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The APC tumor suppressor counteracts b-catenin
activation and H3K4 methylation at Wnt target genes
Sierra et al., Genes & Dev., 2006
The Identification of proteins that bind to the b-cat CTARM domain
Regulatory
Domain
Armadillo Repeat
Activation
Domain
Chromatin-specific activation domain
A
Tutter et al. Genes & Dev., 2001
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
B
SW480 NE
HeLa NE
BAF57: required for H2B ubiquitination
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
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
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
A
Lithium-treated C2C12 cells
C
B
Reveal that cyclic pattern of alternating
coactivator and corepressor complexes
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
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
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
Binding of b-cat to CK1a-phosphorylated APC inhibits LEF-1:b-cat
transcription in vitro
A
B
APC-A,B,C -2,3
Phosphorylation of the APC-2,3 by CK1d enhances its affinity for b-cat
Xing et al., Mol Cell, 2004
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
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
Continued
A
B
The full-length APC protein preferentially interacts with CtBP
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
Post-translational modifications govern the interactions between
different Wnt transcriptional regulators