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A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

Lee C, Hong S, Lee MH, Koo HS - PLoS ONE (2015)

Bottom Line: PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation.In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway.We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, Republic of Korea.

ABSTRACT
PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs), while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs). In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

No MeSH data available.


Related in: MedlinePlus

Slow dissipation of RPA-1 and RAD-51 foci induced by ICLs in mitotic germ cells of jmjd-1.1 mutant worms.Prolonged accumulations of (A) RPA-1 (C. elegans RPA70 homolog) and (B) RAD-51 (C. elegans RAD51 homolog) foci upon ICL formation in the nuclei of mitotic germ cells of jmjd-1.1 worms. L4 stage worms were exposed to trimethyl psoralen as in Fig 3, and gonads were immuno-stained with the indicated antibodies at 6, 12, 18 and 24 h post treatment (with an additional time point at 9 h in (B)). Scale bar, 10 μm.
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pone.0123865.g004: Slow dissipation of RPA-1 and RAD-51 foci induced by ICLs in mitotic germ cells of jmjd-1.1 mutant worms.Prolonged accumulations of (A) RPA-1 (C. elegans RPA70 homolog) and (B) RAD-51 (C. elegans RAD51 homolog) foci upon ICL formation in the nuclei of mitotic germ cells of jmjd-1.1 worms. L4 stage worms were exposed to trimethyl psoralen as in Fig 3, and gonads were immuno-stained with the indicated antibodies at 6, 12, 18 and 24 h post treatment (with an additional time point at 9 h in (B)). Scale bar, 10 μm.

Mentions: ICLs are converted to DSBs in the FA pathway as a result of DNA cleavages by endonucleases such as XPF, MUS81, SLX1, and FAN1 [17,18]. In order for DSBs to be repaired by HR, extensive end resection of DSBs needs to take place; then RPA binds to the exposed single-strand DNA, and RAD51 replaces RPA prior to DNA strand invasion [14]. Mammalian cells defective in HR exhibit prolonged accumulation (or slow dissociation) of RPA and RAD51 proteins at DNA lesions [19,20]. Therefore, we investigated the accumulation and dissociation kinetics of RPA-1 and RAD-51 (C. elegans RPA70 and RAD51 homologs, respectively) in mitotic germ cells of wild-type and jmjd-1.1(tm3980) worms. Although these proteins accumulated normally at 6 h after ICL treatment (also at 9 h for RAD-51), striking defects in HR were revealed by the subsequent sluggish dissociation of these proteins in the mutant worms (Fig 4): accumulation of RPA-1 and RAD-51 were still observed in the mutant worms at 18 h post treatment, but they disappeared almost completely in wild-type worms. Similar results were observed for the repair of DSBs induced by γ-rays (S3 Fig). The formation of RPA-1 and RAD-51 foci in mitotic germ cells was normal in the mutant, but their dissociation was slowed down (S3 Fig). In summary, JMJD-1.1 is needed for repair of both ICLs and DSBs via HR, specifically in the step following DSB end resection and subsequent RPA-1/RAD-51 loading.


A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

Lee C, Hong S, Lee MH, Koo HS - PLoS ONE (2015)

Slow dissipation of RPA-1 and RAD-51 foci induced by ICLs in mitotic germ cells of jmjd-1.1 mutant worms.Prolonged accumulations of (A) RPA-1 (C. elegans RPA70 homolog) and (B) RAD-51 (C. elegans RAD51 homolog) foci upon ICL formation in the nuclei of mitotic germ cells of jmjd-1.1 worms. L4 stage worms were exposed to trimethyl psoralen as in Fig 3, and gonads were immuno-stained with the indicated antibodies at 6, 12, 18 and 24 h post treatment (with an additional time point at 9 h in (B)). Scale bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390335&req=5

pone.0123865.g004: Slow dissipation of RPA-1 and RAD-51 foci induced by ICLs in mitotic germ cells of jmjd-1.1 mutant worms.Prolonged accumulations of (A) RPA-1 (C. elegans RPA70 homolog) and (B) RAD-51 (C. elegans RAD51 homolog) foci upon ICL formation in the nuclei of mitotic germ cells of jmjd-1.1 worms. L4 stage worms were exposed to trimethyl psoralen as in Fig 3, and gonads were immuno-stained with the indicated antibodies at 6, 12, 18 and 24 h post treatment (with an additional time point at 9 h in (B)). Scale bar, 10 μm.
Mentions: ICLs are converted to DSBs in the FA pathway as a result of DNA cleavages by endonucleases such as XPF, MUS81, SLX1, and FAN1 [17,18]. In order for DSBs to be repaired by HR, extensive end resection of DSBs needs to take place; then RPA binds to the exposed single-strand DNA, and RAD51 replaces RPA prior to DNA strand invasion [14]. Mammalian cells defective in HR exhibit prolonged accumulation (or slow dissociation) of RPA and RAD51 proteins at DNA lesions [19,20]. Therefore, we investigated the accumulation and dissociation kinetics of RPA-1 and RAD-51 (C. elegans RPA70 and RAD51 homologs, respectively) in mitotic germ cells of wild-type and jmjd-1.1(tm3980) worms. Although these proteins accumulated normally at 6 h after ICL treatment (also at 9 h for RAD-51), striking defects in HR were revealed by the subsequent sluggish dissociation of these proteins in the mutant worms (Fig 4): accumulation of RPA-1 and RAD-51 were still observed in the mutant worms at 18 h post treatment, but they disappeared almost completely in wild-type worms. Similar results were observed for the repair of DSBs induced by γ-rays (S3 Fig). The formation of RPA-1 and RAD-51 foci in mitotic germ cells was normal in the mutant, but their dissociation was slowed down (S3 Fig). In summary, JMJD-1.1 is needed for repair of both ICLs and DSBs via HR, specifically in the step following DSB end resection and subsequent RPA-1/RAD-51 loading.

Bottom Line: PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation.In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway.We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, Republic of Korea.

ABSTRACT
PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs), while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs). In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

No MeSH data available.


Related in: MedlinePlus