Genome-wide analysis of 8-oxoguanine DNA glycosylase-1 after oxidative stress exposure. Homo sapiens

We report the application of ChIP-Sequencing for profiling genome-wide distribution of 8-oxoguanine DNA glycosylase1 (OGG1, a DNA base excision repair protein), after oxidative stress exposure mediated by glucose oxidase (GOx) of cells. By obtaining an average of over 18 million of total reads per sample and over 19.5 million of unique reads per sample from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of HEK293 cells. We performed gene ontology and functional pathway analysis of genes associated with peaks to identify regions of the genomes (e.g. promoter, introns, etc) where the peaks tend to occur. The results show that OGG1 is primarily associated with promoter regions in vicinity of transcription factor binding sites 5' of transcription start sites (TSS). This pattern of distribution occurs in spite of genome-wide oxidative modifications of guanines (primarily 8-oxoG). OGG1 increased significantly at regulatory regions of CXC-, CC- chemokines, cytokines and growth factors. In controls, the DNA was chromatin immunoprecipitated using antibody to NF-κB/RelA. As expected NF-κB was enriched on gene regulatory regions including those of proinflammatory,cell proliferation ones. These and other data derived from further analysis, suggest that OGG1 modulates gene expression in coordination with NF-κB. Overall design: FLAG-tagged OGG1 expressing HEK293 cells were stimulated with GOx (1 µU) for 0, 15, 30 and 60 min. After GOx exposure, protein-DNA complexes were crosslinked using formalin according to standard Millipore® protocol. DNA was sheared with 10-sec pulses using a Cole-Parmer GEX 130 ultrasonic processor (average 300 bp) and chromatin was immunoprecipitated (IP) with antibody to FLAG or anti-RelA/p65 antibody and IPs were collected using G-agarose beads (Millipore Corporation). After extraction, the DNA was subjected to high-throughput sequencing using Solexa/Illumina genome analyzer (Ambry Genetics).

本研究报道了在细胞经葡萄糖氧化酶（glucose oxidase, GOx）介导的氧化应激处理后，利用染色质免疫共沉淀测序（ChIP-Sequencing）对8-氧鸟嘌呤DNA糖基化酶1（8-oxoguanine DNA glycosylase1, OGG1，一种DNA碱基切除修复蛋白）的全基因组分布谱进行分析的应用。本研究从染色质免疫沉淀得到的DNA中，每个样本平均获得超过1800万条总测序读段与1950万条唯一测序读段，进而构建了HEK293细胞的全基因组染色质状态图谱。我们对与结合峰相关的基因开展基因本体论（Gene Ontology, GO）与功能通路分析，以明确结合峰偏好富集的基因组区域（如启动子、内含子等）。结果显示，OGG1主要定位于转录起始位点（transcription start site, TSS）5'端的转录因子结合位点附近的启动子区域。尽管全基因组范围内的鸟嘌呤氧化修饰（主要为8-氧鸟嘌呤，8-oxoG）广泛存在，OGG1仍呈现上述分布模式。在CXC趋化因子、CC趋化因子、细胞因子与生长因子的调控区域，OGG1的富集水平显著升高。对照组中，我们使用NF-κB/RelA抗体进行染色质免疫沉淀以获取靶DNA。如预期一致，NF-κB在基因调控区域（包括促炎、细胞增殖相关基因的调控区域）呈现显著富集。上述结果与后续分析得到的其他数据共同表明，OGG1可与NF-κB协同调控基因表达。
实验整体设计：将表达FLAG标签融合OGG1的HEK293细胞以1 µU的GOx分别处理0、15、30与60分钟。GOx处理结束后，按照密理博®（Millipore®）标准操作规程，使用福尔马林对蛋白质-DNA复合物进行交联。随后使用Cole-Parmer GEX 130超声破碎仪以10秒脉冲的方式对DNA进行片段化处理（平均片段长度为300 bp），分别使用FLAG抗体与抗-RelA/p65抗体进行染色质免疫沉淀（immunoprecipitation, IP），并利用G-琼脂糖磁珠（密理博公司，Millipore Corporation）回收免疫沉淀产物。DNA提取完成后，使用Solexa/Illumina基因组分析仪（安百利遗传学公司，Ambry Genetics）进行高通量测序。