Wan X et al. (2010), Negative feedback regulation of Wnt4 signaling ...

XB-ART-41149

PLoS One 2010 Feb 09;52:e9118. doi: 10.1371/journal.pone.0009118.

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Negative feedback regulation of Wnt4 signaling by EAF1 and EAF2/U19.

Wan X , Ji W , Mei X , Zhou J , Liu JX , Fang C , Xiao W .

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Previous studies indicated that EAF (ELL-associated factor) family members, EAF1 and EAF2/U19, play a role in cancer and embryogenesis. For example, EAF2/U19 may serve as a tumor suppressor in prostate cancer. At the same time, EAF2/U19 is a downstream factor in the non-canonical Wnt 4 signaling pathway required for eye development in Xenopus laevis, and along with EAF1, contributes to convergence and extension movements in zebrafish embryos through Wnt maintenance. Here, we used zebrafish embryos and mammalian cells to show that both EAF1 and EAF2/U19 were up-regulated by Wnt4 (Wnt4a). Furthermore, we found that EAF1 and EAF2/U19 suppressed Wnt4 expression by directly binding to the Wnt4 promoter as seen in chromatin immunoprecipitation assays. These findings indicate that an auto-regulatory negative feedback loop occurs between Wnt4 and the EAF family, which is conserved between zebrafish and mammalian. The rescue experiments in zebrafish embryos showed that early embryonic development required the maintenance of the appropriate levels of Wnt4a through the feedback loop. Others have demonstrated that the tumor suppressors p63, p73 and WT1 positively regulate Wnt4 expression while p21 has the opposite effect, suggesting that maintenance of appropriate Wnt4 expression may also be critical for adult tissue homeostasis and prevention against tumor initiation. Thus, the auto-regulatory negative feedback loop that controls expression of Wnt4 and EAF proteins may play an important role in both embryonic development and tumor suppression. Our findings provide the first convincing line of evidence that EAF and Wnt4 form an auto-regulatory negative feedback loop in vivo.

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Species referenced: Xenopus laevis
Genes referenced: actl6a cdkn1a eaf1 eaf2 myc sult2a1 tp63 wnt4 wt1

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	Figure 1. Both zebrafish eaf1 and eaf2/u19 genes are downstream factors of zebrafish Wnt4a.(A) Zebrafish embryos were injected with Wnt4-MO and wnt4a mRNA and the levels of zebrafish eaf1 mRNA determined by whole-mount in situ hybridization at 12-somite stage. (B) Zebrafish embryos were injected with Wnt4-MO and wnt4a mRNA and the levels of zebrafish eaf/u19 mRNA determined by whole-mount in situ hybridization. 12s, 12 somites. (C) Semi-quantitative RT-PCR analysis of eaf1 expression in zebrafish embryos (12 somites) injected with Wnt4a-MO and wnt4a mRNA. (D) Semi-quantitative RT-PCR analysis of eaf2/u19 expression in zebrafish embryos (12 somites) injected with Wnt4a-MO and wnt4a mRNA.
	Figure 2. Zebrafish wnt4a can activate the zebrafish eaf1 and eaf2/U19 promoters.(A) The sequence of the 1.2 kb zebrafish eaf1 promoter and the positions of primers used for subcloning. The transcriptional initial site is indicated by +1. (B) Zebrafish embryos were co-injected with the eaf1 promoter reporter and either a control vector or a vector expressing Myc-Want4a. The luciferase activity was normalized to Renilla and reported as the mean Â± standard deviation (SD). (C) The sequence of the 1.0 kb zebrafish eaf2/u19 promoter and the primers used for subcloning. The transcriptional initial site is indicated by +1. (D) The expression of Myc-Wnt4a was verified by Western blot using an anti-Myc antibody. (E) Zebrafish embryos were co-injected with the eaf2/u19 promoter reporter and either a control vector or a vector expressing Myc-Wnt4a. The luciferase activity was normalized to Renilla and reported as the mean Â± SD.
	Figure 3. The knockdown of zebrafish Eaf1 and Eaf2/U19 rescues the embryonic defects caused by wnt4a over-expression.(A) Zebrafish embryos were co-injected with wnt4a mRNA (10 pg) and standard morpholino (STD) (8 ng). Defects, indicated by red arrows, included (b) folking notochord, (c) cyclopia, and (d) shortened axis. (B) (a) The embryos were co-injected with wnt4a mRNA and Eaf1/2-MO1to rescue the phenotype resembles wild-type embryos. (b) An example of a rescued embryo. (c) The efficiency of the Eaf1/Eaf2-MO1-mediated rescue is statistically significant (p<0.05) by counting defective embryos.
	Figure 4. Both zebrafish eaf1 and eaf2/u19 suppress zebrafish wnt4a expression.(A) Whole-mount in situ hybridization analysis of zebrafish wnt4a expression in embryos injected with eaf1 mRNA, eaf2/u19 mRNA, Eaf1-MO and Eaf2-MO at 12-somite stage. The embryos without injection were used as control. (B) The expression of zebrafish wnt4a mRNA was suppressed by ectopic expression of zebrafish eaf1 and eaf2/u19 (a) and up-regulated by knockdown Eaf1 and Eaf2/U19 (b) as revealed by semi-quantitative RT-PCR analysis at 12-somte stage.
	Figure 5. Both human EAF2/U19 and EAF1 are Wnt4 downstream factors.(A) The sequence of the 1.0 kb human EAF1 promoter and the positions of primers used for subcloning. The transcriptional initial site is indicated by +1. (B) 293 cells were co-transfected with the EAF1 promoter reporter and either a control vector or a vector expressing HA-Wnt4. The luciferase activity was normalized to Renilla and reported as the mean Â± standard deviation (SD). (C) The expression of Wnt4 was verified by Western blot using an anti-HA antibody. (D) The partial sequence of the 3.6 kb human EAF2/U19 promoter and the primers used for subcloning. The transcriptional initial site is indicated by +1. (E) .293 cells were co-transfected with the EAF2/U19 promoter reporter and either a control vector or a vector expressing HA-Wnt4. The luciferase activity was normalized to Renilla and reported as the mean Â± SD.
	Figure 6. Western blot analysis of human endogenous EAF1 and EAF2/U19 protein expression after the introduction of ectopic Wnt4 expression in 293 cells.(A) Representative Western blot of EAF1 in 293 cells transfected with a control vector or a vector expressing Wnt4 using a polyclonal antibody against human EAF1. (b) EAF1 expression was normalized to Î²-actin. (B) Representative Western blot of EAF2/U19 in 293 cells transfected with a control vector or a vector expressing Wnt4 using a polyclonal antibody against human EAF2/U19. (b) EAF2/U19 expression normalized to Î²-actin.
	Figure 7. Human Wnt4 expression is suppressed by human EAF1 and EAF2/U19 in a human cell line.(A) 293 cells were transfected with human Wnt4 promoter (1.2 kb and 2.6 kb)-reporter constructs and a control vector or vectors expressing EAF1 or EAF2/U19. Luciferase activity was normalized to Renilla and reported as the mean Â± SD. (B) (a) Endogenous Wnt4 expression in 293 cells is suppressed by the over-expression of either EAF1 or EAF2/U19 as revealed by Northern blot analysis. (b) Northern blots were quantitated and then normalized to Î²-actin mRNA.
	Figure 8. Both human EAF1 and EAF2/U19 bind to the human Wnt4 promoter as revealed by chromatin immunoprecipitaion (ChIP) assays.(A) 293 cells were treated with formaldehyde to create cross-links between EAF1 or EAF2/U19 and chromatin. The chromatin was isolated, sheared, and immunoprecipitated (IP) using polyclonal antibodies against human EAF1 and EAF2/U19, or preimmune serum as control. The presence of chromatin fragments corresponding to the Wnt4 gene or to the Î²-actin gene promoter was assessed by PCR using gene-specific primers. The gel shows the recovery of Wnt4 and actin gene fragments from the protein-chromatin input on the lane 3 and 6 (from left to right) as well as those recovered after immunoprecipitation with the anti-EAF1 antibody (lane 1), with the anti-EAF2/U19 antibody (lane4) and with the pre-immune serum (land 2 and 5). (B) Schematic diagram depicting the fragment of the EAF1, EAF2/U19 and actin genes that were amplified. The positions of PCR primers used to detect EAF1, EAF2/U19 and actin promoter fragments are indicated by arrows.
	Figure 9. Schematic diagram of the negative feedback regulation loop between Wnt4 and the Eaf gene family.

References [+] :

Devgan, p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation. 2005, Pubmed