Sierra et al., Genes & Dev. , 2006

1. The APC tumor suppressor counteracts b-catenin activation and H3K4 methylation at Wnt target genes Sierra et al., Genes & Dev., 2006

2. Regulatory Domain Activation Domain Armadillo Repeat The Identification of proteins that bind to the b-cat CTARM domain Chromatin-specific activation domain A Tutter et al. Genes & Dev., 2001 MALDI-TOF Identification of CTARM-interacting Proetin: Subunits fromchromatin remodeling complexes • Subunits of TRRAP/TIP60 HAT complexes • ISW1 • - SET-type complex protein, MLL1/MLL2

3. B SW480 NE HeLa NE BAF57: required for H2B ubiquitination

4. Analysis of HMT or HAT activity using GST-CTARM pulldown fraction C D The b-cat activation domain associates with active histone H3 methylation complexes. MLL2 may contribute to b-cat-mediated induction of c-Myc transcription in vivo. H3K4 Methylation or H2B ubiquitination steps may be important for b-cat activity

5. Ubiquitin is required for b-cat trans-activation of chromatin pBRE templates in vitro The in vitro chromatin-based transcription assay using pBRE A B The CUE domain competes for transcription on chromatin in vitro. Not block the cooperative binding of b-cat and Lef-1 to chromatin CUE domain: bind tightly to monoubiquitin

6. A Lithium-treated C2C12 cells B b-Cat and other Wnt pathway-specific regulators cycle on and off the c-Myc enhacer in LiCl-treated cells To test whether b-cat regulates H3K4 trimethylation at Wnt target genes in vivo C Reveal that cyclic pattern of alternating coactivator and corepressor complexes

7. Continued D H3K4Me incresed strongly at the c-Myc gene APC and b-TrCP appeared together with b-cat Destruction complex subunits may participate in transcription Disassemble the Wnt enhancer complex Mediate the change of coactivators and corepressor complexes between transcription cycles

8. Anaylsis of APC-induced shut-off c-Myc gene transcription in the HT29-APC CRC cell line HT-29 contains no intact APC protein; instead, two C-terminal-truncated APC proteins A MT: zinc-inducible metallothionein promoter B

9. C RNAPII, CDK9, trimethylated H3K4 were present at high levels at the active c-Myc gene b-cat and the associated coactivators do not cycle on and off of the enhancer APC-mediated shut-off of c-Myc transcription proceeds in two step The transient binding of full-length APC and the CtBP corepressor to the enhancer The stable binding of TLE-1 and HDAC to the region, resulting in the repression of the target gene APC acts directly and immediately to facilitate the repression of Wnt target genes

10. APC-A,B,C-2,3 Binding of b-cat to CK1a-phosphorylated APC inhibits LEF-1:b-cat transcription in vitro A B Phosphorylation of the APC-2,3 by CK1d enhances its affinity for b-cat Xing et al., Mol Cell, 2004

11. Test the DNA-binding activity of these complexes using EMSA C D P-APC-2,3 compete for the formation of the b-cat:LEF-1:DNA complex CK1 phosphorylation of APC may induce high-affinity binding to b-cat and trigger its dissociation from LEF-1

12. Wild-type, but not mutant, APC proteins associate with the CtBP corepressor in extract APC interacts directly with CtBP Hamada et al., Dev Cell, 2004 CtBP does not associate with truncated APC proteins on DNA in vivo

13. Continued A B The full-length APC protein preferentially interacts with CtBP

14. Post-translational modifications govern the interactions between different Wnt transcriptional regulators APC may have a nuclear function, seprate from exporting b-catenin, came from the studie of its interaction with CtBP APC sequesters b-catenin in the nucleus and away from Wnt target gene promoters by targeting it to CtBP APC may have evolved a dual mechanism to negatively regulate b-catenin Exporting b-catenin from the nucleus out to the cytoplasm for degradation Directly to counteract b-cat-mediated transcription at Wnt target genes in vivo

15. Post-translational modifications govern the interactions between different Wnt transcriptional regulators