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UHRF1 is a sensor for DNA interstrand crosslinks and recruits FANCD2 to initiate the Fanconi anemia pathway.

Liang CC, Zhan B, Yoshikawa Y, Haas W, Gygi SP, Cohn MA - Cell Rep (2015)

Bottom Line: Knockdown cells display a drastic reduction in FANCD2 foci formation.Based on these results, we describe a mechanism of ICL sensing and propose that UHRF1 is a critical factor that binds to ICLs.In turn, this binding is necessary for the subsequent recruitment of FANCD2, which allows the DNA repair process to initiate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

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UHRF1 Is Rapidly Recruited to ICLs In Vivo and Precedes the Recruitment of FANCD2(A and B) HeLa cells expressing mCherry-tagged UHRF1 and EGFP-tagged FANCD2 were (A) pre-treated with TMP or (B) untreated, and microirradiated at the indicated areas (white arrows). Charts on the right show quantification of mCherry-UHRF1 and EGFP-FANCD2 at the ICL sites. UHRF1 and FANCD2 were recruited to TMP-induced ICLs sites (A), but not to irradiated sites in the absence of TMP (B). Scale bar, 10 μm.(C) HeLa cells expressing EGFP-tagged FANCD2 with or without UHRF1 knockdown were microirradiated at the indicated areas (white arrows). Depletion of UHRF1 abrogates the rapid accumulation of FANCD2 at the ICLs. Scale bar, 10 μm. Charts on the right show quantification of EGFP-FANCD2 at the ICL sites.(D) Model showing how UHRF1 is recruited to the ICL, facilitating the recruitment of FANCD2, which again precedes the recruitment of additional DNA repair factors.See also Figure S1.
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fig5: UHRF1 Is Rapidly Recruited to ICLs In Vivo and Precedes the Recruitment of FANCD2(A and B) HeLa cells expressing mCherry-tagged UHRF1 and EGFP-tagged FANCD2 were (A) pre-treated with TMP or (B) untreated, and microirradiated at the indicated areas (white arrows). Charts on the right show quantification of mCherry-UHRF1 and EGFP-FANCD2 at the ICL sites. UHRF1 and FANCD2 were recruited to TMP-induced ICLs sites (A), but not to irradiated sites in the absence of TMP (B). Scale bar, 10 μm.(C) HeLa cells expressing EGFP-tagged FANCD2 with or without UHRF1 knockdown were microirradiated at the indicated areas (white arrows). Depletion of UHRF1 abrogates the rapid accumulation of FANCD2 at the ICLs. Scale bar, 10 μm. Charts on the right show quantification of EGFP-FANCD2 at the ICL sites.(D) Model showing how UHRF1 is recruited to the ICL, facilitating the recruitment of FANCD2, which again precedes the recruitment of additional DNA repair factors.See also Figure S1.

Mentions: Given that UHRF1 interacts directly with ICLs in vitro and is required for proper foci formation of FANCD2 in vivo, we speculated that UHRF1 itself is recruited to crosslinked DNA in vivo, and that this triggers the chromatin recruitment of FANCD2. To test this hypothesis directly, we turned to live-cell imaging using fluorophore-tagged proteins. UHRF1 and FANCD2 were stably expressed in HeLa cells as fusion proteins with mCherry and EGFP, respectively. We introduced ICLs with a localized laser stripe after incubating the cells with TMP (Thazhathveetil et al., 2007). We observed that UHRF1 was recruited to ICLs very quickly and formed a clear stripe within 30 s. FANCD2 was also recruited, albeit slightly more slowly than UHRF1, and formed a visible stripe within 5 min (Figure 5A). Importantly, there was no recruitment of either one of the proteins in the absence of TMP (Figure 5B). These data encouraged us to directly test, in live cells, whether UHRF1 mediates the recruitment of FANCD2 to ICLs. Using control and UHRF1 knockdown cells, we assessed the recruitment of FANCD2 to ICLs in the presence and absence of UHRF1. Strikingly, we found that knockdown of UHRF1 completely abolished FANCD2 recruitment (Figure 5C).


UHRF1 is a sensor for DNA interstrand crosslinks and recruits FANCD2 to initiate the Fanconi anemia pathway.

Liang CC, Zhan B, Yoshikawa Y, Haas W, Gygi SP, Cohn MA - Cell Rep (2015)

UHRF1 Is Rapidly Recruited to ICLs In Vivo and Precedes the Recruitment of FANCD2(A and B) HeLa cells expressing mCherry-tagged UHRF1 and EGFP-tagged FANCD2 were (A) pre-treated with TMP or (B) untreated, and microirradiated at the indicated areas (white arrows). Charts on the right show quantification of mCherry-UHRF1 and EGFP-FANCD2 at the ICL sites. UHRF1 and FANCD2 were recruited to TMP-induced ICLs sites (A), but not to irradiated sites in the absence of TMP (B). Scale bar, 10 μm.(C) HeLa cells expressing EGFP-tagged FANCD2 with or without UHRF1 knockdown were microirradiated at the indicated areas (white arrows). Depletion of UHRF1 abrogates the rapid accumulation of FANCD2 at the ICLs. Scale bar, 10 μm. Charts on the right show quantification of EGFP-FANCD2 at the ICL sites.(D) Model showing how UHRF1 is recruited to the ICL, facilitating the recruitment of FANCD2, which again precedes the recruitment of additional DNA repair factors.See also Figure S1.
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fig5: UHRF1 Is Rapidly Recruited to ICLs In Vivo and Precedes the Recruitment of FANCD2(A and B) HeLa cells expressing mCherry-tagged UHRF1 and EGFP-tagged FANCD2 were (A) pre-treated with TMP or (B) untreated, and microirradiated at the indicated areas (white arrows). Charts on the right show quantification of mCherry-UHRF1 and EGFP-FANCD2 at the ICL sites. UHRF1 and FANCD2 were recruited to TMP-induced ICLs sites (A), but not to irradiated sites in the absence of TMP (B). Scale bar, 10 μm.(C) HeLa cells expressing EGFP-tagged FANCD2 with or without UHRF1 knockdown were microirradiated at the indicated areas (white arrows). Depletion of UHRF1 abrogates the rapid accumulation of FANCD2 at the ICLs. Scale bar, 10 μm. Charts on the right show quantification of EGFP-FANCD2 at the ICL sites.(D) Model showing how UHRF1 is recruited to the ICL, facilitating the recruitment of FANCD2, which again precedes the recruitment of additional DNA repair factors.See also Figure S1.
Mentions: Given that UHRF1 interacts directly with ICLs in vitro and is required for proper foci formation of FANCD2 in vivo, we speculated that UHRF1 itself is recruited to crosslinked DNA in vivo, and that this triggers the chromatin recruitment of FANCD2. To test this hypothesis directly, we turned to live-cell imaging using fluorophore-tagged proteins. UHRF1 and FANCD2 were stably expressed in HeLa cells as fusion proteins with mCherry and EGFP, respectively. We introduced ICLs with a localized laser stripe after incubating the cells with TMP (Thazhathveetil et al., 2007). We observed that UHRF1 was recruited to ICLs very quickly and formed a clear stripe within 30 s. FANCD2 was also recruited, albeit slightly more slowly than UHRF1, and formed a visible stripe within 5 min (Figure 5A). Importantly, there was no recruitment of either one of the proteins in the absence of TMP (Figure 5B). These data encouraged us to directly test, in live cells, whether UHRF1 mediates the recruitment of FANCD2 to ICLs. Using control and UHRF1 knockdown cells, we assessed the recruitment of FANCD2 to ICLs in the presence and absence of UHRF1. Strikingly, we found that knockdown of UHRF1 completely abolished FANCD2 recruitment (Figure 5C).

Bottom Line: Knockdown cells display a drastic reduction in FANCD2 foci formation.Based on these results, we describe a mechanism of ICL sensing and propose that UHRF1 is a critical factor that binds to ICLs.In turn, this binding is necessary for the subsequent recruitment of FANCD2, which allows the DNA repair process to initiate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

Show MeSH
Related in: MedlinePlus