Supplemental Materials s3

Supplemental Materials:

Methods

Immunoprecipitation and Western blotting. Samples were macerated and sonicated in

40 mM HEPES, 100 mM KCl, 40% glycerol, 2 mM -ME, 0.5% NP-40, immunoprecipitated with Pax3 polyclonal antibody1 and protein A/G agarose beads

(Oncogene Research Products), followed by Western blotting (Western Breeze,

Invitrogen) with Pax3 antibody, or used directly in Western blots with TLE4/Grg4 (Santa

Cruz Biotechnology) or Pax3 polyclonal antibody, or Tubulin (Oncogene

Science/Calbiochem) monoclonal antibody. For Lef1 Immunoprecipitation, in-vitro translated 35S labelled proteins were mixed with 3 fold excess cold protein in buffer (75 mM NaCl, 25 mM Tris pH 7.5, 0.25 mM EDTA, 0.5% Igepal, 0.5 mM DTT, 0.5 mM

PMSF, 0.05% BSA) and precipitated with Lef1 polyclonal antibody (Santa Cruz

Biotechnology).

Immunohistochemistry. Immunohistochemistry was performed on paraffin-embedded tissue fixed in 4% paraformaldehyde. Antigen was exposed using Bull’s Eye reagent

(Biocare Medical) and heated in a pressure cooker. Antibodies utilized were Pax3

(1:3000 for DAB staining, 1:800 for immunofluorescence), Mitf (Vector Laboratories, mouse mAb, 1:10), Sox10 (Chemicon International rabbit pAb, 1:20), -galactosidase

(Promega Corp, mouse mAb, 1:100), Dct/Tryp2 and Grg4 (Santa Cruz Biotechnology

Inc., goat pAb, 1:50 and rabbit pAb 1:100, respectively). Secondary antibodies conjugated with fluorescent tags (Alexa Fluor, Molecular Probes) were used at a dilution of 1:250. For label retention studies, mice were injected subcutaneously with BrdU (10

µg/g body weight) twice daily from P20 to P27. Skin was collected at P58. BrdU antibody (Biocare Medical) was used at a dilution of 1:200.

Constructs. pCMV-Pax3, pCMV-Sox10 and pCMV-Mitf were described previously1.

The Dct 3181 bp promoter2 was amplified by PCR and cloned upstream of a luciferase cassette in SacI/XhoI sites of pGL2 (Promega). Deletions and mutations in the Dct-Luc construct were made by site directed mutagenesis (Stratagene). The pKW2T-mGrg4 construct was provided by M. Busslinger3. The pCMV5B-Lef1-HA 20 construct was provided by Tom Force and Paul Hamel.

Cell Culture, transfection, and ChIP assays. 293T cells and B16 cells (American Type

Culture Collecion) were maintained in DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies). A total of 0.5 µg of DNA was mixed with 10 µl

Effectene (Qiagen Inc.) Luciferase activity (Luciferase assay kit, Promega Corp.,

Madison, WI) was normalized for transfection efficiency using pCMV (BD biosciences/Clontech) and expressed as either fold activation compared to reporter construct alone, or as arbitrary light units. For ChIP assays, transfected cells were fixed in 1% formaldehyde and quenched in 0.125 M glycine, then processed according to manufacturers protocol (Upstate Biotechnology). PCR was performed with primers to the Dct enhancer region-2 GGAGAAGTACTTAGCAATGCACAGG (F) and

AGCCATCATTAAGGGGATTATAACC (R). All ChIP samples were tested for false positive PCR amplification using primers that amplify sequence from the -actin gene (for genomic DNA contamination) and luciferase (reporter construct contamination). In all cases, these amplifications failed to yield product.

Electrophoretic Mobility Shift Assay (EMSA). EMSA was performed as described previously4 in the presence of 125 µg/ml poly-dIdC, 100 mM NaCl and nuclear extracts from transfected cells. Primers used for EMSA are DCT WT : AGT GGC TTC ATT

GTG CAC TTA GGG TCA TGT GCT AAC AAA GAG GAT TTC TCT GGT TAT

AAT CCC CTT; DCT M/P mut: AGT GGC TTC ATT GTG CAC TTA GGA AGC

GGC CGC AAC AAA GAG GAT TCC AGT AGT ATT AAT CCC CTT.

Pax3Cre knockin mice and other mouse lines. Pax3Cre-KI mice were created by targeted recombination so as to replace the coding region of exon 1 of the Pax3 gene with sequences encoding Cre recombinase followed by a stop codon and a poly-adenylation site. Details of the targeting strategy and characterization of the mice will be reported elsewhere (K.E. and J.A.E., submitted) and are available upon request. Pax3Cre knockin mice were crossed with ß-galactosidase reporter mice5. Dct-lacZ 2,6, Wnt1Cre 7 and Z/EG

GFP reporter mice 8 have been described. References

1. Lang, D. & Epstein, J. A. Sox10 and Pax3 physically interact to mediate

activation of a conserved c-RET enhancer. Hum Mol Genet 12, 937-945 (2003).

2. Mackenzie, M. A., Jordan, S. A., Budd, P. S. & Jackson, I. J. Activation of the

receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the

mouse embryo. Dev Biol 192, 99-107 (1997).

3. Eberhard, D., Jimenez, G., Heavey, B. & Busslinger, M. Transcriptional

repression by Pax5 (BSAP) through interaction with corepressors of the Groucho

family. Embo J 19, 2292-2303 (2000).

4. Epstein, J., Cai, J., Glaser, T., Jepeal, L. & Maas, R. Identification of a Pax paired

domain recognition sequence and evidence for DNA-dependent conformational

changes. J. Biol. Chem. 269, 8355-8361 (1994).

5. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain.

Nat Genet 21, 70-71 (1999).

6. Nishimura, E. K. et al. Dominant role of the niche in melanocyte stem-cell fate

determination. Nature 416, 854-860 (2002).

7. Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. & McMahon, A. P.

Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible

form of Cre recombinase. Curr Biol 8, 1323-1326 (1998).

8. Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe, C. G. Z/EG, a double reporter

mouse line that expresses enhanced green fluorescent protein upon Cre-mediated

excision. Genesis 28, 147-155 (2000). 9. Epstein, J. A., Lam, P., Jepeal, L., Maas, R. L. & Shapiro, D. N. Pax3 inhibits

myogenic differentiation of cultured myoblast cells. J Biol Chem 270, 11719-

11722 (1995).