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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 positively regulates the XLF promoter activity in U2OS cells. (A) Inhibition of WRN expression decreased the XLF promoter activity. The putative XLF promoter containing a sequence from −596 to +169, relative to the transcription start site of XLF, was subcloned into the PGL3-basic luciferase vector, and the resulting reporter construct was transfected into WRN-depleted U2OS cells. Triplicate samples were analyzed. (B) WRN bound the XLF promoter region. ChIP assays were performed using two different anti-WRN antibodies. ChIP with α1-WRN antibody did not yield any PCR product, which also served as a negative control. The PCR product corresponds to the position from −360 to −90 of the promoter relative to the transcription start site. XLF, XRCC4-like factor; ChIP, chromatin immunoprecipitation; si-CTR, control siRNA.
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f2-mmr-09-05-1648: WRN positively regulates the XLF promoter activity in U2OS cells. (A) Inhibition of WRN expression decreased the XLF promoter activity. The putative XLF promoter containing a sequence from −596 to +169, relative to the transcription start site of XLF, was subcloned into the PGL3-basic luciferase vector, and the resulting reporter construct was transfected into WRN-depleted U2OS cells. Triplicate samples were analyzed. (B) WRN bound the XLF promoter region. ChIP assays were performed using two different anti-WRN antibodies. ChIP with α1-WRN antibody did not yield any PCR product, which also served as a negative control. The PCR product corresponds to the position from −360 to −90 of the promoter relative to the transcription start site. XLF, XRCC4-like factor; ChIP, chromatin immunoprecipitation; si-CTR, control siRNA.

Mentions: To determine if WRN regulates the promoter activity of XLF, the putative XLF promoter region from −596 to +169 (relative to the putative transcription start site described in the Ensembl protein-coding gene ENSG00000187736) was cloned into the pGL3 basic luciferase reporter vector. The resulting pGL3-XLFpr was identified to have pronounced luciferase reporter activity in U2OS cells (data not shown). It was identified that the XLF promoter activity was significantly downregulated when WRN expression was inhibited by siRNA in U2OS cells (Fig. 2A). The antibodies against WRN were used for ChIP on cross-linked chromatin fragments prepared from U2OS cells. The ChIP-enriched DNA was subjected to PCR analysis using three pairs of primers for amplification of three fragments within the XLF promoter region. The data revealed that the second fragment (from −360 to −90) of the XLF promoter region was detected in the anti-WRN immunoprecipitates (Fig. 2B). These results suggest that WRN resides on the XLF promoter region, positively regulating its activity.


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 positively regulates the XLF promoter activity in U2OS cells. (A) Inhibition of WRN expression decreased the XLF promoter activity. The putative XLF promoter containing a sequence from −596 to +169, relative to the transcription start site of XLF, was subcloned into the PGL3-basic luciferase vector, and the resulting reporter construct was transfected into WRN-depleted U2OS cells. Triplicate samples were analyzed. (B) WRN bound the XLF promoter region. ChIP assays were performed using two different anti-WRN antibodies. ChIP with α1-WRN antibody did not yield any PCR product, which also served as a negative control. The PCR product corresponds to the position from −360 to −90 of the promoter relative to the transcription start site. XLF, XRCC4-like factor; ChIP, chromatin immunoprecipitation; si-CTR, control siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-mmr-09-05-1648: WRN positively regulates the XLF promoter activity in U2OS cells. (A) Inhibition of WRN expression decreased the XLF promoter activity. The putative XLF promoter containing a sequence from −596 to +169, relative to the transcription start site of XLF, was subcloned into the PGL3-basic luciferase vector, and the resulting reporter construct was transfected into WRN-depleted U2OS cells. Triplicate samples were analyzed. (B) WRN bound the XLF promoter region. ChIP assays were performed using two different anti-WRN antibodies. ChIP with α1-WRN antibody did not yield any PCR product, which also served as a negative control. The PCR product corresponds to the position from −360 to −90 of the promoter relative to the transcription start site. XLF, XRCC4-like factor; ChIP, chromatin immunoprecipitation; si-CTR, control siRNA.
Mentions: To determine if WRN regulates the promoter activity of XLF, the putative XLF promoter region from −596 to +169 (relative to the putative transcription start site described in the Ensembl protein-coding gene ENSG00000187736) was cloned into the pGL3 basic luciferase reporter vector. The resulting pGL3-XLFpr was identified to have pronounced luciferase reporter activity in U2OS cells (data not shown). It was identified that the XLF promoter activity was significantly downregulated when WRN expression was inhibited by siRNA in U2OS cells (Fig. 2A). The antibodies against WRN were used for ChIP on cross-linked chromatin fragments prepared from U2OS cells. The ChIP-enriched DNA was subjected to PCR analysis using three pairs of primers for amplification of three fragments within the XLF promoter region. The data revealed that the second fragment (from −360 to −90) of the XLF promoter region was detected in the anti-WRN immunoprecipitates (Fig. 2B). These results suggest that WRN resides on the XLF promoter region, positively regulating its activity.

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