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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.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.

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

jmjd-1.1 mutant worms are hypersensitive to ICLs and DSBs.(A) L4 stage worms were collected and incubated with trimethyl psoralen (TMP, 100 μM) for 40 min and exposed to UVA light (150 J/m2). (B) L4 stage worms were exposed to γ-rays at 60 Gy. In every experiment, eggs were collected between 24 and 40 h post treatment, and their survival was scored 24 h later. Mutations for fcd-2 and brc-1 (C. elegans FANCD2 and BRCA1 homologs, respectively) were used as positive controls for the corresponding type of DNA damage. Error bars indicate SEM. p values were obtained by calculating the difference (Δ) in embryonic survival between 0 and 100 TMP for each strain, and comparing the Δ values in the strains by two-way ANOVA.
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pone.0123865.g002: jmjd-1.1 mutant worms are hypersensitive to ICLs and DSBs.(A) L4 stage worms were collected and incubated with trimethyl psoralen (TMP, 100 μM) for 40 min and exposed to UVA light (150 J/m2). (B) L4 stage worms were exposed to γ-rays at 60 Gy. In every experiment, eggs were collected between 24 and 40 h post treatment, and their survival was scored 24 h later. Mutations for fcd-2 and brc-1 (C. elegans FANCD2 and BRCA1 homologs, respectively) were used as positive controls for the corresponding type of DNA damage. Error bars indicate SEM. p values were obtained by calculating the difference (Δ) in embryonic survival between 0 and 100 TMP for each strain, and comparing the Δ values in the strains by two-way ANOVA.

Mentions: In order to probe the roles of the histone demethylases in DNA damage responses, the jmjd-1.1(tm3980) and jmjd-1.2(tm3713) mutants were subjected to various types of DNA damage at the L4 larval stage (Fig 2). In C. elegans hermaphrodite gonads, proliferating germ cells undergo promeiotic development to produce oocytes and sperms, and after self-fertilization, early-embryos are laid by the worms. Embryos that developed from treated germ cells in the proliferating region of the gonad were scored for hatching to L1 larvae. Besides the two jmjd-1 mutants, two other mutants were also used as positive controls for different types of DNA damage, along with wild-type N2 as a negative control. FCD-2 (a C. elegans FANCD2 homolog) is a key component of the Fanconi anemia (FA) pathway, which participates in the repair of interstrand crosslinks (ICLs) [11]. Therefore, fcd-2(tm1298) mutant worms were used as a positive control for hypersensitivity to ICLs. BRC-1 (a C. elegans BRCA1 homolog) has an indispensable role in promoting end-resection in the initial stage of homologous recombination (HR) [12], and the mutant brc-1(tm1145) was used as a positive control for hypersensitivity to DSBs.


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

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

jmjd-1.1 mutant worms are hypersensitive to ICLs and DSBs.(A) L4 stage worms were collected and incubated with trimethyl psoralen (TMP, 100 μM) for 40 min and exposed to UVA light (150 J/m2). (B) L4 stage worms were exposed to γ-rays at 60 Gy. In every experiment, eggs were collected between 24 and 40 h post treatment, and their survival was scored 24 h later. Mutations for fcd-2 and brc-1 (C. elegans FANCD2 and BRCA1 homologs, respectively) were used as positive controls for the corresponding type of DNA damage. Error bars indicate SEM. p values were obtained by calculating the difference (Δ) in embryonic survival between 0 and 100 TMP for each strain, and comparing the Δ values in the strains by two-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123865.g002: jmjd-1.1 mutant worms are hypersensitive to ICLs and DSBs.(A) L4 stage worms were collected and incubated with trimethyl psoralen (TMP, 100 μM) for 40 min and exposed to UVA light (150 J/m2). (B) L4 stage worms were exposed to γ-rays at 60 Gy. In every experiment, eggs were collected between 24 and 40 h post treatment, and their survival was scored 24 h later. Mutations for fcd-2 and brc-1 (C. elegans FANCD2 and BRCA1 homologs, respectively) were used as positive controls for the corresponding type of DNA damage. Error bars indicate SEM. p values were obtained by calculating the difference (Δ) in embryonic survival between 0 and 100 TMP for each strain, and comparing the Δ values in the strains by two-way ANOVA.
Mentions: In order to probe the roles of the histone demethylases in DNA damage responses, the jmjd-1.1(tm3980) and jmjd-1.2(tm3713) mutants were subjected to various types of DNA damage at the L4 larval stage (Fig 2). In C. elegans hermaphrodite gonads, proliferating germ cells undergo promeiotic development to produce oocytes and sperms, and after self-fertilization, early-embryos are laid by the worms. Embryos that developed from treated germ cells in the proliferating region of the gonad were scored for hatching to L1 larvae. Besides the two jmjd-1 mutants, two other mutants were also used as positive controls for different types of DNA damage, along with wild-type N2 as a negative control. FCD-2 (a C. elegans FANCD2 homolog) is a key component of the Fanconi anemia (FA) pathway, which participates in the repair of interstrand crosslinks (ICLs) [11]. Therefore, fcd-2(tm1298) mutant worms were used as a positive control for hypersensitivity to ICLs. BRC-1 (a C. elegans BRCA1 homolog) has an indispensable role in promoting end-resection in the initial stage of homologous recombination (HR) [12], and the mutant brc-1(tm1145) was used as a positive control for hypersensitivity to DSBs.

Bottom Line: PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation.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.

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