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A ChIP-chip approach reveals a novel role for transcription factor IRF1 in the DNA damage response.

Frontini M, Vijayakumar M, Garvin A, Clarke N - Nucleic Acids Res. (2009)

Bottom Line: Using this approach we identified 202 new IRF1-binding sites with high confidence.In particular, we demonstrate that the mRNA and protein levels of the DNA repair protein BRIP1 [Fanconi anemia gene J (FANC J)] are upregulated after IRF1 over-expression.We also demonstrate that knockdown of IRF1 by siRNA results in loss of BRIP1 expression, abrogation of BRIP1 foci after DNA interstrand crosslink (ICL) damage and hypersensitivity to the DNA crosslinking agent, melphalan; a characteristic phenotype of FANC J cells.

View Article: PubMed Central - PubMed

Affiliation: MRC Clinical Sciences Centre, Faculty of Medicine Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.

ABSTRACT
IRF1 is a transcription factor that regulates key processes in the immune system and in tumour suppression. To gain further insight into IRF1's role in these processes, we searched for new target genes by performing chromatin immunoprecipitation coupled to a CpG island microarray (ChIP-chip). Using this approach we identified 202 new IRF1-binding sites with high confidence. Functional categorization of the target genes revealed a surprising cadre of new roles that can be linked to IRF1. One of the major functional categories was the DNA damage response pathway. In order to further validate our findings, we show that IRF1 can regulate the mRNA expression of a number of the DNA damage response genes in our list. In particular, we demonstrate that the mRNA and protein levels of the DNA repair protein BRIP1 [Fanconi anemia gene J (FANC J)] are upregulated after IRF1 over-expression. We also demonstrate that knockdown of IRF1 by siRNA results in loss of BRIP1 expression, abrogation of BRIP1 foci after DNA interstrand crosslink (ICL) damage and hypersensitivity to the DNA crosslinking agent, melphalan; a characteristic phenotype of FANC J cells. Taken together, our data provides a more complete understanding of the regulatory networks controlled by IRF1 and reveals a novel role for IRF1 in regulating the ICL DNA damage response.

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IRF1 expression is required for BRIP1 expression and foci formation after ICL damage. MRC5 cells transfected with 50 nM of control (A, B) or IRF1 siRNA (C, D) were treated with 500 nM melphalan (B, D) for 16 hrs and stained with either IRF1 or BRIP1 antibody as indicated and with DAPI.
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Figure 5: IRF1 expression is required for BRIP1 expression and foci formation after ICL damage. MRC5 cells transfected with 50 nM of control (A, B) or IRF1 siRNA (C, D) were treated with 500 nM melphalan (B, D) for 16 hrs and stained with either IRF1 or BRIP1 antibody as indicated and with DAPI.

Mentions: The protein BRIP1 is a member of the FA family of proteins, known as FANC J (33). These proteins are involved in the repair of ICLs in DNA and BRIP1 is known to form foci at DNA damage sites in response to ICL reagents (34). We reasoned that if IRF1 were involved in regulating BRIP1 expression in cells, which depletion of IRF1 by small interfering RNA (siRNA) would result in deregulation of BRIP1 expression or foci formation. Therefore, we performed immunofluorescence experiments and confocal microscopy to look at individual cells and the response to melphalan after IRF1 knockdown. We determined the levels of BRIP1 and IRF1 at the single cell level, before and after ICL damage. In cells transfected with control siRNA, we observed a clear nuclear staining for IRF1 (Figure 5A and B), which is consistent with other studies showing IRF1 as a mainly nuclear protein (35). In control cells, BRIP1 also displayed nuclear staining (Figure 5A). We observed no co-localization of these two proteins (see merged, Figure 5). After control cells were treated with melphalan (Figure 5B), we observed a large increase in BRIP1 foci. In contrast, cells transfected with IRF1 siRNA, displayed very weak residual BRIP1 staining (Figure 5C) and no staining for IRF1. After melphalan treatment of IRF1 knockdown cells, we observed no DNA damage induced BRIP1 foci (compare BRIP1 in Figure 5D to B). These results demonstrate that IRF1 is involved in regulating the expression of the BRIP1 protein in undamaged cells and that BRIP1 foci formation after ICL (melphalan) damage is dependent on IRF1 expression. The fact that we observed a very low level residual BRIP1 staining in untreated cells (that did not change after melphalan treatment), argues that there are other factors that may also be responsible for regulating expression of BRIP1 in undamaged, cycling cells such as E2F1, which has recently been shown to regulate its expression (36).Figure 5.


A ChIP-chip approach reveals a novel role for transcription factor IRF1 in the DNA damage response.

Frontini M, Vijayakumar M, Garvin A, Clarke N - Nucleic Acids Res. (2009)

IRF1 expression is required for BRIP1 expression and foci formation after ICL damage. MRC5 cells transfected with 50 nM of control (A, B) or IRF1 siRNA (C, D) were treated with 500 nM melphalan (B, D) for 16 hrs and stained with either IRF1 or BRIP1 antibody as indicated and with DAPI.
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Figure 5: IRF1 expression is required for BRIP1 expression and foci formation after ICL damage. MRC5 cells transfected with 50 nM of control (A, B) or IRF1 siRNA (C, D) were treated with 500 nM melphalan (B, D) for 16 hrs and stained with either IRF1 or BRIP1 antibody as indicated and with DAPI.
Mentions: The protein BRIP1 is a member of the FA family of proteins, known as FANC J (33). These proteins are involved in the repair of ICLs in DNA and BRIP1 is known to form foci at DNA damage sites in response to ICL reagents (34). We reasoned that if IRF1 were involved in regulating BRIP1 expression in cells, which depletion of IRF1 by small interfering RNA (siRNA) would result in deregulation of BRIP1 expression or foci formation. Therefore, we performed immunofluorescence experiments and confocal microscopy to look at individual cells and the response to melphalan after IRF1 knockdown. We determined the levels of BRIP1 and IRF1 at the single cell level, before and after ICL damage. In cells transfected with control siRNA, we observed a clear nuclear staining for IRF1 (Figure 5A and B), which is consistent with other studies showing IRF1 as a mainly nuclear protein (35). In control cells, BRIP1 also displayed nuclear staining (Figure 5A). We observed no co-localization of these two proteins (see merged, Figure 5). After control cells were treated with melphalan (Figure 5B), we observed a large increase in BRIP1 foci. In contrast, cells transfected with IRF1 siRNA, displayed very weak residual BRIP1 staining (Figure 5C) and no staining for IRF1. After melphalan treatment of IRF1 knockdown cells, we observed no DNA damage induced BRIP1 foci (compare BRIP1 in Figure 5D to B). These results demonstrate that IRF1 is involved in regulating the expression of the BRIP1 protein in undamaged cells and that BRIP1 foci formation after ICL (melphalan) damage is dependent on IRF1 expression. The fact that we observed a very low level residual BRIP1 staining in untreated cells (that did not change after melphalan treatment), argues that there are other factors that may also be responsible for regulating expression of BRIP1 in undamaged, cycling cells such as E2F1, which has recently been shown to regulate its expression (36).Figure 5.

Bottom Line: Using this approach we identified 202 new IRF1-binding sites with high confidence.In particular, we demonstrate that the mRNA and protein levels of the DNA repair protein BRIP1 [Fanconi anemia gene J (FANC J)] are upregulated after IRF1 over-expression.We also demonstrate that knockdown of IRF1 by siRNA results in loss of BRIP1 expression, abrogation of BRIP1 foci after DNA interstrand crosslink (ICL) damage and hypersensitivity to the DNA crosslinking agent, melphalan; a characteristic phenotype of FANC J cells.

View Article: PubMed Central - PubMed

Affiliation: MRC Clinical Sciences Centre, Faculty of Medicine Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.

ABSTRACT
IRF1 is a transcription factor that regulates key processes in the immune system and in tumour suppression. To gain further insight into IRF1's role in these processes, we searched for new target genes by performing chromatin immunoprecipitation coupled to a CpG island microarray (ChIP-chip). Using this approach we identified 202 new IRF1-binding sites with high confidence. Functional categorization of the target genes revealed a surprising cadre of new roles that can be linked to IRF1. One of the major functional categories was the DNA damage response pathway. In order to further validate our findings, we show that IRF1 can regulate the mRNA expression of a number of the DNA damage response genes in our list. In particular, we demonstrate that the mRNA and protein levels of the DNA repair protein BRIP1 [Fanconi anemia gene J (FANC J)] are upregulated after IRF1 over-expression. We also demonstrate that knockdown of IRF1 by siRNA results in loss of BRIP1 expression, abrogation of BRIP1 foci after DNA interstrand crosslink (ICL) damage and hypersensitivity to the DNA crosslinking agent, melphalan; a characteristic phenotype of FANC J cells. Taken together, our data provides a more complete understanding of the regulatory networks controlled by IRF1 and reveals a novel role for IRF1 in regulating the ICL DNA damage response.

Show MeSH
Related in: MedlinePlus