PHF19 mediated regulation of proliferation and invasiveness in prostate cancer cells
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Spain
- Universitat Pompeu Fabra (UPF), Spain
- ICREA, Spain
Figures
Figure 1 with 1 supplement
PHF19L associates with PRC2 in prostate cancer cells.
(A) Schematic representation of PHF19L and PHF19S and their domains. (B) Western blot analysis showing expression of PHF19L, PHF19S, EZH2, and GAPDH in RWPE1, PC3, and DU145 cells. *, non-specific …
Figure 1—figure supplement 1
PHF19L associates with PRC2 in prostate cancer cells.
(A) Co-immunoprecipitation (IP) of FLAG-tagged proteins in PC3 cells overexpressing FLAG-PHF19L, FLAG-PHF19S, or FLAG-Empty. IP experiments were performed using anti FLAG-M2 antibody followed by …
Figure 2 with 1 supplement
PHF19L co-localizes with PRC2 complex on chromatin.
(A) Cell fractionation followed by Western blot analysis in DU145 (left) and PC3 (right) cells, showing that PHF19L is mainly present in the chromatin and PHF19S, in the cytoplasm. Total, total cell …
Figure 2—figure supplement 1
PHF19L co-localizes with PRC2 complex in chromatin.
(A) Cell fractionation in DU145 cells overexpressing PHF19S followed by Western blot analysis, showing that PHF19L is present on chromatin, and PHF19S in the cytoplasm. Total, total cell extract; …
Figure 3 with 1 supplement
Increase of PRC2 occupancy upon PHF19L depletion.
(A) Scatter plots showing correlation of EZH2, SUZ12, and H3K27me3 ChIP-seq reads in shCTR versus shPHF19L#4 DU145 cells. Each dot in the plot corresponds to the number of ChIP-seq reads normalized …
Figure 3—figure supplement 1
Increase of PRC2 occupancy after PHF19L depletion.
(A) Scatter plots showing correlation of ChIP-seq reads for the second replicate (R2) of EZH2, SUZ12, and H3K27me3 ChIP-seq experiments in shCTR versus shPHF19L#4 DU145 cells. Each dot in the plot …
Figure 4 with 1 supplement
MTF2 is enriched in chromatin in the absence of PHF19L.
(A) Schematic representation of PRC2.1 and PRC2.2 complexes. (B) RT-qPCR of PRC2-associated factors in control and PHF19L-depleted (shPHF19L#4) DU145 cells. Expression was normalized to that of the …
Figure 4—figure supplement 1
MTF2 is enriched in chromatin in the absence of PHF19L.
(A) Volcano plot of the MTF2 ChIP-seq peaks, showing a significant gain in signal upon PHF19 loss, as reported by DiffBind biological replicates for each condition (shCTR and shPHF19L#4) (P value < …
Figure 5 with 1 supplement
PHF19L regulates the expression of genes essential for tumor growth, invasiveness, and metastasis in prostate cancer cells.
(A) Scatter plot showing changes in gene expression as detected by RNA-seq in PHF19L knockdown (shPHF19L#4) as compared to control (shCTR) DU145 cells. Up- and downregulated genes are highlighted in …
Figure 5—figure supplement 1
PHF19L regulates the expression of genes essential for tumor growth, invasiveness, and metastasis in prostate cancer cells.
(A) Scatter plot showing no significant changes in gene expression detected by RNA-seq in control (shCTR) versus PHF19S knockdown (shPHF19S#168) DU145 cells. Up- and downregulated genes are …
Figure 6 with 1 supplement
Depletion of PHF19L switches the cells to a less proliferative but more aggressive phenotype.
(A) Growth curve comparing cell growth of control and PHF19L knockdown (shPHF19L#1 or shPHF19L#4) DU145 cells. Data are presented as mean ± SD of three biological replicates. (B) Phase contrast …
Figure 6—figure supplement 1
PHF19L depletion switches prostate cancer cells to a less proliferative but more aggressive phenotype.
(A) Growth curve comparing cell growth of control and PHF19L-knockdown PC3 cells. Error bars represent SD of three independent experiments. (B) Effect of PHF19L depletion (shPHF19L#4) on BrdU …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | DU145 | ATCC HTB-81 | RRID:CVCL_0105 | |
| Cell line (human) | PC3 | ATCC CRL-1435 | RRID:CVCL_0035 | |
| Cell line (human) | RWPE1 | ATCC CRL-11609 | RRID:CVCL_3791 | |
| Antibody | PHF19 (rabbit, polyclonal) | Cell Signaling | Cell Signaling #77271; RRID:AB_2799892 | WB (1:1000), ChIP (1:50, 5 µl/chip), IP (1:500, 5 µl/IP) |
| Antibody | EZH2 (rabbit, monoclonal) | Cell Signaling | Cell Signaling #5246; RRID:AB_10694683 | WB (1:1000), ChIP (1:50, 5 µl/chip), IP (1:500, 5 µl/IP) |
| Antibody | SUZ12 (rabbit, monoclonal) | Abcam | Abcam #ab12073; RRID:AB_442939 | ChIP (5 µg/chip) |
| Antibody | H3K27me3 (rabbit, polyclonal) | Millipore | Millipore #07–449; RRID:AB_310624 | ChIP (5 µg/chip) |
| Antibody | MTF2 (rabbit, polyclonal) | Proteintech | Proteintech 16208–1-AP; RRID:AB_2147370 | WB (1:1000), ChIP (5 µg/chip) |
| Antibody | JARID2 (rabbit, polyclonal) | Novus | Novus #NB100-2214; RRID:AB_10000529 | WB (1:1000) |
| Antibody | IgG (rabbit, monoclonal) | Abcam | Abcam #ab172730; RRID:AB_2687931 | ChIP (5 µg/chip) |
| Antibody | EPOP (rabbit polyclonal) | ActiveMotif | Active Motif #61753; RRID:AB_2793758 | WB (1:1000) |
| Antibody | PALI1 (rabbit polyclonal) | Generated in Adrian Bracken's laboratory (Conway et al., 2018) | WB (1:500) | |
| Antibody | Drosophila H2Av | Active Motif | Active Motif #61686; RRID:AB_2737370 | ChIP (1 µg/chip) |
| Antibody | GAPDH (mouse monoclonal) | Santa Cruz Biotechnology | Santa Cruz #sc32233; RRID:AB_627679 | WB (1:5000) |
| Antibody | H3 (rabbit polyclonal) | Abcam | Abcam #ab1791 RRID:AB_302613 | WB (1:2000) |
| Recombinant DNA reagent | Plasmid: MISSION pLKO.1-puro Empty Vector Control | Addgene | Addgene: SHC001 | |
| Recombinant DNA reagent | Plasmid: Plko.1-Puro_ shPHF19L#1 | This study | See Supplementary file 3 | |
| Recombinant DNA reagent | Plasmid: Plko.1-Puro_ shPHF19L#4 | This study | See Supplementary file 3 | |
| Recombinant DNA reagent | Plasmid: Plko.1-Puro_ shPHF19L#B | This study | See Supplementary file 3 | |
| Recombinant DNA reagent | Plasmid: Plko.1-Puro_ shPHF19L#168 | This study | See Supplementary file 3 | |
| Recombinant DNA reagent | Plasmid: Plko.1-Puro_ shPHF19L#55 | This study | See Supplementary file 3 | |
| Sequence-based reagent | RT-qPCR primers | This study | See Supplementary file 3 | |
| Sequence-based reagent | ChIP-qPCR primers | This study | See Supplementary file 3 | |
| Commercial assay or kit | ChIP-IT High Sensitivity Kit | Active Motif | Active Motif #53040 | |
| Commercial assay or kit | QIAquick PCR purification kit | Qiagen | Qiagen #28106 | |
| Commercial assay or kit | RNeasy Plus Mini Kit | Qiagen | Qiagen #74134 | |
| Commercial assay or kit | APC BrdU Flow Kit | BD Pharmingen | BD #552598 | |
| Software, algorithm | Bowtie | PMID:19261174 | RRID:SCR_005476 | |
| Software, algorithm | MACS | PMID:18798982 | RRID:SCR_013291 | |
| Software, algorithm | DiffBind | PMID:22217937 | RRID:SCR_012918 | |
| Software, algorithm | R software | R Development Core Team, 2019 | RRID:SCR_001905 | |
| Software, algorithm | UCSC genome browser | PMID:29106570 | RRID:SCR_005780 | |
| Software, algorithm | Enrichr | PMID:27141961 | RRID:SCR_001575 | |
| Software, algorithm | TopHat | PMID:19289445 | RRID:SCR_013035 | |
| Software, algorithm | Cufflinks | PMID:22383036 | RRID:SCR_014597 | |
| Software, algorithm | SeqCode | http://ldicrocelab.crg.eu/ | RRID:SCR_018070 | Applications to generate ChIP-seq meta-plots, heat maps and boxplots of counts |
Additional files
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Supplementary file 1
Summary table of ChIP-seq data for all ChIPs performed in this study: ChIP-seq peaks and target genes.
- https://cdn.elifesciences.org/articles/51373/elife-51373-supp1-v1.xlsx
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Supplementary file 2
Summary table of RNAseq data: Lists of upregulated and downregulated genes detected in PHF19L knockdown as compared to control DU145 cells.
- https://cdn.elifesciences.org/articles/51373/elife-51373-supp2-v1.xlsx
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Supplementary file 3
Lists of primers for RT-qPCR and ChIP-qPCR, shRNA sequences and antibodies used in this study.
- https://cdn.elifesciences.org/articles/51373/elife-51373-supp3-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/51373/elife-51373-transrepform-v1.pdf
