ReportsDevelopment

Identification of an NKX3.1-G9a-UTY transcriptional regulatory network that controls prostate differentiation

See allHide authors and affiliations

Science  24 Jun 2016:
Vol. 352, Issue 6293, pp. 1576-1580
DOI: 10.1126/science.aad9512
  • Fig. 1 Murine Nkx3.1 respecifies a nonprostatic epithelium to form prostate in vivo.

    (A) Heat map representations of differentially expressed genes from Nkx3.1+/+ and Nkx3.1−/− prostate (6). (B) Gene set enrichment analysis, using as the query gene set differentially expressed genes from seminal vesicle versus prostate, compared with a reference gene signature of Nkx3.1−/− versus Nkx3.1+/+ prostate. (C) Diagram of the tissue recombination assay. Dissociated epithelium from seminal vesicle (SVE) or prostate (PE) is infected with a lentivirus expressing Nkx3.1 (or control). Mesenchyme from rat embryonic urogenital sinus is combined with the epithelium and grown under the renal capsule of host mice. (D and E) Representative tissue recombinants. (D) (Top) Whole-mount images. (Bottom) Hematoxylin and eosin (H&E) images. (E) Confocal images of immunofluorescence using the indicated antibodies. Scale bars represent 50 μm in (D) and 20 μm in (E). A summary of tissue recombinant data is provided in table S4.

  • Fig. 2 Induction of prostate differentiation by NKX3.1 requires the homeodomain.

    (A) Diagram of the experimental design. Human RWPE1 prostate epithelial cells are infected with a lentivirus expressing human NKX3.1, NKX3.1(T164A), or a control, followed by analyses in vitro (B to D) or recombined with mesenchyme and grown under the renal capsule of host mice (E). (B) Western blot analyses. Actin is a control for protein loading. (C) Gel retardation analysis done using nuclear extracts from RWPE1 cells expressing the control vector, NKX3.1, or NKX3.1 (T164A). The arrow indicates the free DNA probe. The experiments in (B) and (C) were each performed with three independent biological replicates; representative data are shown. (D) Heat map representations of selected differentially expressed genes; a complete list is provided in database S4. (E) Tissue recombinants showing whole-mount images, H&E, and immunofluorescence staining. The ruler shows cm scale; scale bars represent 50 μm in the H&E images and 20 μm in the immunofluorescence images. A summary of all tissue recombinants is provided in table S4.

  • Fig. 3 Induction of prostate differentiation by NKX3.1 is mediated through its interaction with the G9a histone methyltransferase.

    (A and B) Nuclear extracts from RWPE1 cells expressing Flag-HA-NKX3.1 or the control were subjected to immunoprecipitation followed by mass spectrometry (A) or Western blot analysis (B) (see fig. S6). (A) Silver stain showing G9a interaction. Markers, as indicated. NS, nonspecific bands. (B) Immunoprecipitation followed by Western blot analysis. Input shows 5% of the total protein, and immunoprecipitation (IP) shows proteins recovered after IP using an antibody to Flag. Experiments were performed with three independent biological replicates; representative data are shown. (C) Diagram of experimental design for (D) and (E). Human RWPE1 prostate epithelial cells were infected with an NKX3.1-expressing lentivirus (expressing red fluorescent protein), followed by infection with an shRNA-expressing lentivirus (expressing green fluorescent protein). Coinfected cells were sorted by fluorescence-activated cell sorting, followed by analyses in vitro (D) or generation of tissue recombinants in vivo. (D) Western blot analysis. Experiments were performed with three independent biological replicates; representative data are shown. (E) Representative tissue recombinants showing whole-mount, H&E, and confocal images of immunofluorescence staining. Indicated is the kidney and the collagen plug (for the recombinants that did not grow) or the tissue recombinant. The ruler shows cm scale; scale bars represent 50μm in the H&E images and 20 μm in the immunofluorescence images. A summary of tissue recombinants is provided in table S4.

  • Fig. 4 Induction of prostate differentiation by NKX3.1 is mediated by UTY, a male-specific paralog of histone demethylase UTX (KDM6c).

    (A) Real-time polymerase chain reaction (PCR) showing expression of NKX3.1 target genes, UTY and EDEM2 (left). Chromatin immunoprecipitation quantitative PCR analysis of NKX3.1 binding (center) and G9a binding (right) to NKX3.1 target genes were performed. Analyses were performed with three independent biological replicates. Statistical analysis was done using a two-tailed t test; data are indicated as mean ± SD. (B) Diagram of the experimental design for (C) to (E). Human RWPE1 cells (C and D) or mouse tissues (E) were infected with an NKX3.1-expressing lentivirus, followed by infection with an shRNA (or controls). Cells were analyzed in vitro [human (C)] or in tissue recombinants [human (D) and mouse (E)]. (D) Western blot analysis of RPWE1 cells. [(D) and (E)] Representative tissue recombinants of human (D) and mouse (E) showing whole-mount, H&E, and confocal images of immunofluorescence staining. The ruler shows cm scale; scale bars represent 50 μm in the H&E images and 20 μm in the immunofluorescence images. A summary of tissue recombinants is provided in table S4.

Supplementary Materials

  • Identification of an NKX3.1-G9a-UTY transcriptional regulatory network that controls prostate differentiation

    Aditya Dutta, Clémentine Le Magnen, Antonina Mitrofanova, Xuesong Ouyang, Andrea Califano, Cory Abate-Shen

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S11
    • Tables S1 to S6
    • References
    Data S1
    Differentially expressed genes comparing Nkx3.1 wild-type and mutant mice at 4 months (p < 0.0001)
    Data S2
    Differentially expressed genes comparing prostate and seminal vesicle at 15 months (p < 0.001)
    Data S3
    Differentially expressed genes comparing mouse tissue recombinants (p < 0.0001)
    Data S4
    Differentially expressed genes comparing control and NKX3.1- expressing RWPE1 cells (p < 0.0001)

Navigate This Article