is in agreement with Patrinos et al. [23] who have shown that deletion of the b-globin promoter alone is not sufficient to alter the 3D configuration of the ACH. Since 59HS5 functions as a border at the primitive stage [11], we next examined the spatial organization of the PAC3K and PACD3K transgenes in embryonic blood samples. The restriction fragment containing 59HS2 [24], a classical enhancer element within the LCR, was used as the fixed fragment for the results presented in Fig. 4. The analysis shows that the spatial organization of the locus is basically unchanged upon deletion of 59HS5 (PACD3K) when compared with the PAC3K transgenic line. In agreement with the notion that the c-globin gene is predominantly expressed in primitive cells, higher relative crosslinking frequencies of the c-globin gene compared to the b-globin gene could be observed (Fig. 4). The results imply that in primitive erythroid cells association of the actively transcribed c-globin genes with the ACH principally remains the same in the absence of 59HS5. For investigating the spatial organization of the chromatin hub (CH), which can be observed in the erythroid progenitor cells without b-like globin gene expression [7], we used the b-globin gene as the fixed primer in 3C analysis (Fig. 5). Similar results were obtained between the two constructs (PAC3K vs. PACD3K). In conclusion, the results strongly indicate that deletion of 59HS5 of the LCR in the human transgene does not have a measurable effect on the 3 dimensional organization of the b-globin locus, neither in the primitive stage where 59HS5 functions as a border or enhancer blocker, nor in the definitive stage where 59HS5 does not have a known function. ChIP analysis of CTCF binding to 59HS5 in primitive and definitive erythroid cells We have shown previously that human 59HS5 has an enhancer blocking function at the primitive stage [11]. However, the mecha- nism by which this function is achieved remains unknown. Although a number of enhancer blocking proteins have been identified in Drosophila [25–27], CTCF is the only protein known so far to mediate enhancer blocking activity in vertebrates [28,29]. Previous studies conducted by Felsenfeld and his colleagues have described a 1.2kb DNA element with strong enhancer blocking activity, located at the 59HS4 of the chicken b-globin gene locus [28,29]. A 250bp core fragment of the cHS4 is fully functional and a single binding site for the protein CTCF, FII, is necessary and sufficient to block the action of an enhancer on a promoter when placed between them [30]. Conserved CTCF binding sites are found at 59HS5 and 39HS1 of both the mouse and human loci. To investigate whether CTCF binds to 59HS5, ChIP analysis using an antibody to CTCF was performed (Fig. 6). The CTCF ChIP DNA enrichment levels of three fragments were tested, namely, mouse endogenous HS62 (mHS62), human bm (the marked b-globin gene) and human 59HS5. In erythroid tissues, DNA enrichment could be detected by real-time PCR. Our results on mHS62 are in agreement with a previous study [14] and confirm that CTCF binds to the endogenous mouse HS62 fragment. Human 59HS5 exhibits only modest enrichment levels of 1.5 fold at primitive and 2.4 fold at definitive stages. These results support the previous in vitro assay data [13] showing the presence of a CTCF binding site in human 59HS5 [13]. Our data suggest that CTCF also binds to human HS5 in vivo. Simultaneously, the bm gene enrichment level was determined using anti-CTCF ChIP assay. Surprisingly, the DNA enrichment level of bm gene is higher than that of the human 59HS5 and mouse HS62 at both developmental stages (Fig. 6). Further studies are needed to characterize the functional role of CTCF binding to the bm gene. h59HS5 Is b-ACh Dispensable Figure 4. 3C analysis of the PAC3K and PACD3K transgenes using a primer from 59HS2 as the fixed primer. Embryonic blood was collected from E10.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. RT: Random template control. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization. doi:10.1371/journal.pone.0002134.g004 Discussion In this study, we have analyzed the role of 59HS5 with respect to the three dimensional configuration of the human b-globin locus. The transgenic lines carried PAC constructs that were derived from the original 185 kb human b-globin PAC [7]. Transgenic lines carrying single copies of four modified PAC constructs, namely PAC1B, PACD1B, PAC3K and PACD3K, were used [15] to determine possible conformational changes of the locus after deletion of the 59HS5 core region. The 3D conformation of the transgenic loci was determined by 3C analysis. A human b-globin promoter deletion in the context of the full LCR was used to determine whether or not the interaction between the b-globin gene and the LCR would be perturbed when both 59HS5 and the b- globin promoter (PACD3K) were deleted, as was previously found when both 59HS3 and the b-globin promoter were deleted [23]. We analyzed the conformation of the globin transgenes from two important genomic sites in the human b-globin locus, namely the b- globin gene and 59HS2. We first examined the integrity of the ACH upon the removal of 59HS5 in the PACD1B transgenic line at the definitive stage. The results show that the three dimensional PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2134