Human heterochromatin protein 1 isoforms HP1(Hsalpha) and HP1(Hsbeta) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation - PubMed (original) (raw)

Human heterochromatin protein 1 isoforms HP1(Hsalpha) and HP1(Hsbeta) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation

Girdhar G Sharma et al. Mol Cell Biol. 2003 Nov.

Abstract

Telomeres are associated with the nuclear matrix and are thought to be heterochromatic. We show here that in human cells the overexpression of green fluorescent protein-tagged heterochromatin protein 1 (GFP-HP1) or nontagged HP1 isoforms HP1(Hsalpha) or HP1(Hsbeta), but not HP1(Hsgamma), results in decreased association of a catalytic unit of telomerase (hTERT) with telomeres. However, reduction of the G overhangs and overall telomere sizes was found in cells overexpressing any of these three proteins. Cells overexpressing HP1(Hsalpha) or HP1(Hsbeta) also display a higher frequency of chromosome end-to-end associations and spontaneous chromosomal damage than the parental cells. None of these effects were observed in cells expressing mutants of GFP-DeltaHP1(Hsalpha), GFP-DeltaHP1(Hsbeta), or GFP-DeltaHP1(Hsgamma) that had their chromodomains deleted. An increase in the cell population doubling time and higher sensitivity to cell killing by ionizing radiation (IR) treatment was also observed for cells overexpressing HP1(Hsalpha) or HP1(Hsbeta). In contrast, cells expressing mutant GFP-DeltaHP1(Hsalpha) or GFP-DeltaHP1(Hsbeta) showed a decrease in population doubling time and decreased sensitivity to IR compared to the parental cells. The effects on cell doubling times were paralleled by effects on tumorigenicity in mice: overexpression of HP1(Hsalpha) or HP1(Hsbeta) suppressed tumorigenicity, whereas expression of mutant HP1(Hsalpha) or HP1(Hsbeta) did not. Collectively, the results show that human cells are exquisitely sensitive to the amount of HP1(Hsalpha) or HP1(Hsbeta) present, as their overexpression influences telomere stability, population doubling time, radioresistance, and tumorigenicity in a mouse xenograft model. In addition, the isoform-specific effects on telomeres reinforce the notion that telomeres are in a heterochromatinized state.

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Figures

FIG. 1.

Expression levels of GFP-HP1Hsα, GFP-HP1Hsβ, and GFP-HP1Hsγ. (A) RT-PCR of GFP-tagged HP1. The primers are specific to GFP. The gel displays the bands obtained after quantitative RT-PCR over 35 cycles to determine the levels of GFP-HP1 in cells. The levels of expression of wild-type and mutant GFP-HP1Hsα, GFP-HP1Hsβ, and GFP-HP1Hsγ are very similar. (B) Western blot analysis of GFP-tagged wild-type (a) and mutant (b) HP1Hsα, HP1Hsβ, and HP1Hsγ using anti-GFP antibody. Note that the levels of expression of HP1Hsα, HP1Hsβ, and HP1Hsγ are very similar.

FIG. 2.

Human telomeric DNA coimmunoprecipitated by an hTERT antibody after in vivo cross-linking in cells expressing GFP-tagged HP1 proteins. ECR-293 cells overexpressing GFP-tagged wild-type (HP1Hsα, HP1Hsβ, or HP1Hsγ) or mutant (ΔHP1Hsα, ΔHP1Hsβ, or ΔHP1Hsγ) HP1 proteins were treated with formaldehyde (+F) or mock treated (−F). Chromatin was isolated and subjected to immunoprecipitation by using an anti-hTERT antibody. ECR-293 are the parental cells; empty vector cells are ECR-293 cells transfected with an empty vector. Deproteinized DNA isolated from the precipitates was denatured and spotted onto a membrane. The following probes were used for hybridization: total human genomic DNA (total DNA), a DNA fragment containing Alu repeats (Alu), or a DNA fragment containing telomeric DNA (CCCTAA). The same blot is shown after consecutive rehybridizations with the different probes. Note a decrease in the amount of telomeric DNA compared to that of the total genomic DNA in cells overexpressing GFP-HP1Hsα or GFP-HP1Hsβ. The results are representative of three independent experiments.

FIG. 3.

Western blot analysis of HP1Hsα, HP1Hsβ, and HP1Hsγ using protein-specific antibodies. (A) Western blot analysis of HP1Hsα. Lane 1, control cells; lane 2, control cells with empty vector; lane 3, cells with overexpression of HP1Hsα. (B) Western blot analysis of HP1Hsβ. Lane 1, control cells; lane 2, cells with overexpression of HP1Hsβ. (C) Western blot analysis of HP1Hsγ. Lane 1, control cells; lane 2, cells with overexpression of HP1Hsγ.

FIG. 4.

Human telomeric DNA coimmunoprecipitated by an hTERT antibody after in vivo cross-linking in cells expressing nontagged HP1 proteins. ECR-293 cells overexpressing nontagged wild-type (HP1Hsα, HP1Hsβ, or HP1Hsγ) proteins were analyzed for hTERT interactions with telomeres, as described in the legend to Fig. 2. Note a decrease in the amount of telomeric DNA compared to that of the total genomic DNA in cells overexpressing nontagged HP1Hsα or HP1Hsβ.

FIG. 5.

hTERT expression, telomerase activity, and TRF2 levels. (A) Levels of hTERT RNA. The gel displays the bands obtained after quantitative RT-PCR over 35 cycles to determine the levels of hTERT RNA in cells with or without overexpression of wild-type GFP-HP1Hsα, GFP-HP1Hsβ, GFP-HP1Hsγ, or GFP-ΔHP1Hsα. HFF is a negative control, RNA derived from human foreskin fibroblast cells (47). hTERT is a band specific for hTERT RNA. α-Actin is a band for alpha-actin RNA as the internal control. Note that no difference in expression of hTERT was found among cells with or without overexpression of GFP-HP1Hsα, GFP-HP1Hsβ, GFP-HP1Hsγ, or GFP-ΔHP1Hsα. (B) Levels of telomerase activity. Telomerase activity measured in extracts from cells with or without overexpression of GFP-HP1Hsα, GFP-HP1Hsβ, or GFP-HP1Hsγ by TRAP enzyme-linked immunosorbent assay. Note that no difference in telomerase activity was observed in cells with or without HP1Hsα, HP1Hsβ, or HP1Hsγ. (C) Levels of TRF2 RNA. The gel displays the bands obtained after quantitative RT-PCR over 35 cycles to determine the levels of TRF2 RNA in cells with or without overexpression of wild-type GFP-HP1Hsα, GFP-HP1Hsβ, GFP-HP1Hsγ, GFP-ΔHP1Hsα, GFP-ΔHP1Hsβ, or GFP-ΔHP1Hsγ. TRF2, band specific for TRF2 RNA; α-actin, band for alpha-actin RNA as the internal control. Note that no difference in levels of TRF2 was found among cells with or without overexpression of wild-type or mutant HP1Hsα, HP1Hsβ, or HP1Hsγ.

FIG. 6.

Single-strand extensions (G tails), terminal restriction fragment sizes, and telomere FISH. (A and B) G tail and terminal restriction fragment sizes in cells overexpressing GFP-fused wild-type (GFP-HP1Hsα, GFP-HP1Hsβ, or GFP-HP1Hsγ) HP1 proteins. (C and D) G tail and terminal restriction fragment sizes in cells overexpressing nontagged wild-type (HP1Hsα, HP1Hsβ, or HP1Hsγ) HP1 proteins. In panels A and C, nondenaturing in-gel hybridizations to genomic DNA digested with restriction enzymes _Hin_fI and _Rsa_I and using a telomeric repeat probe of the C-rich strand are shown. This method allows visualizing G-strand overhangs on telomeres. Signals were quantified by PhosphorImager analysis and corrected for DNA loading by using the rehybridized gel shown in panels B and D. Lane 1, molecular mass standards; lane 2, DNA from parental ECR-293 cells; lanes 3 to 5, DNA from ECR-293 cells overexpressing HP1Hsα, HP1Hsβ, or HP1Hsγ, respectively; lane 6, denatured plasmid single-stranded DNA containing telomeric repeats (positive control); lane 7, double-stranded plasmid DNA used as a negative control (detected only once the DNA is denatured, as seen in panels B and D). Panels B and D show the same gel as in panels A and C after denaturing of the DNA in the gel and rehybridization with the same probe. The arrow in panel B indicates an internal restriction fragment carrying telomeric repeats that was used to correct for DNA loading. Note that cells with overexpression of GFP-fused HP1 shown in panels A and B have effects on G overhangs and telomere size similar to those seen in the cells with overexpression of nontagged HP1 proteins shown in panels C and D. (E) Telomere FISH analysis showing sections of metaphase chromosomal spreads derived from parental ECR-293 cells (a), ECR-293 cells overexpressing HP1Hsα (b and c), or ECR-293 cells overexpressing HP1Hsβ (d). Note the chromosome end associations in panels b and d and an absence of telomeric signals in panels c and d (indicated by arrows). Telomeric signals are present on some telomere fusion sites (indicated by arrows in panel b).

FIG. 6.

FIG. 7.

Influence of overexpression of wild-type or mutant HP1Hsα, HP1Hsβ, or HP1Hsg on cell growth. Cells overexpressing the indicated forms of HP1 proteins were seeded in plates, and cell counts were determined at regular intervals. The actual numbers of cells are plotted against the hours of growth in a semilog diagram. The values shown are the means of the results from three experiments. (A) The effects of wild-type and mutant GFP-HP1Hsα, GFP-HP1Hsβ, and GFP-HP1Hsγ on cell growth. (B) Effects of wild-type nontagged HP1Hsα, HP1Hsβ, and HP1Hsγ on cell growth. Note that the influence of GFP-tagged HP1 proteins on cell growth is similar to the influence of nontagged HP1 proteins.

FIG. 8.

Comparison of cell survival after IR treatment. Dose response curves for cells overexpressing the indicated GFP-tagged HP1 proteins are shown. Cells were treated with ionizing radiation while growing exponentially and asynchronously. Cells overexpressing GFP-ΔHP1Hsα or GFP-ΔHP1Hsβ are more resistant to damage induced by gamma rays than cells overexpressing GFP-HP1Hsα or GFP-HP1Hsβ. The values shown are the means of the results from three to four experiments.

FIG. 9.

G1, S, and G2 chromosomal aberrations after IR treatment. (A) Cells in plateau phase were irradiated with 3 Gy, incubated for 24 h postirradiation, and then subcultured, and metaphases were collected. G1-type aberrations were examined at metaphase. Categories of asymmetric chromosomal aberrations scored included dicentrics, centric rings, interstitial deletions and acentric rings, and terminal deletions. The frequency of chromosomal aberrations was higher in EC-293 cells overexpressing wild-type GFP-HP1Hsα and GFP-HP1Hsβ but not in cells overexpressing GFP-HP1Hsγ. (B) Cells in exponential phase were irradiated with 2 Gy. Metaphases were harvested 6 h following irradiation and examined for chromosomal aberrations. The frequencies of chromatid and chromosomal aberrations were higher in EC-293 cells overexpressing wild-type GFP-HP1Hsα or GFP-HP1Hsβ than in those expressing GFP-HP1Hsγ. (C) Cells in exponential phase were irradiated with 1 Gy. Metaphases were harvested 1 h following irradiation and examined for chromosomal aberrations. The frequency of chromatid aberrations was higher in EC-293 cells overexpressing wild-type GFP-HP1Hsα or GFP-HP1Hsβ but not in those expressing GFP-HP1Hsγ. Note that cells overexpressing GFP-HP1Hsα or GFP-HP1Hsβ have higher frequencies of chromosomal aberrations than those of the parental control cells in all phases of the cell cycle, suggesting a global defective DNA repair.

FIG. 9.

FIG. 10.

Influence of HP1Hsα, HP1Hsβ, and HP1Hsγ on oncogenic transformation in vitro (A and B) and in vivo (C) and determination of colony formation by the agarose-independent anchorage assay. (A) Cells overexpressing GFP-HP1Hsα or GFP-HP1Hsβ (A-1) or nontagged HP1Hsα or HP1Hsβ (A-2) have a lower number of colonies than the parental cells. Cells overexpressing HP1Hsγ did not show any change in colony formation. (B) Cells with expression of mutant GFP-HP1Hsα or GFP-HP1Hsβ form a higher number of colonies than the parental cells. Cells expressing mutant GFP-HP1Hsγ did not show any influence on colony formation. (C) Cells with or without overexpression of wild-type and mutant GFP-HP1Hsα, GFP-HP1Hsβ, or GFP-HP1Hsγ were injected subcutaneously into mice. Tumor growth was measured starting at day 14 after inoculation. Note that tumors derived from cells overexpressing GFP-HP1Hsα or GFP-HP1Hsβ grow more slowly than those of the control, while those derived from cells expressing mutant GFP-HP1Hsα or GFP-HP1Hsβ grow faster. Tumors derived from cells overexpressing wild-type or mutant GFP-HP1Hsγ did not show any change in growth compared to that for the control.

References

1. Aagaard, L., M. Schmid, P. Warburton, and T. Jenuwein. 2000. Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres. J. Cell Sci. 113:817-829. - PubMed
1. Blobel, G. 1985. Gene gating: a hypothesis. Proc. Natl. Acad. Sci. USA 82:8527-8529. - PMC - PubMed
1. Braunstein, M., A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach. 1993. Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev. 7:592-604. - PubMed
1. Cenci, G., G. Siriaco, G. D. Raffa, R. Kellum, and M. Gatti. 2003. The Drosophila HOAP protein is required for telomere capping. Nat. Cell Biol. 5:82-84. - PubMed
1. Cheutin, T., A. J. McNairn, T. Jenuwein, D. M. Gilbert, P. B. Singh, and T. Misteli. 2003. Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science 299:721-725. - PubMed

Human heterochromatin protein 1 isoforms HP1(Hsalpha) and HP1(Hsbeta) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation - PubMed (original) (raw)