Department of Pathology, Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China.
School of Basic Medical Sciences, Central South University, Changsha, Hunan, People's Republic of China.
Clin Exp Metastasis. 2020 Aug;37(4):477-487. doi: 10.1007/s10585-020-10045-z. Epub 2020 Jul 1.
MACC1 (metastasis associated in colon cancer 1) is a key driver that induces metastasis in colon cancer. However, the mechanisms by which MACC1 expression is transcriptionally regulated and the factors enriched at the MACC1 promoter remain largely unknown. The binding of proteins to specific DNA sites in the genome is a major determinant of genomic maintenance and the regulation of specific genes. The study herein utilized two methods to study the binding proteins of the MACC1 promoter region in colon cancer. Specifically, we adopted CRISPR-based chromatin affinity purification with mass spectrometry (CRISPR-ChAP-MS) and a biotin-streptavidin pulldown assay coupled with MS to identify the specific proteome bound to the MACC1 promoter in two colon cell lines with different metastatic potential. A total of 24 proteins were identified by CRISPR-ChAP-MS as binding to the MACC1 promoter, among which c-JUN was validated by ChIP-PCR. A total of 739 binding protein candidates were identified by biotin-streptavidin pulldown assays coupled with MS, of which HNF4G and PAX6 were validated and compared for their binding to the same promoter sites in the two cell lines. Our studies suggest distinctive proteomic factors associated with the MACC1 promoter in colon cells with different metastatic potential. The dynamic regulatory factors accumulated at the promoter of MACC1 may provide novel insights into the regulatory mechanisms of MACC1 transcription.
MACC1（结肠癌转移相关基因 1）是诱导结肠癌转移的关键驱动因子。然而，MACC1 表达转录调控的机制以及在 MACC1 启动子富集的因子在很大程度上仍然未知。蛋白质与基因组中特定 DNA 位点的结合是基因组维持和特定基因调控的主要决定因素。本研究采用两种方法研究了具有不同转移潜能的两种结肠癌细胞系中 MACC1 启动子区域的结合蛋白。具体来说，我们采用基于 CRISPR 的染色质亲和纯化与质谱（CRISPR-ChAP-MS）和生物素-链霉亲和素下拉测定与 MS 相结合的方法，鉴定与 MACC1 启动子结合的特定蛋白质组。CRISPR-ChAP-MS 鉴定出 24 种与 MACC1 启动子结合的蛋白质，其中 c-JUN 通过 ChIP-PCR 进行验证。生物素-链霉亲和素下拉测定与 MS 相结合共鉴定出 739 种结合蛋白候选物，其中 HNF4G 和 PAX6 被验证并比较了它们在两种细胞系中与相同启动子位点的结合。我们的研究表明，在具有不同转移潜能的结肠细胞中，MACC1 启动子与独特的蛋白质组学因素有关。在 MACC1 启动子上积累的动态调节因子可能为 MACC1 转录的调节机制提供新的见解。
