Dedicated Epithelial Recipient Cells. Determine Pigmentation .... site 1, the probe was 5â²-CTG TCT CCC GCA CCC TAT CCT TAC AC; the primers were 5â²-.
Cell, Volume 130 Supplemental Data Dedicated Epithelial Recipient Cells Determine Pigmentation Patterns Lorin Weiner, Rong Han, Bianca M. Scicchitano, Jian Li, Kiyotaka Hasegawa, Maddalena Grossi, David Lee, and Janice L. Brissette
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Immunofluorescence For analyses of AE13, sections were blocked using the M.O.M. kit (Vector Laboratories). For analyses of Tyrp1, primary antibody was visualized using fluorescein-labeled goat antiserum to rabbit IgG (Pierce Biotechnology). For analyses of Fgf2 and Kit, tyrosinase and peroxidase were quenched with 15% H2O2/PBS for 30 minutes (min.) at room temperature following section re-hydration. Quenching reactions were stopped by five washes with 0.1% NP-40/PBS. Sections were then blocked and probed with antibodies as described (Weiner and Green, 1998). Following the antibody incubations, sections were washed three times with 0.1% NP-40/PBS and probed with streptavidin-horseradish peroxidase conjugates (Pierce Biotechnology) for one hour at room temperature. After three additional washes with 0.1% NP-40/PBS, sections were incubated with biotinyl tyramide from the Individual Indirect Tyramide Reagent Pack (Perkin Elmer Life Science Products); the biotinyl tyramide was used as recommended by the manufacturer for the Tyramide Signal Amplification technique. The tyramide/peroxidase reaction was stopped by washing five times rapidly with 0.1% NP-40/PBS. Sections were then
probed with streptavidin-CY3 conjugates (Sigma-Aldrich, Inc.) and counterstained with Hoechst dye 33258 (Fluka) as described (Weiner and Green, 1998).
RNA Quantitation by Real-Time RT-PCR Real-time PCR was performed with the Bio-Rad iCycler-MyiQ System. PCR products were quantitated using probes dual-labeled with 6-FAM and BHQ1. For normalization, Hprt expression was assayed in parallel with Fgf2 expression. The Fgf2 reactions contained cDNA from 0.24-3.8 μg of RNA; the Hprt reactions used cDNA from 30-500 ng of RNA. For Fgf2, the probe was 5′-CAC TCC CTT GAT AGA CAC AAC TCC TC; the primers were 5′-GAG AAG AGC GAC CCA CAC GTC and 5′-GCC AGC AGC CGT CCA TCT TCC. For Hprt, the probe was 5′-CTG GCC TGT ATC CAA CAC TTC GAG AG; the primers were 5′-CTT TCC CTG GTT AAG CAG TAC AG and 5′-CAT ATC CAA CAA CAA ACT TGT CTG G. PCR reactions were performed with AmpliTaq Gold (Applied Biosystems), 0.6 μM primers, 0.25 μM probe, and a three-step cycling program (58°C annealing step, 68°C extension step). Both primer sets spanned introns and thus specifically amplified cDNA.
Chromatin Immunoprecipitation (ChIP) Assays Chromatin was prepared from wild-type primary keratinocytes (~107 cells) infected with recombinant adenoviruses. After 18 hours of infection, methanol-free formaldehyde (Polysciences, Inc.) was added directly to the culture medium to a final concentration of 1%. After a 10-min. incubation at room temperature, cultures were treated with 125 mM glycine for 5 min., washed twice with PBS, frozen on the dish with liquid nitrogen, and stored at -80°C. Nuclei were prepared using a modified version of the protocol of Lahiri and Ge (2000). Briefly,
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the attached cells were incubated in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA pH 8.0, 0.1 mM EGTA pH 8.0, 1 mM DTT) plus a protease inhibitor cocktail (Roche Applied Science) for 15 min. on ice. NP-40 was then added from a 10% solution to a final concentration of 0.1%. Cells were scraped off the dish, and nuclei were pelleted by centrifugation (18,400xg for 5 min. at 4°C). Following removal of the supernatant, nuclei were transferred to RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) plus the protease inhibitor cocktail and gently vortexed. Chromatin was then resuspended and fragmented by sonication with a Branson sonifier 250. Nuclei were sonicated on ice using setting 3, constant output cycle, a tapered 1/8th-inch microtip, and 20 bursts lasting 20 seconds each. The lysate was next warmed to room temperature, CaCl2 was added to 2.5 mM, and nucleic acids were fragmented with micrococcal nuclease (100 units/ml final concentration; Takara Biomedicals) and RNase A (300 μg/ml final concentration; Fisher Scientific). After 15 min. at room temperature, the digestion with micrococcal nuclease was stopped by addition of EGTA to 10 mM. This combination of digestion and sonication yielded chromatin fragments with an average size of 500-1000 bp. Insoluble debris was then removed by centrifugation (840xg for 10 min. at 4°C). Lysates were pre-cleared of additional contaminants with protein G-agarose beads (Roche Applied Science). Prior to use, the beads were incubated in RIPA buffer plus BSA (200 μg/ml) and sheared fish sperm DNA (200 μg/ml; Roche Applied Science) for 0.5-1 hour at 4°C. The beads were then incubated with the lysates (0.5-1 hour at 4°C) and removed by centrifugation (840xg for 2 min. at 4°C). An aliquot of the supernatant (4-5% of the total) was set aside for input DNA measurements. The remainder was incubated with anti-Flag M2agarose affinity gel (Sigma-Aldrich, Inc.) overnight at 4°C. Prior to use, the anti-Flag resin was
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coated with BSA and fish DNA as described above. Following incubation with the lysates, the anti-Flag resin was washed twice with RIPA, twice with high salt buffer (100 mM Tris-HCl pH 7.4, 500 mM NaCl), and twice with TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl). Chromatin was removed from the resin by two elutions with 20 mM Tris-HCl pH 6.8, 2% SDS; each elution was performed for 15 min. at room temperature with agitation. After each pair of elutions was combined, all chromatin samples (ChIPed and input) were adjusted to 1% SDS and 300 mM NaCl. Protein was removed by digestion with proteinase K (250 μg/ml) for a minimum of 5 hours at 37°C. Crosslinks were reversed by incubation of the digests at 65°C overnight. The samples were then extracted once with phenol:chloroform, and the aqueous phase was isolated using a phase lock gel (Eppendorf AG). After the addition of glycogen (50 μg) to each sample, DNA was precipitated with ethanol and resuspended in 1X PCR Gold Buffer. In control assays, the anti-Flag resin was replaced with normal mouse IgG (Upstate Biotechnology). Following overnight incubation with the lysates, the IgG was precipitated using protein G-agarose beads coated with BSA and fish DNA. ChIP assay outcomes were determined by real-time PCR. Using multiple probe/primer sets, we measured the levels of various DNA segments located in cis with Fgf2. The tested segments fell into the following categories: 1) sites proximal to the Fgf2 core promoter, and 2) noncoding regions conserved in human FGF2 (70% identity over at least 100 bp; Hardison et al., 1997). Conserved noncoding regions were identified using VISTA (Bray et al., 2003; Dubchak et al., 2000; Mayor et al., 2000). In all, we tested 37 distinct chromosomal regions of Fgf2 — two near the core promoter and 35 at other noncoding sites. Assay results were normalized for input DNA and background precipitation (the precipitation of random chromatin fragments). Background was quantitated with a probe/primer set corresponding to a short segment (169 bp)
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of the Gapd coding sequence. PCR conditions were as described in the previous section. For site 1, the probe was 5′-CTG TCT CCC GCA CCC TAT CCT TAC AC; the primers were 5′GGC TCT TAC GTG TTG AGG ACT C and 5′-CAG TCC CGT AGA GCA CAA GCT G. Relative to the start of Fgf2 transcription, the amplified region extends from position -439 to 582. For site 2, the probe was 5′-CTG TAG ATA CAA TTA ACA AAC TAT GAC CAA G; the primers were 5′-CAC AAC ATC CAT TAT GTA ACC AGA C and 5′-CAC TGT GAA CGT TCA TTT GCA AGC. The amplified region extends from position +6377 to +6546 of Fgf2. For the Gapd segment, the probe was 5′-CTG TAG CCG TAT TCA TTG TCA TAC CAG; the primers were 5′-CTG CGA CTT CAA CAG CAA CTC C and 5′-CCA GGG TTT CTT ACT CCT TGG AG.
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SUPPLEMENTAL REFERENCES
Bray, N., Dubchak, I., and Pachter, L. (2003). AVID: A global alignment program. Genome Res. 13, 97-102.
Dubchak, I., Brudno, M., Loots, G. G., Pachter, L., Mayor, C., Rubin, E. M., and Frazer, K. A. (2000). Active conservation of noncoding sequences revealed by three-way species comparisons. Genome Res. 10, 1304-1306.
Hardison, R. C., Oeltjen, J., and Miller, W. (1997). Long human-mouse sequence alignments reveal novel regulatory elements: a reason to sequence the mouse genome. Genome Res. 7, 959966.
Lahiri, D. K., and Ge, Y. (2000). Electrophoretic mobility shift assay for the detection of specific DNA-protein complex in nuclear extracts from the cultured cells and frozen autopsy human brain tissue. Brain Res. Protoc. 5, 257-265.
Mayor, C., Brudno, M., Schwartz, J. R., Poliakov, A., Rubin, E. M., Frazer, K. A., Pachter, L. S., and Dubchak, I. (2000). VISTA : visualizing global DNA sequence alignments of arbitrary length. Bioinformatics 16, 1046-1047.
Weiner, L., and Green, H. (1998). Basonuclin as a cell marker in the formation and cycling of the murine hair follicle. Differentiation 63, 263-272.
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