Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jun 24;352(6293):1576-80.
doi: 10.1126/science.aad9512.

Identification of an NKX3.1-G9a-UTY Transcriptional Regulatory Network That Controls Prostate Differentiation

Affiliations
Free PMC article

Identification of an NKX3.1-G9a-UTY Transcriptional Regulatory Network That Controls Prostate Differentiation

Aditya Dutta et al. Science. .
Free PMC article

Abstract

The NKX3.1 homeobox gene plays essential roles in prostate differentiation and prostate cancer. We show that loss of function of Nkx3.1 in mouse prostate results in down-regulation of genes that are essential for prostate differentiation, as well as up-regulation of genes that are not normally expressed in prostate. Conversely, gain of function of Nkx3.1 in an otherwise fully differentiated nonprostatic mouse epithelium (seminal vesicle) is sufficient for respecification to prostate in renal grafts in vivo. In human prostate cells, these activities require the interaction of NKX3.1 with the G9a histone methyltransferase via the homeodomain and are mediated by activation of target genes such as UTY (KDM6c), the male-specific paralog of UTX (KDM6a) We propose that an NKX3.1-G9a-UTY transcriptional regulatory network is essential for prostate differentiation, and we speculate that disruption of such a network predisposes to prostate cancer.

Figures

Figure 1
Figure 1. Nkx3.1 re-specifies a non-prostatic epithelium to form prostate in vivo
(A) Heat map representations of differentially expressed genes from Nkx3.1+/+ and Nkx3.1−/− prostate (6). (B) GSEA 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,E) Representative tissue recombinants. (D) (Top) Whole mount images. (Bottom) H&E images. (E) Confocal images of immunofluorescence using the indicated antibodies. Scale bars represent 50 μm. A summary of tissue recombinant data is provided in Table S4.
Figure 2
Figure 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 following by analyses in vitro (B-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 3 independent biological replicates; representative data are shown. (D) Heat map representations of selected differentially expressed genes; a complete list is provided in Dataset S4. (E) Tissue recombinants showing whole mount images, H&E and immunofluorescence staining. The ruler shows cm scale; scale bars represent μm. A summary of all tissue recombinants is provided in Table S4.
Figure 3
Figure 3. Induction of prostate differentiation by NKX3.1 is mediated via its interaction with G9a
(A,B) Nuclear extracts from RWPE1 cells expressing Flag-HA-NKX3.1 or the control were subjected to immunoprecipitation followed by mass spectrometry (A) or immunoprecipitation (B) (see Fig. S6). (A) Silver stain showing G9a interaction. Markers, as indicated. NS, non-specific bands. (B) Immunoprecipitation followed by Western blot analysis. Input shows 5% of the total protein, and IP shows proteins recovered following immunoprecipitation using an anti-Flag antibody. Experiments were performed with 3 independent biological replicates; representative data are shown. (C) Diagram of experimental design for (D,E). Human RWPE1 prostate epithelial cells were infected with an NKX3.1-expressing lentivirus (expressing RFP), followed by infection with an shRNA-expressing lentivirus (expressing GFP). Co-infected cells were FACS sorted followed by analyses in vitro (D) or generation of tissue recombinants in vivo. (D) Western blot analysis. Experiments were performed with 3 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 represent 50 μm. A summary of tissue recombinants is provided in Table S4.
Figure 4
Figure 4. Induction of prostate differentiation by NKX3.1 is mediated by UTY (KDM6c)
(A) Real time PCR showing expression of NKX3.1 target genes, UTY and EDEM2 (left). ChIP-qPCR analysis of NKX3.1 binding (center), and G9a binding (right) to NKX3.1 target genes. Analyses were performed with 3 independent biological replicates. Statistical analysis was done using a 2-tailed t-test; data are indicated as mean +/− SD. (B) Diagram of the experimental design for C–E. Human RWPE1 cells (C,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 (mouse and human, D and E). (D) Western blot analysis of RPWE1 cells. (D,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. A summary of tissue recombinants is provided in Table S4.

Similar articles

See all similar articles

Cited by 22 articles

See all "Cited by" articles

Publication types

Feedback