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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

Schematic representation of human PHF8 together with its C. elegans homologs, and a simplified gene structure of C. elegans jmjd-1.1.(A) Comparison of the amino acid sequences of human histone demethylase PHF8 and its C. elegans homologs. The alignment of conserved domains and similarity scores were obtained using NCBI Protein Blast. (B) The structure of C. elegans jmjd-1.1 (F43G6.6), from exon 1 to 7 (black boxes), and the deletion site in the allele tm3980.
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pone.0123865.g001: Schematic representation of human PHF8 together with its C. elegans homologs, and a simplified gene structure of C. elegans jmjd-1.1.(A) Comparison of the amino acid sequences of human histone demethylase PHF8 and its C. elegans homologs. The alignment of conserved domains and similarity scores were obtained using NCBI Protein Blast. (B) The structure of C. elegans jmjd-1.1 (F43G6.6), from exon 1 to 7 (black boxes), and the deletion site in the allele tm3980.

Mentions: JMJD-1.1 and JMJD-1.2 proteins are very similar in amino acid sequences to human PHF8, which is a histone demethylase associated with X-linked mental retardation. There is more than 35% identity between PHF8 and C. elegans homologs over a 380 amino acid stretch and 55% in the JmjC domain (Fig 1). JMJD-1.1, in particular, has 60% similarity to JMJD-1.2 over the entire length of the polypeptide, suggesting that the two C. elegans proteins are probably paralogs. We obtained jmjd-1.1(tm3980) and jmjd-1.2(tm3713) deletion mutant worms generated by the National Bioresource Project (Japan) and outcrossed them several times with wild-type N2 strain to remove any background mutations. The two mutants have deletions of more than 500 nucleotides containing exons, and are very likely to be mutants. In the case of jmjd-1.1(tm3980), two successive exons are deleted, and the most likely transcript is the second exon joined to the fifth exon. Actually, the transcript was found to encode a prematurely terminated polypeptide of 135 amino acids due to a frameshift, as verified by reverse transcription followed by a gene-specific polymerase chain reaction (S1 Fig). In addition, the level of the transcript in mutant worms amounted to only 20% of that in wild-type N2, as measured by quantitative polymerase chain reaction after reverse transcription (S1 Fig). Therefore, the greatly reduced mRNA level and the premature termination eliminating the JmjC domain suggest that the jmjd-1.1(tm3980) allele is completely .


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

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

Schematic representation of human PHF8 together with its C. elegans homologs, and a simplified gene structure of C. elegans jmjd-1.1.(A) Comparison of the amino acid sequences of human histone demethylase PHF8 and its C. elegans homologs. The alignment of conserved domains and similarity scores were obtained using NCBI Protein Blast. (B) The structure of C. elegans jmjd-1.1 (F43G6.6), from exon 1 to 7 (black boxes), and the deletion site in the allele tm3980.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123865.g001: Schematic representation of human PHF8 together with its C. elegans homologs, and a simplified gene structure of C. elegans jmjd-1.1.(A) Comparison of the amino acid sequences of human histone demethylase PHF8 and its C. elegans homologs. The alignment of conserved domains and similarity scores were obtained using NCBI Protein Blast. (B) The structure of C. elegans jmjd-1.1 (F43G6.6), from exon 1 to 7 (black boxes), and the deletion site in the allele tm3980.
Mentions: JMJD-1.1 and JMJD-1.2 proteins are very similar in amino acid sequences to human PHF8, which is a histone demethylase associated with X-linked mental retardation. There is more than 35% identity between PHF8 and C. elegans homologs over a 380 amino acid stretch and 55% in the JmjC domain (Fig 1). JMJD-1.1, in particular, has 60% similarity to JMJD-1.2 over the entire length of the polypeptide, suggesting that the two C. elegans proteins are probably paralogs. We obtained jmjd-1.1(tm3980) and jmjd-1.2(tm3713) deletion mutant worms generated by the National Bioresource Project (Japan) and outcrossed them several times with wild-type N2 strain to remove any background mutations. The two mutants have deletions of more than 500 nucleotides containing exons, and are very likely to be mutants. In the case of jmjd-1.1(tm3980), two successive exons are deleted, and the most likely transcript is the second exon joined to the fifth exon. Actually, the transcript was found to encode a prematurely terminated polypeptide of 135 amino acids due to a frameshift, as verified by reverse transcription followed by a gene-specific polymerase chain reaction (S1 Fig). In addition, the level of the transcript in mutant worms amounted to only 20% of that in wild-type N2, as measured by quantitative polymerase chain reaction after reverse transcription (S1 Fig). Therefore, the greatly reduced mRNA level and the premature termination eliminating the JmjC domain suggest that the jmjd-1.1(tm3980) allele is completely .

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