The testis protein ZNF165 is a SMAD3 cofactor that coordinates oncogenic TGFβ signaling in triple-negative breast cancer
- Department of Pharmacology, University of Texas Southwestern Medical Center, United States
- Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, United States
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
SMAD3 is enriched at ZNF165 binding sites in TNBC cells.
(A) Venn diagram displaying the overlap between ZNF165 and SMAD3 target genes in WHIM12 cells as identified by GREAT (ver 3.0.0). Significance was determined using the hypergeometric distribution. (B…
Figure 1—figure supplement 1
SMAD3 is enriched at ZNF165 binding sites in TNBC cells.
(A) Venn diagram displaying the peak overlap between ZNF165 binding sites identified in WHIM12 and SUM159 cells. P-value was calculated using the hypergeometric distribution. (B) Venn diagram …
Figure 2 with 1 supplement
ZNF165 and SMAD3 cooperate to modulate TGFβ-responsive gene expression.
(A) WHIM12 cells were transfected with siRNA for 48 hr, and qPCR was used to quantify relative expression (log2 fold change) of ZNF165-SMAD3 target genes upon depletion of ZNF165 (x-axis) or SMAD3 …
Figure 2—figure supplement 1
ZNF165 and SMAD3 activate RRAD expression to drive neoplastic behaviors in TNBC.
(A) Heatmap plotting the log2 fold-change in expression of 65/446 shared ZNF165-SMAD3 target genes (cutoff >0.25) following TGFβ exposure in WHIM12 cells. Expression data were obtained from GSE63986 …
Figure 3
ZNF165 physically associates with SMAD3 in a TGFβ-dependent manner.
(A) Forty-eight hours after transfection with HA-SMAD3 and/or ZNF165-myc cDNA, HEK293T cells were subjected to immunoprecipitation with HA antibody. Immunoblotting was performed with indicated …
Figure 4 with 1 supplement
ZNF446 is an obligate component of the ZNF165-SMAD3 transcriptional complex.
(A) ZNF165 interactors that contain a SCAN domain as identified from a systematic yeast two-hybrid screening approach (interactome.dfci.harvard.edu). A domain map of ZNF165 is shown above for …
Figure 4—figure supplement 1
ZNF446 expression is elevated in breast cancer and associated with poor prognosis.
(A) Box-plot showing ZNF446 expression values (log2 median centered ratio) from normal breast and invasive lobular breast carcinoma. Data were obtained from TCGA and p-value was calculated using an …
Figure 5 with 1 supplement
ZNF446 co-occupies a subset of ZNF165 and SMAD3 binding sites in TNBC cells.
(A) Heatmaps of ChIP-seq data for ZNF165 and ZNF446 in the indicated TNBC cell lines. All peaks within each heatmap are centered ±2.5 kb from the WHIM12 ZNF165 peaks (n = 322). (B) Normalized read …
Figure 5—figure supplement 1
ZNF446 co-occupies a subset of ZNF165 and SMAD3 binding sites in TNBC cells.
(A) Browser tracks of ChIP-seq data showing peaks for ZNF165, SMAD3, and ZNF446 near the ITM2C and ZMPSTE24 loci in WHIM12 or SUM159 cells. (B) Venn diagram displaying the peak overlap between …
Figure 6 with 1 supplement
TRIM27 is essential for ZNF165 transcriptional activity and tumor growth in vivo.
(A) Cartoon maps showing the domains of ZNF446 and TRIM27. The KRAB-binding domain of TRIM27 is highlighted for reference. (B) Forty-eight hours after transfection with indicated cDNA, HEK293T …
Figure 6—figure supplement 1
Analysis of TRIM27 expression and contribution to neoplastic behaviors in TNBC.
(A) WHIM12 cells were transfected with indicated siRNA for 72 hr. Whole cell lysates were collected and immunoblotted for the indicated proteins. Data are representative of two independent …
Figure 7
Model for assembly of a neomorphic ZNF165 transcriptional complex in TNBC.
Aberrant expression of testis-specific ZNF165 in TNBC co-opts somatic transcriptional machinery to modulate TGFβ-dependent transcription. The ZNF165 SCAN domain mediates interaction with ZNF446, and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ; NSG | The Jackson Laboratory | Stock no. 005557; RRID:IMSR_JAX:005557 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216; RRID:CVCL_0063 | |
| Cell line (H. sapiens) | MCF7 | ATCC | HTB-22; RRID:CVCL_0031 | |
| Cell line (H. sapiens) | WHIM12 | Li et al., 2013 | ||
| Cell line (H. sapiens) | SUM159 | Asterand | RRID:CVCL_5423 | |
| Cell line (H. sapiens) | SUM159T-Luciferase | Westcott et al., 2015 | ||
| Antibody | anti-HA (Rat monoclonal) | Roche | Cat #11867423001; RRID:AB_390918 | IB (1:1000), IP (1 μg) |
| Antibody | anti-c-Myc (Rabbit polyclonal) | Santa Cruz Biotechnology | sc-789; RRID:AB_631274 | IB (1:1000) |
| Antibody | anti-V5 (Rabbit monoclonal) | Cell Signaling Technology | Cat #13202; RRID:AB_2687461 | IB (1:1000) |
| Antibody | anti-SMAD3 (Rabbit polyclonal) | Abcam | ab28379; RRID:AB_2192903 | IB (1:1000), PLA (1:200), ChIP-qPCR (1.5 μg), ChIP-seq (10 μg) |
| Antibody | anti-phospho-SMAD3 (Rabbit monoclonal) | Cell Signaling Technology | Cat #9520; RRID:AB_2193207 | IB (1:1000), ChIP (5 μg) |
| Antibody | anti-SMURF2 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-393848 | IB (1:500) |
| Antibody | anti-RRAD (Goat polyclonal) | Thermo Fisher Scientific | PA537885; RRID:AB_2554493 | IB (1:1000) |
| Antibody | anti-ZNF165 (Mouse monoclonal) | Novus Biologicals | H00007718-M02; RRID:AB_1717273 | IB (1:1000) |
| Antibody | anti-ZNF165 (Rabbit polyclonal) | Atlas Antibodies | HPA007247; RRID:AB_2797217 | IB (1:1000), PLA (1:200), IP (5 μg) |
| Antibody | anti-Tubulin (Rabbit monoclonal) | Cell Signaling Technology | Cat #2128; RRID:AB_823664 | IB (1:1000) |
| Antibody | anti-ZNF446 (Rabbit polyclonal) | Proteintech | 16218–1-AP | IB (1:1000), PLA (1:200) |
| Antibody | anti-TRIM27 (Rabbit monoclonal) | Cell Signaling Technology | Cat #15099; RRID:AB_2798707 | IB (1:1000) |
| Antibody | anti-V5 (Mouse monoclonal) | Thermo Fisher Scientific | R960-25; RRID:AB_2556564 | PLA (1:500), IP (1 μg) |
| Antibody | anti-SMAD3 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-101154; RRID:AB_1129525 | PLA (1:200) |
| Antibody | anti-SMAD4 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-7966; RRID:AB_627905 | PLA (1:200) |
| Antibody | anti-TRIM27 (Rabbit polyclonal) | Proteintech | 12205–1-AP; RRID:AB_2256660 | PLA (1:200), IHC (1:200) |
| Antibody | anti-H3K27ac (Rabbit polyclonal) | Abcam | ab4729; RRID:AB_2118291 | ChIP-qPCR (2 μg) |
| Antibody | Normal rabbit IgG (Rabbit polyclonal) | Cell Signaling Technology | Cat #2729; RRID:AB_1031062 | IP (1 μg), ChIP-qPCR (1.5–5 μg) |
| Antibody | anti-V5 (Rabbit polyclonal) | Abcam | ab9116; RRID:AB_307024 | ChIP-seq (5 μg) |
| Peptide, recombinant protein | Human Transforming Growth Factor-β1 | Cell Signaling Technology | Cat #8915 | |
| Chemical compound, drug | SB-431542 | Tocris Bioscience | Cat #1614 | |
| Commercial assay or kit | KAPA HyperPrep Kit | KAPA Biosystems | KK8502 | |
| Commercial assay or kit | Duolink In Situ PLA Probe Anti-Mouse PLUS | Sigma-Aldrich | DUO92001; RRID:AB_2810939 | |
| Commercial assay or kit | Duolink In Situ PLA Probe Anti-Rabbit MINUS | Sigma-Aldrich | DUO92005; RRID:AB_2810942 | |
| Commercial assay or kit | Duolink In Situ Detection Reagent Red | Sigma-Aldrich | DUO92008 | |
| Software, algorithm | ImageJ (Fiji) | Schindelin et al., 2012 | RRID:SCR_002285 | ver 2.0.0 |
| Software, algorithm | Bowtie2 | Langmead and Salzberg, 2012 | RRID:SCR_005476 | ver 2.3.2 |
| Software, algorithm | SAMtools | Li et al., 2009 | RRID:SCR_002105 | ver 1.6 |
| Software, algorithm | DeepTools | Ramírez et al., 2016 | RRID:SCR_016366 | ver 2.3.5 |
| Software, algorithm | Integrative Genomics Viewer | Robinson et al., 2011 | RRID:SCR_011793 | ver 2.3.93 |
| Software, algorithm | HOMER | Heinz et al., 2010 | RRID:SCR_010881 | ver 4.9 |
| Software, algorithm | GREAT | McLean et al., 2010 | RRID:SCR_005807 | ver 3.0.0 |
| Software, algorithm | MEME | Bailey et al., 2009 | RRID:SCR_001783 | ver 5.1.1 |
| Software, algorithm | GSEA | Subramanian et al., 2005 | RRID:SCR_003199 | ver 4.0.3 |
Additional files
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Supplementary file 1
GREAT (ver. 3.0.0) analysis of the 118 ZNF165/SMAD3 co-bound regions determined using default association rules.
- https://cdn.elifesciences.org/articles/57679/elife-57679-supp1-v2.docx
-
Supplementary file 2
Genes included in the EPITHELIAL_MESENCHYMAL_TRANSITION gene set with corresponding enrichment values for WHIM12 cells depleted of ZNF165.
- https://cdn.elifesciences.org/articles/57679/elife-57679-supp2-v2.docx
-
Supplementary file 3
Interactome data for ZNF165 and ZNF446 obtained from the CCSB Interactome Database.
- https://cdn.elifesciences.org/articles/57679/elife-57679-supp3-v2.xls
-
Supplementary file 4
Sequence-based reagents used for gene expression analysis, ChIP, and knockdown experiments.
- https://cdn.elifesciences.org/articles/57679/elife-57679-supp4-v2.xls
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/57679/elife-57679-transrepform-v2.pdf
