Enhancer Function of Integrated Alb Reporters predicted Southern blot bands of integrated constructs P and 1+2+P obtained with KpnI (K) and HpaI (H). The hybridization probe is marked as a bar over the promoter-GFP region, and the promoter and known enhancer are marked in black. D: Southern blot mapping of eight integrated constructs. The blots (left,KpnI; right, HpaI digests) were probed with a 1.2 kb FseI - SmaI segment of plasmid P. The expected band sizes are listed in Table S3. doi:10.1371/journal.pone.0023234.g002 HuH7-9, the L1-HYTK-1L cassette was integrated withina 137- kb intergenic region on Chromosome 7, 57 kb from the 39-end of Origin of Replication Complex Subunit 5 (ORC5L) and 78 kb from the 59-end of Reelin (RELN) (Fig. 2A). Both genes are strongly expressed in liver and HCC cells, although neither is specific for these cell types. Surprisingly, the targeting cassette in HuH7-10 was integrated into the same region of Chromosome 7, in this case within the first intron of RELN, 116 kb from the 59-end. This site is 200 kb from the integration site of HuH7-9. Prior to analysis of gene expression, gancyclovir-resistant isolates were first screened by PCR to demonstrate integration and determine orientation of gene constructs (Fig. 2B, Table S2). Southern blot analysis then discriminated clones with intact single integrations (Fig. 2C, Table S3). Analysis of gene expression To work outanalysis of enhancer function, we studied the albumin promoter alone (plasmid P) or combined with the intact 17.4 kb upstream region (plasmid 1+2+P) that contains well- characterized enhancer E1 (Fig. 3A). Early studies that defined E1 suggested that it functioned poorly in transient assays of large plasmid constructs [25]. Nevertheless, our flow cytometry analysis of transient transfection demonstrated 16-fold enhancement of gene expression (Fig. 3B, C). This result, however, required establishment of conditions that gave a linear measurement of gene expression. Several transient transfection reagents oversatu- rated gene expression, i.e., they stimulated expression to a plateau level that was insensitive to plasmid concentration or the strength of the reporter gene. In contrast, simple lipofectamine transfection, under conditions that stimulated gene expression in only ,10% of cells, gave weaker transfection signals that showed a linear relationship between plasmid copy number and level of measured gene expression (data not illustrated). Transcription oftransiently transfected plasmids is controlled only by regulatory elements within the plasmid construct. In contrast, chromosomal integration allows interaction with tran- scription control elements outside of the construct. To control for such positional effects, constructs were compared at different integration sites—HuH7-9 or Huh7-10—and in two orientations, designated O1 and O2 (Fig. 3D–G). By flow cytometry, integrants gave a single symmetrical distribution of gene expression with correspondence of mean and peak values. The promoter and enhancer regions functioned in all integrants. However, expression of the promoter and relative stimulation by enhancers showed significant positional differences. In HuH7-9, the promoter hadsimilar strength in both orientations, while the enhancer region gave 10-fold stimulation in O1 and4- fold in O2. In HuH7-10 cells, the promoter gave 6-fold higher expression than in HuH7-9, with only 2 to 3-fold additional stimulation added by enhancer region. Nevertheless, the combined enhancer and promoter gave gene expression comparable to O1 in HuH7-9 cells. The results in HuH7-10 suggested promoter stimulation by a non-Alb enhancer, so subsequent analysis was limited to HuH7-9, comparing O1 and O2 to discriminate positional effects. Survey of a 35-kb region for regulatory elements Prior studies localizedE1 from 210.1 to 29.2 kb [25,27]. Conserved non-coding regions within both the 17.4-kb upstream and the 16.5-kb gene suggested additional regulatory elements (Fig. 1A). To test their transcriptional function and interactions among regulatory elements, we first surveyed large gene regions (Fig. 4). Transient transfection demonstrated that Regions 1, 2, and ALB each significantly enhanced gene expression, 10-, 4-, and 12- fold, respectively (Fig. 4B). The contribution of Region 2 was additive when combined with either Region 1 or ALB. Transfection of the largest plasmid 1+2+P+ALB (40 kb) was more variable, but its activity was much less than the sum of contributions from individual enhancers. When integrated, Region 1 and ALB each caused significant enhancement in either orientation (Figs. 4C, D). Gene expression was generally stronger in O1 than O2, particularly stimulation by Region 1. Region 2 had complex effects. It was a weak enhancer by itself, and its small contribution probably added to stimulation by ALB. However, Region 2 inhibited stimulation by Region 1, especially in O1. The largest plasmid construct, 1+2+P+ALB, combined all of the regulatory regions.Its large size led to low efficiency of integration, and we obtained only a single integrant, in O1. The combined stimulation by Regions 1, 2 and ALB in this clone was not greater than individual component segments, an effect also noted in transient expression. Thus, transient transfection and stable integration in either orientation demonstrated enhancer function. However, genomic integration revealed complex relationships among regulatory elements within the constructs, andpositional effects suggested interaction with additional elements outside of the constructs. Mapping of novel enhancers The regional analysis and pattern of sequence conservation both suggested enhancers throughout the 35-kb region. We first focused on Region 1. Deletion mapping—with constructs analyzed by both transient transfection and stable integration—identified a new enhancer, E2 (Fig. 5). E1 and E2 were of similar strength and together accounted for all of the enhancing activity within Region 1. The two enhancers functioned in their normal positions or when moved close to the promoter, and their enhancer function was independent of each other and of Region 2. Deletion mapping localized E2 to the 212.8 to 211.9 interval that contained 2 regions of conserved sequence. In stable integration, both regions were required for enhancer function, although only the more proximal conserved region was required for enhancer function in transient assays. By itself, Region 2 (3.7 kb) acted as a weak enhancer (‘‘E3’’) in transient assays, where it added to the stimulation by Region 1. However, deletion mapping did not discretely localize an enhancer within Region 2 (not illustrated). Moreover, in stable integration, Region 2 had an inhibitory effect when combined with Region 1. The 16.5-kb Alb gene itself caused strong transcriptional enhancement, and contains conserved noncoding regions within introns 2 and 4 (Fig. 6). Deletion mapping, analyzed by both transient transfection and stable integration, localized a strong enhancer in a proximal 4.4-kb segment. Analysis of additional deletions by transient transfection localized the strong enhancer to intron 2. Two other segments, within pALBD1 and pE4D3, mediated weak enhancement, but the responsible elements were not further resolved. PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23234