h59HS5 Is b-ACh Dispensable Figure 3. 3C analysis of the PAC1B and PACD1B transgenic lines using a primer from the human b-globin gene in combination with primers from other parts of the locus. Fetal livers were collected from E14.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization. The histograms are the average of at least three separate experiments, with each PCR performed in triplicate. doi:10.1371/journal.pone.0002134.g003 globin locus, yolk sac-derived primitive erythroid cells express the human embryonic e- and fetal c-globin genes [16]. b-like globin gene expression switches to the fetal c- and adult d- and b-globin genes during early definitive erythropoiesis between E12.5 and E14.5. The LCR plays an important role in the regulation of expression of the b-like globins, and the order and distance of the genes relative to the LCR are important parameters for the developmental switch [8,9,20]. These developmental switches of hematopoiesis are accompanied by changes in chromatin structure and spatial organization of regulatory elements throughout the locus [6,7,21]. In a previous study with the human b-globin PAC transgenes we have shown that 59HS5 functions as an enhancer blocker in embryonic erythroid cells [11]. Therefore, it is interesting to determine the possible conformational changes of the locus without 59HS5 in embryonic erythroid cells. PAC3K transgenic lines, in which the promoter of the b-globin gene in the normal position is deleted, have been used for the 3C analysis. The rationale behind the usage of the PAC3K construct for performing the 3C analysis is that in this configuration, the bm gene located upstream of the LCR is maximally activated in definitive cells, while expression in primitive cells is only detectable after deletion of 59HS5. We therefore compared the locus-wide crosslinking frequencies of the PAC3K and PACD3K (PAC construct with deletion of 59HS5) transgenes at different developmental stages. In the fetal liver, we observed that the 3D conformation of the PAC3K and PACD3K transgenes is virtually identical to that observed for the PAC1B and PACD1B transgenes (data not shown). This implies that neither 59HS5 nor the b-globin promoter is required for structural changes in the ACH occurring during the process of b-globin gene switching [7,18,22]. The latter PLoS ONE | www.plosone.org 4 May 2008 | Volume 3 | Issue 5 | e2134