title: Systematic targeted integration to study Albumin gene control elements.
abstract: To study transcriptional regulation by distant enhancers, we devised a system of easily modified 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 localized by deletion. All constructs were also analyzed by transient transfection assays. In addition to the known albumin gene enhancer (E1 at -10 kb), we demonstrated two new enhancers, E2 at -13, 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 -0.7 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.
authors.affiliation: Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors.forename: Sanchari
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: Jianmin
authors.initials: J
authors.lastname: Tian
authors.forename: Eric E
authors.initials: EE
authors.lastname: Bouhassira
authors.forename: Joseph
authors.initials: J
authors.lastname: Locker
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D000418:Albumins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014162:Transfection
chemical_list: D000418:Albumins; D016895:Culture Media, Serum-Free; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins