The APC tumor suppressor counteracts -cat enin activation and H3K4 methylation at Wn t target genes Sierra et al., Genes & Dev., 2006
Dec 31, 2015
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