h59HS5 Is b-ACh Dispensable Figure 2. 3C analysis of the PAC1B transgene 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. WT: PAC8.1wt; RT: Random template control. (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.g002 Effect of the deletion of the 59HS5 on ACH formation To study the effect of 59HS5 on the spatial organization of the b-globin locus, we compared the PAC1B line with the PACD1B line, in which 59HS5 had been deleted by the action of Cre recombinase [15]. It has been proposed that transcriptional activation of b-like globin genes at each stage of development is a multi-step process [5]. An LCR holocomplex is formed to allow the accessibility of transcription and chromatin remodeling factors to the locus, thus providing a high local concentration of the relevant trans-acting factors for efficient transcription. The resultant functional active chromatin hub (ACH) comprises the LCR holocomplex interacting directly with the transcribed genes [6,7,18]. This model implies that maintaining the integrity of the ACH is the key to create a chromatin domain permissive for transcriptional regulation. To examine the possible consequences of deletion of 59HS5 on ACH formation, we compared the 3D structures of the PAC1B and PACD1B transgenes in definitive erythroid cells isolated from 14.5 dpc fetal livers with the 3C technique (Fig. 3). The results show that the overall 3D of the b-globin locus has remained the same when 59HS5 is deleted (PACD1B). However, the relative crosslinking frequency of the 59HS6, the distal hypersensitive site located about 6 kb 59 upstream to the HS5 [19], with the b-globin gene is increased by upon the removal of 59HS5 (Fig. 3B, primer 418). This increase in association frequency with the ACH is presumably due to the closer proximity of 59HS6 to the LCR after Cre-mediated excision of 59HS5. 59HS6 is located 800bp closer to the LCR in the PACD1B line. Besides, in the absent of HS5, the b- gene promoter interacts strongly with the A-c promoter. In mammals, two developmental switches of hematopoiesis occur during ontogeny. In transgenic mice carrying the human b- PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2134