Modeling long-distance transcriptional regulation by targeted integration

Abstract

In higher eukaryotes, varied enhancers frequently regulate transcription over long distances. Standard methods, including transient transfection and random stable integration lead to sub-optimal expression from transgenes due to copy number and position effects. We therefore built a system to study single-copy integration of constructs at unique chromosomal locations, retaining genomic spacing of regulatory elements.;The 72-kb Albumin-alpha-fetoprotein (Alb-Afp ) locus contains strong enhancers that regulate both of these closely related genes in mammalian liver. A series of reporter plasmids -- GFP and DsRed under control of the Alb and Afp promoter respectively -- were integrated to test enhancer function from normal position, and interaction among enhancers in an extended gene region. The experiments utilized Recombinase Mediated Cassette Exchange (RMCE), which enabled single-copy integration of reporter constructs in human hepatocellular carcinoma (HCC) cell lines and comparison to transient transfection..;We identified two novel strong Alb enhancers: E2, at -13 kb, and E4, at +2 kb within the second intron, and also observed competition among them. Both methods measured enhancer activity although specific enhancers varied in strength in an orientation-dependent fashion. Interestingly, we observed that growth arrest enhanced gene expression of several integrated constructs, suggesting that cell proliferation inhibits some enhancer-promoter interactions.;Afp has three upstream enhancers within a 6-kb region. RMCE showed heterogeneity of enhancer-driven gene expression within some clones, and the proportion of cells with maximal gene expression was orientation specific. However, a 29-kb upstream segment, including the Alb gene, stabilized maximal gene expression. Moreover, gene expression was stimulated by blocking DNA methylation, independent of orientation.;Integration of dual reporters containing the Afp enhancers at normal distance from Alb-GFP and Afp-DsRed promoters showed strong expression of both proteins, indicating enhancer sharing. In prolonged culture, however, a clone containing the dual reporter showed promoter switching that was stably inherited by daughter cells.;To analyze the Alb-Afp locus, we developed a general system of plasmids, RMCE and cell lines that allow systematic modification and comparison of single integrated constructs in unique chromosomal positions. Our system therefore allows detailed study of specific mechanisms of enhancer-promoter interactions within the locus.