The APC tumor suppressor counteracts -catenin activation and H3K4 methylation at Wnt target genes

Sierra et al., Genes & Dev., 2006

Regulatory Domain

Activation DomainArmadillo Repeat

Tutter et al. Genes & Dev., 2001

The Identification of proteins that bind to the b-cat CTARM domain

MALDI-TOF Identification of CTARM-interacting Proetin:

Subunits from chromatin remodeling complexes

- Subunits of TRRAP/TIP60 HAT complexes

- ISW1

- SET-type complex protein, MLL1/MLL2

Chromatin-specific activation domainA

BAF57: required for H2B ubiquitination

HeLa NE

SW480 NEB

MLL2 may contribute to -cat-mediated induction of c-Myc transcription in vivo.

The -cat activation domain associates with active histone H3 methylation complexes.

H3K4 Methylation or H2B ubiquitination steps may be important for -cat activity

Analysis of HMT or HAT activity using GST-CTARM pulldown fraction

C D

Ubiquitin is required for -cat trans-activation of chromatin pBRE templates in vitro

CUE domain: bind tightly to monoubiquitin

The CUE domain competes for transcription on chromatin in vitro.

The in vitro chromatin-based transcription assay using pBRE

Not block the cooperative binding of b-cat and Lef-1 to chromatin

A B

-Cat and other Wnt pathway-specific regulators cycle on and off the c-Myc enhacer in LiCl-treated cells

To test whether -cat regulates H3K4 trimethylation at Wnt target genes in vivo

Lithium-treated C2C12 cellsA

B

Reveal that cyclic pattern of alternating coactivator and corepressor complexes

C

H3K4Me incresed strongly at the c-Myc gene

APC and -TrCP appeared together with -cat

Destruction complex subunits may participate in transcription

Continued

Disassemble the Wnt enhancer complex

Mediate the change of coactivators and corepressor complexes between transcription cycles

D

Anaylsis of APC-induced shut-off c-Myc gene transcription in the HT29-APC CRC cell line

MT: zinc-inducible metallothionein promoter

B

A

HT-29 contains no intact APC protein; instead, two C-terminal-truncated APC proteins

RNAPII, CDK9, trimethylated H3K4 were present at high levels at the active c-Myc gene

-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

C

Binding of -cat to CK1a-phosphorylated APC inhibits LEF-1:-cat transcription in vitro

APC-A,B,C -2,3

Phosphorylation of the APC-2,3 by CK1 enhances its affinity for -cat

BA

Xing et al., Mol Cell, 2004

Test the DNA-binding activity of these complexes using EMSA

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

DC

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

The full-length APC protein preferentially interacts with CtBP

Continued

B

A

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 -catenin

Exporting -catenin from the nucleus out to the cytoplasm for degradation

Directly to counteract -cat-mediated transcription at Wnt target genes in vivo

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