Enhancer Function of Integrated Alb Reporters Figure 4. Survey of a 35-kb region for novel regulatory elements. A: Map of plasmid constructs. Conserved noncoding regions, black; Alb gene exons, gray; E1, the previously characterized Alb enhancer. B: Transient transfection analysis. Mean fluorescence intensity and standard deviation werecalculatedfrom triplicatetransfections in at least three separate experiments. C: Expression of stable integrants in O1. D: Expression of stable integrants in O2. C, D: Mean and standard deviation of expression from at least 2 integrant clones per construct in each orientation, except for a single integrant of 1+2+P+ALB in O1. Fluorescence intensity was normalized to the value obtained for the promoter. doi:10.1371/journal.pone.0023234.g004 Modification of gene expression We next evaluated the effects of growth arrest on enhancer- driven gene expression. Because it is likely that DNA replication and cell division will disrupt enhancer-promoter interaction, the expectation was that gene expression would increase when the cell does not divide, and the magnitude of the effect will depend on how long it takes to reestablish looping. This is consistent with two observations. Constitutively proliferating HuH7 cells strongly express albumin mRNA, but at 40% of the level of normal liver [34], while Alb mRNA in normal liver decreases significantly when hepatocytes are stimulated to proliferate by partial hepatectomy [35]. HuH7 is a contact-inhibited cell line, so we compared growth arrest induced by confluenceor serum starvation. Analysis showed that HuH7 cells arrested at 60–72 hr after plating at 25% confluence, or 24 hr after serum starvation (data not illustrated). The two arresting conditions had significantly different effects on gene expression (Fig. 7). Confluence caused a moderate increase, less than 2-fold, in the expression of two constructs, 1+P and 1+2+P+ALB. In contrast, serum starvation stimulated all Orientation 1 integrants to some extent, up to 4-fold by 96 hr. Strong stimulation was observed in long constructs 1+P and 1+2+P+ALB, and in short construct E2+P, and was thus selective for E2. The presence of Region 2 had an inconsistent effect. It reduced stimulation of 1+2+P compared to 1+P, but did not affect stimulation of 1+2+P+ALB. Stimulation of Orientation 2 clones was more moderate, with little selectivity for E2. The difference between confluence and serum starvation could reflect that the latterarrests growthmore effectively, or alternatively, that serum contains a factor that selectively inhibits E2. Discussion We set out to create an experimental system of gene integration into a controlled chromosomal location with the eventual goal of resolving specific molecular interactions within a complex locus. The model of gene expression was Alb, a distinctive high-level phenotypic marker of hepatocytes. Our analysis exploited the hepatocyte-like phenotype of Alb-expressing HuH7 cells, where function of Alb transcriptional regulatory elements is similar— though perhaps not identical—to normal hepatocytes. The analysis validated the gene targeting system by demonstra- tingtwo novel Alb enhancers, E2 and E4. The weak activity of the previously characterized E1 has seemed inadequate to explain the very strong expression of Alb in hepatocyte-derived cells, so the new observations eliminate a significant discrepancy. Comparative sequence analysis showed that E2 and E4 have features typical of hepatocyticenhancers, with putative sites for their characteristic PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23234