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. 2014 Jul 15:3:e02564.
doi: 10.7554/eLife.02564.

The Hippo effector Yorkie activates transcription by interacting with a histone methyltransferase complex through Ncoa6

Yun Qing  1 , Feng Yin  1 , Wei Wang  1 , Yonggang Zheng  1 , Pengfei Guo  1 , Frederick Schozer  2 , Hua Deng  1 , Duojia Pan  1
Affiliations

The Hippo effector Yorkie activates transcription by interacting with a histone methyltransferase complex through Ncoa6

Yun Qing et al. Elife. .

Abstract

The Hippo signaling pathway regulates tissue growth in Drosophila through the transcriptional coactivator Yorkie (Yki). How Yki activates target gene transcription is poorly understood. Here, we identify Nuclear receptor coactivator 6 (Ncoa6), a subunit of the Trithorax-related (Trr) histone H3 lysine 4 (H3K4) methyltransferase complex, as a Yki-binding protein. Like Yki, Ncoa6 and Trr are functionally required for Hippo-mediated growth control and target gene expression. Strikingly, artificial tethering of Ncoa6 to Sd is sufficient to promote tissue growth and Yki target expression even in the absence of Yki, underscoring the importance of Yki-mediated recruitment of Ncoa6 in transcriptional activation. Consistent with the established role for the Trr complex in histone methylation, we show that Yki, Ncoa6, and Trr are required for normal H3K4 methylation at Hippo target genes. These findings shed light on Yki-mediated transcriptional regulation and uncover a potential link between chromatin modification and tissue growth.

Keywords: D. melanogaster; Hippo signaling; developmental biology; growth control; histone methylation; stem cells; transcription factors.

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Conflict of interest statement

DP: Reviewing editor, eLife.

The other authors declare that no competing interests exist.

Figures

Figure 1.
Figure 1.. Ncoa6 physically interacts with Yki and regulates HRE activity.
(A) Schematic protein structure of Drosophila Ncoa6 and its human orthologue, which contain three and two PPxY motifs, respectively. (B) S2R+ cells expressing the indicated constructs were subjected to immunoprecipitation as indicated. Note the physical interactions between Ncoa6 and Yki, and absence of interactions between Ncoa63m and Yki or between Ncoa6 and YkiWM. (C) Drosophila S2R+ cells co-transfected with HA-Yki and FLAG-Ncoa6 or FLAG-Ncoa63m constructs were stained for the indicated epitopes. Cells with or without FLAG expression are marked by arrowheads and arrows, respectively. Both FLAG-Ncoa6 and FLAG-Ncoa63m were localized to the nucleus (arrowheads), while HA-Yki was more concentrated in the cytoplasm (arrows). FLAG-Ncoa6, but not FLAG-Ncoa63m, induced nuclear accumulation of HA-Yki (compare arrowheads in the merged channel). (D) Luciferase activity was measured in triplicates in Drosophila S2R+ cells transfected with the indicated constructs. Ncoa6, but not Ncoa63m, enhanced Yki/Sd-mediated activation of HRE-luciferase reporter. This enhancement was suppressed by co-expression of Wts. Error bars represent standard deviations. (E) Drosophila S2R+ cells expressing FLAG-tagged Ncoa6 were subjected to ChIP analysis using control IgG, antibodies against FLAG or antibodies against endogenous Yki. The enrichment of HRE at the endogenous diap1 locus was measured by real-time PCR. Both Yki and FLAG-Ncoa6 bound to the diap1 HRE. DOI: http://dx.doi.org/10.7554/eLife.02564.003
Figure 1—figure supplement1.
Figure 1—figure supplement1.. Identification of Ncoa6 as a positive regulator of the HRE activity from cell-based RNAi screen.
(A) Identification of Ncoa6 as a positive regulator of HRE activity from the primary RNAi screen. The scatter plot highlights genes whose RNAi resulted in a decrease in Yki/Sd-induced HRE reporter activity, with each gene represented by a single dot. The locations of Ncoa6 (CG14023), sd, and yki are marked. (B) Validation of Ncoa6 as a positive regulator of HRE activity. Luciferase activity was measured in triplicates in Drosophila S2R+ cells transfected with Yki, Sd, HRE-Luciferase, and Pol III-Renilla expression vectors, together with dsRNA of GFP (control) or Ncoa6. Error bars represent standard deviations. (C) A list of primary hits from the RNAi screen with Z-scores of less than −2.26. DOI: http://dx.doi.org/10.7554/eLife.02564.004
Figure 2.
Figure 2.. Ncoa6 and Trr are required for normal tissue growth and expression of Hippo target genes in Drosophila imaginal discs.
(A) RNAi knockdown of Ncoa6 and Trr by dpp-Gal4 resulted in decreased area of the dpp expression domain in adult wings. The pictures were taken under the same magnification. The graph shows quantification of the dpp expression domain (green area in the schematic drawing) relative to the entire wing area (mean ± SEM, n = 14, ***p<0.001). The complete genotypes are: UAS-Dicer2; dpp-Gal4 UAS-GFP (control), UAS-Dicer2; dpp-Gal4 UAS-GFP/UAS-Ncoa6RNAi (Ncoa6 RNAi), and UAS-Dicer2; dpp-Gal4 UAS-GFP/UAS-trrRNAi (trr RNAi). (B–G) RNAi knockdown of Ncoa6 or Trr resulted in decreased expression of Hippo target genes. Wing discs expressing UAS-GFP only (B and E), UAS-GFP plus Ncoa6 RNAi (C and F), or UAS-GFP plus trr RNAi (D and G) were stained for Diap1 (B–D) or fj-lacZ (E–G). Note the reduction of Diap1 and fj-lacZ levels upon Ncoa6 or Trr RNAi. The complete genotypes are: UAS-Dicer2; dpp-Gal4 UAS-GFP (B), UAS-Dicer2; dpp-Gal4 UAS-GFP/UAS-Ncoa6RNAi (C), UAS-Dicer; dpp-Gal4 UAS-GFP/UAS-trr RNAi (D), UAS-Dicer2; fj-lacZ; dpp-Gal4 UAS-GFP (E), UAS-Dicer2; fj-lacZ; dpp-Gal4 UAS-GFP/UAS-Ncoa6 RNAi (F), and UAS-Dicer2; fj-lacZ; dpp-Gal4 UAS-GFP/UAS-trr RNAi (G). DOI: http://dx.doi.org/10.7554/eLife.02564.005
Figure 3.
Figure 3.. Genetic interactions between Ncoa6-Trr and the Hippo pathway.
Adult eye images of the indicated genotypes, all taken under the same magnification. (A) GMR-Gal4/+. Wild-type control. (B) GMR-Gal4/+; UAS-Ncoa6 RNAi/+. RNAi knockdown of Ncoa6 resulted in a mild decrease in eye size (compare B to A). (C) UAS-Dicer2/+; GMR-Gal4/+; UAS-trr RNAi/+. RNAi knockdown of Trr resulted in no visible effects on eye size (compare C to A). (D) GMR-Gal4 UAS-Yki/+. Overexpression of Yki resulted in an increase in eye size (compare D to A). (E) GMR-Gal4 UAS-Yki/+; UAS-Ncoa6 RNAi/+. RNAi knockdown of Ncoa6 suppressed eye overgrowth induced by Yki overexpression (compare E to D). (F) UAS-Dicer2/+; GMR-Gal4 UAS-Yki/+; UAS-trr RNAi/+. RNAi knockdown of Trr suppressed eye overgrowth induced by Yki overexpression (compare F to D). (G) UAS-Wts RNAi/+; GMR-Gal4/+. RNAi knockdown of Wts resulted in an increase in eye size (compare G to A). (H) UAS-Wts RNAi/+; GMR-Gal4/ UAS-Ncoa6 RNAi. RNAi knockdown of Ncoa6 suppressed eye overgrowth induced by Wts knockdown (compare H to G). (I) UAS-Dicer2/+; UAS-Wts RNAi/+; GMR-Gal4/ UAS-trr RNAi. RNAi knockdown of Trr did not obviously suppress eye overgrowth caused by Wts knockdown. (J) GMR-Gal4 UAS-Sd/+. Overexpression of Sd resulted in a decrease in eye size (compare J to A). (K) GMR-Gal4 UAS-Sd/+; UAS-Ncoa6 RNAi/+. RNAi knockdown of Ncoa6 enhanced the small eye phenotype caused by Sd overexpression (compare K to J). (L) UAS-Dicer2/+; GMR-Gal4 UAS-Sd/+; UAS-trr RNAi/+. RNAi knockdown of Trr enhanced the small eye phenotype caused by Sd overexpression (compare L to J). DOI: http://dx.doi.org/10.7554/eLife.02564.006
Figure 4.
Figure 4.. Ncoa6 and Trr are required for Hippo-mediated target gene expression.
Wing discs containing GFP-marked MARCM clones were stained for Diap1 (red). For each genotype, the left most panel shows low magnification view of the wing disc (Hoechst + GFP), while the remaining three panels show higher magnification view of the same wing disc (GFP, Diap1 and GFP + Diap1). (A–F) Wing discs containing GFP-marked MARCM clones (green) of WT control (A), hpo mutant (B), Ncoa6 RNAi (C), hpo mutant with Ncoa6 RNAi (D), Trr RNAi (E), and hpo mutant with Trr RNAi (F). Note the increased Diap1 levels in hpo mutant clones and the decreased Diap1 levels in Ncoa6 RNAi or Trr RNAi clones. Also note the decreased Diap1 levels in hpo mutant clones with Ncoa6 RNAi or Trr RNAi. DOI: http://dx.doi.org/10.7554/eLife.02564.007
Figure 5.
Figure 5.. Fusion of Ncoa6 with the DNA binding domain of Sd bypasses Yki to stimulate Hippo target gene and tissue growth.
(A–E) Wing discs containing GFP-marked MARCM clones (green) of WT control (A), ykiB5 (B), SdDB-Ncoa6 overexpression (C), ykiB5 with SdDB-Ncoa6 overexpression (D), and ykiB5 with Ncoa6 overexpression (E),were stained for Diap1 (red). For each genotype, the left most panel shows low magnification view of the wing disc (Hoechst + GFP), while the remaining three panels show higher magnification view of the same wing disc (GFP, Diap1, and GFP + Diap1). Note the decreased Diap1 expression and undergrowth of ykiB5 clones (B) or ykiB5 clones with Ncoa6 overexpression (E). SdDB-Ncoa6 overexpression resulted in elevated Diap1 levels in ykiB5 clones (D). (F) Luciferase activity was measured in triplicates in Drosophila S2R+ cells transfected with the indicated constructs. Error bars represent standard deviations. Note the Wts-insensitive stimulation of the HRE-luciferase reporter by SdDB-Ncoa6. DOI: http://dx.doi.org/10.7554/eLife.02564.008
Figure 6.
Figure 6.. Yki modulates local H3K4 methylation at Hippo target genes.
(A) Schematic view of diap1 and ex genomic loci analyzed by ChIP. Transcriptional start site is labeled as +1, and p1–p5 are a series of primer sets encompassing following regions of diap1 and ex: diap1: p1: −1951 ∼ −1813, p2: +228 ∼ +377, p3: +3993 ∼ +4104; ex: p4: −749 ∼ −608, p5: +249 ∼ +393. Note that p3 covers the diap1 HRE. Also shown are the profiles of H3K4me1 (blue line) and H3K4me3 (red line) binding derived from a previously published ChIP-Seq analysis in S2 cells (Herz et al., 2012). (B and C) RNAi knockdown of Yki, Ncoa6 or Trr resulted in decreased H3K4me3 (B) and H3K4me1 (C) modification on Hippo target genes. ChIP analysis of H3K4me1 or H3K4me3 were performed in Drosophila S2R+ cells treated with dsRNA of GFP (control), Yki, Ncoa6, or Trr. Chromatins were precipitated by control IgG or antibodies against H3K4me1 and H3K4me3. The enrichment of ChIP products on diap and ex was measured by real-time PCR using the indicated primers. ***p<0.001, **p<0.01,*p<0.05. DOI: http://dx.doi.org/10.7554/eLife.02564.009
Figure 6—figure supplement1.
Figure 6—figure supplement1.. Ncoa6, but not Yki, regulates global levels of H3K4 methylation.
(A–C) Wing discs with Ncoa6 RNAi in the posterior compartment were stained for mono-, di-, and tri-methylation of H3K4 as indicated. Note the subtle decrease of H3K4me1 (A) and H3K4me3 (C), but not H3K4me2 (B), in the GFP-marked posterior compartment. The complete genotype is: UAS-Dicer2; en-Gal4 UAS-GFP; UAS-Ncoa6RNAi. (D–F) Wing discs containing ykiB5 mutant clones were stained for H3K4me1, H3K4me2, and H3K4me3 as indicated. Note the normal levels of H3K4me1, H3K4me2, and H3K4me4 in yki mutant clones (arrows, GFP-negative) compared to the wild-type neighbors (GFP-positive). DOI: http://dx.doi.org/10.7554/eLife.02564.010
Author response image 1.
Author response image 1.
(A) Cell fractionation of S2R+ cells showing that RNAi knockdown of Ncoa6 modestly decreased HA-Yki in the nucleus, with a concurrent increase in the cytoplasm.(B) RNAi knockdown of Ncoa6 caused no obvious change in subcellular fractionation of endogenous Yki. Sd, which has been reported to promote Yki nuclear localization, was included as a control. Knockdown of Sd led to a modest decrease of nuclear Yki. (C-D) RNAi knockdown of Ncoa6 did not obviously affect the expression of Ex. Wing discs expressing UAS-GFP only (C) or UAS-GFP plus Ncoa6 RNAi (D) were stained for Ex. The complete genotypes are: UAS-Dicer2; dpp-Gal4 UAS-GFP (C), UAS-Dicer2; dpp-Gal4 UAS- GFP/UAS-Ncoa6RNAi (D).
See this image and copyright information in PMC

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