Abstract：
Gonadal steroid hormones, namely, testosterone, progesterone, and estrogens, influence the physiological state of an organism through the regulation of gene transcription. Steroid hormones activate nuclear hormone receptor (HR), transcription factors (TFs), which bind DNA in a tissue- and cell type-specific manner to influence cellular function. Identifying the genomic binding sites of HRs is essential to understanding mechanisms of hormone signaling across tissues and disease contexts. Traditionally, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been used to map the genomic binding of HRs in cancer cell lines and large tissues. However, ChIP-seq lacks the sensitivity to detect TF binding in small numbers of cells, such as genetically defined neuronal subtypes in the brain. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) resolves most of the technical limitations of ChIP-seq, enabling the detection of protein-DNA interactions with as few as 100-1000 cells. In this chapter, we provide a stepwise CUT&RUN protocol for detecting and analyzing the genome-wide binding of estrogen receptor α (ERα) in mouse brain tissue. The steps described here can be used to identify the genomic binding sites of most TFs in the brain.
摘要：
性腺类固醇激素,即,睾丸激素，黄体酮,和雌激素，通过调节基因转录来影响生物体的生理状态。类固醇激素激活核激素受体（HR），转录因子（TFs），以组织和细胞类型特异性的方式结合DNA以影响细胞功能。鉴定HR的基因组结合位点对于理解跨组织和疾病背景的激素信号传导机制至关重要。传统上,染色质免疫沉淀随后进行测序（ChIP-seq）已用于绘制癌症细胞系和大组织中HR的基因组结合图。然而,ChIP-seq缺乏检测少量细胞中TF结合的敏感性，比如大脑中基因定义的神经元亚型。靶标下的切割和核酸酶下的释放（CUT和RUN）解决了ChIP-seq的大多数技术限制，能够检测100-1000个细胞的蛋白质-DNA相互作用。在这一章中,我们提供了一个逐步的CUT和RUN方案，用于检测和分析小鼠脑组织中雌激素受体α（ERα）的全基因组结合。本文描述的步骤可用于鉴定脑中大多数TF的基因组结合位点。
