Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity

Abstract

We investigated the influence of telomere proximity and composition on the expression of an EGFP reporter gene in human cells. In transient transfection assays, telomeric DNA does not repress EGFP but rather slightly increases its expression. In contrast, in stable cell lines, the same reporter construct is repressed when inserted at a subtelomeric location. The telomeric repression is transiently alleviated by increasing the dosage of the TTAGGG repeat factor 1 (TRF1). Upon a prolongated treatment with trichostatin A, the derepression of the subtelomeric reporter gene correlates with the delocalization of HP1alpha and HP1beta. In contrast, treating the cells with 5 azacytidin, a demethylating agent, or with sirtinol, an inhibitor of the Sir2 family of deacetylase, has no apparent effect on telomeric repression. Overall, position effects at human chromosome ends are dependent on a specific higher-order organization of the telomeric chromatin. The possible involvement of HP1 isoforms is discussed.

Figure 1

Transient expression assays reveals a transactivation activity of telomeric repeats. (A) Schematic map of the two reporter plasmids used in this study. pCMVTelo contains a stretch of 1.6 kb TTAGGG in close proximity to the reporter EGFP gene driven by a CMV promoter. At 1.8 kb from the TTAGGG repeats, we introduced the fusion gene between hygromycin phosphotransferase and HSV1 thymidine kinase (HyTK) conferring the resistance to hygromycin as a selectable marker. The control vector lacking the telomeric 1.6 kb stretch was named pCMV. (B) Transient expression assays with various amount of pCMV or pCMVTelo circular DNA. A time course of the EGFP expression after transfection was determined. The percentage of EGFP-positive cells is corrected for transfection efficiency determined by CAT assay. The values correspond to the average of at least three independent experiments. We estimated the standard error to be <20%. (C) Ratio of the percentage of EGFP-positive cells in pCMVTelo transfection to that in pCMV transfections.

Figure 2

Telomeric silencing in populations of transfected cells. The pCMV and pCMVTelo DNA were linearized with NotI (Figure 1A) and used to transform C33-A cells. By co-transfection with pBLCat, we estimated that the efficiencies were roughly similar in all situations (data not shown). The percentage of EGFP-positive cells was analyzed by Facscan. Three independent transfections are shown (1, 2 and 3). Three days after transfection, hygromycin (400 μg/ml) was added. The boxes show an enlarged view of the beginning of the kinetics for 2 and 3.

Figure 3

Telomeric silencing in clones. (A) Localization of the EGFP gene at 16p by chromosome 16 painting (image a) and FISH with an EGFP probe (image b); the position of 16p is marked by arrows. (B) The percentage of EGFP-positive cells in clones presenting a single insertion of the reporter gene. These clones were obtained from three independent transfections with either pCMV or PCMVTelo. (C) The percentage of EGFP-positive cells plotted versus the length of the EGFP-linked telomere [eTRF in kb of (TTAGGG)n]. The eTRF value was determined by Southern blotting after probing HindIII-cut genomic DNA with an EGFP probe.

Figure 4

Overexpression of TRF1 antagonizes telomeric silencing. The pITRF1 or pIRES plasmid DNAs were transfected in the 3B10 and 2A3 pCMV clones and the 4C4 and 4C11 pCMVTelo clones. Four days after transfection, a neomycin selection was applied (t0) and cells were cultured for >30 MPDs. (A) TRF1 expression was determined in 4C11 cells transfected with pIRES or pITRF1 by immunoblotting using either anti-FLAG antibodies (a monoclonal anti-M2 antibody; Sigma) or anti-TRF1 antibodies. Anti-tubulin antibodies were used to evaluate the loading variations. (B) The same type of immunoblotting experiments as in (A) but with 2A3 transfected cells. (C) The entire population of neomycin resistant clones was analyzed by Facscan for EGFP expression.

Figure 5

TSA alleviates telomeric silencing. Populations of 4C11, 2A3 or 3B10 cells sorted for a deprivation of EGFP-positive cells were incubated for 3 days with a culture medium containing the indicated drug at T1; then the medium was changed with one of identical composition (T2).