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Supplemental Figure Legends Supplemental Figure Legends Supplementary Figure S1 Generation and characterization of Frat-knockout mice (A) Frat/GBP orthologues are conserved amongst vertebrate species, including Xenopus (xl) zebrafish (dr), mice (mm), rats (rn) and humans (hs). Whereas lower organisms such as Xenopus and zebrafish contain one Frat/GBP gene, the human genome harbors two and the mouse genome three Frat homologues. mmFrat1 and mmFrat3 are 84% homologous on the protein level, whereas mmFrat1 and mmFrat2 share 68% amino-acid identity. A multiple- species alignment of Frat proteins reveals conserved domains in the N– and C-terminal regions. The IKEA-box, which is required for binding to GSK-3β, is conserved in all Frat family members. (B) Frat2– and Frat3-knockout mice were generated by replacing most of the coding sequence with a targeting cassette existing of a promoterless lacZ-reporter gene, followed by a PGK-promoter driven Hygromycin resistance marker to allow selection of targeted ES cells. Homologous recombination was analyzed by Southern Blot analysis using 3’ flanking probes. For Frat2 probe A allows detection of the shift of an endogenous 12 kb Xba fragment to a 9 kb fragment after homologous recombination; Probe B for Frat3 detects a shift in the endogenous 24 kb EcoRV fragment to an 18 kb knockout fragment after homologous recombination. (C) Since Frat1 and Frat2 are located on chromosome 19 only 15 kb apart in opposite orientations, a separate targeting vector was constructed to generate Frat1/Frat2 double- knockout mice. Frat2 was targeted with a PGK-Puromycin selection cassette in Frat1+/lacZ ES cells. Homologous recombination was verified by Southern Blot analysis. Clones in which the Frat1-knockout allele had undergone consecutive targeting of the Frat2 gene (Detectable with probe A as a shift from a 35 kb to a 17 kb EcoRV fragment) were used to generate double- knockout mice. (D) RT-PCR analysis on bone marrow (BM) or mouse embryo fibroblasts (MEF) with 3’ UTR primers reveals read-through from the targeting cassettes into the Frat1 and Frat2 3’UTR (upper two panels). RT-PCR analysis with gene-specific primers shows that Frat1, Frat2 and Frat3 are no longer expressed in Frat triple-knockout mice. GAPDH serves as a control. Lanes 1-4 RT-PCR; Lanes 5-8 no-RT control; Lanes 1,4,5,8 triple-heterozgygotes; Lanes 2,3,6,7 Frat triple-knockout mice. Supplementary Figure S2 Analysis of Frat3 imprinting status (A) Frat3 is located in the Prader-Willi syndrome region on mouse chromosome 7 (Chai et al. 2001; Kobayashi et al. 2002). It has acquired an imprinted status, causing Frat3 to be expressed solely from the paternal allele. Using Southern blot analysis similar to (Chai et al. 2001) we analyzed whether targeting of the Frat3 gene had left the imprinting status of the region intact. Indeed, the EagI site remains imprinted upon transmission of a maternal wildtype allele (and a paternal knockout allele, PKO) but not upon transmission of a paternal wildtype allele (and a maternal knockout allele, MKO). XbaI digest: wildtype allele 6.9 kb; knockout allele 4 kb XbaI/EagI digest: wildtype allele imprinted 6.9 kb; wildtype allele not imprinted 2.8 kb; knockout allele 4 kb. (B) Genomic DNA isolated from Frat3+/lacZ mice was digested with methylation-sensitive restriction enzymes (NruI or HpaII) or a methylation-insensitive enzyme as a control (MspI) and used as input for a Frat3-specific PCR that allows detection of a 380 bp wildtype and a 400 bp knockout band. In Frat3+/lacZ mice the wildtype allele was only detected upon maternal transmission of a wildtype allele (and a paternal knockout allele, PKO) and not upon transmission of a paternal wildtype allele (and a maternal knockout allele, MKO), indicating that only in the first case, methylation at the HpaII and NruI sites had protected the wildtype allele from restriction enzyme digestion. The knockout allele has become methylated, since it can be detected upon both paternal and maternal transmission following digestion with HpaII (but not following digestion with MspI). This has likely caused the lacZ reporter to be silenced, since we could not detect lacZ expression following X-gal staining of wholemount embryos or frozen sections of E11.5-E14.5, during which stages Frat3 expression is readily detected by RT-PCR. Thus, although imprinting at the wildtype allele remains intact, the knockout allele appears to have been silenced. However, since Frat3-deficient mice were also viable, healthy and fertile, we conclude that Frat3 is not involved in the Prader-Willi syndrome in mice. Supplementary Figure S3 Frat is expressed at sites that display active Wnt/β-catenin activity Since lacZ expression in Frat2+/lacZ mice is driven by the endogenous Frat2 promoter, analysis of the lacZ expression pattern provides a direct readout for Frat2 expression in vivo. Cryosections from developing embryos show that Frat2 is predominantly expressed in spinal ganglia (A) and brain ventricles (B) from early development onward. At later stages of development, lacZ activity was also detected in areas of the developing heart (C), the retina (D), the edge of the limbs (E) and in developing hair follicles and skin (F). Many of these sites overlap with areas where Wnt/β-catenin signaling has been reported to take place during mouse development (Maretto et al. 2003). Supplementary Figure S4 Frat is expressed in early lymphoid progenitors To characterize Frat expression during lymphoid differentiation, we used the fluorigenic substrate FDG to visualize β-galactosidase activity encoded by the lacZ knock-in allele present in both Frat1 and Frat2 heterozygous animals by flowcytometric detection. Staining patterns in immature B– en T-lymphocytes between Frat1+/lacZ and Frat2+/lacZ were identical (data not shown) and largely overlap with stages that display β-catenin/TCF activity. (A) A large proportion of differentiating T-cells in the thymus is FDG-positive (49%). During differentiation, immature T-cells rearrange the genomic loci encoding the β and α chains of the T-cell receptor (TCR). In both instances, successful rearrangement induces a burst of cellular proliferation. The earliest thymic population of CD4/CD8 double negative CD25-/CD44+ cells (DN1) does not express Frat. However, Frat expression is strongly induced in CD25+/CD44+ cells (DN2), which is the first subpopulation of cells committed to the T-cell lineage. Frat expression is maintained throughout DN3 and DN4. In DN3, T-cells rearrange the TCRβ locus and enter a proliferative phase after β selection. Subsequently, immature T-cells differentiate into CD4/CD8 double-positive (DP) T-cells and initiate TCRα rearrangements. DP T-cells express high levels of Frat. Upon positive selection, T-cells will downregulate CD4/CD8 expression, inititate CD69 expression and commit to either the CD4 or the CD8 lineage. Remarkably, the highest Frat expression (76% of the cells) is observed in the first subpopulation to be distinguished after the initiation of positive selection, the TCRβlo CD69+ DP T-cells, indicating that Frat might be further induced as an early immediate gene after the positive selection event. During lineage commitment to CD4 and CD8 SP T-cells, Frat expression is maintained in CD4 SP T-cells to a low extent. (B) During B-cell differentiation, Frat expression is mainly observed in immature B-cells (B220lo, CD43-) that do not yet express IgM on their cell surface. DN double-negative; DP double-positive; SP single-positive Supplementary Figure S5 T– and B-cell differentiation is not affected by the loss of Frat Flowcytometric analysis of single cell suspensions derived from bone marrow, thymus and spleen from young as well as old Frat-deficient mice did not reveal any subpopulations that were affected by the loss of Frat when compared to littermate controls. Both overall cell numbers as well as the process of B– and T-cell differentiation as measured by the presence of specific subpopulations were not perturbed in the absence of Frat (also data not shown). Mice deficient for Tcf1 or for both Tcf1 and Lef1 show defects in specific stages of T-cell differentiation (Staal and Clevers 2000). Tcf1-/- animals show a progressive decline in T-cell maturation, due to specific blockades at the stage of actively proliferating CD4-CD8- (double- negative, DN) thymocytes (Verbeek et al. 1995; Schilham et al. 1998). Lef1-/- mice display defects in pro-B-cell proliferation (Reya et al. 2000) and in Tcf1-/-;Lef1-/- mice, T-cell differentiation is completely impaired from DN3 onward (Okamura et al. 1998). We therefore paid specific attention to these populations, but here, as in other subpopulations, we found no differences between Frat-TKO mice and control littermates. Additional References Chai, J.H., D.P. Locke, T. Ohta, J.M. Greally, and R.D. Nicholls. 2001. Retrotransposed genes such as Frat3 in the mouse Chromosome 7C Prader- Willi syndrome region acquire the imprinted status of their insertion site. Mamm Genome 12: 813-21. Kobayashi, S., T. Kohda, H. Ichikawa, A. Ogura, M. Ohki, T. Kaneko-Ishino, and F. Ishino. 2002. Paternal expression of a novel imprinted gene, Peg12/Frat3, in the mouse 7C region homologous to the Prader-Willi syndrome region. Biochem Biophys Res Commun 290: 403-8. Maretto, S., M. Cordenonsi, S. Dupont, P. Braghetta, V. Broccoli, A.B. Hassan, D. Volpin, G.M. Bressan, and S. Piccolo. 2003. Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A 100: 3299- 304. Okamura, R.M., M. Sigvardsson, J. Galceran, S. Verbeek, H. Clevers, and R. Grosschedl. 1998. Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1. Immunity 8: 11-20. Reya, T., M. O'Riordan, R.
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