Unknown,Transcriptomics,Genomics,Proteomics

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DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts

ABSTRACT: High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered. Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (M-NM-3H2AX), which occurs around DNA double-strand breaks and contributes to their repair. By chromatin immunoprecipitation using anti-M-NM-3H2AX antibody, followed by massive parallel sequencing (ChIP-Seq), we find that during repair of doubleM-bM-^@M-^Sstrand breaks induced by high NaCl, M-NM-3H2AX is predominantly localized to regions of the genome devoid of genes (M-bM-^@M-^\gene desertsM-bM-^@M-^]), indicating that the high NaCl-induced double-strand breaks are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation and oxidants. We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes. ChIP-Seq experiment to find locations of M-NM-3H2AX in mouse genome

ORGANISM(S): Mus musculus

SUBMITTER: Iouri Chepelev

PROVIDER: E-GEOD-32882 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts.

Dmitrieva Natalia I NI Cui Kairong K Kitchaev Daniil A DA Zhao Keji K Burg Maurice B MB

Proceedings of the National Academy of Sciences of the United States of America 20111121 51

High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered. The exact nature of the breaks, and their location, has not been entirely clear, and it has not been evident how cells survive, replicate, and maintain genome integrity in environments like the renal inner medulla in which cells are constantly exposed to high NaCl concentration. Repair o ...[more]

PMID: 22106305

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Project description:Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. ChIP-seq, combined with RNA-seq, indicating that PRC2 is also associated with active genes, but most of these are not regulated by PRC2. These results were complemented by in vitro binding assays measuring the binding constant of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 and irrelevant transcripts from ciliates and bacteria. PRC2 binding is size-dependent, with lower affinity for shorter RNAs. These findings support a model in which promiscuous binding of PRC2 to RNA transcripts allows it to scan for target genes that have escaped repression, leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2. Cell culture: HEK293T/17 cells were cultured in DMEM with 10% FBS for no longer than 15 passages. ON-TARGETplus SMARTpool for human SUZ12 (Thermo Scientific, Dharmacon cat # L-006957-00-0005) was used to knockdown SUZ12 (siSUZ12) and ON-TARGETplus Non-targeting Pool (Dharmacon cat # D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using LipofectamineM-bM-^DM-" RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturerM-bM-^@M-^Ys recommendations. RNA-seq: Polyadenylated RNA was purified from 4 ug of RNA. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer. ChIP-seq: 30 million cells at 80% to 90% confluent culture were crosslinked in 1% v/v formaldehyde for 10 min, quenched in 150 mM glycine, washed with cold 1xPBS and harvested by scraping. Cells were lysed in Lysis Buffer (1% w/v SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with 1x CompleteM-BM-. protease inhibitors (Roche). Cells were sonicated for 10 to 15 min using a BioruptorM-bM-^DM-" UCD-200 (Diagenode) with 30 sec pulses at maximum power. Lysates were diluted to 10 ml in IP Buffer (0.01% w/v SDS, 1.1% v/v Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCL pH 8.0, 167 mM NaCl) and 10 to 20 ng of antibodies were added and incubated overnight at 4 M-bM-^AM-0C with rotation. Antibodies were immunoprecipitated with 60 M-BM-5l protein G Plus/protein A Agarose Suspension (Calbiochem cat # IP05) and washed sequentially with 1 ml Low Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), 1 ml High Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 ml LiCl Wash Buffer (0.25 M LiCl, 1% v/v Nonidet P-40, 1% w/v deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and 1 ml TE buffer (Qiagen). DNA was eluted with 0.4 ml of 0.1 M NaHCO3 and 1% w/v SDS. Eluent was transferred into a new tube and NaCl was added to a final concentration of 200 mM. Crosslinks were reversed by incubation at 65 M-bM-^AM-0C for 2 hours. Next, proteins and RNA were digested by adding 33 M-BM-5l of Digestion Reagent (0.6 M Tris-HCl pH 6.5, 152 mM EDTA, 61 ng/ml RNase A (Invitrogen cat # AM2274) and 0.61 mg/ml Proteinase K (NEB cat # P8102)) following by 1 hour incubation at 37 M-bM-^AM-0C. DNA was extracted by phenol:chloroform and ethanol precipitated. DNA was resuspended in Milli-Q pure water and concentration was measured using QubitM-bM-^DM-" (Invitrogen). At least 10 ng of recovered DNA was used to synthesize sequencing libraries using the ChIP-seq Sample Preparation kit (Illumina). Between 6 and 10 pmoles were used for sequencing on the HiSeq2000 sequencer.

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Dataset Information

DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts

Publications

DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts.

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