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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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GO functional enrichment. The table shows the statistical enrichment of GO categories assigned to the target gene list with P-value <0.10. Enrichment was calculated using the non-redundant list of genes on the CGI array as the background set (from the UHN CGI Microarray database) and the IRF1 target gene list (see Supplementary Figure 3). Statistical analysis was performed using the DAVID software. From right to left; column 1—GO term name from DAVID; column 2—number of genes on the CGI array mapping to the GO term; column 3—number of IRF1 bound genes mapping to the GO term; column 4—percentage of GO-mapped genes bound by IRF1 (column 3/column 2); column 5—statistical significance (P-value).
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Figure 3: GO functional enrichment. The table shows the statistical enrichment of GO categories assigned to the target gene list with P-value <0.10. Enrichment was calculated using the non-redundant list of genes on the CGI array as the background set (from the UHN CGI Microarray database) and the IRF1 target gene list (see Supplementary Figure 3). Statistical analysis was performed using the DAVID software. From right to left; column 1—GO term name from DAVID; column 2—number of genes on the CGI array mapping to the GO term; column 3—number of IRF1 bound genes mapping to the GO term; column 4—percentage of GO-mapped genes bound by IRF1 (column 3/column 2); column 5—statistical significance (P-value).

Mentions: An important aspect of this high-throughput approach is that we can assign new role(s) for IRF1 in different biological processes by looking for statistically significant functional categories in our list of genes. In order to do this, we functionally classified the ChIP–chip enriched genes into Gene Ontology (GO) categories. We performed this analysis using the DAVID functional annotation tool. Functional categorization revealed a diverse set of GO biological processes that were statistically significant in our list of genes compared to the background list, P-value < 0.10. Enriched categories included DNA metabolism, antigen processing, protein transport, response to DNA damage and transcription. Strikingly, we found that 8 out of the 92 genes (∼9%) on the CGI array mapping to the GO DNA damage category were bound by IRF1 in our study (Figure 3 and Supplementary Figure 5). It is known that IRF1 is involved in the response of cells to DNA damage such as γ-irradiation (25,26) and may play a role in DNA repair processes (27). However, the full nature of its involvement and/or which IRF1 target genes might be involved in this process was not known. Moreover, it is known that IRF1 expression is stabilized in cells treated with DNA damaging agents (28) resulting in elevated levels of IRF1 similar to induction by IFN. Taken together the data support the idea that IRF1 plays a significant role in DNA damage/repair pathways via direct transcriptional regulation of genes involved in these processes.Figure 3.


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)

GO functional enrichment. The table shows the statistical enrichment of GO categories assigned to the target gene list with P-value <0.10. Enrichment was calculated using the non-redundant list of genes on the CGI array as the background set (from the UHN CGI Microarray database) and the IRF1 target gene list (see Supplementary Figure 3). Statistical analysis was performed using the DAVID software. From right to left; column 1—GO term name from DAVID; column 2—number of genes on the CGI array mapping to the GO term; column 3—number of IRF1 bound genes mapping to the GO term; column 4—percentage of GO-mapped genes bound by IRF1 (column 3/column 2); column 5—statistical significance (P-value).
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Related In: Results  -  Collection

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Figure 3: GO functional enrichment. The table shows the statistical enrichment of GO categories assigned to the target gene list with P-value <0.10. Enrichment was calculated using the non-redundant list of genes on the CGI array as the background set (from the UHN CGI Microarray database) and the IRF1 target gene list (see Supplementary Figure 3). Statistical analysis was performed using the DAVID software. From right to left; column 1—GO term name from DAVID; column 2—number of genes on the CGI array mapping to the GO term; column 3—number of IRF1 bound genes mapping to the GO term; column 4—percentage of GO-mapped genes bound by IRF1 (column 3/column 2); column 5—statistical significance (P-value).
Mentions: An important aspect of this high-throughput approach is that we can assign new role(s) for IRF1 in different biological processes by looking for statistically significant functional categories in our list of genes. In order to do this, we functionally classified the ChIP–chip enriched genes into Gene Ontology (GO) categories. We performed this analysis using the DAVID functional annotation tool. Functional categorization revealed a diverse set of GO biological processes that were statistically significant in our list of genes compared to the background list, P-value < 0.10. Enriched categories included DNA metabolism, antigen processing, protein transport, response to DNA damage and transcription. Strikingly, we found that 8 out of the 92 genes (∼9%) on the CGI array mapping to the GO DNA damage category were bound by IRF1 in our study (Figure 3 and Supplementary Figure 5). It is known that IRF1 is involved in the response of cells to DNA damage such as γ-irradiation (25,26) and may play a role in DNA repair processes (27). However, the full nature of its involvement and/or which IRF1 target genes might be involved in this process was not known. Moreover, it is known that IRF1 expression is stabilized in cells treated with DNA damaging agents (28) resulting in elevated levels of IRF1 similar to induction by IFN. Taken together the data support the idea that IRF1 plays a significant role in DNA damage/repair pathways via direct transcriptional regulation of genes involved in these processes.Figure 3.

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