Systematic Targeted Integration to Study Control Elements AlbuminGene Sanchari Bhattacharyya1, Jianmin Tian1,2, Eric E. Bouhassira3, Joseph Locker1,2* 1 Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 The Marion Bessin Liver Center, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America Abstract To study transcriptional regulation by distant enhancers, we devised a system of easilymodified reporter plasmids for integration into single-copy targeting cassettes in clones of HuH7, a human hepatocellular carcinoma. The plasmid constructs tested transcriptional function of a 35-kb region that contained the rat albumin gene and its upstream flanking region. Expression of integrants was analyzed in two orientations, and compared to transient expression of non-integrated plasmids. Enhancers were studied in their natural positions relative to the promoter and localizedby deletion. All constructs were also analyzed by transient transfection assays. In addition to the known albumin gene enhancer (E1 at 210 kb), we demonstrated two new enhancers, E2 at 213, and E4 at +1.2 kb. All three enhancers functioned in both transient assays and integrated constructs. However, chromosomal integration demonstrated several differences from transient expression. For example, analysis of E2 showed that enhancer function within the chromosome required a larger gene region than in transient assays. Another conserved region, E3 at 20.7 kb, functioned as an enhancer in transient assays but inhibited the function of E1 and E2 when chromosomally integrated. The enhancers did not show additive or synergistic behavior,an effect consistent with competition for the promoter or inhibitory interactions among enhancers. Growth arrest by serum starvation strongly stimulated the function of some integrated enhancers, consistent with the expected disruption of enhancer-promoter looping during the cell cycle. Citation: Bhattacharyya S, Tian J, Bouhassira EE, Locker J (2011) Systematic Targeted Integration to Study Albumin Gene Control Elements. PLoS ONE 6(8): e23234. doi:10.1371/journal.pone.0023234 Editor: Alfred Lewin, University of Florida, United States of America Received March 30, 2011; Accepted July 12, 2011; Published August 12, 2011 Copyright: 2011 Bhattacharyya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grants, CA68440, CA76394, and CA104292 to JL and HL088467 to EEB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: joseph.locker@einstein.yu.edu Introduction In addition to the core promoter and its proximal regulatory element, many genes have enhancers, which vary in size, location, strength, and mode of action. Ranging from 50–1500 bp, enhancers can activate their target promoters from great distances (up to 1 mb) and may reside within introns or even on different chromosomes [1,2]. Distant enhancers physically associate with promoters by looping out large intervening sequences [3]. Theselong-range interactions regulate developmental and tissue- specific gene expression. However, the variety of enhancers and the large genomic distances separating them from promoters have hindered mechanistic study of their interactions within chromo- somes. Individual enhancers may vary in promoter specificity and strength, and combinations may additively, synergistically, or competitively stimulate transcription [4,5,6]. In addition, many enhancers can activate heterologous weak promoters, a property exploited in enhancer trapping [7,8]. Promoters may compete for enhancers [9,10], and there are also a few cases where multiple promoters share enhancers [11,12]. The study of integrated activity in an intact gene therefore goes beyond simple demonstrations of enhancer function. There has been great progress in finding enhancers. Conserved or ultraconserved regions in non-coding DNA frequently identify enhancers, but do not always correlate with transcriptional function [13]. Active enhancers are marked by monomethylated lysine 4 on histone 3 (H3K4) in contrast to H3K4 trimethylation of active promoters. DNAseI hypersensitive sites, transcription factor binding, and coactivator binding also correlate with enhancer function [3,14,15,16]. Chromatin Conformation Capture and its updated versions have shown physical association of distant regulatory regions to target promoters [17,18,19,20,21]. These contemporary approaches have predicted 105–106 enhancers in the genome, i.e., an enhancer every 3–30 kb [8]. Nevertheless,the approximate and predictive enhancer localization by these methods is not equivalent to functional assessment [22]. Since the discovery of enhancers, transient assay in reporter plasmids has provided a standard test of function independent of chromosomal context. The limitations of this approach are exemplified by the b-globin locus control region (LCR), a complex of enhancers and other functional elements that render position- independence to transgenes [23,24]. Some LCR components regulatetranscription in transient assays whileothers require integration [8]. However, integration of transgenesis subject tovariable copy number and chromosomal position, which makes it difficult to compare different integrated constructs. Wetherefore derived a system for analyzing sets of single copy gene constructs in a consistent chromosomal position, and then used this system to study the Albumin gene. Prior studies of Alb—a classical marker of the liver phenotype— established paradigms for gene expression controlled by distant PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23234