CG methylated microarrays identify novel methylated sequence bound by the CEBPB|ATF4 heterodimer that are active in vivo. Mus musculus

To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. EMSA confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50X methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. mRNA-seq of primary female mouse dermal fibroblasts with and without thapsigargin identified differentially expressed genes. Genes that are commonly bound by CEBPB and ATF4 to TGAT|GCAA (the best-bound 8-mer in the array) at the promoters were highly expressed and up-regulated or remained unchanged in the thapsigargin treated primary female mouse dermal fibroblasts. Overall design: RNA-Seq: Examination of whole genome transcriptome profiles (RNA-seq) of primary mouse dermal fibroblasts with and without Thapsigargin treatment ChIP-Seq: Examination of transcription factor binding in dermal fibroblasts with and without Thapsigargin teratment BS-Seq: Determination of whole genome DNA methylation profiles (BS-seq) of primary mouse dermal fibroblasts

为以高通量方式评估CG甲基化对序列特异性B-ZIP转录因子（transcription factors, TFs）DNA结合活性的影响，我们对蛋白质结合微阵列（protein binding microarrays, PBMs）上CG二核苷酸中的胞嘧啶进行了酶促甲基化修饰。借助这一新型技术，我们发现CG甲基化可增强CEBPA与CEBPB的结合能力，同时抑制CREB、ATF4、JUN、JUND、CEBPD及CEBPG的结合能力。CEBPB|ATF4异二聚体对新型基序CGAT|GCAA的结合亲和力在甲基化后提升了10倍。电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA）验证了上述结果。
我们使用覆盖度达50X（每条链）的原代雌性小鼠皮肤成纤维细胞甲基化组数据开展CEBPB染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）分析，结果表明，在微阵列上结合能力较强的甲基化序列同样可在体内结合。CEBPB可结合小鼠基因组中39%的甲基化经典10聚体序列ATTGC|GCAAT。在毒胡萝卜素（thapsigargin）诱导ATF4蛋白表达、引发内质网应激（endoplasmic reticulum stress, ER stress）后，CEBPB可在体内结合甲基化的CGAT|GCAA基序，这与我们在微阵列上观察到的结果一致。
对经毒胡萝卜素处理与未处理的原代雌性小鼠皮肤成纤维细胞进行mRNA测序（messenger RNA sequencing, mRNA-seq），鉴定出差异表达基因。在启动子区域同时被CEBPB与ATF4结合至TGAT|GCAA（该基序为微阵列上结合能力最强的8聚体）的基因，在经毒胡萝卜素处理的原代雌性小鼠皮肤成纤维细胞中呈高表达、上调或表达水平无变化。
整体实验设计：
- RNA测序（mRNA-seq）：分析经毒胡萝卜素处理与未处理的原代小鼠皮肤成纤维细胞的全基因组转录组谱；
- 染色质免疫共沉淀测序（ChIP-seq）：分析经毒胡萝卜素处理与未处理的皮肤成纤维细胞的转录因子结合情况；
- 亚硫酸氢盐测序（bisulfite sequencing, BS-seq）：测定原代小鼠皮肤成纤维细胞的全基因组DNA甲基化谱。