Specific boundary activities of Gal-Pro at telomeric transgenes. (A and B) Percentile of fluorescing cells from cell clones having integrated a reporter construct at a telomeric locus and which display little or no basal expression of the transgenes (A) or results from clones generated similarly but having detectable levels of GFP or DsRed fluorescence (B). B-labeled (B05, B09, B10, and B23) and D-labeled (D17, D26, D31, and D34) clones were generated using the convergent or divergent reporter constructs containing telomeric repeats, respectively. Each clone was transiently transfected with an empty expression vector (control) or with the Gal-DBD, Gal-Pro, or Gal-VP16 expression vector. Values represent the average of the percentile of cells expressing DsRed, GFP, or both DsRed and GFP (dsRed + GFP) among 1,000 BFP-expressing cells, determined as illustrated in Fig. 1. Each error bar shows the standard error of the mean of at least three independent experiments. (C) Clones generated without telomeric repeats and showing the internal integration site were transfected and processed as for panels A and B.

Telomeric histones H3 and H4 are hypoacetylated. Chromatin immunoprecipitation was performed on two telomeric clones (B09 and D17) and one clone with nontelomeric integration (cD06). Chromatin fragments were immunoprecipitated using antibodies specific for acetylated H3 and H4 (A and B), trimethylated H3K9 (C), or the histone variant H2A.Z (D), and the precipitated DsRed and GFP genomic sequences were assayed by real-time PCR and normalized to values obtained by amplifying the GAPDH gene. Means and standard errors of the means of three independent experiments with at least two independent chromatin preparations are indicated.

Gal-Pro protects transgenes from telomeric position silencing effects. (A) Vectors used to assay the silencing of telomeric transgenes. Constructions were designed to place the GFP-coding gene in a telomere-proximal position and DsRed in a telomere-distal position relative to four binding sites for the yeast GAL4 protein. Transcription of the reporter genes is either convergent or divergent (green and red arrows), and the positions of the quantitative PCR amplicons used to probe chromatin structure are shown as a bar under each arrow. Not depicted here is an antibiotic selection gene located to the left of the DsRed gene. Control plasmids used for random integration at internal chromosomal locations were deleted of the telomeric repeats, which are shown by arrowheads. (B to G) Examples of cytofluorometric analysis of DsRed and GFP fluorescence in clones B09 (B, C, and D) and D17 (E, F, and G), carrying at a telomeric position shown in panel A the convergent or divergent reporter construct, respectively. Each clone was transiently cotransfected with a plasmid encoding the GAL4 DNA-binding domain alone (Gal-DBD) (B and E) or fused to the CTF1 proline-rich (Gal-Pro) (C and F) or VP16 (Gal-VP16) (D and G) activation domain and with a BFP expression vector. The panels depict the GFP and DsRed fluorescence of 1,000 BFP-expressing cells. Quadrant regions were set for each clone according to the basal DsRed and GFP fluorescence in Gal-DBD-expressing cells so as to obtain 99% of the cells in the bottom left region. The percentile of cells in each quadrant is indicated.

Native CTF1 acts as boundary at human cell telomeres. HeLa cells were transfected with a plasmid reporter construct as for Fig. 1A except that it carried seven CTF/NF1 binding sites instead of GAL4 sites inserted between the DsRed and GFP genes, which are divergently transcribed. The integration site of clones generated with reporter constructs containing (A and B) or devoid (C and D) of telomeric repeats was verified by FISH analysis by probing telomeric repeats (green) or the reporter vector (red) (A and C). Boundary activity was evaluated by comparing DsRed and GFP expression as described in the legend for Fig. 2 in clones transiently cotransfected with a control plasmid (pBS) or with the CTF1-encoding expression plasmid (CTF) (B and D). *, P = 0.06 (Student's t test).

Effect of the Gal-Pro boundary on telomeric chromatin structure. Chromatin immunoprecipitations were performed on telomeric clones B09 and D17 stably expressing Gal-DBD or Gal-Pro or treated with the HDAC inhibitor TSA. Antibodies were specific for acetylated H3 and H4 (A and B), trimethylated H3K9 (C), or the histone variant H2A.Z (D), and precipitated sequences were processed as for Fig. 4. *, P < 0.05; **, P < 0.01 (Student's t test).