolute value of log2-fold change > 0.58, the p-adj < 0.05 B GSEA showing the significant enrichment of gene sets associated with cell proliferation (left panel), and apoptosis (right panel). NES, normalized enrichment score. C Cell proliferation assay for PHF13-WT and PHF13-KO Panc1. Image of crystal violet staining (left panel). The confluency of stained cells in each well was calculated by ImageJ (right panel). The data are shown as the mean ± SD. *** p < 0.001, calculated by two-side t test. all experiments were performed in triplicate. D Colony-forming assay for PHF13-WT and PHF13-KO Panc1 cells. The left panel indicates the image of crystal violet staining for the colonies. ImageJ was utilized to calculate the numbers of stained colonies in each well (right panel). Biological triplicate experiments were performed. The data are represented as the mean ± SD. *** p < 0.001, calculated by two-side t test. E Representative graphs showing the size of tumors formed by transplanting PHF13 -WT and PHF13 -KO Panc1 cells into the right groin of the mouse to establish an orthotopic model. Error bars represent the standard error of the mean between the six biological replicates. *** p < 0.001, calculated by two-side t test. F The pictures showed the tumors formed by transplanting PHF13-WT and PHF13-KO Panc-1 cells. The xenografts were harvested at 30 days. G boxplot showing the weight of tumors implanting PHF13-WT and PHF13-KO Panc1 cells for 30 days. P value was calculated by the unpaired Wilcoxon–Mann–Whitney Test.

Article Snippet: In brief, guide RNAs targeting exon 3 of the PHF13 genes were designed using the MIT CRISPR design software and further cloned into a pSpCas9(BB)-2A-GFP (PX458) plasmids from Feng Zhang lab (Addgene plasmid #48138).

Techniques: Proliferation Assay, Staining, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: PHF13 epigenetically activates TGFβ driven epithelial to mesenchymal transition

doi: 10.1038/s41419-022-04940-4

Figure Lengend Snippet: A The top 15 significantly enriched MSigDB Hallmark gene sets via GSEA for the transcriptomic profiles of PHF13-depleted and control cells. FDR false discovery rate. B Heatmap showing the statistical significance of correlations between each subtype of pancreatic cancers, PHF13 expression levels, and gene modules identified by WGCNA. Bailey et al. determined the four molecular subtypes of pancreatic cancers: pancreatic progenitor (PP), immunogenic (IMG), aberrantly differentiated endocrine exocrine (ADEX), and squamous (SQ) . The significantly correlated cells contain Student’s asymptotic P values and Pearson correlations. Transwell migration assay showing a significant decrease in migration potential of TGFβ-treated cells in response to PHF13 depletion ( C ) or CRISPR/Cas9-mediated knockout ( D ). The migrated cells were stained with 0.1% (w/v) crystal violet (scale bar 100 μm). The experiment was performed in duplicate. E Heatmaps showing the significantly regulated genes upon TGFβ treatment in Panc-1 cells. K-means clustering ( K = 6) was performed based on the normalized expression level using the option scale = “row” during the following conditions: Control (NC), Control with TGFβ treatment (NCT), PHF13 knockdown (PHF), and TGFβ-treated cells depleted for PHF13 (PHFT). The color scale bar shows the row-wise normalized value. GSEA showing a significant decrease in the enrichment of TGFβ targets ( F ), KRAS targets ( G ), and squamous-specific genes ( H , I ) in PHF13-depleted Panc-1 cells. Squamous-specific genes were generated from the published data . J RT-qPCR analysis of PHF13 knockdown efficiency, epithelial ( CDH1 and EpCAM ), and mesenchymal ( CDH2 and MMP9 ) markers in Panc-1 cells in response to TGFβ treatment and PHF13 depletion. The data represent the mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001, and calculated by two-side t test. K western blots showing the protein level of PHF13, CDH2, and EpCAM in Panc-1 following TGFβ induction and PHF13 knockdown. The level of ACTB was set as a loading control. L RT-qPCR analysis of epithelial ( CDH1 and EpCAM ) and mesenchymal ( CDH2 and MMP9 ) markers in PHF13 -WT and PHF13 -KO Panc1 following TGFβ treatment. M Western blot analysis of the protein level of PHF13, CDH1, and CDH2 in the given samples. ACTB was set as a loading control.

Article Snippet: In brief, guide RNAs targeting exon 3 of the PHF13 genes were designed using the MIT CRISPR design software and further cloned into a pSpCas9(BB)-2A-GFP (PX458) plasmids from Feng Zhang lab (Addgene plasmid #48138).

Techniques: Control, Expressing, Transwell Migration Assay, Migration, CRISPR, Knock-Out, Staining, Knockdown, Generated, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: PHF13 epigenetically activates TGFβ driven epithelial to mesenchymal transition

doi: 10.1038/s41419-022-04940-4

Figure Lengend Snippet: A Distribution of PHF13 occupancy on distinct chromatin elements. B Heatmaps show the occupancy of chromatin assembly (ATAC-seq), CpG island, H3K4me3, H3K27ac, H3K4me1, and H3K27me3 surrounding the center of PHF13 peaks (±5 kb). The color key of each heatmap is shown on the right side. C Pairwise heatmaps show the normalized density and distribution of the differentially changed H3K27ac, H3K4me3, and H3K27me3 in siNC (NC), siNC + TGFβ (NCT), and siPHF13 + TGFβ(PHFT) Panc1 cells. The differentially changed chromatin sites upon TGFβ treatment were identified by DiffBind (Supplementary Fig. ). The region flanking ±5 kb of each peak center is shown. The right color heatmap shows the row-wise normalized signal of H3K27ac, H3K4me3, and H3K27me3. D ChromHMM was used to learn and characterize chromatin states in Panc1 cells. The first table from left to right shows the emission parameters determined de novo with ChromHMM from the given chromatin markers over the entire genome. The second table indicates the distribution of each characterized chromatin state on the genome. The third table displays the relative fold enrichment of each chromatin state on several chromatin elements, including CpG islands, TSS, and promoters (surrounding TSS ± 3 kb). Chromatin annotations for each state are shown on the rightest table. Boxplots compare the occupancy of H3K4me3, H3K27ac, and H3K27me3 at the genomic regions corresponding to the identified states, including active promoter ( E ), poised promoter ( F ), weak promoter ( G ), strong enhancer ( H ), weak transcribed ( I ), poised enhancer ( J ), and polycomb-repressed ( K ) chromatin regions, in siNC (NC), siNC + TGFβ (NCT), and siPHF13 + TGFβ(PHFT) Panc1 cells. p value was calculated using the unpaired Wilcoxon–Mann–Whitney Test. ns nonsignificant difference; ** p value greater than 0.001 but less than 0.01; **** p value less than 0.0001.

Article Snippet: In brief, guide RNAs targeting exon 3 of the PHF13 genes were designed using the MIT CRISPR design software and further cloned into a pSpCas9(BB)-2A-GFP (PX458) plasmids from Feng Zhang lab (Addgene plasmid #48138).

Techniques: MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: PHF13 epigenetically activates TGFβ driven epithelial to mesenchymal transition

doi: 10.1038/s41419-022-04940-4

Figure Lengend Snippet: A Aggregate profiles show the occupancy of H3K4me3, H3K27me3, and H3K27ac surrounding the center of PHF13-occupied poised chromatin regions (±5 kb) in siNC, siNC + TGFβ (NCT), and siPHF13 + TGFβ (PHFT) Panc-1 cells. P values were calculated by Wilcoxon–Mann–Whitney Test. ns nonsignificant difference; **0.01 < p < 0.001; *** p < 0.001. B Heatmaps show gene expression of TGFβ-activated poised genes in siNC (NC), siPHF13 (PHF), siNC + TGFβ (NCT), and siPHF13 + TGFβ (PHFT) Panc1 cells. The color scale bar shows the row-wise normalized expression signal. C GO analysis of the TGFβ-activated poised genes. The top 10 significantly enriched gene terms were shown. Western blot analysis of nuclear extract inputs, anti-PHF13 IP ( D ), anti-EZH2 IP ( E ), and anti-IgG IP from Panc1 nuclear extract. F Western blot showing the level of H3K27me3 in the given samples. The protein TBP was set as a loading control. The down numbers indicated the normalized signal of H3K27me3. The signal of each band was measured using ImageJ. The signal of H3K27me3 was normalized to TBP and represented as fold change relative to the control cells. G Genome browser tracks the normalized signal of chromatin assembly (ATAC-seq), EZH2, PHF13, H3K27me3, H3K4me3 occupancy, and mRNA-seq data at PCDHB8 an d PAPLN lo cus from siNC (NC), siPHF13 (PHF), siNC + TGFβ (NCT), siPHF13 + TGFβ (PHFT) Panc1 cells. H ChIP-qPCR analysis of H3K27me3 occupancy near the TSS of the PCDHB8 and PAPLN genes in siControl treated, PHF13-depleted, TGFβ-treated, and PHF13-depleted TGFβ-treated Panc-1 cells. p value was calculated using a two-side t test. * p < 0.05; *** p < 0.001. ChIP-qPCR for IgG was set as a negative control. The dotted line indicated the average signal of IgG.

Article Snippet: In brief, guide RNAs targeting exon 3 of the PHF13 genes were designed using the MIT CRISPR design software and further cloned into a pSpCas9(BB)-2A-GFP (PX458) plasmids from Feng Zhang lab (Addgene plasmid #48138).

Techniques: MANN-WHITNEY, Gene Expression, Expressing, Western Blot, Control, ChIP-qPCR, Negative Control

Journal: Cell Death & Disease

Article Title: PHF13 epigenetically activates TGFβ driven epithelial to mesenchymal transition

doi: 10.1038/s41419-022-04940-4

Figure Lengend Snippet: A The heatmaps show H3K4me3, H3K27me3, and H3K27ac on the regions flanking −5 kb to 10 kb around the TSS of all genes in Panc1 cells. All TSS were clustered based on the occupancy of the given histone modifications: the broad H3K4me3 occupied TSS (broad, defined based on Supplementary Fig. ), the typical H3K4me3 occupied TSS (active), H3K4me3 and H3K27me3 co-occupied TSS (poised), only H3K27me3-occupied TSS (H3K27me3 only), and non-histone modifications marked TSS (neither). The color key at the right side of each heatmap indicates the occupied density. B Observed/Expected enrichment of regulated and unchanged H3K4me3 occupied sites calculated from siNC_TGFβ versus siNC (up panel), siPHF13_TGFβ versus siNC_TGFβ (middle panel), and siPHF13_TGFβ versus siNC (down panel) at “broad”, “active”, and “poised” TSS regions. Aggregate profiles compare H3K4me3 ( C ) and H3K27ac ( D ) occupancy in siNC, siNC_TGFβ, and siPHF13_TGFβ Panc-1 cells at the regions from upstream 5 kb to downstream 10 kb of the broad H3K4me3-occupied TSS. E Boxplot compares the length of the broad H3K4me3 and H3K27ac peaks in siNC, siNC_TGFβ, and siPHF13_TGFβ Panc-1 cells. The unpaired Wilcoxon–Mann–Whitney Test was used to calculate p values. F Boxplot compares the log 2 -fold changes in the expression of broad H3K4me3-occupied genes in the following conditions: siPHF13 versus siNC (PHF/NC), siNC + TGFβ versus siNC (NCT/NC), and siPHF13 + TGFβ versus siNC + TGFβ (PHFT/NCT). G Venn diagram displaying the numbers of overlap and unique broad H3K4me3-occupied genes between siNC, siNC_TGFβ, and siPHF13_TGFβ Panc-1 cells. The broad H3K4me3-occupied genes in various conditions were obtained according to Supplementary Fig. . H GO analysis of the TGFβ-gained broad H3K4me3-linked genes (correlated to Fig. 6G, the “231” and “28” genes). The enrichment map displays the connection of the top 25 significantly enriched gene sets. Four functional biological modules have been identified, including NF-kappaB signaling, cell migration, mRNA process, and endoderm formation. I Genome browser tracks the normalized signal of chromatin assembly (ATAC-seq), PHF13, H3K27ac, H3K4me3 occupancy, and mRNA-seq data at SNAI1 locus from siNC (NC), siPHF13 (PHF), siNC + TGFβ (NCT), siPHF13 + TGFβ (PHFT) Panc-1 cells. J Western blot analysis shows the expression of SNAI1 in Panc-1 cells following TGFβ treatment and PHF13 depletion. K Western blots analysis shows the expression of SNAI1 in PHF13-overexpressed Panc-1 cells.

Article Snippet: In brief, guide RNAs targeting exon 3 of the PHF13 genes were designed using the MIT CRISPR design software and further cloned into a pSpCas9(BB)-2A-GFP (PX458) plasmids from Feng Zhang lab (Addgene plasmid #48138).

Techniques: MANN-WHITNEY, Expressing, Functional Assay, Migration, Western Blot

Journal: Cell Death & Disease

Article Title: PHF13 epigenetically activates TGFβ driven epithelial to mesenchymal transition

doi: 10.1038/s41419-022-04940-4

Figure Lengend Snippet: A Pairwise heatmaps show the occupancy and distribution of PHF13 and H3K27ac over differential changed enhancers in siNC, siNC + TGFβ, and siPHF13 + TGFβ Panc-1 cells. Enhancers clustered according to the DiffBind analysis results (Supplementary Fig. ). TGFβ-activated enhancers, increased in siNC + TGFβ compared to siNC; PHF13-dependent enhancers, decreased in siPHF13 + TGFβ compared to siNC + TGFβ. The region flanking ±5 kb of each enhancer center is shown. The color scale bar shows the row-wise normalized signal of H3K27ac. B The de novo motif sequence of PHF13-dependent enhancers. DNA sequence-based motif analysis was performed on the PHF13-dependent enhancers (the 1201 PHF13 peaks identified in Supplementary Fig. ). The most significantly enriched motif is shown here. C ReMap analysis shows the top 12 transcription factors that hit PHF13-dependent enhancers. D Observed/Expected enrichment of regulated and unchanged H3K27ac occupied sites calculated from siNC_TGFβ versus siNC (left panel), siPHF13_TGFβ versus siNC_TGFβ (middle panel), and siPHF13_TGFβ versus siNC (right panel) at TEs and SEs. E Metagene profiles show the normalized signal of H3K27ac at typical enhancers (TE) and super-enhancers (SE) in siNC, siNC + TGFβ, and siPHF13 + TGFβ Panc-1 cells. The length of enhancers (from “start” to “end”) was calculated relative to the median length (TE, 1.4 kb; SE, 33 kb). F Pairwise heatmaps show the normalized density and distribution of H3K27ac over SEs in siNC, siNC + TGFβ, and siPHF13 + TGFβ Panc-1 cells. SEs were clustered using K -means ( K = 3) based on the normalized occupancy of H3K27ac. The regions flanking ±100 kb of each SEs center are shown. The right color heatmap shows the row-wise normalized signal of H3K27ac and highlights the PHF13-dependent and TGFβ-activated SEs identified by K -means clustering. G GO analysis of PHF13-dependent SEs associated genes. The GREAT tool was used to predict SE annotated genes. The top 10 significantly enriched gene terms are shown. H Ranking of enhancers based on the normalized signal of H3K27ac in siNC, siNC + TGFβ, and siPHF13 + TGFβ Panc-1 cells. SEs are annotated by a GREAT online tool. The positions of SEs associated with SOX9 , MMP17 , ITGA2 , and COL1A1 , as well as the unique SEs in siNC + TGFβ or siPHF13 + TGFβ Panc-1 cells, are highlighted (red points). I Genome browser tracks the normalized signal of chromatin assembly (ATAC-seq), PHF13, H3K27ac, H3K4me3 occupancy, and mRNA-seq data at SOX9 locus from siNC (NC), siPHF13 (PHF), siNC + TGFβ (NCT), siPHF13 + TGFβ (PHFT) Panc1 cells. One of the enhancers has been zoon out.

Article Snippet: In brief, guide RNAs targeting exon 3 of the PHF13 genes were designed using the MIT CRISPR design software and further cloned into a pSpCas9(BB)-2A-GFP (PX458) plasmids from Feng Zhang lab (Addgene plasmid #48138).

Techniques: Sequencing