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Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination.

Yin B, Savic V, Juntilla MM, Bredemeyer AL, Yang-Iott KS, Helmink BA, Koretzky GA, Sleckman BP, Bassing CH - J. Exp. Med. (2009)

Bottom Line: Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation.Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation.We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre-B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-alpha/delta locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.

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No accumulation of Jκ coding ends in the absence of H2AX phosphorylation along RAG-cleaved Igκ DNA strands. (a) Shown are schematic diagrams of the Jκ cluster of the Igκ locus in the uncleaved (GL) and cleaved but not repaired (CE) configurations. Open boxes represent the Jκ segments and triangles their recombination signal sequences. Indicated are the relative positions of the EcoRI and SacI sites and 3′Jκ probe used for Southern blot analysis. (b) Southern blot analysis of recombination products generated in H2ax−/− and Artemis−/− abl pre–B cell lines, either untreated or treated with STI571 for 72 h. EcoRI–SacI-digested genomic DNA was hybridized with the 3′Jκ probe. The bands corresponding to Jκ loci of the GL and CE configurations are indicated. The STI571-treated H2ax−/− cells harbor a band that likely represents a predominant VκJκ rearrangement. Blots were stripped and then probed with a TCR-β probe as a control for DNA content. These data are representative of experiments performed >10 independent times. (c) Schematic diagram of the mouse Igκ locus and graphical representation of γ-H2AX densities as determined by ChIP at locations along DNA strands within and adjacent to Igκ in Artemis−/− abl pre–B cells treated with STI571 for 96 h. The 0-kb value of the x-axis corresponds to the 3′ end of the Jκ5 coding segment. The negative and positive values represent the distances centromeric and telomeric, respectively, from the 3′ end of Jκ5. Red and green bars indicate the approximate genomic locations to which the 5′ Vκ (RP24-243E11) and 3′ Cκ (RP23-341D5) BACs hybridize. The lengths of these bars are not drawn to scale. These data are representative of experiments performed >20 independent times. Error bars indicate standard deviation of three independent experiments.
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fig2: No accumulation of Jκ coding ends in the absence of H2AX phosphorylation along RAG-cleaved Igκ DNA strands. (a) Shown are schematic diagrams of the Jκ cluster of the Igκ locus in the uncleaved (GL) and cleaved but not repaired (CE) configurations. Open boxes represent the Jκ segments and triangles their recombination signal sequences. Indicated are the relative positions of the EcoRI and SacI sites and 3′Jκ probe used for Southern blot analysis. (b) Southern blot analysis of recombination products generated in H2ax−/− and Artemis−/− abl pre–B cell lines, either untreated or treated with STI571 for 72 h. EcoRI–SacI-digested genomic DNA was hybridized with the 3′Jκ probe. The bands corresponding to Jκ loci of the GL and CE configurations are indicated. The STI571-treated H2ax−/− cells harbor a band that likely represents a predominant VκJκ rearrangement. Blots were stripped and then probed with a TCR-β probe as a control for DNA content. These data are representative of experiments performed >10 independent times. (c) Schematic diagram of the mouse Igκ locus and graphical representation of γ-H2AX densities as determined by ChIP at locations along DNA strands within and adjacent to Igκ in Artemis−/− abl pre–B cells treated with STI571 for 96 h. The 0-kb value of the x-axis corresponds to the 3′ end of the Jκ5 coding segment. The negative and positive values represent the distances centromeric and telomeric, respectively, from the 3′ end of Jκ5. Red and green bars indicate the approximate genomic locations to which the 5′ Vκ (RP24-243E11) and 3′ Cκ (RP23-341D5) BACs hybridize. The lengths of these bars are not drawn to scale. These data are representative of experiments performed >20 independent times. Error bars indicate standard deviation of three independent experiments.

Mentions: Because RAG-dependent formation of γ-H2AX occurs at Jκ segments and over adjacent sequences extending away from the Igκ locus (Savic et al., 2009), we also considered that H2AX might be required for resolution of Jκ CEs. The mouse Igκ locus resides on chromosome 6 and is composed of 140 Vκs spanning 2 Mb and four functional Jκs spanning 1.8 kb and residing 60 kb from the Vκs. Igκ locus V(D)J recombination occurs through the coupled cleavage and subsequent joining of a Vκ and Jκ segment. Because of the small size of the Jκ cluster, Southern blot analysis with a 3′ Jκ probe can be used to monitor and quantify the induction of RAG-generated Igκ locus DSBs (Fig. 2 a). Thus, we next conducted Southern blotting of H2ax−/− pre–B cells either untreated or treated with STI571 for 72 h. As a control for the accumulation of unrepaired Jκ CEs, we also conducted Southern blot analysis of previously described Artemis−/− cells (Helmink et al., 2009). We observed decreased intensity of the Jκ GL band in both H2ax−/− and Artemis−/− cells treated with STI571, and appearance of bands corresponding to Jκ CEs in STI571-treated Artemis−/− cells but not in STI571-treated H2ax−/− cells (Fig. 2 a). These data indicate that H2AX deficiency does not result in the detectable accumulation of unrepaired Igκ coding ends in G1-phase cells. Consequently, we conclude that H2AX function is not required for chromosomal end joining during V(D)J recombination in G1-phase lymphocytes.


Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination.

Yin B, Savic V, Juntilla MM, Bredemeyer AL, Yang-Iott KS, Helmink BA, Koretzky GA, Sleckman BP, Bassing CH - J. Exp. Med. (2009)

No accumulation of Jκ coding ends in the absence of H2AX phosphorylation along RAG-cleaved Igκ DNA strands. (a) Shown are schematic diagrams of the Jκ cluster of the Igκ locus in the uncleaved (GL) and cleaved but not repaired (CE) configurations. Open boxes represent the Jκ segments and triangles their recombination signal sequences. Indicated are the relative positions of the EcoRI and SacI sites and 3′Jκ probe used for Southern blot analysis. (b) Southern blot analysis of recombination products generated in H2ax−/− and Artemis−/− abl pre–B cell lines, either untreated or treated with STI571 for 72 h. EcoRI–SacI-digested genomic DNA was hybridized with the 3′Jκ probe. The bands corresponding to Jκ loci of the GL and CE configurations are indicated. The STI571-treated H2ax−/− cells harbor a band that likely represents a predominant VκJκ rearrangement. Blots were stripped and then probed with a TCR-β probe as a control for DNA content. These data are representative of experiments performed >10 independent times. (c) Schematic diagram of the mouse Igκ locus and graphical representation of γ-H2AX densities as determined by ChIP at locations along DNA strands within and adjacent to Igκ in Artemis−/− abl pre–B cells treated with STI571 for 96 h. The 0-kb value of the x-axis corresponds to the 3′ end of the Jκ5 coding segment. The negative and positive values represent the distances centromeric and telomeric, respectively, from the 3′ end of Jκ5. Red and green bars indicate the approximate genomic locations to which the 5′ Vκ (RP24-243E11) and 3′ Cκ (RP23-341D5) BACs hybridize. The lengths of these bars are not drawn to scale. These data are representative of experiments performed >20 independent times. Error bars indicate standard deviation of three independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig2: No accumulation of Jκ coding ends in the absence of H2AX phosphorylation along RAG-cleaved Igκ DNA strands. (a) Shown are schematic diagrams of the Jκ cluster of the Igκ locus in the uncleaved (GL) and cleaved but not repaired (CE) configurations. Open boxes represent the Jκ segments and triangles their recombination signal sequences. Indicated are the relative positions of the EcoRI and SacI sites and 3′Jκ probe used for Southern blot analysis. (b) Southern blot analysis of recombination products generated in H2ax−/− and Artemis−/− abl pre–B cell lines, either untreated or treated with STI571 for 72 h. EcoRI–SacI-digested genomic DNA was hybridized with the 3′Jκ probe. The bands corresponding to Jκ loci of the GL and CE configurations are indicated. The STI571-treated H2ax−/− cells harbor a band that likely represents a predominant VκJκ rearrangement. Blots were stripped and then probed with a TCR-β probe as a control for DNA content. These data are representative of experiments performed >10 independent times. (c) Schematic diagram of the mouse Igκ locus and graphical representation of γ-H2AX densities as determined by ChIP at locations along DNA strands within and adjacent to Igκ in Artemis−/− abl pre–B cells treated with STI571 for 96 h. The 0-kb value of the x-axis corresponds to the 3′ end of the Jκ5 coding segment. The negative and positive values represent the distances centromeric and telomeric, respectively, from the 3′ end of Jκ5. Red and green bars indicate the approximate genomic locations to which the 5′ Vκ (RP24-243E11) and 3′ Cκ (RP23-341D5) BACs hybridize. The lengths of these bars are not drawn to scale. These data are representative of experiments performed >20 independent times. Error bars indicate standard deviation of three independent experiments.
Mentions: Because RAG-dependent formation of γ-H2AX occurs at Jκ segments and over adjacent sequences extending away from the Igκ locus (Savic et al., 2009), we also considered that H2AX might be required for resolution of Jκ CEs. The mouse Igκ locus resides on chromosome 6 and is composed of 140 Vκs spanning 2 Mb and four functional Jκs spanning 1.8 kb and residing 60 kb from the Vκs. Igκ locus V(D)J recombination occurs through the coupled cleavage and subsequent joining of a Vκ and Jκ segment. Because of the small size of the Jκ cluster, Southern blot analysis with a 3′ Jκ probe can be used to monitor and quantify the induction of RAG-generated Igκ locus DSBs (Fig. 2 a). Thus, we next conducted Southern blotting of H2ax−/− pre–B cells either untreated or treated with STI571 for 72 h. As a control for the accumulation of unrepaired Jκ CEs, we also conducted Southern blot analysis of previously described Artemis−/− cells (Helmink et al., 2009). We observed decreased intensity of the Jκ GL band in both H2ax−/− and Artemis−/− cells treated with STI571, and appearance of bands corresponding to Jκ CEs in STI571-treated Artemis−/− cells but not in STI571-treated H2ax−/− cells (Fig. 2 a). These data indicate that H2AX deficiency does not result in the detectable accumulation of unrepaired Igκ coding ends in G1-phase cells. Consequently, we conclude that H2AX function is not required for chromosomal end joining during V(D)J recombination in G1-phase lymphocytes.

Bottom Line: Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation.Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation.We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre-B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-alpha/delta locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.

Show MeSH
Related in: MedlinePlus