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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

Retarded relaxation of heterochromatin structure upon ICL formation in mitotic germ cells of jmjd-1.1 mutant worms.L4 stage worms were collected and treated with trimethyl psoralen as in Fig 3. (A) The gonads were immuno-stained with antibody against histone H3K9me3 as an indicator of heterochromatin at 3, 6, 9, 12 and 24 h post treatment. Scale bar, 10 μm. (B) After DNA damage induction, worms were grown for a further 6 h, and extracts were prepared. Proteins were separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After probing the membrane for histone H3K9me3 and α-tubulin, band intensities were measured and plotted in the bar graph. Each bar represents an average of 9 measurements, obtained in 3 independent experiments each with 3 technical repetitions of the gel electrophoresis. p values were obtained by Student’s t-test.
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pone.0123865.g005: Retarded relaxation of heterochromatin structure upon ICL formation in mitotic germ cells of jmjd-1.1 mutant worms.L4 stage worms were collected and treated with trimethyl psoralen as in Fig 3. (A) The gonads were immuno-stained with antibody against histone H3K9me3 as an indicator of heterochromatin at 3, 6, 9, 12 and 24 h post treatment. Scale bar, 10 μm. (B) After DNA damage induction, worms were grown for a further 6 h, and extracts were prepared. Proteins were separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After probing the membrane for histone H3K9me3 and α-tubulin, band intensities were measured and plotted in the bar graph. Each bar represents an average of 9 measurements, obtained in 3 independent experiments each with 3 technical repetitions of the gel electrophoresis. p values were obtained by Student’s t-test.

Mentions: Spatiotemporal regulation of chromatin structure is critical for the DNA repair machinery to perform its work efficiently [21,22]. We therefore examined the level of histone H3 tri-methyl Lys9 (H3K9me3), a well-established heterochromatin marker, in mitotic germ cells after ICL or DSB formation. The immuno-signal for H3K9me3 became weaker after DNA damage induction, but it recovered gradually with the progression of DNA repair in both wild-type and mutant cells (Fig 5A and S4 Fig). However, the amount of heterochromatin in the mitotic germ cells of mutant worms was higher than in wild-type worms both before and after the induction of ICLs and DSBs. The differences in H3K9me3 level were also evident in a western analysis of worm extracts (Fig 5B), where the signal for H3K9me3 in the jmjd-1.1 mutant was about 1.5 fold that in the wild type both before and after ICL treatment. The higher level of heterochromatin in the mutant is consistent with the kinetics of RPA-1 and RAD-51 dissociation from DNA lesions in Fig 4 and S3 Fig.


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

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

Retarded relaxation of heterochromatin structure upon ICL formation in mitotic germ cells of jmjd-1.1 mutant worms.L4 stage worms were collected and treated with trimethyl psoralen as in Fig 3. (A) The gonads were immuno-stained with antibody against histone H3K9me3 as an indicator of heterochromatin at 3, 6, 9, 12 and 24 h post treatment. Scale bar, 10 μm. (B) After DNA damage induction, worms were grown for a further 6 h, and extracts were prepared. Proteins were separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After probing the membrane for histone H3K9me3 and α-tubulin, band intensities were measured and plotted in the bar graph. Each bar represents an average of 9 measurements, obtained in 3 independent experiments each with 3 technical repetitions of the gel electrophoresis. p values were obtained by Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123865.g005: Retarded relaxation of heterochromatin structure upon ICL formation in mitotic germ cells of jmjd-1.1 mutant worms.L4 stage worms were collected and treated with trimethyl psoralen as in Fig 3. (A) The gonads were immuno-stained with antibody against histone H3K9me3 as an indicator of heterochromatin at 3, 6, 9, 12 and 24 h post treatment. Scale bar, 10 μm. (B) After DNA damage induction, worms were grown for a further 6 h, and extracts were prepared. Proteins were separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After probing the membrane for histone H3K9me3 and α-tubulin, band intensities were measured and plotted in the bar graph. Each bar represents an average of 9 measurements, obtained in 3 independent experiments each with 3 technical repetitions of the gel electrophoresis. p values were obtained by Student’s t-test.
Mentions: Spatiotemporal regulation of chromatin structure is critical for the DNA repair machinery to perform its work efficiently [21,22]. We therefore examined the level of histone H3 tri-methyl Lys9 (H3K9me3), a well-established heterochromatin marker, in mitotic germ cells after ICL or DSB formation. The immuno-signal for H3K9me3 became weaker after DNA damage induction, but it recovered gradually with the progression of DNA repair in both wild-type and mutant cells (Fig 5A and S4 Fig). However, the amount of heterochromatin in the mitotic germ cells of mutant worms was higher than in wild-type worms both before and after the induction of ICLs and DSBs. The differences in H3K9me3 level were also evident in a western analysis of worm extracts (Fig 5B), where the signal for H3K9me3 in the jmjd-1.1 mutant was about 1.5 fold that in the wild type both before and after ICL treatment. The higher level of heterochromatin in the mutant is consistent with the kinetics of RPA-1 and RAD-51 dissociation from DNA lesions in Fig 4 and S3 Fig.

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