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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

FCD-2 focus formation is unaffected upon ICL induction in mitotic germ cells of jmjd-1.1 worms.L4 stage worms of wild type, jmjd-1.1(tm3980), and fcd-2(tm1298) were exposed to photoactivatable trimethyl psoralen (TMP, 200 μM) for 40 min and then to UVA light (150 J/m2). (A) Gonads were dissected, fixed, and immuno-stained with antibody against FCD-2 (C. elegans FANCD2 homolog) at 6, 12, 18 and 24 h post treatment. FCD-2 foci were observed in germ cells in the mitotically proliferating region of the gonad, and were not present in the negative control strain, fcd-2(tm1298). Scale bar, 10 μm. (B) The focal plane with the maximum number of FCD-2 foci was chosen for each nucleus of the germ cells, and the numbers of FCD-2 foci per focal plane were compared in wild-type and jmjd-1.1 worms at various time points (n = 100 for each bar). p values were obtained by Student’s t-test.
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pone.0123865.g003: FCD-2 focus formation is unaffected upon ICL induction in mitotic germ cells of jmjd-1.1 worms.L4 stage worms of wild type, jmjd-1.1(tm3980), and fcd-2(tm1298) were exposed to photoactivatable trimethyl psoralen (TMP, 200 μM) for 40 min and then to UVA light (150 J/m2). (A) Gonads were dissected, fixed, and immuno-stained with antibody against FCD-2 (C. elegans FANCD2 homolog) at 6, 12, 18 and 24 h post treatment. FCD-2 foci were observed in germ cells in the mitotically proliferating region of the gonad, and were not present in the negative control strain, fcd-2(tm1298). Scale bar, 10 μm. (B) The focal plane with the maximum number of FCD-2 foci was chosen for each nucleus of the germ cells, and the numbers of FCD-2 foci per focal plane were compared in wild-type and jmjd-1.1 worms at various time points (n = 100 for each bar). p values were obtained by Student’s t-test.

Mentions: In mammalian cells, the FA pathway participates in the initial stage of ICL repair, which involves the recognition of ICLs and their conversion to DSBs. FANCD2 is a key player in the FA pathway, shunting the DSB intermediate to the HR pathway [13–15]. The accumulation of nuclear foci of FCD-2, a FANCD2 homolog in C. elegans, at ICL sites is a good indicator of successful activation of the FA pathway [16]. We found that the extent of FCD-2 focus formation after ICL treatment in the proliferating mitotic germ cells of jmjd-1.1(tm3980) and wild-type worms was not significantly different (Fig 3, p>0.09 at all the time points, Student’s t-test). In addition, the immuno-signal declined gradually to similar extents in the two strains until 24 h. These results indicate that JMJD-1.1 does not affect FCD-2 activation and has a role downstream of the FA pathway in dealing with ICLs.


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

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

FCD-2 focus formation is unaffected upon ICL induction in mitotic germ cells of jmjd-1.1 worms.L4 stage worms of wild type, jmjd-1.1(tm3980), and fcd-2(tm1298) were exposed to photoactivatable trimethyl psoralen (TMP, 200 μM) for 40 min and then to UVA light (150 J/m2). (A) Gonads were dissected, fixed, and immuno-stained with antibody against FCD-2 (C. elegans FANCD2 homolog) at 6, 12, 18 and 24 h post treatment. FCD-2 foci were observed in germ cells in the mitotically proliferating region of the gonad, and were not present in the negative control strain, fcd-2(tm1298). Scale bar, 10 μm. (B) The focal plane with the maximum number of FCD-2 foci was chosen for each nucleus of the germ cells, and the numbers of FCD-2 foci per focal plane were compared in wild-type and jmjd-1.1 worms at various time points (n = 100 for each bar). p values were obtained by Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123865.g003: FCD-2 focus formation is unaffected upon ICL induction in mitotic germ cells of jmjd-1.1 worms.L4 stage worms of wild type, jmjd-1.1(tm3980), and fcd-2(tm1298) were exposed to photoactivatable trimethyl psoralen (TMP, 200 μM) for 40 min and then to UVA light (150 J/m2). (A) Gonads were dissected, fixed, and immuno-stained with antibody against FCD-2 (C. elegans FANCD2 homolog) at 6, 12, 18 and 24 h post treatment. FCD-2 foci were observed in germ cells in the mitotically proliferating region of the gonad, and were not present in the negative control strain, fcd-2(tm1298). Scale bar, 10 μm. (B) The focal plane with the maximum number of FCD-2 foci was chosen for each nucleus of the germ cells, and the numbers of FCD-2 foci per focal plane were compared in wild-type and jmjd-1.1 worms at various time points (n = 100 for each bar). p values were obtained by Student’s t-test.
Mentions: In mammalian cells, the FA pathway participates in the initial stage of ICL repair, which involves the recognition of ICLs and their conversion to DSBs. FANCD2 is a key player in the FA pathway, shunting the DSB intermediate to the HR pathway [13–15]. The accumulation of nuclear foci of FCD-2, a FANCD2 homolog in C. elegans, at ICL sites is a good indicator of successful activation of the FA pathway [16]. We found that the extent of FCD-2 focus formation after ICL treatment in the proliferating mitotic germ cells of jmjd-1.1(tm3980) and wild-type worms was not significantly different (Fig 3, p>0.09 at all the time points, Student’s t-test). In addition, the immuno-signal declined gradually to similar extents in the two strains until 24 h. These results indicate that JMJD-1.1 does not affect FCD-2 activation and has a role downstream of the FA pathway in dealing with ICLs.

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