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Telomere Position Effect in Human Cells
Joseph A. Baur, Ying Zou, Jerry W. Shay, and Woodring E. Wright
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Supplementary Material
Supplemental Figure 1. Generation of clones containing telomeric luciferase reporters. A plasmid containing a 1.6-kb tract of telomere repeats adjacent to a luciferase reporter was linearized using Not I and transfected into HeLa cells (
1-
4). Clones were recovered in which integration of the plasmid had caused the truncation of a chromosome and subsequent extension of the plasmid sequences to form a new telomere. (TetO)
7 represents seven tet operator sites fused to create a bidirectional tetracycline-responsive promoter. The indicated Stu I sites were used to analyze chromosomal insertion sites. The plasmid was designed to have a tet-inducible promoter driving both the puromycin selectable marker and a luciferase reporter. This would permit drug selection in the presence of a strong (induced) promoter, followed by growth and establishment of potential TPE under noninduced (weak promoter) conditions. The results shown in Web Fig. 2A were obtained after continuous growth under induced conditions. No significant change in the dose response or magnitude of the TPE was observed after selected telomeric and internal clones were cultured for 1 month in the presence of the tetracycline analog doxycycline (uninduced) and then tested for luciferase activity under graded levels of induction. We thus did not find any evidence for an influence of promoter strength on TPE in human cells within the range of our tet-off system, in contrast to yeast TPE which is abolished in the presence of a strong promoter (
5,
6). The remainder of the experiments were therefore carried out under doxycycline-free (fully induced) conditions.
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Supplemental Figure 2. Characterization of transgene integrations. (A) Additional clones were analyzed as described in Web Fig. 1. From left to right, they are a clone with both a telomeric and an internal integration, a clone with a single internal integration, and a clone with two internal integrations. Genomic DNA was digested with Stu I, leaving the luciferase gene attached to the plasmid telomere sequences (Web Fig. 1). Telomeres were then separated from bulk genomic DNA by first annealing a biotinylated (CCCTAA)6 oligonucleotide to the single-stranded overhang and then retrieving the telomeres using streptavidin beads as has been described previously (7). Both the telomere and supernatant (bulk genomic DNA) fractions were analyzed by Southern blotting. The fact that the signal seen in the telomere fraction for the first clone appears as a smear provides additional confirmation that the clone is telomeric, because the attached telomere sequences are heterogenous in length. (B) Multiple bands appear in some clones. Genomic DNA was digested using Stu I and analyzed by Southern blotting without prior separation of telomeres. Four telomeric clones with no internal sites (lanes 1, 5, 6, and 7), a single internal integration site (lane 4), two internal integration sites (lane 3), and three internal integration sites (lane 2) are shown.
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References and Notes
1. The puromycin resistance gene was first inserted into the Pvu II site of the bidirectional tet-off vector pBI-2 by blunt cloning. The Bam H1-Bgl II fragment from pSXneo, containing 1.6 kb of telomeric repeats, proved unstable when cloned next to the luciferase gene of pBI-2 but was stable when placed into the Bgl II site next to the puromycin gene. The relative location of puromycin and luciferase was then flipped in order to obtain the final plasmid, pBX, in which the telomeric repeats were adjacent to luciferase. This was accomplished by ligation of the Nhe I-Hpa I fragment containing the luciferase/puromycin cassette from the intermediate plasmid to the Spe I-Bgl II fragment containing the rest of the plasmid backbone. A control vector for generating internal insertions was created by excising the T2AG3 repeats from pBX by Bst X1-Not I, blunting, and religating the plasmid. HeLa cells expressing the tetracycline-responsive transcriptional activator (tTA, tet-off) were used throughout these experiments. Transfections were carried out using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions.
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7. W. E. Wright, V. M. Tesmer, K. E. Huffman, S. D. Levene, J. W. Shay, Genes Dev. 11, 2801 (1997).
