Supplementary Experimental Procedures
SUPPLEMENTARY EXPERIMENTAL PROCEDURES
GST-pull down
We cloned mouse Bcl9 (from amino acid 1 to 372, a region that includes both HD1 and HD2) into the pGEX-KG vector in frame with the GST coding sequence. In order to produce equivalent plasmids encoding for the Bcl9-DHD1 and Bcl9-DHD2 fragment proteins we amplified the corresponding region using cDNA from Bcl9-DHD1 and Bcl9-DHD2 mutant mice, respectively. pGEX-KG allows the expression and production of proteins in bacteria. We incubated these proteins with protein extractions from wild-type mouse embryos. Briefly, wild-type embryos at 12.5 dpc were minced in cold PBS to single cell suspension, and then lysed with a hypotonic lysis buffer (20 mM Tris-HCl; 75 mM NaCl; 1.5 mM MgCl2; 1 mM EGTA; 0.5% NP-40; 5% Glycerol). A complete lysis was achieved via sonicating the sample. The embryonic protein extracts obtained this way were incubated over night at 4°C with GST-Bcl9, GST-Bcl9DHD1, or GST-Bcl9DHD2), together with Glutathione Sepharose beads (GE Healthcare) on a rotating wheel. The beads were then spun down, and washed three times in lysis buffer. All steps were performed on ice, and all buffers were supplemented with fresh protease inhibitors (Complete, Roche) and 1 mM PMSF.
Western blot
The pulled down beads were boiled at 85°C for 15’ in “Laemmli buffer“, and subjected to SDS–PAGE separation and blotting. Immunoblotting was performed to detect the presence of Pygo2, Pygo1 and b-catenin using the following antibodies: anti-Pygo2 (Novus Biological, NBP1-46171, and Santa Cruz, sc-74878); anti-b-catenin (BD, 610154); anti-Pygo1 (Santa Cruz, H-162, sc98662).
Immunohistochemistry
A biotinylated anti-rabbit secondary antibody was added for 1 hour at room temperature to slides previously treated with a specific primary antibody; the signal is amplified via the Elite Vectastain ABC kit (Vector Laboratories). Peroxidase reaction is performed at room temperature using DAB-substrate kit (Vector Laboratories). Please note that the slides were previously treated for 20’ at room temperature with 6% H2O2 to inhibit endogenous peroxidases.
Quantitative RT-PCR
Total RNA was extracted using TRIzol (Invitrogen), and retro-transcribed in cDNA using Transcription High Fidelity cDNA Synthesis Kit (Roche, REF. 05 091 284 001). Quantitative real-time SYBR Green based PCR reactions were performed in triplicate and monitored with the ABI Prism 7900HT system (Applied Biosystem). Specific primers (see below) were designed to amplify 70 to 150-bp intron-spanning coding regions, and were based on sequences from the Ensemble database (http://www.ensembl.org/Mus_musculus/Info/Index). Dissociation curves confirmed the homogeneity of PCR products. Samples from three or more independent animals were analyzed, each in triplicate.
Primers for RT-PCR:
Pax6
F 5’ ATCCGAGACAGATTATTATCCG 3’
R 5’ GAACTCTGTTTATTGATGACAC 3’
Pygo1
F 5' GTAATGTCAGCGGAACAGGAC 3’
R 5' GCGTACTCTGACAGCGGAGG 3’
Pygo2
F 5' GCGAAGAAAGTCCAATACTCAG 3’
R 5' AAAAGGGTTAGAAGCGACCAG 3’
Bcl9
F 5' CAATCAATGCATCCCAGTAACC 3’
R 5' AGATGGGGACATCACTGTAGG 3’
Bcl9l
F 5’ ACTCCATCCCTCGATTCAGA 3’
R 5’ CCCACTGTACGGCTGCTT 3’
Cryaa
F 5’ CACGGCAAACACAACGAGAGG 3’
R 5’ AGGGCGGACTGGTCCACATTGG 3’
Housekeeping genes:
b-actin
F 5’ GATCTGGCACCACACCTTCT 3’
R 5’ GGGGTGTTGAAGGTCTCAAA 3’
Gapdh
F 5’ AACTTTGGCATTGTGGAAGG 3’
R 5’ ATCCACAGTCTTCTGGGTGG 3’
Sdha
F 5’ AAGGCAAATGCTGGAGAAGA 3’
R 5’ TGGTTCTGCATCGACTTCTG 3’
Chromatin Immunoprecipitation (ChIP)
Immunoprecipitation of the chromatin was performed after overnight incubation with 5 μg of α-Pax6 (Covance and Millipore) and IgG (Santa Cruz), followed by addition of protein A agarose beads (Upstate biotechnology). Immunoprecipitated DNA was then analyzed by quantitative PCR. The enrichment obtained with the α-Pax6 is normalized using the ratio α-Pax6 /IgG; the α-Pax6/IgG ratio obtained for a negative region has been set to 1.
Primers used in the ChIP experiments:
Cryaa-promoter
F 5’ CTACCTCTCCCCACCTGTGA 3’
R 5’ GCCAAGGGACATCACTGTTT 3’
Pygo2_ -0.3 Kb from TSS
F 5’ CACCGGACAAGCTGGAG 3’
R 5’ AACCGTTCCATCCGAGGTTCC 3’
Pygo2_ -0.95 Kb from TSS
F 5’ TCTGGAAAGTCTGATTGATGGG3’
R 5’ CCTTGCAGATACGTCCCTACC 3’
Bcl9_ -1 Kb from TSS
F 5’ TGGCCAGTTGTGAGTTTTGC 3’
R 5’ CAGTGCTGTCGTAAAGGATGG 3’
Bcl9_ -1.7 Kb from TSS
F 5’ CACAGAGTTGCTGGAGTAGC 3’
R 5’ TGTATAAACACGAGTGGAGAGC 3’
Bcl9_ -5 Kb from TSS
F 5’ ATATCCAAACTCAGGTCCACG 3’
R 5’ CCCACCTACTCTGAGAACCTA 3’
Bcl9L_ -0.3 Kb from TSS
F 5’ CGTGAGGGATTCCCGAATCC 3’
R 5’ TCACTAATAGGCTTGGTCGC 3’
Bcl9L_ -0.85 Kb from TSS
F 5’ GTCTTTGTCTCCCTGTCCACG 3’
R 5’ ACAGACACATGGACAAGTCCTCC 3’
Bcl9L_ -1.6 Kb from TSS
F 5’ CTCCCACCCGTAGCTTATTG 3’
R 5’ GCTAAGGCAGGAGATCACAAA 3’
Bcl9L_ -5 Kb from TSS
F 5’ GCTGTCTGCATGTCAACGTA3’
R 5’ AGACACATCCTCCATCCCTT 3’
Prm2-promoter
F 5' GGGTGGGAACAATCAATCAGG 3'
R 5' TCAGAGGGCTCATATATAAAGG 3’