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The cellular phenotype of Roberts syndrome fibroblasts as revealed by ectopic expression of ESCO2 - PubMed

  • ️Thu Jan 01 2009

The cellular phenotype of Roberts syndrome fibroblasts as revealed by ectopic expression of ESCO2

Petra van der Lelij et al. PLoS One. 2009.

Abstract

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Functional complementation of Roberts syndrome fibroblasts by epitope tagged ESCO2.

Stable VU1199-F SV40 cell lines expressing V5- or GFP-tagged ESCO2 were obtained by transfection and neomycin selection. (A) Whole cell extracts were analyzed for ESCO2 or GFP expression by Western blotting with an ESCO2- or GFP-specific antibody. Tubulin served as a loading control. The VU1199-F SV40 cell line stably transfected with a GFP construct served as a control for cells expressing GFP protein only. (B) Nuclear localization of V5-ESCO2, GFP-ESCO2 and GFP-ESCO2 (W539G) in the stably transfected VU1199-F SV40 fibroblasts. Cells were fixed with 4% methanol-free formaldehyde solution and the V5-ESCO2 expressing cell line was probed with an anti-V5 antibody. Nuclei were stained with ToPro3. (C) Railroad chromosomes in RBS immortal fibroblasts and complemented cell lines. Fifty metaphases per cell line were scored for the presence of railroad chromosomes, from coded slides; the percentage of metaphases containing one or more railroad chromosome was calculated.

Figure 2
Figure 2. Ectopic ESCO2 expression during the cell cycle.

VU1199-F SV40 cells stably expressing V5-ESCO2 were synchronized by a double aphidicolin block and mitotic shake-off and samples were analyzed for (A) ESCO2 expression on Western blot and (B) DNA content by flow cytometry. (C) Cell cycle distribution related to GFP-ESCO2 expression was measured by flow cytometry in VU1199-F SV40 cells stably expressing wild type or mutant GFP-ESCO2. Cells were fixed in 70% ethanol and DNA was stained with ToPro3. Asterisk indicates a protein detected by the ESCO2 antibody which is supposed to represent a degradation product of ESCO2.

Figure 3
Figure 3. ESCO2 levels are regulated by proteasomal degradation.

(A) Fluoresence microscopy showing stabilization of ESCO2 protein by proteasome inhibitors in VU1199-F SV40 cells stably expressing wild type or mutant ESCO2. Cells were treated for 6 h with 50 µM MG-132 or 100 nM bortezomib and fixed with 100% cold methanol. Nuclei were stained with DAPI. (B) Ectopic ESCO2 is stabilized by proteasome inhibitors as shown by Western blotting. Cell lines were exposed to proteasome inhibitors for 6 h with 50 µM MG-132 or 100 nM bortezomib and analyzed for ESCO2 expression by Western blotting. Tubulin was used as loading control. (C) Endogenous ESCO2 levels in synchronized U2OS cells. Cells were arrested in G1/S phase by a thymidine block and released for 9 h to obtain cells in G2 phase. Mitotic cells were harvested by treatment with nocodazole followed by mitotic shake-off. These cells were released for 90 min to obtain early G1 phase cells. Cell lysates were analyzed for ESCO2, securin, cyclin B1 and APC expression by Western blotting. Asterisk indicates an aspecific band considered as loading control. (D) Proteasome inhibitor MG-132 stabilizes ESCO2 levels in M phase cells. U2OS cells were cultured for 4 h in the presence of 5 µM MG-132 before M phase cells were isolated. APC3 phosphorylation is shown as a control for mitotic cells. Asterisk indicates an aspecific band as loading control. (E) ESCO2 levels in unsynchronized wild type fibroblasts treated with proteasome inhibitors. LN9SV was treated for 6 h with 50 µM MG-132 or 1 µM bortezomib.

Figure 4
Figure 4. Survival of RBS and stably transfected fibroblasts after treatment with various DNA-damaging agents.

Data are averages of at least 2 or 3 independent experiments; error bars represent standard error of the mean. VU1199-F SV40 cell line, V5-ESCO2-transfected VU1199-F SV40, GFP-ESCO2-transfected VU1199-F SV40, GFP-ESCO2 mutant (W539G)-transfected VU1199-F SV40 and wild type fibroblasts LN9SV (figures A to E) or VH10 SV40 (figures F and G) were grown for 10–12 days after treatment with X-rays or UV-C irradiation, or after continuous exposure to the indicated DNA-damaging agents. (A) Clonogenic survival after continuous MMC exposure. SV40-immortalized fibroblasts of a Fanconi anemia patient (EUFA1341, FA-N) are shown as a hypersensitive control (open circles). Clonogenic survival after continuous exposure to (B) camptothecin, (C) etoposide, (D) aphidicolin, (E) hydroxyurea. Clonogenic survival after (F) X-ray or (G) UV-C exposure.

Figure 5
Figure 5. Formation of Rad51 foci and sister chromatid exchanges in RBS cells.

(A) Rad51 foci in normal and RBS cells, as determined 6 and 24 h after treatment with X-ray (12Gy) or MMC treatment (7 µM for 1 h). The percentages of cells containing more than five nuclear foci were determined. Data are the means of at least three experiments; error bars represent the standard error of the mean. (B) SCE induction in RBS cells after MMC treatment. Wild type (LN9SV), RBS (VU1199-F SV40) and RBS cells expressing V5-ESCO2, GFP-ESCO2 or GFP-ESCO2 (W539G) were either mock-treated or treated by continuous exposure to 50 nM MMC. Numbers of SCEs were counted and normalized against the number of chromosomes scored.

Figure 6
Figure 6. Chk-1 phosphorylation status in RBS cells.

VU1199-F SV40 fibroblasts, VU1199-F corrected with V5-ESCO2 and wild type fibroblasts LN9SV were left untreated or exposed to MMC, camptothecin (CPT) or hydroxyurea (HU) for 24 h and whole cell extracts were obtained to investigate the phosphorylation of Chk-1. Vinculin and Chk-1 served as loading controls.

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