h59HS5 Is b-ACh Dispensable Figure 5. 3C analysis of the PAC3K and PACD3K transgenes using a primer from the human b-globin gene as the fixed primer. Embryonic blood was collected from E10.5 embryo for this set of 3C experiment. (A) Representative expamples of the PCR fragments resulting from the 3C experiments. RT: Random template control. (B) Histograms of the relative crosslinking efficiencies after quantitation and normalization. doi:10.1371/journal.pone.0002134.g005 structure of the b-globin locus has essentially remained the same when 59HS5 is deleted. Mild increments of the association frequency of 59HS6 to the LCR could be explained by distance effects [9]: 59HS6 interacts more frequently with the ACH once it moves closer to the LCR after Cre-mediated excision of 59HS5. This is consistent with our previous data on human b-globin transgenic mice which showed that the increase in expression of the bm gene, which is located 59 upstream of the LCR, is likely due to the shorter distance of the gene relative to the LCR [11]. Our previous study on the human b-globin PAC transgenes has shown that 59HS5 is a stage-specific enhancer blocking element that functions at the primitive stage of erythropoiesis [11]. Surprisingly, we did not observe any significant alterations in the structure of the b-globin locus by 3C analysis primitive embryonic blood when 59HS5 was deleted. The results obtained from both the definitive and primitive stages imply that spatial organization of the locus is essentially unchanged upon deletion of 59HS5 (PACD1B) or in the double deletion of HS5 and the b-globin promoter (PACD3K). We can not completely discount the possible influence of 59HS5 on b- globin locus configuration as this might not be detectable due to limitations of the resolution achieved with the 3C technique. However, we can conclude that the enhancer blocking properties of 59HS5 in primitive erythroid cells is not accompanied by major effects on the spatial organization of the b-globin locus. Interestingly this study shows that CTCF, the first transcription factor found to have insulating activity [28] and enhancer blocking function [13,14] in mammalian cells, binds to the 59HS5 core region at both the primitive and definitive stages in erythroid cells in vivo. This indicates that CTCF could be involved in the border function of 59HS5 in the primitive cells. Since it also binds to the 59HS5 region in definitive cells, this suggests that CTCF, most likely in conjunction with other proteins, can have opposing effects on gene expression. Filippova et al. [31] proposed that CTCF could be a transcriptional repressor of the c-Myc oncogene in vertebrates. In contrast, the CTCF protein can bind to the promoter of the amyloid b protein precursor and activate transcription [32]. PLoS ONE | www.plosone.org 6 May 2008 | Volume 3 | Issue 5 | e2134