Enhancer Function of Integrated Alb Reporters Figure 5. Localization of E2, a novel upstream Alb enhancer. A: Maps of deletion constructs. Large deletions of 1+2+P (top) were constructed with restriction enzymes ApaI and BstXI. Plasmid E2+P was further deleted by PCR (bottom; see Table S2). Black, regions highly conserved between rat and human genes. B: Analysis of plasmids containing intact enhancers. The plots show analysis of transient assays and integrated constructs in two orientations (O1, O2). C: Analysis of plasmids for fine mapping of E2, as in panel B. For each construct, mean and standard deviation of fluorescence intensity were calculated from 3 separate transient transfection experiments, or from multiple integrant clones. doi:10.1371/journal.pone.0023234.g005 binding factors, HNF4, FOXA/HNF3, and C/EBP (Fig. 8). Moreover, ENCODE databases derived from analysis of HepG2 cells showed that the human counterpart regions had hypersen- sitive sites and bound both HNF4 and C/EBP (http://genome. ucsc.edu/cgi-bin/hgTracks). Phylogenetic comparisons demon- strated that enhancers have conserved regions flanked by specific binding sites that may be poorly conserved [36,37]. Nevertheless, enhancers from different species tend to bind the same transcription factors near the conserved regions, sometimes in different positions. Thus, prediction of transcription factor binding sites and genomic analysis of human counterpart regions are both highly consistent with our functional characterization of these enhancers. Prior RMCE studies have analyzed orientation-dependent effects of flanking sequences on transgenes, position-dependent silencing of the b-globin LCR, and transcriptional interference at a complex locus [33,38,39]. We adapted RMCE to examine enhancer-mediated regulation over long distances. Comparison of integrated constructs with transient transfection showed that each method has limitations: transient assays give a simplified view of gene expression, but integratedconstructs can interactwith unrelated regulatory elements. The latter effects, however, can be constant if integration site, orientation, and copy number are eliminated as experimental variables. Both transient and RMCE analyses revealed that Region 1 and ALB mediate independent and comparable enhancement of gene expression. However, the behavior of smaller regions and the intact Region 2 exemplifies the differences between transient and integrated regulatory elements.By itself, Region 2 functioned as an enhancer in transient assays, where it caused 5-fold stimulation of gene expression and was additive with either Region 1 or ALB. Moreover, Region 2 had been previously shown to contain a hypersensitive site [25]. We therefore considered Region 2 to contain a third enhancer (E3), which we did not resolve further because of itsweak activity. Integrationof Region 2 gave a different perspective, since it produced little transcriptional stimulation by itselfand reduced stimulation by Region 1 in larger constructs. Thus Region 2 contains regulatory elements, but their specific function and contribution to Alb gene expression require further investigation. Another difference between transient and integrated expression was revealed by deletion mapping of E2, which was localized to an 0.8 kb genomic interval that contains two conserved regions.The E2D3 construct eliminated one conserved region and still functioned as an enhancer in transient assays. In contrast, enhancer function after integration required both conserved regions, revealing an essential activity that is dispensable for transient expression from a plasmid. PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23234