ChIP-seq analysis of genome-edited MCF7 and T47D ESR1 mutant cell models

This study is designed to comprehensively characterize the cistromes of Y537S and D538G mutated ER versus WT ER in breast cancer cells. Genome-edited MCF7 and T47D cells were hormone deprived and treated with or without E2 for 45 minuts. Chromatin DNA was then extracted from each sample. The immunoprecipitation was performed using ERa (Santa Cruz Biotechnologies, sc543) antibody. Pooled DNA samples from individual clones were sent to sequencing with Illumina Hiseq 2500 Platform. ChIP-seq reads were aligned to either hg38 genome assembly using Bowtie 2.0, and peaks were called using MACS2.0 with p value below 10E-5. DiffBind was used to perform principle component analysis, identify differentially expressed binding sites and analyze intersection ratios with other data sets. Genomic feature distribution were called using ChIPseeker. Overall design: Individual clones of genome-eidted MCF7 and T47D cell lines with knock-in of WT/Y537S/D538G ER were hromone deprived and treated with veh or 1 nM E2 for 45 minutes. Chromatin DNA was then extracted from each sample. The immunoprecipitation was performed using ERa (sc543) antibody (Santa Cruz Biotechnologies). Pooled DNA samples from individual clones were sent to sequencing with Illumina Hiseq 2500 Platform.

本研究旨在全面表征乳腺癌细胞中Y537S、D538G突变型雌激素受体（ER）相较于野生型（WT）ER的顺式调控组（cistrome）。采用基因编辑的MCF7与T47D细胞系，经激素剥夺处理后，分别施加17β-雌二醇（E2）或溶剂对照处理45分钟。随后提取各样本的染色质DNA，使用ERα（Santa Cruz Biotechnologies，货号sc543）抗体开展免疫沉淀实验。将各单克隆细胞株的混合DNA样本送至Illumina Hiseq 2500平台进行高通量测序。使用Bowtie 2.0将染色质免疫共沉淀测序（ChIP-seq）读数比对至hg38基因组组装版本，并通过MACS2.0识别结合峰，设置p值阈值为10^-5。采用DiffBind软件开展主成分分析、差异结合位点识别及与其他数据集的交集比例分析；使用ChIPseeker注释基因组特征的分布情况。本研究的整体实验设计如下：将携带野生型（WT）/Y537S/D538G ER的基因编辑MCF7与T47D单克隆细胞株经激素剥夺处理后，分别以溶剂（veh）或1 nM E2处理45分钟。后续实验步骤与前述一致：提取各样本的染色质DNA，使用ERα（Santa Cruz Biotechnologies，货号sc543）抗体进行免疫沉淀，将各单克隆细胞株的混合DNA样本送至Illumina Hiseq 2500平台完成测序。