h59HS5 Is b-ACh Dispensable Table 1. Sequence of 3C-PCR primers. Table 2. PCR primers used in the Anti-CTCF ChIP Real-time PCR experiments. Target sequence: primer # 59Olfactory Receptor gene: Marked b-globin gene 474 (bm): Target gene locus Human b-globin Human b-globin Human b-globin HS5: Human b-globin HS4 Human b-globin HS3 Human b-globin HS2 Human b-globin Ac-globin gene (Ac): Human b-globin b-globin gene (b): Human b-globin 39HS1: Mouse XPB XPB Forward: Reverse: doi:10.1371/journal.pone.0002134.t001 Sequence (59-39) 318 CAGCTTGGGTCAAATACAG ACCACCCTTTCCACAGTC CTGCACACTTTCAGTCCG CAAATGGGTGACTGTAGGG GCCTGCATTTATTGTTGTG GAACTGCTCATGCTTGGAC 442 260 261 262 321 266 424 GAAGAAAGACCTCTATAGGACAGG TGGTTATGGTCAGAGCCTC ACATGGATAATACTGTTCCCC 341&343 TGACCTCCACACTCCTGAC ATGCGCAATTAGAAACTGC immunoprecipitated sequences, including 59HS5, bm (marked b- globin gene), mHS62 (mouse HS62) and a fragment of the mouse Necdin gene (control), were determined by real-time PCR. Fold enrichment was calculated by comparing the ratio to Necdin in pull- down-eluate against the value from input-starting material. The sequences of the ChIP PCR primers are shown in Table 2. The location of the bm gene primers were shown in Fig. 1B. Supporting Information Figure S1 Alignment of the cHS4 CTCF binding site with the bm promoter. Alignment of the cHS4 CTCF binding site with the bm promoter (1). The bm gene cap site (+1) are indicated with a blue References 1. Grosveld F, Antoniou M, van Assendelft GB, de Boer E, Hurst J, et al. (1987) The regulation of expression of human beta-globin genes. Prog Clin Biol Res 251: 133–144. 2. Forrester WC, Takegawa S, Papayannopoulou T, Stamatoyannopoulos G, Groudine M (1987) Evidence for a locus activation region: the formation of developmentally stable hypersensitive sites in globin-expressing hybrids. Nucleic Acids Res 15: 10159–10177. 3. Fraser P, Pruzina S, Antoniou M, Grosveld F (1993) Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev 7: 106–113. 4. Bungert J, Tanimoto K, Patel S, Liu Q, Fear M, et al. (1999) Hypersensitive site 2 specifies a unique function within the human beta-globin locus control region to stimulate globin gene transcription. Mol Cell Biol 19: 3062–3072. 5. Levings PP, Bungert J (2002) The human beta-globin locus control region. Eur J Biochem 269: 1589–1599. 6. Tolhuis B, Palstra RJ, Splinter E, Grosveld F, de Laat W (2002) Looping and interaction between hypersensitive sites in the active beta-globin locus. Mol Cell 10: 1453–1465. Target sequence: primer name ChIP-bm Forward (bm-F): Reverse (bm-R): ChIP-HS5 Forward: Reverse: ChIP-mHS62 Forward: Reverse: ChIP-necdin Forward: Reverse: Amplicon length (bp) Sequence (59-39) TCCTAAGCCAGTGCCAGAAGAG 236 TGTCCTCCTCTGTGAAATGAC TAGCTGAAGCTGCTGTTATGACCAC 149 CCAGATGTCCTGTCCCTGTAAGGT TGCCGTAGTTCTCTAGTGTAGCCAC 170 TATGGGGTGTGGGTATTTGTAAGAG AGTTCTGTGCCATACAGGAGAC AGAGGAAGTGCGCTTTACTGAG 212 doi:10.1371/journal.pone.0002134.t002 letters. The proposed CTCF binding sites in the bm sequence are indicated with red letters and the cHS4 CTCF binding sequence is shown on top of each proposed binding site. Conserved binding sequences are highlighted with a gray background. The alignment was performed with the ClustalX program (2) Reference List 1. Farrell CM, West AG, Felsenfeld G (2002) Conserved CTCF insulator elements flank the mouse and human beta-globin loci. Mol Cell Biol 22: 3820-3831. 2. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876-4882. Found at: doi:10.1371/journal.pone.0002134.s001 (0.04 MB DOC) Acknowledgments We would like to thank Dr. Roy Drissen and Dr. Laura Gutierrez for their assistance throughout this study. Author Contributions Conceived and designed the experiments: SP PC KT. Performed the experiments: PC. Analyzed the data: SP PC KT. Contributed reagents/ materials/analysis tools: SP KT AW. Wrote the paper: SP PC KT. 7. Palstra RJ, Tolhuis B, Splinter E, Nijmeijer R, Grosveld F, et al. (2003) The beta-globin nuclear compartment in development and erythroid differentiation. Nat Genet 35: 190–194. 8. Hanscombe O, Whyatt D, Fraser P, Yannoutsos N, Greaves D, et al. (1991) Importance of globin gene order for correct developmental expression. Genes Dev 5: 1387–1394. 9. Dillon N, Trimborn T, Strouboulis J, Fraser P, Grosveld F (1997) The effect of distance on long-range chromatin interactions. Mol Cell 1: 131–139. 10. Tanimoto K, Liu Q, Bungert J, Engel JD (1999) Effects of altered gene order or orientation of the locus control region on human beta-globin gene expression in mice. Nature 398: 344–348. 11. Wai AW, Gillemans N, Raguz-Bolognesi S, Pruzina S, Zafarana G, et al. (2003) HS5 of the human beta-globin locus control region: a developmental stage- specific border in erythroid cells. EMBO J 22: 4489–4500. 12. Hebbes TR, Clayton AL, Thorne AW, Crane-Robinson C (1994) Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J 13: 1823–1830. 13. Farrell CM, West AG, Felsenfeld G (2002) Conserved CTCF insulator elements flank the mouse and human beta-globin loci. Mol Cell Biol 22: 3820–3831. PLoS ONE | www.plosone.org 8 May 2008 | Volume 3 | Issue 5 | e2134