We generated a Pygo2 knock-in mutant allele by introducing a two-nucleotide change (CT>GC) so that the Leucine in position 368-within the plant homology domain (PHD) of Pygo2-is replaced by an Alanine (Figure A). The homologous mutation in pygo (L789A) leads to lethality in Drosophila melanogaster . The mice are genotyped with primers spanning the remaining FRT sequence that follows the NEO-cassette excision (Figure B, see Materials and Methods section for a detailed description). The presence of the mutation is confirmed by DNA sequencing (Figure B). Initially, three independent breedings between heterozygous mice (Pygo2L368A/+ X Pygo2L368A/+) gave rise to mutant homozygous Pygo2L368A/L368A animals (6/44 [13.6%], representing half of the expected Mendelian ratio). The homozygous mutant mice obtained were then interbred, giving rise to healthy animals, thereby showing that homozygous Pygo2-L368A mutant mice reach adulthood without any malformation and are fertile. To rule out any compensatory mechanism due to potential redundancy with Pygo1, we brought the Pygo2-L368A mutant allele into a Pygo1-knockout background. By crossing Pygo2 heterozygous, Pygo1-knockout mice (Pygo1-/-; Pygo2L368A/+), we obtained Pygo1/2 double mutant animals (Pygo1-/-; Pygo2L368A/L368A) in a proportion of 4/15 (26%-the expected Mendelian ratio is 1/4). A double mutant male mouse (Pygo1-/-; Pygo2L368A/368A) from this progeny was successively bred twice with a Pygo1-/-; Pygo2L368A/+ female and gave rise to a total of 10/23 double mutant mice (43.5%-the expected Mendelian ratio is 1/2). Both male and female double mutant mice could breed, allowing us to establish a colony of healthy Pygo1-/-; Pygo2L368A/L368A animals.
The effect of the L368A mutation on the Pygo2-Bcl9 interaction was confirmed by in vitro GST pull-down experiments. We extracted proteins from adult wild-type and mutant kidneys, an abundant source for Pygo2 protein. We incubated wild-type and mutant protein extracts with a recombinant GST-Bcl9 protein fragment (amino acids 1-372 of mouse Bcl9, that includes the region spanning two relevant domains: HD1 [Pygo-binding] and HD2 [beta-catenin-binding]). GST-GFP was used as negative control. With glutathione-conjugated sepharose beads, we pulled down the GST-proteins and performed Western blot analysis to detect Pygo2 in the pull-down reactions. Whereas the GST-Bcl9 can strongly interact with the wild-type Pygo2, it fails in significantly pulling down the Pygo2-L368A mutated protein (Figure C, compare the bands indicated by the two white asterisks).
Pygo1/2 double knockout mice die during embryonic development between 13.5 dpc and birth due to a series of developmental defects. However, Pygo2-L368A embryos are, in their superficial appearance, indistinguishable from wild-type littermates (Figure D). Out of 25 embryos analyzed, we scored six mutants (24%, close to the expected Mendelian ratio of 1/4). Moreover, histological analyses of the tissues affected during development in Pygo1/2-knockout mice (mainly the lens, the lungs, and the kidney) reveal no obvious alterations at 15.5 dpc (Figure E).
The complete abrogation of the interaction between Bcl9/9l and Pygo1/2-via the deletion of the HD1 domain in both Bcl9 and Bcl9l-leads to embryonic lethality at 13.5 dpc, with a striking “Pygo knockout” phenotype. These results appear contradictory (see also the Alternative Explanations paragraph). It is possible that the deletion of the full Pygo-interacting HD1 domain of Bcl9/9l completely abrogates their interaction, while the single amino acid substitution L368A in Pygo2 strongly reduces their binding, but leaves some residual interaction. Thus, the residual binding between Pygo2-L368A and Bcl9 would be capable of fulfilling all the developmental functions for which this interaction is required. It is possible, in fact, that this interaction is weak and dynamic in nature in vivo. The extent to which the mutation L368A decreases the affinity between Pygo2 and Bcl9 remains to be determined. In conclusion, we cannot observe any developmental or homeostatic defect in mutant Pygo1-/-; Pygo2L368A/L368A mice: they reach adulthood healthy and fertile. Because several reports have previously shown that the interaction between Pygo1/2 and Bcl9/9l is necessary for proper development, we propose that only a weak interaction between these factors is required.