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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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H2AX suppresses separation of RAG-cleaved Igκ locus DNA strands. (a) Shown are representative fluorescent light microscopy images of 2C-FISH analysis conducted on G1-phase nuclei of Rag2−/−, Artemis−/−, and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. Nuclei were hybridized with the 5′ Vκ (red) and 3′ Cκ (green) BACs and stained with DAPI to visualize DNA. The representative Rag2−/− image shows a nucleus with coincident probe hybridization signals on both Igκ alleles. The top nucleus contains paired Igκ alleles and the bottom unpaired Igκ alleles. The representative Artemis−/− and Artemis−/−H2ax−/− images each shows nuclei with coincident probe signals on both Igκ alleles and paired Igκ alleles (top) or noncoincident probe signals on one Igκ allele, overlapping probe hybridization on the other Igκ allele, and unpaired Igκ alleles (bottom). Bars, ∼3 µm. (b) Shown are representative scatter plots depicting the distances between red and green signals on allele 1 (shorter distance) and allele 2 (longer distance) in G1-phase nuclei of Rag2−/−, Artemis−/−, and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. The numbers of nuclei assayed to generate the representative data are indicated. These data are representative of experiments performed three independent times. (c) Shown are bar graphs depicting in arbitrary units the nuclei with separated RAG-cleaved Igκ DNA strands normalized to the extent of cutting in three experiments conducted on cells of independent Artemis−/− and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. To obtain these data, the percentage of nuclei with separated signals was divided by the percentage of RAG-cleaved Igκ alleles within the population of treated cells. The graphs use either 1 µm (left) or 1.5 µm (right) as the cutoff for distinction between coincident or overlapping versus noncoincident probe hybridization signals. The p-values for comparison between cells of different genotypes are indicated. These data were obtained from the same experiment performed three independent times. Error bars indicate standard deviation of three independent experiments.
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fig3: H2AX suppresses separation of RAG-cleaved Igκ locus DNA strands. (a) Shown are representative fluorescent light microscopy images of 2C-FISH analysis conducted on G1-phase nuclei of Rag2−/−, Artemis−/−, and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. Nuclei were hybridized with the 5′ Vκ (red) and 3′ Cκ (green) BACs and stained with DAPI to visualize DNA. The representative Rag2−/− image shows a nucleus with coincident probe hybridization signals on both Igκ alleles. The top nucleus contains paired Igκ alleles and the bottom unpaired Igκ alleles. The representative Artemis−/− and Artemis−/−H2ax−/− images each shows nuclei with coincident probe signals on both Igκ alleles and paired Igκ alleles (top) or noncoincident probe signals on one Igκ allele, overlapping probe hybridization on the other Igκ allele, and unpaired Igκ alleles (bottom). Bars, ∼3 µm. (b) Shown are representative scatter plots depicting the distances between red and green signals on allele 1 (shorter distance) and allele 2 (longer distance) in G1-phase nuclei of Rag2−/−, Artemis−/−, and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. The numbers of nuclei assayed to generate the representative data are indicated. These data are representative of experiments performed three independent times. (c) Shown are bar graphs depicting in arbitrary units the nuclei with separated RAG-cleaved Igκ DNA strands normalized to the extent of cutting in three experiments conducted on cells of independent Artemis−/− and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. To obtain these data, the percentage of nuclei with separated signals was divided by the percentage of RAG-cleaved Igκ alleles within the population of treated cells. The graphs use either 1 µm (left) or 1.5 µm (right) as the cutoff for distinction between coincident or overlapping versus noncoincident probe hybridization signals. The p-values for comparison between cells of different genotypes are indicated. These data were obtained from the same experiment performed three independent times. Error bars indicate standard deviation of three independent experiments.

Mentions: We first conducted 2D-2C-FISH using these 5′ Vκ and 3′ Cκ probes on G1 interphase nuclei prepared from three independent Rag2−/− abl pre–B cells treated with STI571 for 96 h (Fig. 3 a). We measured the distances between 5′ Vκ (red) and 3′ Cκ (green) signals on both alleles in ∼200 nuclei of each cell line assayed, designated the shorter distance from allele 1 and the longer distance from allele 2, and plotted these values onto scatter plots. We observed overlapping or coincident probe hybridization signals (<1 µm apart) on both alleles in >95% of nuclei and noncoincident signals on one allele in <4% of nuclei (Fig. 3 b). Using three independent Artemis−/− cell lines, we observed overlapping or coincident probe signals on both alleles in ∼80% of nuclei and noncoincident probe signals on a single allele in ∼20% of nuclei (Fig. 3 b). With three independent Artemis−/−H2ax−/− cell lines, we observed overlapping or coincident probe signals on both alleles in ∼60% of nuclei and noncoincident probe signals on a single allele in ∼40% of nuclei (Fig. 3 b). Although similar levels of unrepaired Igκ locus CEs accumulated in all Artemis−/− and Artemis−/−H2ax−/− cells assayed (not depicted), we also normalized the percentage of nuclei with noncoincident probe hybridization signals to the extent of Igκ locus cleavage (Fig. 3 c). These data show that RAG-cleaved Igκ locus DNA strands physically separate in a significantly higher percentage of Artemis−/−H2ax−/− cells than in Artemis−/− cells. Similar results were obtained using a larger distance (>1.5 µm) to score noncoincident hybridization (Fig. 3 c). Consequently, we conclude that γ-H2AX–mediated chromatin changes suppress physical separation of RAG-cleaved antigen receptor loci in G1-phase cells to prevent their irreversible disassociation or aberrant joining.


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)

H2AX suppresses separation of RAG-cleaved Igκ locus DNA strands. (a) Shown are representative fluorescent light microscopy images of 2C-FISH analysis conducted on G1-phase nuclei of Rag2−/−, Artemis−/−, and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. Nuclei were hybridized with the 5′ Vκ (red) and 3′ Cκ (green) BACs and stained with DAPI to visualize DNA. The representative Rag2−/− image shows a nucleus with coincident probe hybridization signals on both Igκ alleles. The top nucleus contains paired Igκ alleles and the bottom unpaired Igκ alleles. The representative Artemis−/− and Artemis−/−H2ax−/− images each shows nuclei with coincident probe signals on both Igκ alleles and paired Igκ alleles (top) or noncoincident probe signals on one Igκ allele, overlapping probe hybridization on the other Igκ allele, and unpaired Igκ alleles (bottom). Bars, ∼3 µm. (b) Shown are representative scatter plots depicting the distances between red and green signals on allele 1 (shorter distance) and allele 2 (longer distance) in G1-phase nuclei of Rag2−/−, Artemis−/−, and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. The numbers of nuclei assayed to generate the representative data are indicated. These data are representative of experiments performed three independent times. (c) Shown are bar graphs depicting in arbitrary units the nuclei with separated RAG-cleaved Igκ DNA strands normalized to the extent of cutting in three experiments conducted on cells of independent Artemis−/− and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. To obtain these data, the percentage of nuclei with separated signals was divided by the percentage of RAG-cleaved Igκ alleles within the population of treated cells. The graphs use either 1 µm (left) or 1.5 µm (right) as the cutoff for distinction between coincident or overlapping versus noncoincident probe hybridization signals. The p-values for comparison between cells of different genotypes are indicated. These data were obtained from the same experiment performed three independent times. Error bars indicate standard deviation of three independent experiments.
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Related In: Results  -  Collection

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Show All Figures
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fig3: H2AX suppresses separation of RAG-cleaved Igκ locus DNA strands. (a) Shown are representative fluorescent light microscopy images of 2C-FISH analysis conducted on G1-phase nuclei of Rag2−/−, Artemis−/−, and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. Nuclei were hybridized with the 5′ Vκ (red) and 3′ Cκ (green) BACs and stained with DAPI to visualize DNA. The representative Rag2−/− image shows a nucleus with coincident probe hybridization signals on both Igκ alleles. The top nucleus contains paired Igκ alleles and the bottom unpaired Igκ alleles. The representative Artemis−/− and Artemis−/−H2ax−/− images each shows nuclei with coincident probe signals on both Igκ alleles and paired Igκ alleles (top) or noncoincident probe signals on one Igκ allele, overlapping probe hybridization on the other Igκ allele, and unpaired Igκ alleles (bottom). Bars, ∼3 µm. (b) Shown are representative scatter plots depicting the distances between red and green signals on allele 1 (shorter distance) and allele 2 (longer distance) in G1-phase nuclei of Rag2−/−, Artemis−/−, and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. The numbers of nuclei assayed to generate the representative data are indicated. These data are representative of experiments performed three independent times. (c) Shown are bar graphs depicting in arbitrary units the nuclei with separated RAG-cleaved Igκ DNA strands normalized to the extent of cutting in three experiments conducted on cells of independent Artemis−/− and Artemis−/−H2ax−/− abl pre–B cells treated with STI571 for 96 h. To obtain these data, the percentage of nuclei with separated signals was divided by the percentage of RAG-cleaved Igκ alleles within the population of treated cells. The graphs use either 1 µm (left) or 1.5 µm (right) as the cutoff for distinction between coincident or overlapping versus noncoincident probe hybridization signals. The p-values for comparison between cells of different genotypes are indicated. These data were obtained from the same experiment performed three independent times. Error bars indicate standard deviation of three independent experiments.
Mentions: We first conducted 2D-2C-FISH using these 5′ Vκ and 3′ Cκ probes on G1 interphase nuclei prepared from three independent Rag2−/− abl pre–B cells treated with STI571 for 96 h (Fig. 3 a). We measured the distances between 5′ Vκ (red) and 3′ Cκ (green) signals on both alleles in ∼200 nuclei of each cell line assayed, designated the shorter distance from allele 1 and the longer distance from allele 2, and plotted these values onto scatter plots. We observed overlapping or coincident probe hybridization signals (<1 µm apart) on both alleles in >95% of nuclei and noncoincident signals on one allele in <4% of nuclei (Fig. 3 b). Using three independent Artemis−/− cell lines, we observed overlapping or coincident probe signals on both alleles in ∼80% of nuclei and noncoincident probe signals on a single allele in ∼20% of nuclei (Fig. 3 b). With three independent Artemis−/−H2ax−/− cell lines, we observed overlapping or coincident probe signals on both alleles in ∼60% of nuclei and noncoincident probe signals on a single allele in ∼40% of nuclei (Fig. 3 b). Although similar levels of unrepaired Igκ locus CEs accumulated in all Artemis−/− and Artemis−/−H2ax−/− cells assayed (not depicted), we also normalized the percentage of nuclei with noncoincident probe hybridization signals to the extent of Igκ locus cleavage (Fig. 3 c). These data show that RAG-cleaved Igκ locus DNA strands physically separate in a significantly higher percentage of Artemis−/−H2ax−/− cells than in Artemis−/− cells. Similar results were obtained using a larger distance (>1.5 µm) to score noncoincident hybridization (Fig. 3 c). Consequently, we conclude that γ-H2AX–mediated chromatin changes suppress physical separation of RAG-cleaved antigen receptor loci in G1-phase cells to prevent their irreversible disassociation or aberrant joining.

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