Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13 - PubMed (original) (raw)

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13

Inés Carrera et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Wnt target gene transcription is mediated by nuclear translocation of stabilized beta-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. We show here that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of beta-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. We propose that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

skd and kto are required for the expression of Wg target genes. Third instar wing discs (A–H and K–P) and third instar eye disc (I and J) are shown. Anterior is to the left and dorsal is up in this and subsequent figures. The phenotypes of skd and kto mutations are indistinguishable (30, 31), so only one genotype is shown in each experiment. Clones homozygous for skdT413 (A–F), ktoT241 (G and H), skdT606 (I and J), ktoT555 (K and L), cdk8K185 (M and N), or cycCY5 (O and P) are marked by the absence of GFP (green in B, D, F, H, J, L, N, and P). (A and B) Wg staining in magenta; the arrow in B indicates a clone at the wing margin. (C, D, O, and P) β-gal staining reflects Dll-lacZ expression in magenta. (E, F, M, and N) Sens staining is in magenta. (G and H) β-gal staining reflects vgQ-lacZ expression in magenta. (I and J) β-gal staining reflects ds-lacZ expression in magenta. (K and L) β-gal staining reflects sal-lacZ expression in magenta. Although Wg is still expressed at the wing margin in skd or kto mutant clones, expression of Wg target genes is lost. However, Wg target genes are unaffected in cdk8 or cycC mutant clones.

Fig. 2.

Fig. 2.

skd and kto act downstream of Arm and Pygo. Third instar wing discs (A–D) and third instar antennal discs (E–K) are shown. (A and B) Clones expressing ArmΔN are marked by coexpression of GFP (green in B). (C and D) Clones expressing ArmΔN and homozygous for ktoT631 are marked by coexpression of GFP (green in D). β-gal staining reflecting Dll-lacZ expression is shown in magenta in A–D. ArmΔN can activate Dll expression in wild-type, but not kto mutant, cells. (E–H) HA staining (blue in E and H) shows expression of _hs_-HAPygoGAL4 after a 2-h heat shock and a 2-h recovery. UAS-GFP expression is shown in green in F and H. Clones homozygous for skdT413 are marked by a lack of arm-lacZ expression (β-gal staining in red in G and H). Loss of skd does not affect the expression of _hs_-HAPygoGAL4, but abolishes its ability to activate UAS-GFP. (I and K) Clones homozygous for ktoT555 (arrowheads) are marked by lack of arm-lacZ expression (β-gal staining in magenta in I and K). UAS-GFP expression driven by _tub_-GAL4 is shown in green in J and K. GAL4 can activate UAS-GFP in the absence of kto.

Fig. 3.

Fig. 3.

skd and kto are required for the expression of a Wg reporter in cultured cells. (A) Ratio of TCF firefly luciferase to the transfection control Pol III-RL in Kc cells treated with the indicated dsRNAs. The TCF luciferase reporter is strongly activated when axin is knocked down by RNAi, but this activation is reduced 80- to 100-fold by knocking down skd or kto in addition to axin. In Kc cells transfected with _actin_-GAL4 and UAS-luciferase, knocking down skd or kto reduces activation of the reporter by ≈3-fold. Error bars indicate the standard deviation between the triplicate samples tested for each dsRNA. This figure is a representative example of three independent experiments. (B) Western blot showing the levels of Skd and Kto protein in Kc cells treated with cdk8 (control), skd, or kto dsRNA. skd knockdown also partially reduces the level of Kto protein. The bottom blot shows a band that cross-reacts with the Kto antibody and serves as a loading control. (C–H) Wing imaginal discs with clones homozygous for ktoT241 (C–E) or skdT606 (F–H) marked by the absence of GFP (green in E and H) and stained with anti-Kto (C and F; blue in E and H) and anti-Skd (D and G; red in E and H). Kto protein is reduced in skd mutant cells, but Skd protein is unaffected in kto mutant cells.

Fig. 4.

Fig. 4.

Pygo physically interacts with Skd. (A) Anti-Skd immunoprecipitations (IPs) of extracts from embryos expressing UAS-HAPygo or UAS-HAPygoΔNHD (58) with the ubiquitous drivers daughterless (da)-GAL4 or tubulin (tub)-GAL4. Input lanes show 1% of the input for the IP. HAPygoΔNHD is less efficiently coimmunoprecipitated with anti-Skd than full-length Pygo. The lower blot shows that the unrelated nuclear protein PCNA does not coimmunoprecipitate with anti-Skd. (B) Coimmunoprecipitation of HAPygo with anti-Skd from Kc cells treated with lacZ or skd dsRNA. Removing Skd protein greatly reduces Pygo coimmunoprecipitation, demonstrating the specificity of the Skd antibody. Input lanes show 0.5% of the input, and control lanes show IPs with Protein A beads but no primary antibody. (C) Coimmunoprecipitation of a Pygo construct that lacks the PHD domain, HA-PygoΔPHDGAL4, with anti-Skd. Input lane shows 1% of the input, and the control lane shows an IP with no primary antibody. This figure shows that the interaction with Skd is independent of Pygo binding to the Lgs/Arm/TCF complex. (D) Model consistent with our results. Pygo, one of the most downstream components of the Wg-responsive transcriptional complex, may recruit the mediator complex through interactions of its NHD with Skd/Med13 and Kto/Med12, leading to transcriptional activation of Wg target genes. The C-terminal domain of Arm also directly interacts with Med12 (63), enhancing binding to the mediator complex; this interaction may explain why skd and kto have a stronger effect than pygo on Wg target genes.

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References

    1. Bjorklund S, Gustafsson CM. The yeast Mediator complex and its regulation. Trends Biochem Sci. 2005;30:240–244. - PubMed
    1. Conaway RC, Sato S, Tomomori-Sato C, Yao T, Conaway JW. The mammalian Mediator complex and its role in transcriptional regulation. Trends Biochem Sci. 2005;30:250–255. - PubMed
    1. Kim YJ, Lis JT. Interactions between subunits of Drosophila Mediator and activator proteins. Trends Biochem Sci. 2005;30:245–249. - PubMed
    1. Thompson CM, Koleske AJ, Chao DM, Young RA. A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast. Cell. 1993;73:1361–1375. - PubMed
    1. Kim YJ, Bjorklund S, Li Y, Sayre MH, Kornberg RD. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994;77:599–608. - PubMed

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