The testis protein ZNF165 is a SMAD3 cofactor that coordinates oncogenic TGFβ signaling in triple-negative breast cancer

  1. Zane A Gibbs
  2. Luis C Reza
  3. Chun-Chun Cheng
  4. Jill M Westcott
  5. Kathleen McGlynn
  6. Angelique W Whitehurst  Is a corresponding author
  1. Department of Pharmacology, University of Texas Southwestern Medical Center, United States
  2. Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, United States
  3. Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, United States
7 figures, 1 table and 5 additional files

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 …

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 …

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
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Mus musculus)NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ; NSGThe Jackson LaboratoryStock no. 005557; RRID:IMSR_JAX:005557
Cell line (Homo sapiens)HEK293TATCCCRL-3216; RRID:CVCL_0063
Cell line (H. sapiens)MCF7ATCCHTB-22; RRID:CVCL_0031
Cell line (H. sapiens)WHIM12Li et al., 2013
Cell line (H. sapiens)SUM159AsterandRRID:CVCL_5423
Cell line (H. sapiens)SUM159T-LuciferaseWestcott et al., 2015
Antibodyanti-HA (Rat monoclonal)RocheCat #11867423001; RRID:AB_390918IB (1:1000), IP (1 μg)
Antibodyanti-c-Myc (Rabbit polyclonal)Santa Cruz Biotechnologysc-789; RRID:AB_631274IB (1:1000)
Antibodyanti-V5 (Rabbit monoclonal)Cell Signaling TechnologyCat #13202; RRID:AB_2687461IB (1:1000)
Antibodyanti-SMAD3 (Rabbit polyclonal)Abcamab28379; RRID:AB_2192903IB (1:1000), PLA (1:200), ChIP-qPCR (1.5 μg), ChIP-seq (10 μg)
Antibodyanti-phospho-SMAD3 (Rabbit monoclonal)Cell Signaling TechnologyCat #9520; RRID:AB_2193207IB (1:1000), ChIP (5 μg)
Antibodyanti-SMURF2 (Mouse monoclonal)Santa Cruz Biotechnologysc-393848IB (1:500)
Antibodyanti-RRAD (Goat polyclonal)Thermo Fisher ScientificPA537885; RRID:AB_2554493IB (1:1000)
Antibodyanti-ZNF165 (Mouse monoclonal)Novus BiologicalsH00007718-M02; RRID:AB_1717273IB (1:1000)
Antibodyanti-ZNF165 (Rabbit polyclonal)Atlas AntibodiesHPA007247; RRID:AB_2797217IB (1:1000), PLA (1:200), IP (5 μg)
Antibodyanti-Tubulin (Rabbit monoclonal)Cell Signaling TechnologyCat #2128; RRID:AB_823664IB (1:1000)
Antibodyanti-ZNF446 (Rabbit polyclonal)Proteintech16218–1-APIB (1:1000), PLA (1:200)
Antibodyanti-TRIM27 (Rabbit monoclonal)Cell Signaling TechnologyCat #15099; RRID:AB_2798707IB (1:1000)
Antibodyanti-V5 (Mouse monoclonal)Thermo Fisher ScientificR960-25; RRID:AB_2556564PLA (1:500), IP (1 μg)
Antibodyanti-SMAD3 (Mouse monoclonal)Santa Cruz Biotechnologysc-101154; RRID:AB_1129525PLA (1:200)
Antibodyanti-SMAD4 (Mouse monoclonal)Santa Cruz Biotechnologysc-7966; RRID:AB_627905PLA (1:200)
Antibodyanti-TRIM27 (Rabbit polyclonal)Proteintech12205–1-AP; RRID:AB_2256660PLA (1:200), IHC (1:200)
Antibodyanti-H3K27ac (Rabbit polyclonal)Abcamab4729; RRID:AB_2118291ChIP-qPCR (2 μg)
AntibodyNormal rabbit IgG (Rabbit polyclonal)Cell Signaling TechnologyCat #2729; RRID:AB_1031062IP (1 μg), ChIP-qPCR (1.5–5 μg)
Antibodyanti-V5 (Rabbit polyclonal)Abcamab9116; RRID:AB_307024ChIP-seq (5 μg)
Peptide, recombinant proteinHuman Transforming Growth Factor-β1Cell Signaling TechnologyCat #8915
Chemical compound, drugSB-431542Tocris BioscienceCat #1614
Commercial assay or kitKAPA HyperPrep KitKAPA BiosystemsKK8502
Commercial assay or kitDuolink In Situ PLA Probe Anti-Mouse PLUSSigma-AldrichDUO92001; RRID:AB_2810939
Commercial assay or kitDuolink In Situ PLA Probe Anti-Rabbit MINUSSigma-AldrichDUO92005; RRID:AB_2810942
Commercial assay or kitDuolink In Situ Detection Reagent RedSigma-AldrichDUO92008
Software, algorithmImageJ (Fiji)Schindelin et al., 2012RRID:SCR_002285ver 2.0.0
Software, algorithmBowtie2Langmead and Salzberg, 2012RRID:SCR_005476ver 2.3.2
Software, algorithmSAMtoolsLi et al., 2009RRID:SCR_002105ver 1.6
Software, algorithmDeepToolsRamírez et al., 2016RRID:SCR_016366ver 2.3.5
Software, algorithmIntegrative Genomics ViewerRobinson et al., 2011RRID:SCR_011793ver 2.3.93
Software, algorithmHOMERHeinz et al., 2010RRID:SCR_010881ver 4.9
Software, algorithmGREATMcLean et al., 2010RRID:SCR_005807ver 3.0.0
Software, algorithmMEMEBailey et al., 2009RRID:SCR_001783ver 5.1.1
Software, algorithmGSEASubramanian et al., 2005RRID:SCR_003199ver 4.0.3

Additional files

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

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