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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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WRN regulates mRNA levels of XLF. (A) XLF protein levels decreased in WRN-deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A (WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by siRNA resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.
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f1-mmr-09-05-1648: WRN regulates mRNA levels of XLF. (A) XLF protein levels decreased in WRN-deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A (WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by siRNA resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.

Mentions: A candidate gene approach was employed to identify DDR factors in the human fibroblast cell line AG11395A, which was originally isolated from a WRN patient bearing a nonsense mutation in the WRN gene. It was identified that the endogenous XLF protein level was lower in AG11395A cell line than in the control fibroblast cell line GM00637G (Fig. 1A). In U2OS cells, inhibition of WRN expression by two independent siRNA oligos resulted in a decrease of XLF protein levels (Fig. 1B), which were not restored by treatment with the proteasome inhibitor MG132 (Fig. 1C). Real-time RT-PCR assays demonstrated that the mRNA levels of XLF were significantly decreased upon depletion of WRN by siRNA (Fig. 1D). Taken together, these data suggest that WRN is a positive regulator of XLF at the transcriptional level.


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)

WRN regulates mRNA levels of XLF. (A) XLF protein levels decreased in WRN-deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A (WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by siRNA resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-mmr-09-05-1648: WRN regulates mRNA levels of XLF. (A) XLF protein levels decreased in WRN-deficient fibroblasts. Whole cell lysates were prepared from human fibroblasts GM00637G (normal) and AG11395A (WRN deficient) and subjected to immunoblotting analysis for XLF and β-actin. (B) Inhibition of WRN expression by siRNA resulted in a decrease of XLF protein levels. U2OS cells were transfected with si-CTR, XLF siRNA (si1-XLF or si2-XLF) or WRN siRNA (si1-WRN or si2-WRN). Total cell lysates were harvested 48 h following transfection and subjected to immunoblotting analysis with the antibodies indicated. (C) MG132 treatment did not restore the decrease of XLF protein levels caused by the depletion of WRN. U2OS cells were treated as described in (B) except that MG132 was added to cells 4 h prior to harvest. (D) Inhibition of WRN expression led to a decrease of XLF mRNA. U2OS cells were treated as described in (B). Total RNA was extracted and subjected to real-time RT-PCR assays for XLF mRNA. XLF, XRCC4-like factor; si-CTR, control siRNA; RT-PCR, reverse transcription PCR.
Mentions: A candidate gene approach was employed to identify DDR factors in the human fibroblast cell line AG11395A, which was originally isolated from a WRN patient bearing a nonsense mutation in the WRN gene. It was identified that the endogenous XLF protein level was lower in AG11395A cell line than in the control fibroblast cell line GM00637G (Fig. 1A). In U2OS cells, inhibition of WRN expression by two independent siRNA oligos resulted in a decrease of XLF protein levels (Fig. 1B), which were not restored by treatment with the proteasome inhibitor MG132 (Fig. 1C). Real-time RT-PCR assays demonstrated that the mRNA levels of XLF were significantly decreased upon depletion of WRN by siRNA (Fig. 1D). Taken together, these data suggest that WRN is a positive regulator of XLF at the transcriptional level.

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