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Werner syndrome protein positively regulates XRCC4-like factor transcription.

Liu D, Deng X, Yuan C, Chen L, Cong Y, Xu X - Mol Med Rep (2014)

Bottom Line: Depletion of WRN in HeLa cells led to a decrease of XLF mRNA and its promoter activity.Chromatin immunoprecipitation assays demonstrated that WRN was associated with the XLF promoter.Taken together, the results suggest that XLF is a transcriptional target of WRN and may be involved in the regulation of cellular senescence.

View Article: PubMed Central - PubMed

Affiliation: Beijing Key Laboratory of DNA Damage Response, College of Life Science, Capital Normal University, Beijing 100048, P.R. China.

ABSTRACT
XRCC4-like factor (XLF) is involved in non-homologous end joining-mediated repair of DNA double-strand breaks (DSBs). Mutations in the WRN gene results in the development of Werner syndrome (WS), a rare autosomal recessive disorder characterized by premature ageing and genome instability. In the present study, it was identified that XLF protein levels were lower in WRN-deficient fibroblasts, compared with normal fibroblasts. Depletion of WRN in HeLa cells led to a decrease of XLF mRNA and its promoter activity. Chromatin immunoprecipitation assays demonstrated that WRN was associated with the XLF promoter. Depletion of XLF in normal human fibroblasts increased the percentage of β-galactosidase (β-gal) staining-positive cells, indicating acceleration in cellular senescence. Taken together, the results suggest that XLF is a transcriptional target of WRN and may be involved in the regulation of cellular senescence.

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XLF suppresses cellular senescence. (A) Inhibition of XLF expression in WI38 cells. WI38 cells at passage 39 PD were transfected with si-CTR or si-XLF. Total cell lysates were extracted 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (B) Depletion of XLF inhibited cell proliferation. XLF expression was inhibited by transfection with si-XLF as described in (A) and cell numbers were determined every 24 h. Six duplicate samples were analyzed at each time point. (C and D) Inhibition of XLF expression accelerated cellular senescence. XLF-depleted WI38 cells were assayed for β-gal. Representative images of stained cells are shown in (C) and quantitative data in (D). PD, population doubling; XLF, XRCC4-like factor; β-gal, β-galactosidase; si-CTR, control siRNA.
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f3-mmr-09-05-1648: XLF suppresses cellular senescence. (A) Inhibition of XLF expression in WI38 cells. WI38 cells at passage 39 PD were transfected with si-CTR or si-XLF. Total cell lysates were extracted 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (B) Depletion of XLF inhibited cell proliferation. XLF expression was inhibited by transfection with si-XLF as described in (A) and cell numbers were determined every 24 h. Six duplicate samples were analyzed at each time point. (C and D) Inhibition of XLF expression accelerated cellular senescence. XLF-depleted WI38 cells were assayed for β-gal. Representative images of stained cells are shown in (C) and quantitative data in (D). PD, population doubling; XLF, XRCC4-like factor; β-gal, β-galactosidase; si-CTR, control siRNA.

Mentions: The prominent biological function of XLF is to repair DSBs by NHEJ, while the central effect of WRN deficiency is premature cellular senescence. It is well known that defects in NHEJ-mediated DSB repair contribute to cellular senescence. Therefore, we hypothesized that defects in XLF would lead to premature senescence. Indeed, inhibition of XLF expression by transfection with two independent siRNA oligos resulted in a decrease of cell growth in the normal human fibroblasts WI38 (Fig. 3A and B), while an increase in the percentage of β-gal-positive cells as compared with the mock transfectants (Fig. 3C). Therefore, it was concluded that XLF is critical in promoting cell proliferation and suppressing cellular senescence.


Werner syndrome protein positively regulates XRCC4-like factor transcription.

Liu D, Deng X, Yuan C, Chen L, Cong Y, Xu X - Mol Med Rep (2014)

XLF suppresses cellular senescence. (A) Inhibition of XLF expression in WI38 cells. WI38 cells at passage 39 PD were transfected with si-CTR or si-XLF. Total cell lysates were extracted 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (B) Depletion of XLF inhibited cell proliferation. XLF expression was inhibited by transfection with si-XLF as described in (A) and cell numbers were determined every 24 h. Six duplicate samples were analyzed at each time point. (C and D) Inhibition of XLF expression accelerated cellular senescence. XLF-depleted WI38 cells were assayed for β-gal. Representative images of stained cells are shown in (C) and quantitative data in (D). PD, population doubling; XLF, XRCC4-like factor; β-gal, β-galactosidase; si-CTR, control siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4020486&req=5

f3-mmr-09-05-1648: XLF suppresses cellular senescence. (A) Inhibition of XLF expression in WI38 cells. WI38 cells at passage 39 PD were transfected with si-CTR or si-XLF. Total cell lysates were extracted 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (B) Depletion of XLF inhibited cell proliferation. XLF expression was inhibited by transfection with si-XLF as described in (A) and cell numbers were determined every 24 h. Six duplicate samples were analyzed at each time point. (C and D) Inhibition of XLF expression accelerated cellular senescence. XLF-depleted WI38 cells were assayed for β-gal. Representative images of stained cells are shown in (C) and quantitative data in (D). PD, population doubling; XLF, XRCC4-like factor; β-gal, β-galactosidase; si-CTR, control siRNA.
Mentions: The prominent biological function of XLF is to repair DSBs by NHEJ, while the central effect of WRN deficiency is premature cellular senescence. It is well known that defects in NHEJ-mediated DSB repair contribute to cellular senescence. Therefore, we hypothesized that defects in XLF would lead to premature senescence. Indeed, inhibition of XLF expression by transfection with two independent siRNA oligos resulted in a decrease of cell growth in the normal human fibroblasts WI38 (Fig. 3A and B), while an increase in the percentage of β-gal-positive cells as compared with the mock transfectants (Fig. 3C). Therefore, it was concluded that XLF is critical in promoting cell proliferation and suppressing cellular senescence.

Bottom Line: Depletion of WRN in HeLa cells led to a decrease of XLF mRNA and its promoter activity.Chromatin immunoprecipitation assays demonstrated that WRN was associated with the XLF promoter.Taken together, the results suggest that XLF is a transcriptional target of WRN and may be involved in the regulation of cellular senescence.

View Article: PubMed Central - PubMed

Affiliation: Beijing Key Laboratory of DNA Damage Response, College of Life Science, Capital Normal University, Beijing 100048, P.R. China.

ABSTRACT
XRCC4-like factor (XLF) is involved in non-homologous end joining-mediated repair of DNA double-strand breaks (DSBs). Mutations in the WRN gene results in the development of Werner syndrome (WS), a rare autosomal recessive disorder characterized by premature ageing and genome instability. In the present study, it was identified that XLF protein levels were lower in WRN-deficient fibroblasts, compared with normal fibroblasts. Depletion of WRN in HeLa cells led to a decrease of XLF mRNA and its promoter activity. Chromatin immunoprecipitation assays demonstrated that WRN was associated with the XLF promoter. Depletion of XLF in normal human fibroblasts increased the percentage of β-galactosidase (β-gal) staining-positive cells, indicating acceleration in cellular senescence. Taken together, the results suggest that XLF is a transcriptional target of WRN and may be involved in the regulation of cellular senescence.

Show MeSH
Related in: MedlinePlus