Figure 6. CTCF-ChIP assay analysis of the binding of CTCF protein to the mouse HS62 (mHS62), bm and human 59HS5 in the primitive (A) and definitive (B) stage. Fold enrichment of the test sequence in bound with CTCF versus input/starting material is shown on the y axis. doi:10.1371/journal.pone.0002134.g006 Thus, CTCF could be a ‘‘dual functionality’’ protein that has divergent effects on different gene regulation systems. In addition, we show that CTCF binds to the bm gene in both primitive and definitive stage. Three putative CTCF binding sites can be identified at 2133 to 2149, 21005 to 2990 and 21396 to 21412 bp relative to the cap site of the bm gene (Figure S1). Collectively, our results indicate that CTCF may have a more general role in the regulation of the b-globin locus than previously anticipated. Splinter et al. (2006) [33] have recently shown that in CTCF cells, there were reduced DNA-DNA interaction frequencies between the sites that normally bind CTCF in the mouse b-globin gene locus. Interest- ingly, no changes were seen in the interaction of the surrounding olfactory genes (MOR5B1-3 and MOR3B1-4 [33]), or the expression level of the b-globin genes. It has been proposed previously that CTCF and the chick HS4 insulator sequence are highly concentrated in Matrix Attachment Regions (MARs) which create separate ‘‘loop’’ domains that prevent the interaction between an enhancer and promoter [34]. Collectively, these data suggested that CTCF participates in more than one pathway in the regulation of the globin genes. Materials and Methods Transgenic mice PAC transgenic constructs (Fig. 1) containing the b-globin locus were generated using homologous recombination in Escherichia coli according to Iman et al. (2000) [15]. Modified PACs were used to generate transgenic mice; these mice have been characterized extensively in Wai et al. (2003) [11]. Tissues used for 3C Plugged transgenic mice were sacrificed and the embryos were collected at 10.5 and 14.5 dpc (day post coitum) respectively. Fetal liver and brain were collected from 14.5 dpc embryos, and embryonic blood and head were collected at 10.5 dpc. as previously described [6,7,35]. Preparation of 3C templates The 3C protocol was adapted from Tolhuis et al. (2002) [6]. 16107 cells was defined as 1 unit of sample in a 10ml crosslinking reaction (2% formaldehyde in DMEM with 10% FCS). The samples were cross-linked for 10 minutes at room temperature with gentle mixing. The reaction was quenched by the addition of glycine to 0.125M. Nuclei were harvested by lysis of the cells in 5ml ice-cold lysis buffer (10mM Tris-Cl, 10mM NaCl, 0.2% NP-40 pH8.0) containing h59HS5 Is b-ACh Dispensable protease inhibitors (pefabloc, Roche) for 10 minutes on ice. Nuclei were resuspended in 0.5ml 16 HindIII restriction buffer (Roche) containing 0.3% SDS and incubated for 1 hour at 37uC with agitation. Triton X-100 was added to 2% (v/v), and the nuclei were further incubated for 1 hour at 37uC to sequester the SDS. The crosslinked DNA was digested overnight at 37uC with 400U HindIII. The restriction enzyme was inactivated by the addition of SDS to 1.6% and incubated at 65uC for 20 minutes with agitation. The reaction was diluted with 7ml 16ligation buffer (Promega) and incubated for 1 hour at 37uC. The chromatin was ligated with 100U T4 DNA ligase (Promega) for 4 hours at 16uC followed by 30 minutes at room temperature. 600 mg of Proteinase K was added, and samples were incubated overnight at 65uC to reverse the cross-links. Next, the samples were incubated for 30 minutes at 37uC with 1500 mg of DNase-free RNaseA, and the DNA was purified by phenol extraction and ethanol precipitation. The DNA pellet was resuspended in 150 ml 10 mM Tris-Cl (pH7.5). Preparation of Random Template control In order to normalize the amplification efficiency of different primer sets, the PAC185 [15] plasmid containing the human b- globin locus was used for preparation of the random template control (RTC). Theoretically, this random template control contains all possible ligation products in equimolar amounts. Wild-type mouse genomic DNA was mixed with PAC185 plasmid and a 60–70kb PAC containing the mouse XPB locus (PAC Clone #443-C18, MRC gene services). The genomic DNA/PAC clones mixture was processed according to the 3C technique [6]. The processed products were serially diluted with wild-type mouse genomic DNA until the PAC DNA was present in a molar ratio similar to single-copy b-globin locus transgenic DNA (line PAC8.1), as assessed by quantitative PCR [6,7]. PCR products were run on a 2% agarose gel and quantified with a Typhoon 9200 imager (Amersham). Typically 300ng of DNA was subsequently used for each 3C-PCR reaction. Each PCR reaction was performed in triplicate and repeated at least three times. Sequences of the 3C PCR primers are shown in Table 1. Anti-CTCF ChIP assays 16107 cells were defined as 1 unit of sample for 10ml ChIP crosslinking reaction (2% formaldehyde and 40mM HEPES pH7.9 in DMEM with 10% FCS). The samples were crosslinked for 20 minutes at room temperature with gentle mixing. The reaction was quenched by the addition of glycine to 0.125M. The cells were harvested by centrifugation at 1500rpm for 5 minutes at 4uC. The cells were first washed with PBS buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml aprotinin and 1 mg/ml pepstatin A) and centrifuged at 1500rpm for 5 minutes. 10 ml of Triton-wash buffer (0.25% Triton-X 100, 10mM Na-EDTA, 0.5 mM Na-EGTA and 10 mM Tris-Cl pH8.0) was added and the samples were incubated at room temperature for 10 minutes. The cells were pelleted by centrifugation at 1500rpm for 5 minutes and they were further washed with 10ml NaCl-wash (200mM NaCl, 1 mM Na-EDTA, 0.5mM Na-EGTA and 10mM Tris-Cl pH8.0) buffer at room temperature for 10 minutes. After centrifugation at 1500rpm for 5 minutes, the pellet was resuspended in 500 ml sonication buffer (1mM Na-EDTA, 0.5mM Na-EGTA and 10mM Tris-Cl pH8.0) and kept on ice for 5 minutes. DNA was sheared by sonication to lengths between 200 and 1000bp, keeping the sample cold on ice. Cell debris was removed by centrifugation. ChIP reactions were performed according to the Upstate Biotechnology chromatin immunoprecipitation protocol with 1 mg of anti-CTCF antibody (Upstate Biotechnology) per IP reaction. Enrichment of specific PLoS ONE | www.plosone.org 7 May 2008 | Volume 3 | Issue 5 | e2134