ChIP-seq analysis of the C. elegans CEBP-1 protein. Caenorhabditis elegans

Purpose: The goal of this study is to identify genes selectively associated with C. elegans CEBP-1. Methods: We generated transgenic animals expressing FLAG-tagged CEBP-1 in a cebp-1(tm2807) mutant background (cebp-1(0); juIs418 [Pcebp-1::FLAG::CEBP-1::cebp-1 3’UTR]) and then immunoprecipitated FLAG-CEBP-1-associated DNA fragments using anti-FLAG antibodies (M2 anti-FLAG magnetic beads; Sigma). We collected mixed stage worms grown at 20˚C on NGM plates followed by 2% formaldehyde and sonicated the samples as described (Mukhopadhyay et al., 2008). We next generated ChIP-seq DNA libraries. Briefly, both ChIPed DNA and input genomic DNA were ligated to specific adaptors and amplified by barcode primers followed by sequencing on the Illumina HiSeq-2000 platform. We performed two independent ChIP-seq experiments, with parallel genomic DNA controls prepared from the same strain. We conducted peak-calling using CLC genomics workbench 6.0 (CLCbio). To define genes associated with the peaks, we used the annotation of transcription start site (TSS) and transcription end site (TES) from WS220 and annotated the peak if it overlapped the gene or the 3 kb upstream of the TSS. We then manually confirmed the peaks and associated genes using UCSC browser and update to WS252. Results: We found 209 CEBP-1 ChIP-seq peaks in the genome that were associated with 212 coding genes. Through motif discovery tools, we also found that CEBP-1 binds conserved DNA motifs. Conclusions: The conservation of C.elegans CEBP-1 binding motif supports functional parallels between C. elegans CEBP-1 and vertebrate C/EBPs. Overall design: CEBP-1 ChIP and input sample

**研究目的**：本研究旨在筛选出与秀丽隐杆线虫CEBP-1特异性相关的基因。
**实验方法**：我们在cebp-1(tm2807)突变体背景（cebp-1(0); juIs418 [Pcebp-1::FLAG::CEBP-1::cebp-1 3’UTR]）中构建了表达FLAG标签CEBP-1的转基因动物，随后使用抗FLAG抗体（Sigma公司M2抗FLAG磁珠）免疫沉淀与FLAG-CEBP-1结合的DNA片段。我们收集了在20℃条件下于NGM培养基平板上培养的混合发育阶段线虫，经2%甲醛固定后，按照已发表方法（Mukhopadhyay等，2008）对样本进行超声破碎。接下来我们制备了染色质免疫共沉淀测序（ChIP-seq）DNA文库：简言之，将免疫沉淀得到的DNA与输入基因组DNA分别连接特异性接头，通过条码引物进行扩增，随后在Illumina HiSeq-2000测序平台上完成测序。本研究共开展两次独立的ChIP-seq实验，并设置了来自同一菌株的平行基因组DNA对照。我们使用CLC生物公司的CLC基因组工作台6.0进行峰识别分析。为定义与峰相关的基因，我们采用WS220版本的转录起始位点（TSS）和转录终止位点（TES）注释，若峰与基因区域或转录起始位点上游3kb区域重叠，则判定该峰与该基因相关。随后我们通过UCSC基因组浏览器手动验证了峰及对应的关联基因，并将注释版本更新至WS252。
**实验结果**：我们在全基因组范围内鉴定出209个与CEBP-1结合的ChIP-seq峰，这些峰对应212个编码基因。通过基序发现工具分析，我们还发现CEBP-1可结合保守的DNA基序。
**研究结论**：秀丽隐杆线虫CEBP-1的结合基序具有保守性，这提示秀丽隐杆线虫CEBP-1与脊椎动物C/EBPs存在功能上的相似性。
**整体实验设计**：CEBP-1染色质免疫共沉淀样本及输入对照样本