same transgenic lines in which 59HS5 had been removed by Cre- mediated excision. The first insulator element reported in vertebrates is 59HS4 (cHS4) in the chicken b-globin locus [12]. Binding of the 11 Zn- finger protein CTCF to the FII region of the cHS4 is thought to play an important role in the activity of cHS4 [13,14]. To investigate the potential activity of CTCF as an enhancer blocking protein on human 59HS5 in vivo, we performed chromatin immunoprecipitation assays using antibodies directed against CTCF. Results PAC constructs for 3C analysis of the human b-globin locus To examine the potential conformational changes of the ACH in the presence or absence of 59HS5 in the context of the human b-globin locus, we employed transgenic mice carrying modified human b-globin PAC transgenes (Fig. 1, [11]). The transgenes were derived from PAC185 which contains the entire human b- globin gene cluster. Homologous recombination in E. coli DH10B cells [15] was used for generation of the constructs. To determine the effect of deletion of 59HS5 on the three dimensional (3D) organization of the locus, 59HS5 was flanked by loxP sites to enable Cre-mediated deletion of 59HS5 (PACD1B). The basal promoter and 59 untranslated regions of the b-globin gene, from 2139 to +49 relative to the cap site of the gene, has been removed to generate the b-globin promoter deletion (PAC3K; Fig. 1). h59HS5 Is b-ACh Dispensable Intactness of the transgenes was carefully mapped by Southern blot analysis using a total of 11 different restriction enzymes and hybridization with cosmid LCRe, -ccdb probes [16], and smaller probes along the b-globin locus [11]. 3C analysis of the PAC constructs with the bm gene and 59HS5 flanked by loxP sites Previous studies on human b-globin transgenes in mice have shown that the b-globin gene interacts with the ACH when it is transcribed in definitive erythroid cells [7]. In these studies, the Chromosome Conformation Capture (3C) technique [6,7,17] was used to examine the three dimensional organization of the b-globin locus. First, we determined whether introduction of the b-marked (bm) gene, which is an artificially modified b-globin gene with part of its exon 2 replaced by equivalent Ac-globin gene [9] (Fig. 1B), and flanking 59HS5 by loxP sites per se caused conformational changes of the b-globin locus. We performed 3C analysis on the PAC transgenes and compared this directly to the results obtained with the wild type locus (PAC8.1; Fig. 2). In the wild type locus, 59HS1-5 of the LCR and the HS in the 39 flanking region (39HS1) interact with each other to form the ACH of the b-globin locus. The results of 3C analysis on the PAC1B transgene show that the spatial structure of the locus is basically unaltered when compared with that of wild type PAC8.1. In brain, a non-erythroid tissue, the PAC transgene shows an essentially linear conformation of the b- globin locus (data not shown). This is consistent with the notion that the formation of ACH is tissue specific [6]. Figure 1. Schematic presentation of the human b-globin locus in PAC transgenic constructs. A) Arrows on top of the locus depict the individual hypersensitive sites within the LCR. b-like globin genes are indicated by large triangles with different colors. The b-mark globin gene was indicated by light blue rectangle with bm labeled on top of it. LTR located at the 59-upstream of the bm gene was indicated by the small rectangle with black in color. The HS5 is flanked by loxP sites indicated by small triangle on the locus. PACD constructs were generated by homologous recombination according to Iman et al., 2000. The white boxes indicate the olfactory receptor (OR) genes which flank the b-globin locus. HindIII restriction sites (small arrows) and the DNA fragments used in the 3C analysis (solid rectangles) are shown below the PAC1B locus. Distances are in kb counting from the transcription initiation site of the e-globin gene. B) Structure of the marked b-globin gene (bm) and the ChIP-bm primers location. doi:10.1371/journal.pone.0002134.g001 PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2134