Protection against telomeric position effects by the chicken cHS4 beta-globin insulator

Abstract

Epigenetic silencing of genes relocated near telomeres, termed telomeric position effect, has been extensively studied in yeast and more recently in vertebrates. However, protection of a transgene against telomeric position effects by chromatin insulators has not yet been addressed. In this work we investigated the capacity of the chicken beta-globin insulator cHS4 to shield a transgene against silencing by telomeric heterochromatin. Using telomeric repeats, we targeted transgene integration into telomeres of the chicken cell line HD3. When the chicken cHS4 insulator is incorporated to the transgene, we observe a sustained gene expression of single-copy integrants that can be maintained for >100 days of continuous culture. However, uninsulated single-copy clones showed an accelerated gene expression extinction profile. Unexpectedly, telomeric silencing was not reversed with trichostatin A or nicotidamine. In contrast, significant reactivation was obtained with 5-aza-2'-deoxycytidine, consistent with the subtelomeric DNA methylation status. Strikingly, insulated transgenes integrated into telomeric regions were enriched in histone methylation, such as H3K4me2 and H3K79me2, but not in histone acetylation. Furthermore, the cHS4 insulator counteracts telomeric position effects in an upstream stimulatory factor-independent manner. Our results suggest that this insulator has the capacity to adapt to different chromatin propagation signals in distinct insertional epigenome environments.

Fig. 1.

The cHS4 insulator protects against CPE. (A) Scheme of the vectors used for stable transfections. (B) Stable integrant of HD3 cells transfected with an uninsulated or insulated GFP transgene was maintained in continuous cell culture for 100 days (d100). FACS analysis was performed for each clone every 2 weeks. Representative FACS profiles are shown for several single or multicopy integrants. (C) Reactivation assays were performed with silenced uninsulated clones by incubating with TSA or 5-azadC for 24 and 48 h, respectively.

Fig. 2.

Telomeric insertion of insulated and uninsulated transgenes. (A) Transgene vector with the telomeric (TTGAAA)_n repeats. (B) Colocalization of transgene with telomeric repeats. In situ hybridization was performed with the clones 613 and 615 and the random integrated clone 1001. Transgene signal was amplified and detected with a FITC-labeled antibody against anti-digoxigenin (green). Telomeric repeats were hybridized with biotinylated oligonucleotides and identified with streptavidin coupled to Alexa Fluor 568 (red). Cells were counterstained with DAPI. Arrows indicate the location of the transgene. (C) Sequence specificity of the purification of telomeres. DraIII-digested HD3 genomic DNA was annealed to telomeric-specific biotinylated oligonucleotides, and telomere–oligonucleotides complexes were captured with streptavidin-coated magnetic beads. The bound DNA was resolved on an agarose gel and probed with a ³²P-labeled GFP probe or with an ³²P-labeled (TTAGGG)₇ oligonucleotide.

Fig. 3.

The cHS4 insulator protects against TPE. HD3 cells were stably transfected with uninsulated (A) or insulated (B) transgenes. Isolated single-copy and multicopy clones were analyzed by FACS. TR, telomeric (TTGAAA)_n repeats.

Fig. 4.

Telomeric repeats cause DNA methylation-dependent transgene silencing. (A) Clones with a silenced uninsulated transgene were incubated with TSA, nicotinamide (NAM), and/or 5-azadC, as is reported in Materials and Methods. (B) As an additional example, clone 805 is shown that is sensitive to TSA. Its behavior is representative of several independent clones, like the 861 and 862 clones. Clone 805 was treated as above, and the fluorescence mean (Upper) and the percentage of positive cells (Lower) are shown.

Fig. 5.

Histone modifications over cHS4-insulated transgenes. (A) FACS profiles of the lines with telomeric (613) and random (1001) integration, respectively. (B) Duplex PCRs were used to evaluate the relative enrichment of open chromatin marks. (C and D) ChIP assays were performed with insulated telomeric (C) and random (D) integrants. Relative enrichment of histone modification was plotted, and the average enrichment values of at least two independent immunoprecipitations are shown. Each PCR was performed at least twice, and standard error is presented.

Fig. 6.

Protection against TPE by the cHS4 does not require USF. Stable clones with the core cHS4-insulated transgene both in random or telomeric regions were stably transfected with a mixture of five USF1-specific RNAi-expressing plasmids (13). (A) Knockdown of USF1 was addressed by Western blotting. (B) RNAi-expressing subclones were maintained for 3 weeks, and GFP expression was analyzed by FACS. Representative FACS profiles of three transfections are shown.