TY - JOUR
T1 - Systematic targeted integration to study Albumin gene control elements
AU - Bhattacharyya, Sanchari
AU - Tian, Jianmin
AU - Bouhassira, Eric E.
AU - Locker, Joseph
PY - 2011
Y1 - 2011
N2 - 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 -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.
AB - 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 -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.
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U2 - 10.1371/journal.pone.0023234
DO - 10.1371/journal.pone.0023234
M3 - Article
C2 - 21858039
AN - SCOPUS:80051611722
SN - 1932-6203
VL - 6
JO - PloS one
JF - PloS one
IS - 8
M1 - e23234
ER -