The chromatin remodelling factor Brg-1 interacts with beta-catenin to promote target gene activation

Abstract

Wnt-induced formation of nuclear Tcf-beta-catenin complexes promotes transcriptional activation of target genes involved in cell fate decisions. Inappropriate expression of Tcf target genes resulting from mutational activation of this pathway is also implicated in tumorigenesis. The C-terminus of beta-catenin is indispensable for the transactivation function, which probably reflects the presence of binding sites for essential transcriptional coactivators such as p300/CBP. However, the precise mechanism of transactivation remains unclear. Here we demonstrate an interaction between beta-catenin and Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodelling complexes. A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells is enhanced activity of a Tcf-responsive reporter gene. Consistent with this, stable expression of inactive forms of Brg-1 in colon carcinoma cell lines specifically inhibits expression of endogenous Tcf target genes. In addition, we observe genetic interactions between the Brg-1 and beta-catenin homologues in flies. We conclude that beta-catenin recruits Brg-1 to Tcf target gene promoters, facilitating chromatin remodelling as a prerequisite for transcriptional activation.

Fig. 1. β-catenin interacts specifically with Brg-1. (A) Schematic representation of the β-catenin domain structure. The N-terminal domain (grey stripes) contains four conserved serine/threonine phosphorylation sites for GSK-3β, which are essential for mediating destruction of free β-catenin. The central domain comprises 12 imperfect repeats of 42 amino acids (denoted Armadillo repeats 1–12; note the presence of an insertion within repeat 10), which are responsible for mediating many of the interactions between β-catenin and its binding partners. The C-terminal domain (shaded grey) contains potent transcriptional activation elements that are essential for the signalling activity of β-catenin. The regions of β-catenin responsible for mediating interaction with other proteins are indicated by curly brackets. (B) Mapping of the Brg-1 domain responsible for mediating interaction with β-catenin. I–IV denote regions of sequence conservation between Brg-1 and Drosophila brm (Khavari et al., 1993). Brg-1 deletion constructs were co-transformed with the β-catenin Arm1–12 bait into the Y190 reporter yeast strain and positive interactions quantified by measuring the activity of a β-galactosidase reporter gene. The asterisk denotes background β-galactosidase activity, as determined by co-transfection of empty prey vector with the β-catenin bait. (C) Mapping of Armadillo repeats mediating interaction with Brg-1. Baits comprising overlapping regions of the β-catenin Armadillo repeats were co-transformed with the Brg-C1 prey plasmid into the Y190 yeast strain and positive interactions quantified by measuring the activity of a β-galactosidase reporter gene.

Fig. 1. β-catenin interacts specifically with Brg-1. (A) Schematic representation of the β-catenin domain structure. The N-terminal domain (grey stripes) contains four conserved serine/threonine phosphorylation sites for GSK-3β, which are essential for mediating destruction of free β-catenin. The central domain comprises 12 imperfect repeats of 42 amino acids (denoted Armadillo repeats 1–12; note the presence of an insertion within repeat 10), which are responsible for mediating many of the interactions between β-catenin and its binding partners. The C-terminal domain (shaded grey) contains potent transcriptional activation elements that are essential for the signalling activity of β-catenin. The regions of β-catenin responsible for mediating interaction with other proteins are indicated by curly brackets. (B) Mapping of the Brg-1 domain responsible for mediating interaction with β-catenin. I–IV denote regions of sequence conservation between Brg-1 and Drosophila brm (Khavari et al., 1993). Brg-1 deletion constructs were co-transformed with the β-catenin Arm1–12 bait into the Y190 reporter yeast strain and positive interactions quantified by measuring the activity of a β-galactosidase reporter gene. The asterisk denotes background β-galactosidase activity, as determined by co-transfection of empty prey vector with the β-catenin bait. (C) Mapping of Armadillo repeats mediating interaction with Brg-1. Baits comprising overlapping regions of the β-catenin Armadillo repeats were co-transformed with the Brg-C1 prey plasmid into the Y190 yeast strain and positive interactions quantified by measuring the activity of a β-galactosidase reporter gene.

Fig. 2. β-catenin and Brg-1 interact in vivo. (A) 293T cells were transfected with plasmids expressing N-terminal Flag-tagged Brg-C1 clone (amino acids 56–587) and N-terminal Myc-tagged β-catenin. Whole-cell lysates were prepared 24 h later. Extracts were immuno precipitated with anti-Flag antibody or control anti-CD3 antibody as indicated. Precipitated protein was then immunoblotted with anti-Myc antibody to visualize the exogenous tagged β-catenin protein. (B) Expression of full-length K798R Brg-1 protein was induced in DK11 cells by treatment with doxycycline for 24 h. Non-induced (–) and induced (+) cells were then lysed and K798R protein immuno precipitated (IP) with an anti-HA-epitope monoclonal antibody. Precipitated protein was immunoblotted with either anti-HA or anti-β-catenin antibodies. Input lanes show that levels of β-catenin did not differ significantly between non-induced and induced samples, while induction of HA-tagged K798R Brg-1 is clearly visible in lysates before and after immunoprecipitation.

Fig. 3. Brg-1 enhances Tcf–β-catenin transcriptional activity. (A) SW13 cells were transfected with 1 µg of wild-type (black bars) or mutant Siamois reporter plasmid (grey bars) together with the expression constructs indicated. Cells were harvested after 48 h and luciferase activity determined. (B) A partial Brg-1 protein lacking the ATPase domain inhibits constitutive Tcf–β-catenin signalling in DLD1 colon carcinoma cells. A 1 µg aliquot of TOPFLASH (black bars) or FOPFLASH (grey bars) reporter plasmids was transfected into DLD1 cells in the presence or absence of Brg-C1 or full-length Brg-1 expression constructs. Luciferase activities were assayed 48 h later.

Fig. 4. (A) Induction of K798R Brg-1 expression in stable colon carcinoma cell line transfectants. Induction of K798R Brg-1 expression in LS174T (LK1 and LK3) and DLD1 (DK11 and DK19) transfectants by treatment with doxycycline for 24 h was assessed by western blotting using an anti-HA antibody directed against a C-terminal HA tag. (B) Stable expression of dominant-negative Tcf or K798R Brg-1 specifically down-regulates Tcf target genes. Cells with the indicated inducible expression constructs or parental LS174T TR4 and DLD1 TR7 cells were treated with doxycycline (+) or with vehicle alone (–) and RNA isolated 24 h later. RNA, resolved by electrophoresis and transferred to a nylon membrane, was probed with radiolabelled cDNA fragments of the indicated genes.

Fig. 5. Genetic interactions between Brahma complex components and Armadillo in Drosophila. Normal fly eye (A) and wing (F), compared with eyes from GMR.Arm* transformants (B–E) and wings from Engrailed.Gal4 UAS.Cad-I transformants (G–J) in different genetic backgrounds; (B and G) y w; (C and H) dTC3²/+; (D and I) brm²/+; (E and J) mor^x/+. Note the strong suppression of the Arm* phenotype in the eye, and the strong enhancement of the Arm^under phenotype in the wing due to reduced levels of endogenous Brahma. Similar though less pronounced modifications of the same phenotypes are caused by reducing dTcf and Moira levels.