Enhancer Function of Integrated Alb Reporters Figure 6. Localization of intronic enhancer E4. A: Large deletions clones of P+ALB were constructed with restriction enzymes SgfI, XmaI, and EagI (top). Clone ALBD2 was then further deleted by PCR (bottom; see Table S2). B: Analysis of the larger constructs by transient transfection. C: Analysis of the larger constructs by integration in two orientations (O1, O2). D: Localization of a strong enhancer toIntron 2 by transient transfection assays. B, D; Mean and standard deviation of fluorescence intensity were calculated from two transient transfection experiments. C; Average and standard deviation of measurements from integrated constructs. doi:10.1371/journal.pone.0023234.g006 Transfection of the largest plasmid, 1+2+P+ALB, was challeng- ing due to its size and low copy number, but even this large plasmid functioned in transient assays at a level indicating enhancer-stimulated gene expression. Both transient assay and chromosomal integration produced intermediatereporter stimula- tion, indicating that the enhancer regions were not additive or synergistic. The level was much less than the maximum capacity of the promoter since the 1+P construct was about 3-fold stronger. Because each can separately stimulatetranscription, the lack of additive stimulation suggestedenhancer competition. This could result from competition for binding to the same element within the promoter or competition for a limiting trans factor. Alternatively, the effect could represent binding of the enhancers to each other in competition with their binding of the promoter, or an indirect boundary function where one enhancer establishes a local chromatin structure that obstructs access of the other to the promoter. Any of these functions might modulate during development, so they are all possible mechanisms for enhancer switching. Transgenic studies of the b-globin gene cluster and its LCR led to the concept that position independence is an ideal criterion for demonstrating that a regulatory region is intact [24]. In this paper, however, we demonstrated that an extended region of the rat Alb locus does not show complete position independence. Perhaps additional elements outside the 35 kb region contribute to higher- order Alb regulation [40]. Alternatively, position-independence may be an artificial concept, since each gene has been selected to function in its natural position, surrounded by local and distant regulatory elements. From another perspective, demonstration of position effects in our system has revealed functional cis elements that are obscured by simpler assays of gene expression. It is well established that promoters and enhancers loop together, but looping represents different kinds of interactions. The distant LCR dynamically oscillates between b- and c-globin genes by the distant LCR, transitory looping within a more stable chromatin hub [41,42,43]. In contrast, the HNF4a-gene promoter and enhancer require two days to form a stable loop after growth arrest of Caco2 cells [44]. In either type of looping, the higher order chromatin architecture will be disrupted by DNA replica- tion. We therefore investigated whether growth arrest increased the effects of enhancers, particularly synergy of multiple enhancers in the full 35-kb Alb region. Natural Alb expression is consistent PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23234